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The Role of Mitochondrial DNA Mutations in Sarcopenia

Permanent Link: http://ufdc.ufl.edu/UFE0021067/00001

Material Information

Title: The Role of Mitochondrial DNA Mutations in Sarcopenia Implications for the Mitochondrial 'Vicious Cycle' Theory and Apoptosis
Physical Description: 1 online resource (153 p.)
Language: english
Creator: Hiona, Asimina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: apoptosis, chain, cycle, dysfunction, electron, membrane, mitochondrial, muscle, oxidative, oxygen, potential, reactive, sarcopenia, skeletal, species, stress, transport, vicious
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Aging results in a progressive loss of skeletal muscle, a condition termed sarcopenia which can have significant effects on physical function and quality of life as aging commences. At the cellular level, the aging process can activate stress-associated signal transduction pathways that result in mitochondrial dysfunction and apoptosis. Because the mitochondrion contains its own DNA, a central role for mitochondrial DNA (mtDNA) mutations in mammalian aging has been postulated. In fact, mtDNA mutations have been shown to accumulate with aging in skeletal muscle fibers of various species. The purpose of my dissertation project was to determine whether mtDNA mutations are causal to sarcopenia. The central hypothesis tested was that mutations in mitochondrial DNA, known to be associated with aging in many post mitotic tissues, play a causal role in skeletal muscle loss, possibly by inducing mitochondrial dysfunction, leading to the activation of a mitochondrial-mediated apoptotic program. In order to demonstrate a causal relationship between mtDNA mutations and skeletal muscle loss with age, we used a transgenic mouse model that expresses a proofreading-deficient version of the mitochondrial DNA polymerase gamma (PolgD257A), resulting in increased spontaneous mutation rates in mtDNA. The causal role of mtDNA mutations in mammalian aging is supported in this mouse model by the observation that mice with the PolgD257A (D257A) phenotype develop several aging phenotypes among which, is skeletal muscle loss. We specifically hypothesized that the accumulation of mtDNA mutations in skeletal muscle will lead to compromised mitochondrial bioenergetics. We found that D257A mice have decreased protein content of complexes I, III and IV, all of which contain subunits encoded by mitochondrial DNA, compared to wild type (WT) mice at 11-mo of age. Mitochondrial dysfunction was also evident in D257A mice by decreased mitochondrial oxygen consumption, lower membrane potential during both state 3 (phosphorylative state) and state 4 (resting state), and lower ATP content. However, this dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. In fact, we detected a decrease in the rate of H2O2 production by intact D257A mitochondria and no difference in mtDNA oxidative modification measured by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), compared to WT. This is in contrast to the mitochondrial 'Vicious Cycle' theory of aging which suggests that mtDNA mutations may lead to mitochondrial dysfunction via further increases in mitochondrial ROS production. We further hypothesized that mitochondrial dysfunction will result in mitochondrial-mediated apoptosis, which would be responsible for the loss of skeletal muscle mass we have observed in D257A mice. We detected DNA laddering and an increase in the amount of cytosolic mono- and oligo-nucleosomes in D257A mice compared to WT, indicative of apoptosis. Concurrently, we demonstrated increased activity of both, the initiator caspase-9, and the effector caspase-3, as well as an increase in cleaved (activated) caspase-3 content. This suggests that apoptosis in mutant mice is mitochondrial-mediated, and is conferred upon mitochondrial dysfunction. Thus, mutations in mtDNA play a causal role in sarcopenia, through enhancing apoptosis induced by mitochondrial dysfunction.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Asimina Hiona.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Leeuwenburgh, Christiaan.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021067:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021067/00001

Material Information

Title: The Role of Mitochondrial DNA Mutations in Sarcopenia Implications for the Mitochondrial 'Vicious Cycle' Theory and Apoptosis
Physical Description: 1 online resource (153 p.)
Language: english
Creator: Hiona, Asimina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: apoptosis, chain, cycle, dysfunction, electron, membrane, mitochondrial, muscle, oxidative, oxygen, potential, reactive, sarcopenia, skeletal, species, stress, transport, vicious
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Aging results in a progressive loss of skeletal muscle, a condition termed sarcopenia which can have significant effects on physical function and quality of life as aging commences. At the cellular level, the aging process can activate stress-associated signal transduction pathways that result in mitochondrial dysfunction and apoptosis. Because the mitochondrion contains its own DNA, a central role for mitochondrial DNA (mtDNA) mutations in mammalian aging has been postulated. In fact, mtDNA mutations have been shown to accumulate with aging in skeletal muscle fibers of various species. The purpose of my dissertation project was to determine whether mtDNA mutations are causal to sarcopenia. The central hypothesis tested was that mutations in mitochondrial DNA, known to be associated with aging in many post mitotic tissues, play a causal role in skeletal muscle loss, possibly by inducing mitochondrial dysfunction, leading to the activation of a mitochondrial-mediated apoptotic program. In order to demonstrate a causal relationship between mtDNA mutations and skeletal muscle loss with age, we used a transgenic mouse model that expresses a proofreading-deficient version of the mitochondrial DNA polymerase gamma (PolgD257A), resulting in increased spontaneous mutation rates in mtDNA. The causal role of mtDNA mutations in mammalian aging is supported in this mouse model by the observation that mice with the PolgD257A (D257A) phenotype develop several aging phenotypes among which, is skeletal muscle loss. We specifically hypothesized that the accumulation of mtDNA mutations in skeletal muscle will lead to compromised mitochondrial bioenergetics. We found that D257A mice have decreased protein content of complexes I, III and IV, all of which contain subunits encoded by mitochondrial DNA, compared to wild type (WT) mice at 11-mo of age. Mitochondrial dysfunction was also evident in D257A mice by decreased mitochondrial oxygen consumption, lower membrane potential during both state 3 (phosphorylative state) and state 4 (resting state), and lower ATP content. However, this dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. In fact, we detected a decrease in the rate of H2O2 production by intact D257A mitochondria and no difference in mtDNA oxidative modification measured by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), compared to WT. This is in contrast to the mitochondrial 'Vicious Cycle' theory of aging which suggests that mtDNA mutations may lead to mitochondrial dysfunction via further increases in mitochondrial ROS production. We further hypothesized that mitochondrial dysfunction will result in mitochondrial-mediated apoptosis, which would be responsible for the loss of skeletal muscle mass we have observed in D257A mice. We detected DNA laddering and an increase in the amount of cytosolic mono- and oligo-nucleosomes in D257A mice compared to WT, indicative of apoptosis. Concurrently, we demonstrated increased activity of both, the initiator caspase-9, and the effector caspase-3, as well as an increase in cleaved (activated) caspase-3 content. This suggests that apoptosis in mutant mice is mitochondrial-mediated, and is conferred upon mitochondrial dysfunction. Thus, mutations in mtDNA play a causal role in sarcopenia, through enhancing apoptosis induced by mitochondrial dysfunction.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Asimina Hiona.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Leeuwenburgh, Christiaan.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021067:00001


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316cbb77c01033a1c0d9799727a642a1dcfd0047







THE ROLE OF MITOCHONDRIAL DNA MUTATIONS IN SARCOPENIA: IMPLICATIONS
FOR THE MITOCHONDRIAL "VICIOUS CYCLE" THEORY AND APOPTOSIS






















By


ASIMINA HIONA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































2007 Asimina Hiona



























To my family Thank you for your unwavering love and support.

In the memory of my deceased grandfather Lambros who I cherished so much Thank you for
the great childhood memories.









ACKNOWLEDGMENTS

This work would not have been possible without the support and guidance of several

important people. First, I thank my family for their love and support during these difficult years.

Their support has not been only moral but also financial. Without their help it would have been

impossible for me to complete a Ph.D especially. I also thank my parents for teaching me some

very important values in life: determination, persistence and how to always rely on my abilities

and be an honest and fair person. Without their love and support I never could have come this

far.

From my colleagues I first and foremost thank Dr. Barry Drew for his scientific guidance

during my first years in the lab and his friendship. I appreciate everything that he taught me and

more importantly, his ability to make the lab such an enjoyable environment to work in. He is a

best friend of mine for life. I also thank Stephane Servais for his guidance and great mentorship

regarding my project and my future career. I thank Alberto Sanz from Spain for all his

contributions to my thesis and all the laughs we had during my thesis studies. I want to especially

thank Rizwan Kalani, an outstanding undergraduate student in our lab and current medical

student for always helping me during big studies, whenever I needed him. A special thanks to

Alex Samhan Arias and Miguel Garcia from Spain that have been so helpful to me during their

short stay in our lab. They made me laugh a lot and became great friends. Moreover, I thank

Suma Kendhayia and Evelyn Kowenhoven for their help in the lab. I would also like to thank my

advisor Christiaan Leeuwenburgh for all the opportunities I had in the lab during my Ph.D.

From University of Wisconsin, Madison, I especially thank Dr. Tomas Prolla. Without his

help, guidance and mentoring I wouldn't have the thesis project I have, and great career

opportunities. I also thank Dr. Greg Kujoth, a post-doc in Dr. Prolla's lab for constructing the









transgenic mice I used for my project, and for always being so helpful with providing me with

transgenic mice for my studies and answering my multiple questions.

Importantly, I thank my committee members drs Meyer, Kilberg and Kaushal, for being

such great, insightful, and helpful committee members. I would also like to thank them for their

mentoring and for being so helpful to me during my applications for post-doc positions. They

really made my journey to the final defense much easier. Individually, I thank first and foremost

Dr. Meyer for tremendously helping me with my future career. I think that words do not do

justice to how much I appreciate his help. I thank Dr. Kilberg for being so supportive of my

transfer to the IDP, which I believe was one of the best things out of my Ph.D, since I enjoyed

my classes so much and the extra knowledge, and for all his help regarding my job applications.

Last, but not least, I thank Dr. Kaushal for all his help with my future career and for believing in

me. If I had to choose anyone of the three to be my advisor I would do it in a heartbeat but it

would be a very hard choice.

Finally, and most importantly, I want to thank two very important people I met while in

Gainesville: Giorgos Leonis, who's been an amazing friend all these years and Joel French, for

loving me and for being the most supportive boyfriend a girl could ever have.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..............................................................................................................4

LIST OF FIGURES .................................. .. .. .... .... ................. 10

A B S T R A C T ............ ................... ............................................................ 12

CHAPTER

1 INTRODUCTION AND HYPOTHESES.................................... ............................ ........ 14

Specific Aim 1. Determine the Effect of mtDNA Mutations in Skeletal Muscle
M itochondrial F unction ............................................................................. .................... 15
H y p oth esis 1 ....................................... .. ................. .......... ... ................... 16
Specific Aim 2. Determine Whether Increased Load of mtDNA Mutations Leads to
A poptosis in Skeletal M u scle ................................................................... ..................... 17
H hypothesis 2 .................... .................... .... ...... ........ ........ ..................... 17
Specific Aim 3. Identify the Specific Apoptotic Signaling Pathway Responsible for
Sarcopenia in D 257A M ice ............................................................................... ........... 17
H y p oth esis 3 ............................................................................. 18

2 BACKGROUND AND SIGNIFICANCE.................................... ............................. ....... 19

In tro d u ctio n ................................... ..... ... .......................................................... ............... 19
Age-Related Changes in M itochondrial Function ................................. ... ........................20
Electron Transport Chain Abnormalities and Mitochondrial DNA Mutations in Aging .......21
Suggested Molecular Mechanisms for the Propagation of mtDNA Mutations, and
Potential Reasons for the Greater Occurrence of mtDNA Mutations Compared to
N nuclear D N A w ith A ge.................................... .... ..... .......................................24
Mitochondrial DNA Damage and the Mitochondrial 'Vicious Cycle' Theory of Aging.......26
Challenging the Mitochondrial 'Vicious Cycle' Theory of Aging.......................................28
Direct Evidence for a Causal Role of mtDNA Mutations in Aging: D257A Mice................29
Mitochondrial Dysfunction, Apoptosis and Skeletal Muscle Aging/Sarcopenia .................30
Mitochondrial-Mediated Pathways of Apoptosis. ............................................................34

3 M A TER IA L S A N D M ETH O D S ........................................ .............................................41

Experim mental D design ........................ ........ ...... .. ............ .....41
General Procedures.............................. .. .... .... ..................41
Animals................................................. 41
M itochondrial and Cytosolic Isolation ........................................ ........................ 42
S p e cific M eth o d s ................................................................ ..... ................... .................4 3
Specific Aim 1. Effect of mtDNA Mutations on Skeletal Muscle Mitochondrial
F u n ctio n ............................................................................ 4 3
R atio n ale ...................................... ................................................. 4 4









Experim ental approach.................................................. ............................... 45
M itochondrial H202 generation............. .............................. 45
M itochondrial respiration........................................................... ............... 47
A TP content and production ............................................................................. 47
M itochondrial m embrane potential .............. .... .. ............ ................. ....48
Blue native page (BN-page) for determination of content and enzymatic
activity of respiratory com plexes..................................................... .................. 49
Determination of protein content of selected mitochondrial- and nuclear-
encoded subunits from ETC complexes I, II, III and IV ....................................50
Determ nation of m itochondrial protein yield ....................................................... 51
Determination of MnSOD and Catalase mRNA expression by RT-PCR ................52
O xidative dam age to m tD N A ................................................................................... 52
Specific Aim 2. mtDNA Mutations and Apoptosis in Skeletal Muscle..........................53
R atio n a le ................... ...................5...................4..........
Experimental approach.................. .................................... 54
Determination of cleaved caspase-3 content.........................................................54
Enzymatic measurement of caspase 3 activity .....................................................55
Determination of cytosolic mono- and oligonucleosomes .............. ...............56
D N A laddering ...................... ............. ..... ..................... ....... ............... ...... 56
Specific Aim 3. Identification of the Specific Apoptotic Signaling Pathway
Responsible for Skeletal Muscle Loss in D257A Mice................... ....... .........57
R a tio n a le ................................................................................................................... 5 7
Experimental approach................................................... 58
Determination of cytochrome c content by Western Blotting..............................58
Enzymatic measurement of caspase-9 activity............... ....................................59
Statistical A analyses ............... ........... .......................... ............................59

4 R E SU L T S ...........................................................................................6 1

Mouse Characterization Data from Dr. Prolla's Lab: Generation and Phenotype of
D 2 5 7 A M ice .......................................................................... 6 1
D ata from O ur Lab ....................................................... ...... ...... .. ............ .. 62
Results for Specific Aim 1 .............. ......... ....... .... ............................................ 62
Impaired mitochondrial bioenergetics in 11-month-old D257A mice ...................62
D257A mice display decreased content of ETC Complexes I, III, and IV that
contain mtDNA-encoded subunits.............................. ... ....................... 63
Electron transport chain complex specific activity remains unaffected by
mtDNA mutations in D257A mice................ ...... .. .................64
D257A mice show decreased content of both nuclear-encoded and
m itochondrial-encoded ETC subunits ........................................ .....................64
D257A mice display decreased ATP content...................................... ...............65
Mitochondrial membrane potential is significantly lower in D257A mice..............65
Mitochondrial protein yield is reduced in skeletal muscle of D257A mice.............66
Skeletal muscle mitochondria from D257A mice produce significantly less
R O S ...................................................................................... 6 6
D257A mitochondria produce less ROS in both main ROS generators of the
E TC : C om plex I and C om plex III ............................................. .....................67









No difference in antioxidant enzyme mRNA expression between genotypes .........68
Mitochondrial DNA mutations cause aging phenotypes in the absence of
increased oxidative stress .................................. .....................................69
R results for Specific A im 2 ...................................... .............. ... ........................69
D257A mice display significant skeletal muscle loss by 11-mo of age...................69
Apoptosis in D257A skeletal muscle is evident by an increase in cytosolic
m ono- and oligo-nucleosom es.................................... ........................... ......... 70
DNA laddering is evident in skeletal muscle of D257A mice ...............................70
Caspase-3 cleavage and activation is up-regulated in D257A mice and
resembles caspse-3 activation during normal aging ..........................................70
Results for Specific Aim 3 ............................................ .......................... ...............72
Cytochrome c release in the cytosol of D257A and WT skeletal muscle ..............72
Caspase-3 and caspase-9 activities are significantly higher in D257A mice:
Evidence for induction of the mitochondrial, caspase-dependent pathway of
a p o p to sis ............................................................................................................... 7 2

5 D ISC U S SIO N ............................................................................... 89

O verview of P principal F indings.................................................................................... ... 89
Hypothesis One: The Effect of mtDNA Mutations in Skeletal Muscle Mitochondrial
F u n c tio n ........................................ ....... .. ............................................. .................... .9 0
Mitochondrial DNA Mutations Cause Profound Deficiencies in Mitochondrial
F u n ctio n ......................................... ........ ....... .................. .............................. 9 1
Mitochondrial DNA Mutations Cause Mitochondrial Dysfunction in the Absence of
Increased ROS Production or Oxidative Damage to mtDNA: Implications for the
Mitochondrial "Vicious Cycle" Theory of Aging ....................................................94
Mitochondrial DNA Mutations Lead to Mitochondrial Dysfunction, Via Alterations
of ETC Com plex Com position .............................................................................. 101
Total Skeletal Muscle Mitochondrial Protein Yield Continuously Decreases as
Time Progresses in M utant M ice ................................ .. ... .. ..................106
Hypothesis Two: the Effect of mtDNA Mutations on Skeletal Muscle Apoptosis.............. 109
A poptosis w ith A ging................................ .......... .................... ............... 110
M itochondrial DNA M stations and Apoptosis ...........................................................111
Disruption of Mitochondrial Membrane Potential and Role for Apoptosis ................13
Apoptosis is Evident in Skeletal Muscle of D257A Mice..........................................15
Hypothesis Three: Identify the Specific Apoptotic Signaling Pathway Responsible for
Sarcopenia in D 257A M ice .......................................................................... ................ 116
Proposed Mechanism for the Skeletal Muscle Loss Induced by High Load of Somatic
m tD N A M u station s ....... .......... .... ...................................................... .......... .. .. 1 19
S y n o p sis .....................................................................12 1
C onclu sions.. .........................................................123
F u tu re D ire ctio n s ............................................................................................................ 12 4

APPENDIX: ADDITIONAL FIGURES .................................................... 128

LIST O F R EFEREN CE S ....................................................................................................132









B IO G R A PH IC A L SK E T C H ............................................................................... ............... ..... 153









LIST OF FIGURES


Figure pe

2-1 Contributions of the mitochondrial and nuclear DNA to protein subunits of the
com plexes of the ETC ............................................................... .......... 39

2-2 The mitochondrial 'vicious cycle' theory. ........................................................................39

2-3 M itochondrial-m ediated apoptosis............................................................................... ....40

3-1 Experimental design and summary of the parameters measured in specific aims 1, 2
a n d 3 .......................................................... ............................... . 6 0

4-1 D257A mice display a premature aging phenotype. ................... .................74

4-2 Mitochondrial respiration is compromised in skeletal muscle of D257A mice................74

4-3 D257A mice display decreased content of ETC Complexes I, III and IV that contain
m tD N A -encoded subunits ...................... .... ......... .... ........................ ............... 75

4-4 Statistical analysis of ETC complex I, II, III, IV and the Fl domain of the ATPase
content measured by Blue Native Page in skeletal muscle of 11-mo old WT and
D 2 57A m ice.. ............................................................................... 76

4-5 Electron transport chain complex activity in skeletal muscle of 11-mo old WT and
D 257A m ice. ...............................................................................77

4-6 Statistical analysis of ETC complex activity in skeletal muscle of 11-mo old WT and
D 257A m ice. ...............................................................................78

4-7 D257A mice show decreased content of both nuclear-encoded and mitochondrial-
encoded ETC subunits. ................................ ......... ......................79

4-8 D257A mice display decreased ATP content. ............... ................... ...................80

4-9 Mitochondrial membrane potential (Ay) drop in D257A mice.................. ...............80

4-10 Mitochondrial yield is reduced in D257A skeletal muscle............... .....................81

4-11 D257A mitochondria produce less reactive oxygen species (ROS) during state 4 ...........81

4-12 D257A mitochondria produce less ROS in both main ROS generators: Complex I
and Com plex III............................ ......... .............. ............. .......... ......... 82

4-13 D257A mice show no difference in antioxidant enzyme mRNA expression ...................83

4-14 Mitochondrial DNA oxidation in skeletal muscle of WT and D257A mice .....................84









4-15 D257A mice display significant skeletal muscle loss by 11-mo of age compared to
age-m watched W T ................................................................. ....... .........84

4-16 Apoptosis evident in D257A muscle by increase in cytosolic mono- and oligo-
n u cleo so m e s ........................................................ ................ 8 5

4-17 DNA laddering evident in skeletal muscle of D257A mice ................... ........ ........85

4-18 Caspase-3 activation in skeletal muscle of D257A mice resembles caspase-3
activation during norm al aging. ...... ........................... .......................................86

4-19 Cytochrome c release in the cytosol of D257A and WT skeletal muscle..........................87

4-20 Caspase-3 and -9 activities are elevated in D257A muscle: Proof of activation of the
mitochondrial caspase-dependent pathway of apoptosis ................................................88

4-21 Caspase-3 and caspase-9 activity Pearson correlations in WT and D257A mice..............88

5-1 Proposed mechanism for the skeletal muscle loss induced by high load of somatic
m tDN A m stations ...................................................... .......... ..... ......... 127

A-i Skeletal muscle mass gastrocnemiuss) in 3-mo old (N=8 per group), and 11-mo old
(N=11 per group), W T and D257A mice.................................... ......................... 128

A-2 Caspase-3 activation in gastrocnemius muscle..................................... ............... 128

A-3 Mitochondrial respiration in skeletal muscle of 3-mo old mice .................................... 129

A-4 Reactive oxygen species production during state 4 in isolated mitochondria from 3-
m o o ld m ice ............................................................................13 0

A-5 Protein expression of nuclear-encoded and mitochondrial-encoded ETC subunits in
skeletal muscle of 3-mo old and 11-mo old WT and D257A mice ...............................131








Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLE OF MITOCHONDRIAL DNA MUTATIONS IN SARCOPENIA: IMPLICATIONS
FOR THE MITOCHONDRIAL "VICIOUS CYCLE" THEORY AND APOPTOSIS

By

ASIMINA HIONA

August 2007

Chair: Christiaan Leeuwenburgh
Major: Medical Sciences--Biochemistry and Molecular Biology

Aging results in a progressive loss of skeletal muscle, a condition termed sarcopenia

which can have significant effects on physical function and quality of life as aging commences.

At the cellular level, the aging process can activate stress-associated signal transduction

pathways that result in mitochondrial dysfunction and apoptosis. Because the mitochondrion

contains its own DNA, a central role for mitochondrial DNA (mtDNA) mutations in mammalian

aging has been postulated. In fact, mtDNA mutations have been shown to accumulate with aging

in skeletal muscle fibers of various species. The purpose of my dissertation project was to

determine whether mtDNA mutations are causal to sarcopenia. The central hypothesis tested was

that mutations in mitochondrial DNA, known to be associated with aging in many post mitotic

tissues, play a causal role in skeletal muscle loss, possibly by inducing mitochondrial dysfunction,

leading to the activation of a mitochondrial-mediated apoptotic program. In order to demonstrate a

causal relationship between mtDNA mutations and skeletal muscle loss with age, we used a

transgenic mouse model that expresses a proofreading-deficient version of the mitochondrial

DNA polymerase gamma (PolgD257A), resulting in increased spontaneous mutation rates in

mtDNA. The causal role of mtDNA mutations in mammalian aging is supported in this mouse









model by the observation that mice with the PolgD257A (D257A) phenotype develop several

aging phenotypes among which, is skeletal muscle loss.

We specifically hypothesized that the accumulation of mtDNA mutations in skeletal

muscle will lead to compromised mitochondrial bioenergetics. We found that D257A mice have

decreased protein content of complexes I, III and IV, all of which contain subunits encoded by

mitochondrial DNA, compared to wild type (WT) mice at 11-mo of age. Mitochondrial

dysfunction was also evident in D257A mice by decreased mitochondrial oxygen consumption,

lower membrane potential during both state 3 (phosphorylative state) and state 4 (resting state),

and lower ATP content. However, this dysfunction was not accompanied by an increase in

mitochondrial reactive oxygen species (ROS) production or oxidative damage. In fact, we

detected a decrease in the rate of H202 production by intact D257A mitochondria and no

difference in mtDNA oxidative modification measured by 8-oxo-7,8-dihydro-2'-deoxyguanosine

(8-oxodG), compared to WT. This is in contrast to the mitochondrial "Vicious Cycle" theory of

aging which suggests that mtDNA mutations may lead to mitochondrial dysfunction via further

increases in mitochondrial ROS production. We further hypothesized that mitochondrial

dysfunction will result in mitochondrial-mediated apoptosis, which would be responsible for the

loss of skeletal muscle mass we have observed in D257A mice. We detected DNA laddering and

an increase in the amount of cytosolic mono- and oligo-nucleosomes in D257A mice compared

to WT, indicative of apoptosis. Concurrently, we demonstrated increased activity of both, the

initiator caspase-9, and the effector caspase-3, as well as an increase in cleaved (activated)

caspase-3 content. This suggests that apoptosis in mutant mice is mitochondrial-mediated, and is

conferred upon mitochondrial dysfunction. Thus, mutations in mtDNA play a causal role in

sarcopenia, through enhancing apoptosis induced by mitochondrial dysfunction.









CHAPTER 1
INTRODUCTION AND HYPOTHESES

Aging individuals lose muscle mass at a rate of 1-2% per year past the age of 50 (1, 2).

This age-related muscle atrophy, termed sarcopenia, is associated with muscle weakness and can

have significant effects on an individual's health and quality of life. Sarcopenia affects a growing

population, occurring in 10-25% of individuals under the age of 70 and in more than 40% of the

elderly over the age of 80 (1, 2). The annual cost of treating sarcopenia is greater than the

amount spent due to osteoporosis, yet little effort is made to increase public awareness to prevent

sarcopenia (3). Thus, sarcopenia and the subsequent loss of physical function is a significant

public health problem.

Mitochondria are the main source of cellular ATP and play a central role in a variety of

cellular processes, including fatty acid P-oxidation, calcium signaling, reactive oxygen species

(ROS) generation and apoptosis. The aging process can introduce a variety of stressors that result

in the collapse of mitochondrial function, causing apoptotic cell death. Because the

mitochondrion contains its own -16-kilobase circular DNA, that is also intron-less and histone-

less, and close to the main ROS generator in the cell: the electron transport chain (ETC), a

central role for mitochondrial DNA (mtDNA) mutations in aging has been postulated (4-6).

Indeed, mtDNA mutations have been shown to accumulate with aging in several tissues of

various species (7-12), including skeletal muscle.

The central hypothesis tested in my research project is that mutations in mitochondrial

DNA, known to be associated with aging in many post mitotic tissues, play a causal role in

sarcopenia, possibly through enhancing apoptosis mediated by mitochondrial dysfunction.

Previous studies have provided strong experimental support for an association between

mitochondrial DNA mutations and tissue dysfunction, particularly in long-lived post-mitotic









cells such as cardiomyocytes, skeletal muscle fibers and neurons (7, 9-12). However, such

association studies can provide only correlative data. In order to determine whether mtDNA

mutations underlie the aging process, we used a genetically engineered mouse model that

expresses a proofreading-deficient version of the mitochondrial DNA polymerase gamma

(PolgD257A), resulting in increased spontaneous mutation rates in mitochondrial DNA. Previously

(13), we have characterized accelerated aging in D257A mice and found that these mice

exhibited various age-related phenotypes including thymic involution, loss of bone mass, cardiac

dysfunction and skeletal muscle loss. In mitochondria from the tissues examined we showed that

mtDNA mutations do not lead to increases in ROS production or oxidative stress, contrary to the

"free radical theory of aging." Importantly, in most tissues examined, including skeletal muscle,

we demonstrated increased levels of cleaved (activated) caspase-3, which is indicative of

apoptosis (13). Hence, the accumulation of mtDNA mutations may be associated with the

induction of apoptosis irrespective of elevations in ROS production and oxidative stress in

mitochondria with age. The D257A mouse provides an in vivo model to study the mechanisms of

apoptosis in skeletal muscle with age, specifically, the contribution of mtDNA mutations. Our

hypotheses have implications for both the basic biology of aging and clinical approaches to age-

related diseases of skeletal muscle, such as sarcopenia.

Specific Aim 1. Determine the Effect of mtDNA Mutations in Skeletal Muscle
Mitochondrial Function

We, as well as, others have shown that mice with a mitochondrial mutator phenotype

develop several aging phenotypes (13, 14). We found that D257A mice have significant skeletal

muscle loss by 11-mo of age compared to WT, which is indicative of sarcopenia. However, our

findings thus far, also indicate that despite increased mutational load, mitochondria from D257A

mice do not show increased levels of oxidative stress in all currently examined tissues (13).









These observations do not support the idea that mtDNA mutations contribute to increased ROS

production and oxidative stress in mitochondria with age, placing the mitochondrial "vicious

cycle" theory of aging in question. In this aim we determined whether mtDNA mutations lead to

mitochondrial dysfunction and further investigated the mechanism by which mutations induce

mitochondrial dysfunction. Since these mice have elevated levels of mtDNA mutations, we

expected that the structure and/or function of proteins encoded by mitochondrial DNA would be

affected, ultimately affecting mitochondrial bioenergetics, and leading to mitochondrial

dysfunction. The concentration, and enzymatic activity of respiratory complexes I, II, III, IV, and

F1Fo ATPase, mitochondrial respiration, basal oxidant production, ATP content and production,

mitochondrial membrane potential and oxidative damage to mtDNA were determined.

Hypothesis 1

We hypothesized that the accumulation of mtDNA mutations would lead to mitochondrial

dysfunction, due to alterations in the content and/or activity of the respiratory complexes I, III,

IV and F1Fo ATPase, which contain subunits encoded by mtDNA, leading to compromised ETC

activity in skeletal muscle of 11-mo-old D257A mice compared to WT. Decrease in ETC activity

would lead to a more extensive decrement in state 3 respiration, since this is the active state of

the mitochondria when electron flux is highest. We expected that the decrease in state 3

respiration would be associated with reduced ATP content and production in skeletal muscle

mitochondria, ultimately leading to mitochondrial dysfunction. We further hypothesized that

mitochondrial dysfunction would not be associated with increases in basal ROS production in the

D257A mice, compared to WT, and specifically, with increases in ROS production at the main

ROS generators of the ETC, Complex I and Complex III. Rather, mitochondrial dysfunction

would lead to loss of mitochondrial membrane potential and greater susceptibility of

mitochondria to apoptosis.









Specific Aim 2. Determine Whether Increased Load of mtDNA Mutations Leads to
Apoptosis in Skeletal Muscle

We have already demonstrated that D257A mice lose skeletal muscle and exhibit

significantly greater content of cleaved (activated) caspase-3 in skeletal muscle by 11-mo of age,

compared to WT mice (13). However, additional measures are needed in order to determine that

apoptosis is indeed a central mechanism responsible for skeletal muscle loss associated with the

accumulation of mtDNA mutations. In this aim, we wanted to corroborate and more extensively

investigate apoptosis in skeletal muscle by performing specific apoptotic measures. We further

measured caspase-3 activity, the content of mono- and oligonucleosomes released in the cytosol

following apoptotic DNA fragmentation, and DNA laddering, evident on agarose gel during

apoptotic nucleosomal fragmentation.

Hypothesis 2

We hypothesized that the levels of apoptosis would be elevated in skeletal muscle of

D257A mice compared to WT by 11-mo of age, which may explain the decline in skeletal

muscle mass we observed in D257A animals.

Specific Aim 3. Identify the Specific Apoptotic Signaling Pathway Responsible for
Sarcopenia in D257A Mice

The significant skeletal muscle loss in conjunction with the elevated cleaved caspase-3

levels in the D257A animals suggest that loss of critical, irreplaceable cells through apoptosis

may be a central mechanism of tissue dysfunction associated with the accumulation of mtDNA

mutations. In this aim we determined the pathway of skeletal muscle apoptosis in the D257A

mice. We speculated that the signaling pathway responsible for induction of apoptosis in D257A

skeletal muscle has to be intrinsic to the mitochondria since loss of muscle mass in these animals

is conferred upon accumulation of mtDNA mutations. Moreover, the mitochondrial pathway

may encompass both caspase-dependent and -independent induction. Since the caspase-









independent path still remains to be elucidated, we assessed the activation of key proteins from

the main mitochondrial-mediated, caspase-dependent pathway to evaluate whether this path is

activated in response to the increase in mtDNA mutational load.

Hypothesis 3

We hypothesized that mitochondrial dysfunction in D257A mice would ultimately lead to

mitochondrial outer membrane permeability and efflux of cytochrome c into the cytosol.

Cytochrome c release would instigate formation of the apoptosome, leading to cleavage and

activation of the initiator caspase-9. Caspase-9 would further cleave and activate the final

effector caspase-3 which is responsible for carrying out the actual proteolytic events that result in

cellular breakdown, leading to mitochondrial-mediated apoptosis.









CHAPTER 2
BACKGROUND AND SIGNIFICANCE

Introduction

Over the past two decades, increasing evidence suggests that mitochondrial dysfunction

may play a causal role in the aging process. The essential role of mitochondria in cellular ATP

production, the generation of reactive oxygen species (ROS), and the induction of apoptosis

suggest a number of mechanisms for mitochondrial pathology. There is now strong evidence that

age induces alterations in the mitochondrial genome that lead to defects in mitochondrial

function, especially in tissues with high energy requirements such as the heart, liver, brain and

skeletal muscle (15-18).

It was proposed that during an individual's life span, ROS, by-products of oxidative

metabolism, accumulate and alter cell components (19). Mitochondria, one of the primary

sources of ROS, are particularly affected, leading to changes in their structure as well as in the

genetic information of mtDNA. The observed alterations of mtDNA include oxidative damage to

DNA bases, point mutations and large scale deletions or duplications. MtDNA mutations are

known to have deleterious effects on oxidative phosphorylation, especially in patients with

mitochondrial diseases (20, 21), and tissues that rely heavily on oxidative phosphorylation are

expected to be more affected. We chose skeletal muscle as the focus tissue in this project

because this tissue is highly dependent on oxidative phosphorylation and suffers marked age-

related degeneration (sarcopenia).

The background information presented in this section first focuses on the changes that are

induced in mitochondrial function as a result of aging, and the cellular impact of mtDNA

mutations, by providing direct evidence for a causal role of mtDNA mutations in the aging

process. This is followed by an explanation of how mitochondrial dysfunction can lead to









apoptosis initiated by the mitochondria, and an examination of the specific pathways and

functions of the molecules involved in mitochondrial-mediated apoptosis. Although much of the

evidence suggests an important role for mtDNA mutations in aging, this evidence was largely

correlative until 2004 when Trifunovic et al. published the first results on the POLG mouse

model (14). We propose that this mouse model (also used in the present study), with altered

mitochondrial mutation rates, represents a valuable tool to critically assess in vivo the role of

mtDNA mutations on sarcopenia.

Age-Related Changes in Mitochondrial Function

The mitochondrial genome is a double-stranded, circular DNA molecule of 16,569 bp,

lacking histones and compactly organized (i.e., no introns) that, apart from a 1.1 kbp non-coding

D-loop, encodes for 13 protein components of the ETC (Fig 2-1), 22 tRNAs and 2 rRNAs. There

are approximately 2 to 10 copies of the mitochondrial genome per mitochondrion (22) and 10's to

100's of mitochondria per cell, depending on the cell's energy requirements (23).

Biochemical analyses of ETC complex activities performed in tissue homogenates from

humans, rhesus monkeys and rodents have, in general, identified age-associated decreases in the

activities of complexes I and IV. Those tissues in which robust biochemical declines have been

repeatedly detected are the highly oxidative, non-replicating tissues such as skeletal muscle, heart

and brain (16, 24-31). Commonly used markers for mitochondrial ETC abnormalities include the

loss of cytochrome c oxidase (COX) activity and the concomitant increase in succinate

dehydrogenase (SDH) activity (SDH hyperreactive regions, also known as ragged red fibers

(RRF)). In situ histochemical studies of human and monkey myocardial tissue have focused on

detection of cells deficient in COX activity (32, 33); in these studies, the number of

cardiomyocytes displaying defects in the ETC COX enzyme was found to increase with age in

humans from 2-3 defects/cm2 in the second and third decades, to 50 defects/cm2 in the fifth to ninth









decades (32). Recent cytochemical-immunocytochemical studies of oxidative phosphorylation

enzymes in monkeys (10-25 years of age) showed complex III, complex IV and complex V defects

in skeletal muscles, diaphragm, myocardium and extraocular muscles of 25-year-old animals.

These defects were randomly distributed and not associated with a loss of complex II, which is all

nuclear encoded (33). Furthermore, in rats, aged mitochondria exhibit lower mitochondrial

membrane potential (34), reduced cardiolipin levels (35-37), and a decrease in the activity of

camitine acetyltransferase, a key mitochondrial enzyme for fuel utilization (38). In general, the

main feature of these age-related alterations in post-mitotic tissues is the development of a shift

in activity ratios among different complexes, such that it would tend to hinder the ability of

mitochondria to effectively transfer electrons down the respiratory chain and thus, adversely

affect oxidative phosphorylation. In accordance with the above, energy depletion in the

mitochondria during aging is also evident; Our laboratory recently found that mitochondrial ATP

content and production in gastrocnemius muscle from aged rats significantly decreased, although

H202 production and mtDNA 8-oxodGuo levels were unchanged compared to young animals

(39). This decline observed in skeletal muscle may be a factor in the process of sarcopenia,

which increases in incidence with advancing age (39). Consistent with our findings, a decline in

human skeletal muscle mitochondrial ATP production with advancing age has recently been

observed (40). Eventually, energy depletion could impair important cellular functions including

damage repair/removal mechanisms, and also trigger apoptosis (41).

Electron Transport Chain Abnormalities and Mitochondrial DNA Mutations in Aging

There is growing evidence that the accumulation of mitochondrial mutations and deletions,

associated with aging, result in tissue dysfunction. For example, in the rat, the levels of a

particular deletion in mtDNA (4834 deletion) in the dorsal root ganglion were about 300-fold

higher in old compared to young rats. The abundance of this particular mtDNA deletion in









dorsal root ganglia from individual rats correlated strongly with their decline in function (42). In

normal aging, impaired respiratory function and oxidative phosphorylation in muscle fibers is

becoming increasingly evident (39, 40), and point-mutations and deletions in mtDNA have been

found to correlate with this reduced capacity (43-45). Cao et al., showed that fibers from the

femoris muscle of 38- month-old rats, with electron transport system abnormalities, also had

large mtDNA deletions (4.4-9.7 kb), whereas normal ETC fiber regions had wild-type mtDNA.

Deletions occurred at the major arc of the mtDNA spanning the origin of replication, and were

clonal within the fibers, with different deletions between the fibers (43). Similarly, Wanagat et

al, have demonstrated an age-related increase in skeletal muscle fibers that display ETC

abnormalities, and that, mtDNA deletion mutations, co-localize with segmental ETC

abnormalities (7). Specifically, they showed that the proportion of ETC abnormal fibers that

displayed the RRF phenotype (i.e., loss of COX activity with concomitant hyperactivation of

SDH activity) increased significantly with age, and there were no ETC abnormal fibers with the

RRF phenotype observed in the 5-month-old muscles, whereas 42% of the total ETS abnormal

fibers in the 38-month-old animals displayed the RRF phenotype. They further detected shorter

than wild type genomes in all of the RRFs, while mtDNA deletion mutations were not detected

in ETS normal fibers from the same sections. Multiple microdissections along the same RRF

amplified identically sized products, supporting the clonal nature of the mtDNA deletion (7).

Moreover, human studies also provide evidence for an increase in mtDNA mutations with

aging, and a correlation between mtDNA mutations and the occurrence of skeletal muscle

abnormalities with advancing age (44-46). Michikawa et al., showed that human fibroblast

mtDNA from normal old individuals, revealed high copy point mutations at specific positions in

the control region for replication, which was not evident in young subjects (47). Furthermore, in









longitudinal studies, one or more mutations appeared in an individual only at an advanced age.

Most strikingly, a T414G transversion was found, in a generally high proportion (up to 50%) of

mtDNA molecules, in 8 of 14 individuals above 65 years of age (57 percent) but was absent in

13 younger individuals (47). Wang et al., showed that muscle-specific mutations accumulate

with aging in critical human mtDNA control sites for replication; specifically, they demonstrated

that most of 26 individuals 53 to 92 years old, without a known history of neuromuscular

disease, exhibited an accumulation of two new point mutations, i.e., A189G and T408A, at

mtDNA replication control sites in muscle which were absent or marginally present in 19 young

individuals. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years

of age (T408A), or were present only in three subjects in very low amounts (A189G)(12). The

investigators suggested that the striking tissue specificity of the muscle mtDNA mutations

detected, and their mapping at critical sites for mtDNA replication, strongly point to the

involvement of a specific mutagenic machinery and to the functional relevance of these

mutations.

Latest experimental evidence also suggests that randomly deleted mtDNA appears mainly

in skeletal muscle of healthy old subjects (beyond 80 years old), affecting up to 70% of mtDNA

molecules, and coincides with a decrease in the activities of complexes III and IV of the ETC,

which contain subunits encoded by mtDNA (Fig 2-1) (48). Most importantly, high levels of

clonally expanded mtDNA point mutations were identified in cytochrome c oxidase deficient

muscle fibers, from old individuals without muscle disease, but in none of the normal fibers (49).

Immunohistochemical experiments showed that the majority of the cytochrome c oxidase

deficient muscle fibers expressed reduced levels of subunit II of cytochrome c oxidase, which is

encoded by mitochondrial DNA, whereas there was normal or increased expression of subunit IV









of cytochrome c oxidase, which is encoded by nuclear DNA (49). The authors concluded that

mtDNA point mutations are associated with cytochrome c oxidase deficient muscle fiber

segments in aging, the focal accumulation of which may cause significant impairment of

mitochondrial function in individual cells in spite of low overall levels of mitochondrial DNA

mutations in muscle (49). Indeed, although only a few cells develop COX deficiency, the

resultant cellular dysfunction might have substantial effects, especially if the cell is part of a

complex network-e.g., the central nervous system (50). It is therefore, likely that, in skeletal

muscle of aged individuals, normal mtDNA devoid of deletions or point mutations may represent

a minority of the total mtDNA pool. There is also evidence that the rate of mitochondrial

mutagenesis is faster in mice than humans per unit time (51), a necessary condition if

mitochondrial mutations are causally linked to aging. When taken as a whole, these studies

provide compelling evidence for an important role of mitochondrial DNA mutations in aging.

Suggested Molecular Mechanisms for the Propagation of mtDNA Mutations, and Potential
Reasons for the Greater Occurrence of mtDNA Mutations Compared to Nuclear DNA,
with Age

The cellular and physiological ramifications of mtDNA disruption have been first made

clear from studies of a broad class of neuromuscular disorders known as mitochondrial

myopathies and encephalomyopathies (reviewed by Wallace, 1999; DiMauro, 1993) (52, 53).

These diseases are, clinically and biochemically, a diverse group of disorders affecting primarily

those tissues having the highest energy demands: brain, skeletal muscle and heart. The mtDNA

abnormalities associated with these disorders were shown to range from point mutations (21, 54)

to large mtDNA deletions in the mitochondrial encephalomyopathies (53, 55). The mtDNA

abnormalities found in mitochondrial myopathies have been linked to many oxidative defects in

cells, with a very common defect being the RRF phenotype which is also common in skeletal

muscle fibers of aged individuals.









Mathematical models suggest that the same basic cellular mechanisms are responsible for

the amplification of mutant mtDNA in aging and in mtDNA diseases (50); Using an in silicon

model of mitochondrial genetic processes within individual non-dividing cells, which was based

on a contemporary understanding of "relaxed replication" of mtDNA (unlike nuclear DNA,

which replicates once during the cell cycle, mitochondrial DNA is degraded and replaced

continuously, even in non-dividing cells such as skeletal muscle fibers and central neurons),

Chinnery et al. introduced a copy-error (mutation) rate, and measured the proportion of

individual simulated cells over time (50). With this approach they were able to show that, even

for very rare somatic mutations, "relaxed replication" leads to random drift of the amount of

mutant mtDNA within the cell. This powerful mechanism alone leads to the clonal expansion of

mutant mtDNA during the lifetime of a person, and the accumulation of COX-negative cells at a

similar rate to that seen in vivo (50). They further showed that random genetic drift was also the

mechanism for the clonal expansion of mutant mtDNA in progressive mtDNA disease. The

mechanism proposed in this study is interesting, yet the actual process of propagation of somatic

mtDNA mutations remains to be determined.

Several other hypotheses regarding the propagation of mtDNA mutations and thus, of the

mutant mitochondria, have also been suggested. De Grey suggested in 1997 that it is precisely

the loss of superoxide production that gives mutant mitochondria and their DNA a selective

advantage and drives their clonal expansion: in this "survival of the slowest" hypothesis,

mitochondrial turnover by autophagy is driven by self-inflicted free radical damage to the

mitochondrial membranes, so a mutant mitochondrion is "less suicidal" and is more often

replicated simply because it is more long-lived (56). Another hypothesis, known as the "crippled









mitochondrion" hypothesis, states that, the internal biochemistry of mutant mitochondria

somehow stimulates them to replicate (57).

Furthermore, the greater occurrence of point-mutations and deletions observed in mtDNA

compared to nuclear DNA could be due to: a) a greater mtDNA exposure to reactants, b) the lack

of protective histones, and c) to a less advanced DNA repair system (58-60). In fact,

mitochondria are believed to entirely lack nucleotide excision repair (NER), which constitutes a

major nuclear defense system acting on various nDNA lesions including pyrimidine dimmers

(58, 59). Mitochondria also have other discrepancies and may also have less advanced mismatch

repair (MMR) (59). Like crosslinks between DNA bases (such as thymine dimmers), DNA-

protein crosslinks, or bulky DNA-adducts can cause a stall during mtDNA replication which can

induce DNA double-strand brakes (61, 62), contributing to the occurrence of mtDNA deletions

with aging, and as previous research suggests, it is likely for mtDNA containing deletions, to

acquire a replicative advantage over longer wild type mtDNA (43).

Mitochondrial DNA Damage and the Mitochondrial 'Vicious Cycle' Theory of Aging

Despite the fact that in animal cells mtDNA comprises only 1-3% of genetic material, several

lines of evidence suggest that its contribution to cellular physiology could be much greater than

would be expected from its size alone (63). For instance, (i) it mutates at higher rates than

nuclear DNA, which may be a consequence of its close proximity to the ETC (electron transfer

chain); (ii) it encodes either polypeptides of ETC or components required for their synthesis and,

therefore, any coding mutations in mtDNA will affect the ETC as a whole; this could affect both

the assembly and function of the products of numerous nuclear genes in ETC complexes; (iii)

defects in the ETC can have pleiotropic effects because they affect cellular energetic as a whole

(63).









Mitochondria have been shown to accumulate high levels of lipophilic carcinogens such as

polycyclic aromatic hydrocarbons (64, 65) which can preferentially damage mtDNA (66). Other

mutagenic chemicals also have been shown to preferentially target mtDNA (64, 67-69).

Therefore, it is conceivable that life-long exposure to certain environmental toxins could result in

a preferential accumulation of mtDNA damage and accelerate aging. However, by far, the

predominant kind of insult to which mtDNA is exposed is oxidative damage. The susceptibility

of the mitochondrial genome to oxidative DNA damage may be due to a number of factors

including: 1) its close proximity to the ETC, whose complexes I and III are believed to be the

predominant sites of ROS production inside the cell, 2) lack of protective histones and, 3) the

compactness of its genetic information is such that damage at any point in the genome will likely

occur in a gene. The phenotypic implications of mtDNA mutations are dependent on which gene

product is disrupted and one might predict that damage may occur in those complexes to which the

mitochondrial genome makes the greatest contributions.

The free radical theory of aging first put forward by Harman (4, 5, 70, 71) states that it is

the mitochondrial production of ROS, such as superoxide and H202, and the resulting

accumulation of damage to macromolecules that causes aging. Cumulative damage to biological

macromolecules was proposed to overwhelm the capacity of biological systems to repair

themselves, resulting in an inevitable functional decline (63). The mitochondrial 'vicious cycle'

theory of aging can be considered as an extension and refinement of the free radical theory (63).

Its major premise is that mtDNA mutations accumulate progressively during life, as a side effect

of respiration, and are directly responsible for a measurable deficiency in cellular oxidative

phosphorylation activity, leading to an enhanced ROS production (63). In turn, increased ROS

production results in an increased rate of mtDNA damage and mutagenesis, thus causing a









'vicious cycle' of exponentially increasing oxidative damage and dysfunction, which ultimately

culminates in death (Fig 2-2) (63).

Challenging the Mitochondrial 'Vicious Cycle' Theory of Aging

Bandy and Davison were the first to put forward a mechanistic elaboration of what later

became known as the mitochondrial 'vicious cycle' theory (72). While they showed that mtDNA

mutations may have the same effect on the respiratory chain as small-molecule inhibitors of

respiration, that is, to stimulate the one-electron reduction of molecular oxygen to superoxide

(therefore, increasing ROS production), they also carefully noted that not all mutations stimulate

superoxide production. Specifically, they pointed out that a mutation preventing the synthesis of

cytochrome b would actually abolish any superoxide production at complex III that normal

mitochondria might exhibit, because without cytochrome b in place, complex III cannot be

assembled (72). Later studies reported that respiration-deficient cells of several tissues possessed

mutations that would indisputably preclude assembly of both the enzyme complexes known to be

responsible for mitochondrial ROS production, complexes I and III (73-76). These mutations

were large deletions, which eliminated the genes for at least a couple of respiratory chain

subunits, but also removed at least one tRNA gene. There is no redundancy of tRNA genes in the

mtDNA, so the loss of any such gene abolishes the synthesis of all 13 mtDNA-encoded proteins

(77, 78). These findings are highly relevant to the plausibility of the 'vicious cycle' theory in

normal aging.

Recently, there is an increasing body of evidence challenging the vicious cycle theory of

aging (63, 78-80), and our results provide direct proof against the theory (13) (Figs 4-11, 4-12, 4-

14). In our specific aim #1 we tested the hypothesis that the accumulation of mtDNA mutations

indeed leads to impaired synthesis of mtDNA-encoded ETC subunits and loss of activity of the

mitochondrial complexes, which could explain the decrease in 02 consumption in conjunction









with the decrease in H202 production we have shown in the D257A skeletal muscle mitochondria

(Figs 4-2, 4-11, 4-12).

Direct Evidence for a Causal Role of mtDNA Mutations in Aging: D257A Mice

As already discussed, there is an ever growing body of research that supports an important

role for mtDNA mutations in aging by providing experimental support for an association

between mtDNA mutations, apoptosis and tissue dysfunction, particularly in long-lived post

mitotic cells such as cardiomyocytes and skeletal muscle fibers (7, 81). However, most of the

studies to date are correlative in nature. In particular, until recently, it has been unclear whether

mtDNA mutations are simply associated with aging in various tissues, or if they actually cause

alterations in tissue function. In 2004, Trifunovic et al. published the first experimental evidence

providing a causative link between mtDNA mutations and mammalian aging (14). They showed

that homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the

nucleus-encoded catalytic subunit of mtDNA polymerase, develop a mtDNA mutator phenotype

with a threefold to fivefold increase in the levels of point mutations, as well as increased

amounts of deleted mtDNA. This increase in somatic mtDNA mutations was associated with

reduced lifespan and premature onset of aging-related phenotypes such as alopecia, kyphosis,

osteoporosis, and heart enlargement. A year later we corroborated Trifunovic's findings

regarding the impact of mtDNA mutations in aging, using mice with the same mutation, and we

also showed that the accumulation of mtDNA mutations was not associated with increased levels

of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of

apoptotic markers, suggesting that accumulation of mtDNA mutations that promote apoptosis

may be a central mechanism driving mammalian aging (13).

Moreover, Zassenhaus and colleagues studied mice that express a proofreading-deficient

POLy specifically in the heart, and develop cardiac mtDNA mutations, in order to determine









whether low frequency mitochondrial mtDNA mutations are pathogenic. They found that

sporadic myocytic death occurred in all regions of the heart, due to apoptosis as assessed by

histological analysis and TUNEL staining (82). They also pointed out that cytochrome c was

released from mitochondria and concluded that mtDNA mutations are pathogenic, and seem to

trigger apoptosis through the mitochondrial pathway (82). The use of D257A mice in the present

investigation allowed us to elucidate in vivo, the contribution of mtDNA mutations to the aging

phenotype in skeletal muscle, and their role in apoptosis.

Mitochondrial Dysfunction, Apoptosis and Skeletal Muscle Aging/Sarcopenia

An age-related loss of muscle mass and function occurs in skeletal muscle of a variety of

mammalian species; this process is referred to as sarcopenia, and is reflected by 25% to 35%

decreases in the cross-sectional area of several limb muscles due to muscle fiber atrophy and loss

(83). The public health ramifications of this large decline are evident in the clinical presentation,

which includes decreased mobility and respiratory function. These declines have significant

effects on individual health and quality of life, affecting the ability of elderly people to live

independently.

In humans, specific skeletal muscles may undergo a -40% decline in muscle mass between

the ages of 20 and 80 years (84). What is more, a 25% decrease in cross-sectional area of vastus

lateralis (VL) (the most studied muscle in the context of sarcopenia) is consistently seen in

comparisons of 70- to 75-year-olds with 20- to 30-year-olds (85, 86). Large declines also occur in

the number of fibers in the VL. Progressive muscle wasting has also been demonstrated in

murine and nonhuman primates. These sarcopenic changes are evidenced by a significant

reduction in muscle cross sectional area, muscle mass loss and fiber number loss over time. In

the Fischer 344 x Brown Norway (FBN) hybrid rat, the difference between the rectus femoris

muscles of 18- and 38-month-old animals is striking. Muscle cross sectional area is reduced by









30% in the older animals and the muscle composition is more heterogeneous including an

increase in fibrotic tissue (87). A significant reduction in muscle mass (45%) is observed

between 18- and 36/38-months of age, as well as, a significant (27%) loss of muscle fibers (87).

Another study in male FxBNF1 rats found that atrophy occurs from 9 to 31 months of age in the

soleus (13%), EDL (15%), plantaris (22%), and gastrocnemius (25%) (88). In C57BL/6 mice (the

same background strain used in this study) skeletal muscle mass also decreases with aging with

percent atrophy reported ranging from 15% to more than 30% (89). When we compared aged

WT C57BL/6 mice (30-mo) to young WT (5-mo old) animals we also found significant muscle

atrophy (Fig 4-15).

Although the specific characteristics of sarcopenia in murine depend on the strain, gender,

muscle and age groups studied, the relative magnitude of muscle atrophy in old animals resembles

that of old persons (90). We have observed a similar degree of atrophy in the skeletal muscle of 11-

mo-old D257A mice (24% atrophy in gastrocnemius and 19% atrophy in quadriceps) that very

closely resembles sarcopenia during aging (Fig 4-15). Although the molecular events responsible

for sarcopenia are unknown, the muscle mass loss is due to both fiber atrophy (84, 91, 92) and

fiber loss (84, 93, 94), and proposed mechanisms for fiber loss include mtDNA mutations and

altered apoptotic signaling (87). It is also important to point out that skeletal muscle does not

possess the high repair capacities that occur in more mitotically active tissues, which makes it more

susceptible to age-induced deterioration; although satellite cells are capable of replacing lost

muscle fibers, both the percentage of satellite cells and their proliferative capacities decrease with

aging (95). Therefore, skeletal muscle degenerates with aging in both humans and murine, and

may represent an important target for age-related mitochondrial dysfunction.









The consequences on skeletal muscle of mtDNA disruption, ranging from point mutations

to large deletions, are clear from neuromuscular disorders known as mitochondrial myopathies

(96). The mtDNA abnormalities associated with these disorders cause ETC dysfunctions in

muscle fibers (97). Moreover, mutations affecting the mitochondrial genome can increase the

susceptibility of cells to apoptosis (98). Several prominent examples include Friedreich's Ataxia,

a neurodegenerative disease in which mtDNA mutation sensitizes the cells to undergo apoptosis

(99, 100) and Leigh syndrome, the most common neurodegenerative disorder affecting oxidative

phosphorylation in children, in which mtDNA mutations also increase mitochondrial-mediated

apoptosis (101). In the normally aged skeletal muscle of rats it was clearly shown that segmental

mitochondrial abnormalities colocalize with mtDNA deletion mutations (7). Importantly, these

muscle fibers harboring mitochondrial abnormalities displayed a striking decrease in cross

sectional area indicative of atrophy, and fiber splitting, strongly suggesting a causal role for age-

associated mitochondrial DNA deletion mutations and mitochondrial dysfunction in sarcopenia

(7). The same group also showed that the vastus lateralis muscle, which undergoes a high degree

of sarcopenia, exhibited more ETS abnormalities and associated fiber loss than the soleus and

adductor longus muscles, which are more resistant to sarcopenia, suggesting a direct association

between ETS abnormalities and fiber loss (102). Moreover, Prolla and co-workers have

demonstrated that aging of specific organs, including skeletal muscle, is associated with specific

patterns of transcriptional alterations that serve as molecular biomarkers to indicate

mitochondrial dysfunction in the aging process (103).

There is evidence indicating that deregulation of apoptosis plays a key role in the

pathophysiology of skeletal muscle cell loss. Indeed, accelerated skeletal muscle apoptosis has

been documented to occur with aging (104, 105). Our laboratory has previously shown that in









the gastrocnemius muscle of old Fischer-344 rats, apoptosis is significantly elevated compared to

young rats and this also coincided with a significant increase in the levels of cleaved caspase-3

(105). These findings also agree with the elevated caspase-3 levels we have detected in the

gastrocnemius of aged, 30-mo old WT mice, and in the gastrocnemius of 11-mo old D257A

mice, a time-point when the sarcopenic phenotype is evident in these mice (13) (Fig 4-18).

Despite a very likely role of apoptosis in sarcopenia, there are only sparse reports on the

occurrence of skeletal muscle cell apoptosis in humans with normal aging. In 1999, Strasser et al.

showed that, in humans, an age-dependent increase in apoptosis of the striated muscle fibers of

the rhabdosphincter led to a dramatic decrease in the number of striated muscle cells (106); in a

5-week-old neonate, 87.6% of the rhabdosphincter consisted of striated muscle cells, in striking

contrast with only 34.2% in a 91-year-old subject. To our knowledge this was the first report on

the role of apoptosis in human skeletal muscle atrophy with age. Results from a very recent study

in humans indicate that apoptosis appears to be a contributing pathway to skeletal muscle

wasting in healthy older adults compared to healthy young adults (107). This was marked by

significant increases in TUNEL positive cells stained for DNA fragmentation (older adults

showing an increase of 87% over young adults) in the vastus lateralis (107).

Several investigations have implicated the mitochondria as key mediators involved in

sarcopenia. Cortopassi and others (100, 108, 109) suggested that mitochondrial dysfunction

could induce the mitochondrial permeability transition pore (PTP), the release of cytochrome c,

and eventually initiation of apoptosis. Furthermore, Fitts et al (110) showed increases in

glycolysis and glycogen utilization during contractile activity in aged rats, suggesting an

increased reliance on energy production from glycolytic processes possibly as a consequence of

an age-related mitochondrial dysfunction. Importantly, in the white gastrocnemius of Fischer-









344 rats, it was recently demonstrated that aging significantly increased DNA fragmentation and

cleaved caspase-3 content and this also coincided with a 35.4% lower mean fiber cross- sectional

area in the old sedentary rats versus the young sedentary controls (111). Additionally, pro-

apoptotic Bax was increased in the old rats compared to young, while anti-apoptotic Bcl-2

protein expression declined in both white gastrocnemius and soleus muscles (111), suggesting

that mitochondria may be a target for skeletal muscle aging and alterations in mitochondrial

apoptotic regulatory proteins may be responsible for the observed age-induced muscle fiber

atrophy. Moreover, Leeuwenburgh et al., showed that the soleus muscle weight and cross

sectional area from 32-mo-old rats were 24% and 26% lower, respectively, compared to 6-mo-

old animals, and in the old rats there was a six-fold higher incidence of total TUNEL-positive

nuclei compared to young rats (112). Interestingly, Endonuclease G translocation from the

mitochondria to the nucleus also occurred in old, but not in young animals, implicating the

mitochondrial mediated apoptosis in the loss of skeletal muscle mass with age.

In summary, our review of the literature suggests that: a) aging is undoubtedly associated

with loss of skeletal muscle mass, b) aging is associated with mitochondrial abnormalities in

skeletal muscle of several species, including humans, and c) age-associated mtDNA mutations,

leading to mitochondrial dysfunction, may be important contributors to sarcopenia. Furthermore,

mitochondrial-mediated apoptosis appears to be a likely mechanism for sarcopenia, but this still

remains to be substantiated.

Mitochondrial-Mediated Pathways of Apoptosis.

Apoptosis is a cell suicide program that is highly conserved among species. Under

physiological conditions, apoptosis is essential for embryonic development, tissue homeostasis

and removal of cells whose persistence would be detrimental to the organism (e.g. self-reactive

immune cells, neoplastic cells, virus-infected cells) (113). Apoptosis is up-regulated in many









tissues with age. Accelerated apoptosis in mitotic tissues during aging, such as liver and white

blood cells, is most likely beneficial as it may serve, respectively, to prevent age-associated

tumorigenesis and to maintain overall control of immunocompetent cells. However, excessive

apoptosis in post mitotic cells, such as skeletal muscle fibers and cardiomyocytes may lead to a

decline in function and the development of pathological conditions, such as sarcopenia and

cardiac dysfunction, since these types of cells have a limited ability to regenerate.

Apoptosis is executed via activation of specific signaling pathways which are tightly

regulated. Hence, particular morphological, biochemical, and molecular events occur, such as

DNA fragmentation, nuclear condensation, and formation of apoptotic bodies, which are then

engulfed by macrophages or neighboring cells without initiating an inflammatory response (60).

Recent evidence has implicated mitochondria as key regulators of apoptosis (114-116).

Although other apoptotic pathways exist, this proposal focuses exclusively on the main

mitochondrial-mediated pathway, since mtDNA mutations are expected to affect mitochondrial

function and possibly mitochondrial outer membrane permeability (MOMP). MOMP can lead to

1) release of molecules implicated in the activation of caspases that orchestrate downstream

events associated with apoptosis, 2) release of molecules involved in caspase-independent cell

death, and 3) loss of mitochondrial functions essential for cell survival. The key step in the

initiation of mitochondrial-mediated apoptosis is the release of pro-apoptotic proteins from the

mitochondrial inter-membrane space into the cytosol.

Mitochondrial-mediated apoptosis entails both caspase-dependent and caspase-independent

modes (Fig 2-3). Caspases normally exist in an inactivated state, called procaspases, in the

cytoplasm but can be activated by dimerization or proteolytic cleavage. Cytochrome c release

from the mitochondrial intermembrane space, in the caspase-dependent pathway, is one of the









most intensively studied pathways of apoptosis. Upon receiving a death-inducing signal, there is

a disruption of the mitochondrial inner transmembrane potential, which results in the opening of

the mitochondrial permeability transition pore (PTP), that involves components of the outer

mitochondrial membrane (VDAC, Bax and Bcl-2), inner mitochondrial membrane (ANT-

adenine nucleotide translocase), and matrix (cyclophilin D). PTP opening results in loss of

membrane potential, osmotic swelling of the mitochondrial matrix, rupture of the outer

membrane, and the release of cytochrome c and other apoptogenic factors from the inter-

membrane space (117, 118).

A second model proposes that at least some of the pro-apoptotic Bcl-2 proteins (e.g Bax

and Bak) are able to form tetrameric outer membrane channels that could also mediate the

release of apoptogenic factors from the inter-membrane space, without the involvement of inner

mitochondrial membrane components (119).

Cytochrome c in the cytosol combines with procaspase-9 and (Apoptotic Protease

Activating Factor 1) Apaf-1, which is constitutively expressed in the cytoplasm, and in the

presence of ATP (which is required for the induction of apoptosis) forms the "apoptosome". This

complex cleaves off the pro-enzyme of caspase-9 into the active form. This allows the molecule

to change conformation, and bind to another cleaved caspase-9 precursor, forming a homodimer.

Caspase-9 is autocatalytic, thus it activates other caspase-9 molecules by cleaving off their N-

terminal prodomain. This is known as the "Caspase Cascade." Caspase-9 also activates caspase-

3, which is not autocatalytic, by cleavage at the C-terminal side of a specific aspartate residue.

Activation of the final apoptosis-effector, caspase-3, which carries out the actual proteolytic

events that result in cellular breakdown will irreversibly commit the cell to suicide (120) (Fig 2-

3).









Alternatively, mitochondria can release apoptosis inducing factor (AIF) and endonuclease

G (EndoG), which have been suggested to function in a caspase-independent fashion (121-124),

as both AIF and EndoG induce apoptotic changes in purified nuclei, even in the presence of

caspase inhibitors (125, 126). Upon release from the mitochondria, both mediators translocate to

the nucleus and may lead to large-scale DNA fragmentation and peripheral chromatin

condensation (Fig 2-3), but not oligonucleosomal DNA laddering. In vitro, AIF appears to be an

essential mitochondrial pathway for cell death since caspase inhibitors block only 40-50% of cell

death (127). Moreover, genetic analyses in C. elegans indicate that AIF cooperates with EndoG

to participate in the regulation of cell death (128), however, it is unclear whether the same occurs

in mammalian cells. Recent data also reveal an important contradiction to the idea that AIF

functions in a strictly caspase-independent manner (128, 129). Arnoult et al. showed that the

mitochondrial release of AIF, occurring in HeLa and Jurkat cell lines treated with general

apoptosis inducers, such as staurosporine or actinomycin D, is suppressed (or at least delayed) by

caspase inhibitors. The authors suggested that AIF would be released only after cytochrome c

release, subsequent to apoptosome-mediated caspase activation (129). However, it has not been

elucidated whether and how AIF can induce DNA fragmentation since it has no reported intrinsic

DNAse activity.

Since all mammalian nucleated cells have the ability to undergo apoptosis (130), several

apoptotic regulatory mechanisms have evolved, some inhibiting mitochondrial release of pro-

apoptotic proteins and others preventing caspase activation in the cytosol (131). One of these, the

Bcl-2 family of proteins consists of both pro- (Bax) and anti-apoptotic (Bcl-2) proteins that are

structurally related, and act to either prevent or promote the release of cytochrome c in the

cytosol (132). It appears that the relative ratios of these proteins influences whether a cell lives or









dies. In the aged rat heart, the Bcl-2/Bax ratio has been shown to decrease while cytosolic

cytochrome c rises, indicating that the heart becomes more sensitive to apoptotic stimuli (133).

A very recent study showed that in the white gastrocnemius, the Bax/Bcl-2 ratio increased by

98% with age and this increase was associated with a dramatic increase in cleaved caspase-3 and

in histone-associated DNA fragmentation (111).

Furthermore, endogenous inhibitors of apoptosis proteins (lAP's), initially discovered in

baculoviruses, also exist in mammalian cells (134-136). Amongst them, the X-linked IAP

(XIAP) is regarded as the most potent suppressor of mammalian cell death. At least one

explanation for the versatile suppression of cell death exhibited by this protein resides in its

ability to bind directly to, and inhibit, caspases in the cytosol (137). Specifically, the BIR2 region

of XIAP is a potent and specific inhibitor of caspase-3, whereas the BIR3 domain is specific for

caspase-9 (137).

Apoptosis mediated by the mitochondria appears to be the most likely pathway

responsible for skeletal muscle loss in the D257A mice, since the observed mitochondrial

dysfunction (Figs 4-2, 4-9) can be a trigger for apoptosis. In our specific aim # 3 we investigated

the impact of mtDNA mutations in the induction of the main caspase-dependent mitochondrial-

mediated pathway of apoptosis.












Inter
membrane
space


H'


TRANSLOCATOR



| [Jjfjj


MtDNA 7 0 3
Nuclear 32 9 4 10 10


Figure 2-1. Contributions of the mitochondrial and nuclear DNA to protein subunits of the
complexes of the ETC. Depicted are the 5 enzymatic complexes of the mitochondrial
ETC embedded in the inner mitochondrial membrane. The number of subunits from
each complex encoded from mtDNA and nuclear DNA are also shown. Note that
complex II is all nuclear encoded while complex IV has the greatest contribution of
mtDNA-encoded subunits. Adapted from Wallace, 1997.


Apoptosis


Figure 2-2. The mitochondrial 'vicious cycle' theory.


I I




















MiLr
E-I-


/ -


i / I ,"
-.1^^
\< ..Sc*


APOPF 'SIS


Figure 2-3. Mitochondrial-mediated apoptosis. Scheme was adapted from Cell Signaling
Technology Inc. and modified.









CHAPTER 3
MATERIALS AND METHODS

Experimental Design

In recent published results we performed experiments at different time points of the

D257A animals' lifespan in order to determine the point at which the D257A phenotype deviates

significantly from the WT phenotype (13). For post mitotic tissues, such as skeletal muscle and

heart, we detected a phenotype that resembles normal aging at 9-10 mo of age in D257A mice,

while at 3-mo of age there was no difference in phenotype between WT and D257A for the

parameters measured, which indicates that the phenotype is age-related and is not due to

developmental defects. To ensure the selection of an ideal time-point for the present dissertation

project, in our pilot study we performed the same experiments at two different time points: 3

months and 11 months, and our results corroborated our previous findings regarding the change

in the phenotype. Indeed, we did not detect any differences in skeletal muscle mass, oxygen

consumption, ROS production, free radical leak or caspase-3 levels, between WT and D257A

mice at 3 months of age (Appendix-a, Figs A-i, A-2, A-3, A-4). Therefore, in our design, we

selected a time point between 11-13-mo old for our experiments in order to ensure that we will

get the age-related phenotype. For all three specific aims the experimental groups are identical.

We used and compared two groups: 11-13 mo-old WT (n = 11) versus 11-13 mo-old D257A (n

11) mice (Fig 3-1).

General Procedures

Animals

C57BL/6 strain WT and D257A male and female mice were obtained at -11 months of age

from University of Wisconsin, Madison, from the lab of our collaborator Dr. Tom Prolla. The

animals were housed in quarantines, in the animal care facility located at the Progress Park









(Specific-pathogen free and accredited facility). The facility is climate- and light-controlled.

After one week of acclimation in the facility the animals were sacrificed by rapid cervical

dislocation followed by extraction of the gastrocnemious and quadriceps muscles and immediate

isolation of mitochondria for the measurements of mitochondrial respiratory and functional

parameters. Cervical dislocation was chosen in order to avoid the influence of other anesthetics

(e.g. volatile gases) on some of the parameters to be measured, primarily mitochondrial

functional parameters (basal mitochondrial respiration, and ROS production) (138, 139). Four

animals a day were sacrificed. The number of animals used per group is n =11 (Fig 3-1). This

number was determined via a power analysis based on detecting a 50% difference, using

previous data from our laboratory. The power was set at 0.90 and alpha level at p<0.05.

Mitochondrial and Cytosolic Isolation

Mitochondrial and cytosolic protein fractions were isolated using differential

centrifugation. Immediately after sacrifice, skeletal muscle (both gastrocnemious and quadriceps

muscles were mixed) was removed, cleaned, and weighed. Skeletal muscle was finely minced

into small pieces and homogenized in (1:5 wt/vol) ice-cold isolation buffer containing 0.21 M

mannitol, 0.07 M sucrose, 0.005 Hepes, 0.001 M EDTA, 0.2% fatty acid free BSA, pH 7.4, using

a Potter-Elvehjem glass homogenizer. The homogenate was centrifuged at 1,000 g for 10 min.

After the first spin, the pellets containing nuclei were frozen for future analysis. The supernatant

was then centrifuged at 14,000 g for 20 min. The supernatant (crude cytosol) was stored at -800C

and the mitochondrial pellet was re-suspended in isolation buffer without BSA and was

centrifuged again at 14,000 g for 10 min. The final mitochondrial pellet was re-suspended in 350

tl of isolation buffer without BSA, and was used immediately for the measurements of

mitochondrial H202 production, oxygen consumption and ATP content and production. All









centrifugation steps were carried out at 40C. Protein concentrations were determined using the

Bradford method (140).

Specific Methods

Specific Aim 1. Effect of mtDNA Mutations on Skeletal Muscle Mitochondrial Function

In this specific aim we investigated the impact of the accumulation of mtDNA mutations

on mitochondrial bioenergetics, specifically, on 02 consumption, ATP content and production,

and ROS generation. Using specific complex inhibitors we also determined maximum ROS

production at the main ROS-generating sites within the ETC, complex I and complex III. In our

preliminary studies we had measured mitochondrial oxygen consumption and our results match

our hypothesis: D257A mitochondria show compromised respiration during state 3, and

uncoupling between oxidation and phosphorylation (Fig 4-2). Interestingly, H202 production and

free radical leak from mutant mitochondria was lower compared to that of WT (Fig 4-11, 4-12).

As previously mentioned, this finding doesn't support the mitochondrial "vicious cycle" theory

of aging, suggesting that mtDNA mutations may lead to mitochondrial dysfunction without

increases in ROS production.

In this aim we are providing additional evidence that oxidative stress levels are not

elevated in response to high mtDNA mutational rate, by demonstrating that 8-oxodGuo levels in

skeletal muscle mtDNA were not significantly different between WT and D257A mice (Fig 4-

14). In this aim we further determined whether the mitochondrial dysfunction we have observed

in the mutant mice leads to loss of membrane potential thus making the mutant mitochondria

more susceptible to apoptosis. We examined the possible mechanism by which this dysfunction

is induced by assessing the content and activity of ETC respiratory complexes I, III, IV and F1Fo

ATPase, which contain subunits encoded by the mitochondrial genome, and compare them to

complex II which is all nuclear-encoded.









Rationale

Part of the focus in this aim was to determine whether mtDNA mutations induce

mitochondrial dysfunction. We have evaluated several mitochondrial functional parameters and

we determined that indeed mitochondrial dysfunction is evident by the reduction in

mitochondrial respiration at state 3, and the higher degree of uncoupling we have observed in the

D257A mice.

As previously mentioned, D257A mice accumulate mutations due to the mutated

exonuclease domain of POLy, which is devoid of proof-reading activity. We expected that

mutated gene-encoding areas of the mitochondrial genome will have a direct effect on protein

transcription and/or translation. Therefore, we anticipated that the proteins encoded by the

mitochondrial genome will be directly affected. Depending on the type of mutations introduced

and the location at which they occur, the proteins encoded by mtDNA may be truncated, or

completely absent, may partially or totally lose activity. We hypothesized that the end result

would be that the content and/or activity of the proteins encoded by mtDNA would be

compromised in the mutant mice. Since the proteins encoded by the mitochondria (total of 13

proteins) are all proteins of the electron transport chain (ETC), we expected that the mtDNA

mutations would have a direct impact on mitochondrial ETC activity and hence, on

mitochondrial function. Differently stated, we hypothesized that the reduction in the content

and/or activity of ETC proteins would be the primary mechanism of the observed mitochondrial

dysfunction associated with mtDNA mutations. We evaluated the total concentration and

maximum activity of the respiratory complexes I, III, IV and F1Fo ATPase. These are the ETC

complexes that contain subunits encoded by mtDNA. We also evaluated the content of selected

individual subunits from these complexes that are either nuclear- or mitochondrial-encoded.









These measures helped us understand and explain how the mitochondrial dysfunction we have

observed in the D257A mice is induced.

Furthermore, the measurement of mitochondrial membrane potential was critical to help us

understand why mutant mitochondria produce less ROS (evident by the decrease in H202

generation), and explain the induction of apoptosis in the mutant mice. Importantly, loss of

membrane potential represents a link between mitochondrial dysfunction and apoptosis and

hence, a link between aim #1 and aims #2, #3 (see also Fig 3-1).

Experimental approach

Mitochondrial H202 generation

The rate of mitochondrial H202 production was assayed in freshly isolated mitochondria

by a highly sensitive fluorometric method according to Barja (141), and adapted to a microplate

reader. H202 generation was monitored by measuring the increase in fluorescence (excitation at

312 nm, emission at 420 nm) due to the oxidation of homovanillic acid by H202 in the presence

of horseradish peroxidase like it is shown in the reaction below:

COOH
CH,



CH, + H202 HRPx Fluorescent dimer
CH30

OH
Homovanillic
acid
The assay was performed in incubation buffer (145-mM KC1, 30-m Hepes, 5-mM

KH2PO4, 3-mM MgC12, 0.1-mM EGTA, 0.1% BSA, pH 7.4) at 37oC, and the reaction

conditions were: 0.25 mg of mitochondrial protein per mL, 6 U/mL of horseradish peroxidase,

0.1-mM homovanillic acid and 50-U/mL of superoxide dismutase (SOD). The reaction was

started by the addition of 2.5 mM pyruvate/2.5 mM malate or 5 mM succinate as substrates.









Pyruvate/malate was used to study complex I ROS production, and succinate was used to study

complex III ROS production (for details also see Barja, 1999) (142). We also used inhibitors of the

ETC in order to study maximum rates of H202 production from complexes I and III, since they

represent the main sites of ROS generation (especially complex I) within the mitochondria. For

complex I maximum rate we used 2 pM rotenone added to pyruvate/malate supplemented

mitochondria. For complex III maximum rate we used 2 pM antimycin A plus 2 pM rotenone,

added to succinate supplemented mitochondria. In addition, some of the assays with succinate as

substrate were performed in the presence of 2 pM rotenone alone, in order to avoid the backwards

flow of electrons to Complex I. After 15 min of incubation at 37 C, the reaction was stopped and

the samples were transferred on ice and a stop solution (0.1-M glycine, 25-mM EDTA, pH 12)

was added. Known amounts of H202 generated in parallel by glucose oxidase, with glucose as

substrate, were used as standards. Since the SOD added in excess converts all the 02 produced

(if any) to H202, the measurement represents the total (02" plus H202) rate of mitochondrial

ROS production. All samples were run in duplicate. H202 production and 02 consumption were

measured in parallel in the same muscle mitochondria under similar experimental conditions.

This allowed the calculation of the fraction of electrons out of sequence which reduce 02 to ROS

at the respiratory chain (the percent free radical leak or FRL%) instead of reaching cytochrome

oxidase to reduce 02 to water. Since two electrons are needed to reduce 1 mole of 02 to H202

whereas four electrons are transferred in the reduction of 1 mole of 02 to water, the percent free

radical leak can be calculated as the rate of H202 production divided by two times the rate of 02

consumption, and the result is multiplied by 100.









Mitochondrial respiration

Mitochondrial oxygen consumption was measured at 370C by polarography, with a Clark-

type oxygen electrode (Oxytherm, Hansatech, Norfolk, UK) under the same conditions used

(same mitochondria, buffer composition and substrate concentrations) for H202 production

measurements: incubation buffer (145 mM KC1, 30 mM Hepes, 5 mM KH2PO4, 3 mM MgC12,

0.1 mM EGTA, pH 7.4) with 0.25 mg of mitochondrial protein per ml and 2.5 mM pyruvate/2.5

mM malate as substrates. The assay was performed in the absence (State 4-resting state) and in

the presence (State 3-phosphorylating state) of 500 [tM ADP. Clark-type electrode without (State

4) and with (State 3) saturant ADP allows calculation of the respiratory control ratio (RCR)

(State 3/State 4 oxygen consumption) as an indicator of the degree of coupling and metabolic

activity of the mitochondrial preparations.

ATP content and production

Mitochondria isolated from skeletal muscle were used immediately after isolation to

determine mitochondrial ATP content and production, following the method of Drew (143). This

bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and

its substrate luciferin. Upon addition, ATP combines with luciferin to form luciferyl adenylate

and inorganic pyrophosphate (PPi) on the surface of the luciferase enzyme as shown in reaction

1 below:

Reaction 1 Luciferin + ATP Luciferase, Luciferyl Adenylate + PPi

While bound to the enzyme, luciferyl adenylate combines with 02 to form oxyluciferin and

AMP through a series of enzymatic redox reactions. As oxyluciferin and AMP are released from

the enzyme's surface, a quantum yield of light is emitted in proportion to the ATP concentration

as shown in reaction 2:

Reaction 2 Luciferyl Adenylate + 02 Oxyluciferin + AMP + hv










The light emission (hv) can then be recorded and quantified using a chemiluminometer.

ATP content methodology was modified from a method of Molecular Probes (A-22066, Eugene,

OR). Chemicals used are D-luciferin, luciferase (40 jtL of a 5 mg/mL solution in 25 mM Tris-

acetase, pH 7.8, 0.2 M ammonium sulfate, 15% (v/v) glycerol and 30% (v/v) ethylene glycol),

dithiothreitol (DTT), adenosine 5'-triphosphate (ATP), and a Reaction Buffer (10 mL of 500 mM

Tricine buffer, pH 7.8, 100 mM MgSO4, 2mM EDTA and 2 mM sodium azide). The reagents

and reaction mixture were combined according to the protocol by Molecular Probes. In order to

determine ATP content, freshly isolated mitochondria were added to a cuvette containing

reaction buffer, D-luciferin, luciferase and DTT. In addition, 2.5 mM pyruvate and 2.5 mM

malate were added to the reaction mixture, as substrates for oxidative phosphorylation.

Immediately after the ATP content measurements, 2.5 mM ADP was added to the cuvette

containing the reaction mixture and mitochondria in order to determine the rate of ATP

production. A blank cuvette containing no sample, only reaction mixture, was assayed to account

for background luminescence, and known concentrations of ATP standards were used to

establish a standard curve. The values for ATP content and rate of production were normalized to

total mitochondrial protein concentration. All mitochondrial samples were assayed in duplicate,

and an average of these results was used to calculate final ATP content and rate of production.

Mitochondrial membrane potential

Mitochondrial membrane potential changes in isolated skeletal muscle mitochondria were

followed qualitatively by monitoring the fluorescence of tetramethyl rhodamine methyl ester

(TMRM, Molecular Probes, Eugene, OR), a cationic lipid-soluble probe that accumulates in

energized mitochondria. The method of Scaduto (144) was followed without modification.

Briefly, mitochondria (0.25 mg/ml) were incubated at 370C in a medium composed of 135 mM









KC1, 20 mM MOPS, 5 mM K2HPO4, and 5 mM MgC12 at pH 7.00. The experiment was

initiated by the addition of mitochondria to the medium, also containing 0.33 mM TMRM and

either 5mM succinate or 5mM glutamate + 2.5mM malate in order to record membrane potential

during state 4. Fluorescence at 546 and 573 nm excitation was monitored using an emission

wavelength of 590 nm. This was followed by the addition of ADP (0.17 mM) to record

membrane potential during state 3. Addition of 0.5 mM CCCP followed to serve as a control for

TMRM binding. An increase in fluorescence represents de-quenching of TMRM when the probe

is released into the medium upon mitochondrial depolarization.

Blue native page (BN-page) for determination of content and enzymatic activity of
respiratory complexes

For determination of the content of the ETC complexes we followed the protocol as

described by Schagger et al. with some modification (145). Skeletal muscle was homogenized in

buffer 1 containing 20 mM MOPS, 440 mM saccharose, 1 mM EDTA and 0.5 mM PMSF, pH

7.2 at 40C. The homogenates were centrifuged at 20,000 g for 20 min. The pellet was re-

suspended in 80 [tl of buffer containing 1 M aminocaproic acid, 50 mM Bis-tris and 0.5 mM

PMSF, pH 7.0. The membranes were then solubilized by the addiction of 30 [tl n-

dodecylmaltoside (10 %, prepared fresh). Mitochondrial suspensions were incubated on ice for

30 minutes with vortex mixing every 5 min, followed by ultracentrifugation for 25 min at

100,000 g (Beckman, Optima LE-80K). The supernatant, containing all the solubilized

mitochondrial membrane proteins was used for the BN-page. 7 [tl of 5% w/v coomassie brilliant

blue G-250 in aminocaproic acid (1M) were added to 100 [tl of supernatant. Samples were then

stored on ice for no more than 30 min prior to gel loading. For electrophoresis, a 3-12 % gradient

gel with 4% of stacker was used. The anode buffer was comprised of 50 mM Bis-Tris, pH 7.0.

The cathode buffer was comprised of 50 mM tricine, 15 mM Bis tris, and coomassie brilliant









blue G-250 (0.02% w/v), pH 7.0. Samples were electrophoresed at 90 V for 20 min, and

thereafter at 170 V for 2 h, at 40C. Immediately after electrophoresis gels were incubated in

coomassie brilliant blue G-250 solution (0.1% coomassie in 10% acetic acid and 40% methanol)

for Ih, followed by incubation in de-staining solution (10% acetic acid, 40% methanol) for 2h.

After de-staining gels were photographed and analyzed using the Alpha Innotech FluorChem SP

imaging system. Densitometry values were normalized to total protein loaded per well, measured

by the Bradford assay.

For determination of enzymatic activity, enzymatic colorimetric reactions were performed

on the BN-PAGE. Gels were incubated overnight at room temperature with the following

solutions: Complex I: 2mM Tris-HC1, pH 7.4, 0.1 mg/ml NADH, and 2.5 mg/ml NTB

(nitrotetrazolium blue). Complex II: 4.5mM EDTA, 10mM KCN, 0.2mM phenazine

methasulfate, 84mM succinic acid and 50mM NTB in 1.5mM phosphate buffer, pH 7.4.

Complex IV: 5 mg 3:30-Diamidobenzidine tetrahydrochloride (DAB) dissolved in 9ml

phosphate buffer (0.05 M, pH 7.4), Iml catalase (20[tg/ml), 10 mg cytochrome c, and 750 mg

sucrose. Complex V: 35mM Tris, 270mM glycine, 14mM MgSO4, 0.2% Pb(N03)2, and 8mM

ATP, pH 7.8. Gels were then washed in distilled water and photographed immediately.

Densitometry values for activity were normalized to respective content densitometry values.

Determination of protein content of selected mitochondrial- and nuclear-encoded subunits
from ETC complexes I, II, III and IV

Skeletal muscle tissue was immersed and rinsed in cold homogenization buffer: 50mN

Tris-HCl pH 7.4, 1% Tween 20 (Amersham Biosciences), 0.25% sodium deoxycholate, 150 mM

NaC1, ImM disodium ethylenediaminetetraacetate dehydrate (EDTA), ImM

Diethylenetriaminepenta-acetic acid (DTPAC), 1 lM 2,6-di-tert-butyl-4-methylphenol (BHT), and

1.5% Protease Inhibitor Mix (Amersham Biosciences). This was followed by homogenization in









25 ml homogenization buffer/g of skeletal muscle with a Potter-Elvehjem type homogenizer

system (Glas-Col, Terre Haute, IN). The homogenate was then centrifuged at 500 x g for 5 min

at 40C yielding a pellet corresponding to crude nuclear fraction. Protein concentration was

determined by the Bradford method using BioRad reagent and BSA as standard. Homogenates

were immediately frozen at -800C until further analysis. The protein content of skeletal muscle

mitochondrial respiratory chain complexes was estimated using western blot analysis.

Immunodetection was performed using specific antibodies for the 39KDa (NDUFA9) and 30KDa

(NDUFS3) subunit of complex I (1:1000 and 1:1000, respectively), 70KDa subunit (Flavoprotein)

of complex II (1:500), 48KDa (CORE 2) and 29KDa (Rieske iron-sulfur protein) subunits of

complex III (1:1000 and 1:1000, respectively), and COXI subunit of complex IV (1:1000) (ref

A21344, A21343, Al 1142, Al 1143, A21346 and A6403, respectively; Molecular Probes). An

antibody to porin (1:5000, A31855, Molecular Probes) or beta-actin (1:5000, AB20272, Abcam), as

a loading control for total mitochondrial mass or total protein content, was also used. Appropriate

peroxidase-coupled secondary antibodies and chemiluminescence HRP substrate (Millipore, MA,

USA) were used for primary antibody detection. Signal quantification and recording was performed

with a ChemiDoc equipment (Bio-Rad Laboratories, Inc., Barcelona, Spain). Protein concentration

was determined by the Bradford method. Data were expressed as Arbitrary Units.

Determination of mitochondrial protein yield

In order to determine mitochondrial yield, we first determined the total protein

concentration in each mitochondrial extract by the Bradford assay. We then multiplied each

concentration value by the total volume of each mitochondrial extract. Last, we divided this

product by the skeletal muscle weight used each time to obtain the respective mitochondrial

extract. In this way we were able to normalize and express the total mitochondrial content per

gram of skeletal muscle tissue.









Determination of MnSOD and Catalase mRNA expression by RT-PCR

To extract RNA skeletal muscles (1/10 weight/reagent volume) were homogenized in ImL

of Trizol reagent and the Trizol protocol for RNA isolation was followed. Briefly, after

homogenization samples were centrifuged at 12,000 x g for 10 min in order to remove insoluble

material. To the clear supernatant, 0.2mL of chloroform was added and the supernatant was

centrifuged at 12,000 x g for 15 min. This separates the mixture into 3 phases: a red organic

phase, an interphase and a colorless upper aqueous phase containing RNA. To the aqueous phase

0.6 mL ofisopropanol was added and the mixture was centrifuged again at 12,000 x g for 10

min. The precipitated RNA was washed with 75% ethanol, centrifuged again at 7,500 x g for 5

min and the resulting RNA pellets were dried for 5-10 minutes under a vacuum. 1 [tg of isolated

RNA was reverse transcribed (Eppendorf RT plus PCR kit) using oligo (dT) primer, as described

by the manufacturer's instructions. PCR was performed on 3-rl aliquots from each cDNA

reaction, using primer sets for detecting MnSOD (5'-GGTGGCCTTGAGCGGGGACTTG-3', 5'-

GGTGGGTGGGGAGGTAGGGAGGAT-3', sense and antisense, respectively) and Catalase (5'-

ATGGCCTCCGAGATCTTTTCAATG-3', 5'-GAGCGCGGTAGGGACAGTTCAC-3', sense

and antisense, respectively). The sizes of the amplification products were 611 bp for MnSOD

and 366 bp for Catalase. Conditions for PCR reactions were for MnSOD: 94 C for 30 sec, 58 C

for 30 sec, and 72 C for 30 sec and for Catalase: 94 C for 30 sec, 57.7 C for 30 sec, and 72 C

for 30 sec. PCR amplification was conducted for 29 cycles for both MnSOD and Catalase. RT-

PCR products were analyzed by agarose gel electrophoresis and digital imaging of the ethidium

bromide-stained gel, using the Alpha Innotech FluorChem SP imaging system.

Oxidative damage to mtDNA

Mitochondrial DNA oxidation was measured according to Sanz et al.(146), with

modification. Briefly, mitochondrial DNA, free of nDNA, were isolated by the method of









Latorre et al. (1986), adapted to mammals (Asuncion et al., 1996). After isolation mtDNA was

completely dissolved in 85 ptL of 30 ptM DFOM, DNA was digested with 4 U of Nuclease P1

(dissolved in 300 mM sodium acetate, 0.2 mM ZnC12, pH 5.3), and 5 U of alkaline phosphatase

during 60 min at 500C. After filtering, samples were put into an autosampler vial for HPLC-EC-

UV analysis. 8-oxodG and dG were measured by HPLC with online electrochemical and

ultraviolet detection respectively. For analysis, the nucleoside mixture was injected into two

Delta-Pak (150x3.9mm id, 5 [tm) C-18 reversed-phase columns (Waters, Milford, MA). 8-

oxodG was detected with an electrochemical detector (Coulochem III, ESA Inc, Chelmsford,

MA, USA) with a PEEK filter protected 5011A analytical cell (ESA, 5 nA, screen electrode:

+205 mV analytical electrode: +275), and dG was measured with a Spectra SYSTEM UV1000

detector (Thermo Electron Corp., San Jose, CA, USA) set at 290 nm. Chromatograms were

recorded using EZChrome Elite (Scientific Software INC., Pleasanton, CA, USA). Calibration

curves for dG and 8-oxodG were constructed by injection of each standard 3-4 times. The HPLC

buffer consisted of 9% v/v methanol and 50 mM sodium acetate, set to pH 5.3, with acetic acid

filtered through a CN 0.2 ptm filter from Nalgene Nunc (Rochester, NY, USA).

Specific Aim 2. mtDNA Mutations and Apoptosis in Skeletal Muscle

In out pilot study we had already measured cleaved (activated) caspase-3 content in

skeletal muscle of 3-mo and 11-mo old WT and D257A mice. Proteolytic activation of caspase-3

is a key event in the execution of apoptosis, marking the point at which the cell is committed to

die. We have shown significant elevation in cleaved caspase-3 levels by 11-mo of age in skeletal

muscle of D257A mice compared to WT, which was not evident in D257A mice at 3-mo-of age,

a time-point when the D257A phenotype is also not evident (Figs 4-18, A-2). We also have

found that 30-mo-old WT mice have significant muscle atrophy in concert with elevated









caspase-3 levels compared to young, 5-mo-old mice (Figs 4-15, 4-18) (13). Therefore, the

elevation in cleaved caspase-3 levels, coupled to a significant skeletal muscle loss at 11-mo of

age (Figs 4-15, 4-18), suggests that D257A muscle becomes sarcopenic (13). Together, these

findings suggest that caspase-3-mediated apoptosis may be one of the main pathways responsible

for the decline in skeletal muscle loss associated with mtDNA mutations in the accelerated aging

D257A mice and the same mechanism may also be responsible during normal aging.

Rationale

Although we showed increased levels of one marker of apoptosis (caspase-3), additional

measures are needed in order to corroborate that apoptosis is indeed a central mechanism

responsible for skeletal muscle loss in the D257A mice. Therefore, in this aim, we wanted to

further investigate apoptosis in skeletal muscle, by conducting specific apoptotic measures.

Besides the content of cleaved caspase-3 we further evaluated caspase-3 activity. Furthermore,

we used a quantitative ELISA to measure the amount of mono- and oligo-nucleosomes released

in the cytosol after apoptotic DNA fragmentation. Last, we isolated DNA from skeletal muscle,

performed a DNA laddering-specific ligation PCR in order to amplify apoptotic fragments, and

subjected PCR products to electrophoresis through agarose gel in order to detect

oligonucleosomal fragmentation evident by the formation of specific ladders of -180-200 bps or

multiples in the gel.

Experimental approach

Determination of cleaved caspase-3 content

The active form of caspase-3, cleaved caspase-3, was quantified by Western blotting.

Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated

fragments. The specific antibody used detects endogenous levels of the large fragment (17/19

kDa) of activated caspase-3 resulting from cleavage adjacent to Asp 175. Proteins were separated









using 15% PAGEr Gold pre-cast Tris-glycine gels (Cambrex, USA) under denaturing

conditions, and then transferred to PVDF membranes (0.2 rim, Trans-Blot Transfer Medium,

Bio-Rad Laboratories, CA USA). Protein concentration was determined using the Bradford

assay, and samples were subsequently normalized so that the protein content among samples is

identical. Subsequently, 20 [il of sample were loaded to each well. HL-60 cells induced with

etoposide were also loaded in a well as an appropriate positive control. Membranes were blocked

for 1.5 hrs using a blocking solution containing TBS and 5% milk. Membranes were then

incubated overnight in the 5% blocking solution containing the monoclonal primary antibody

caspase-3 (Cell Signaling, Beverly, MA, USA) in a dilution of 1:500. The following day

membranes were incubated for 1 h at room temperature with IgG horseradish peroxidase-linked

whole secondary antibody (1:1000, Amersham Biosciences UK Ltd, Amersham, UK). Specific

protein bands were visualized using ECL reagent (Amersham, UK). The resulting Western blots

were analyzed using the Alpha Innotech FluorChem SP imaging system. Specific protein bands

were further normalized to b-actin bands. Values were expressed as arbitrary units after

normalizing and expressing samples as % of a control sample that was included in all

membranes.

Enzymatic measurement of caspase 3 activity

Caspase-3 activity was measured using a fluorometric protease assay kit: (Caspase-

3/CPP32, Biovision, Mountain View, CA, USA) according to manufacturer's instructions.

Briefly, the assay is based on detection of cleavage of the substrate DEVD-AFC (AFC: 7-amino-

4-trifluoromethyl coumarin) by caspase-3. DEVD-AFC emits blue light (Xmax = 400 nm); upon

cleavage of the substrate by caspase-3, free AFC emits a yellow-green fluorescence (Xmax = 505









nm), which can be quantified using a fluorescence microplate reader. Samples were run in

triplicate and values were expressed as raw fluorescence units per mg of cytosolic protein.

Determination of cytosolic mono- and oligonucleosomes

Endogenous endonucleases activated during apoptosis cleave double-stranded DNA in the

linker region between nucleosomes to generate mono- and oligonucleosomes of-180 bp or

multiples. Apoptotic DNA fragmentation was quantified in skeletal muscle by measuring the

amount of cytosolic mono- and oligonucleosomes using a Cell Death detection ELISA (Roche

Molecular Biochemicals, Germany). The assay is based on the quantitative sandwich-enzyme-

immunoassay-principle. Briefly, wells were coated with a monoclonal anti-histone antibody.

Nucleosomes in the sample bound to the antibody followed by the addition of anti-DNA-

peroxidase, which reacted with the DNA associated with the histones. The amount of peroxidase

retained in the immunocomplex was determined spectrophotometrically with ABTS (2.2'-azino-

di-[3-ethylbenzthiazoline sulfonate]) as a substrate. All samples were run in triplicate and the

means were expressed as arbitrary OD units normalized to milligrams of cytosolic protein, with

sample protein concentrations determined by the Bradford method.

DNA laddering

To enable detection of nucleosomal ladders in apoptotic cells, the DNA ladder assay was

performed. Skeletal muscle was homogenized in 1 mL DNAzol (Molecular Research Center

Inc., Cincinnati, OH). Proteinase K (Qiagen, Valencia, CA) was added to the homogenates,

which, after a 3 h incubation period, were centrifuged (10,000 x g for 10 min at 40C) and the

supernatants were precipitated and washed with 100% and 75% ethanol, respectively. After

digestion with RNase A, DNA samples were subjected to a DNA ladder-specific ligation PCR,

following the manufacturer's protocol (Maxim Biotech, CA). Briefly, isolated DNA is subjected

to an overnight ligation reaction using de-phosphorylated adaptors (12-mer: 5'-









AGTCGACACGTG-3', 27-mer: 5'-GACGTCGACGTCGTACACGTGTCGACT-3') that are

ligated to the ends of DNA fragments generated during apoptosis, using T4 DNA ligase. In

mammalian cells, such fragments generally have 5'-phosphorylated blunt ends and 3'-OH ends,

thus only the 27-mer is ligated to the DNA fragments. When the mixture is heated to 55 C, the

12-mer is released. Next, the 5' protruding ends of the molecules are filled by Taq polymerase.

The 27-mer then serves as a primer for PCR in which the fragments with adaptors on both ends

are amplified. Conditions for PCR reactions were 72 C for 10 min, 94 C for Imin, 94 C for 1

min, and 70 C for 2 min. PCR amplification was conducted for 30 cycles. PCR products were

electrophoresed through 1% agarose gels containing 0.5 [tg/mL ethidium bromide at 80 V for 1

h, and were examined under UV light for the presence of apoptosis-specific nucleosomal ladders.

Specific Aim 3. Identification of the Specific Apoptotic Signaling Pathway Responsible for
Skeletal Muscle Loss in D257A Mice

In this aim we hypothesized that mitochondrial dysfunction in D257A mutator mice would

lead to mitochondrial outer membrane permeability and leakage of cytochrome c and other pro-

apoptotic factors into the cytosol. Cytochrome c release from the mitochondria may subsequently

activate the caspase-dependent mitochondrial-mediated pathway of apoptosis, leading to the

activation of caspase-9 and downstream cleavage and activation of caspase-3 that we have

observed in these mice (Figs 4-18, 4-20).

Rationale

The demonstration that the effector caspase-3 is cleaved and thus, activated, in D257A

mice does not provide proof of activation of a mitochondrial-mediated pathway of apoptosis,

since other apoptotic pathways, such as, receptor-mediated (extrinsic pathway), and ER-stress-

mediated pathways may also lead to the activation of the final effector caspase-3. Since the

accumulation of mtDNA mutations was expected to cause changes in mitochondrial









bioenergetics, ultimately leading to mitochondrial dysfunction, we hypothesized that the pathway

of apoptosis would be intrinsic to the mitochondria. The D257A mouse model allowed us to

elucidate the relevant mitochondrial pro-apoptotic proteins that are activated in response to

mtDNA mutations. Apoptosis originating from these pathways has been strongly implicated to

be causal in the aging process and is also highly relevant to many clinical conditions in humans

that are associated with mtDNA mutations (147). Since the specific functions of AIF and EndoG

(caspase-independent pathway) in mitochondrial function, as well as, in apoptosis-initiated by

the mitochondria remain to be substantiated, we evaluated the levels of key regulators of the

main caspase-dependent, mitochondrial-mediated pathway of apoptosis: Cytochrome c and the

initiator caspase-9. We further correlated caspase-9 activity levels with caspase-3 activity levels

(Fig 4-21) in order to demonstrate that activation of caspase-9 indeed leads to downstream

activation of caspase-3 and apoptosis.

Experimental approach

Determination of cytochrome c content by Western Blotting

For quantification of cytochrome c content by Western blot analysis, proteins were

separated using 15% PAGEr Gold pre-cast Tris-glycine gels (Cambrex, USA) under

denaturing conditions, and then transferred to PVDF membranes (0.2 rim, Trans-Blot Transfer

Medium, Bio-Rad Laboratories, CA USA). Protein concentration was determined using the

Bradford assay, and was subsequently normalized so that the protein content among samples is

identical. Subsequently, 20 ptl of sample were loaded to each well. 5 ptl of purified human heart

mitochondria were also loaded in a well as an appropriate positive control. Membranes were

blocked for 1.5 hrs using a blocking solution containing TBS and 5% milk. Membranes were

then incubated overnight in the 5% blocking solution containing the cytochrome c monoclonal









primary antibody at a dilution of 1:1000. The following day membranes were incubated for 1 h

at room temperature with IgG horseradish peroxidase-linked whole secondary antibody (1:1000,

Amersham Biosciences UK Ltd, Amersham, UK). Specific protein bands were visualized using

ECL plus reagent (Amersham Pharmacia Biotech, UK). The resulting Western blots were

analyzed using the Alpha Innotech FluorChem SP imaging system. Specific protein bands were

further normalized to tubulin. Values were expressed as arbitrary units after normalizing and

expressing samples as % of a control sample that was included in all membranes.

Enzymatic measurement of caspase-9 activity

Caspase -9 activity was measured using a fluorometric protease assay kit: (Caspase-

9/Mch6, Biovision, Mountain View, CA, USA) according to manufacturer's instructions.

Briefly, the assay is based on detection of cleavage of the substrate LEHD-AFC (AFC: 7-amino-

4-trifluoromethyl Coumarin) by caspase-9. LEHD-AFC emits blue light (Xmax = 400 nm); upon

cleavage of the substrate by caspase-9, free AFC emits a yellow-green fluorescence (Xmax = 505

nm), which can be quantified using a fluorescence microtiter plate reader. Samples were run in

triplicate and values were expressed as raw fluorescence units per mg of cytosolic protein.



Statistical Analyses

All results are expressed as means + SEM and the means obtained were used for

independent t tests. Statistical analyses were carried out using the Graph-Pad Prism 4.0 statistical

analysis program (San Diego, CA, USA). Statistical significance was set at P<0.05.






































Link between mitochondrial d\ sflinction and apoptosis



Figure 3-1. Experimental design and summary of the parameters measured in specific aims 1, 2 and 3.









CHAPTER 4
RESULTS

Mouse Characterization Data from Dr. Prolla's Lab: Generation and Phenotype of D257A
Mice

In order to elucidate the role of mtDNA mutations in skeletal muscle loss, observed with

age, we will use a "knock in" mouse model (PolgD257A) with increased spontaneous mutation

rates in mtDNA. In brief, these mice contain a mutation that results in a functional disruption of

the exonuclease domain of mouse mitochondrial DNA polymerase y, POLG.

Based on yeast, site directed mutagenesis studies, our collaborators constructed a mutation

that corresponds to the D230 substitution in yeast (D257 in mice), which was the substitution

that elicited the strongest mutator phenotype in vivo among the substitutions tested (148). This

residue, D257, is conserved in all POLG proteins identified to date and is involved in dNMP and

divalent ion binding, playing an essential role in the catalytic activity of the 3'-5' exonuclease

(149, 150). This mutation completely abolishes POLG exonuclease activity in yeast and mice,

but has no significant effect on polymerase activity (148).

The mouse POLG locus, PolgA, was cloned and gene targeting in embryonic stem (ES)

cells was used to introduce an AC -> CT two-base substitution that corresponds to positions

1054 and 1055 of the exonuclease-encoding domain (see supporting data on Science online).

This mutation results in a critical residue substitution in the conserved exonuclease domain of

POLG, impairing its proofreading ability (14). Seven correctly targeted ES cell clones were

expanded and the cells were injected into blastocysts derived from B6 female mice. Injected

blastocysts were implanted in pseudo-pregnant females for generation of chimeric mice. Several

chimeric mice were identified as determined by coat color. Of these, six chimeras, representing

four different ES cell clone lines, resulted in germline transmission of the PolgD257A allele when









mated to B6 females. Germline transmission of the mutation produced PolgAD257A+ mice, which

were then intercrossed to generate homozygous PolgAD257A/D257A mice. Mice carrying one copy

of the PolgD257A allele are healthy and fertile, and are continuously used to generate homozygote

Polg257A mice. Young homozygous PolgD257A mice, which are devoid of WT Polg protein, were

indistinguishable from wild-type littermates, however, long-term follow-up revealed a striking

premature aging phenotype beginning at 9 months of age (13). Phenotypes are age-related and

consisted of: hair loss, loss of bone mass, hearing loss, kyphosis, skeletal muscle loss, and

cardiac dysfunction (Fig 4-1). The mutant mice have a significantly reduced life span (for

D257A mice, maximum survival 460 days, median survival 416 days; for wild-type littermates,

maximum and median survival >850 days (Fig 4-1) (13).

Data from Our Lab

In our pilot study we had obtained data on both 3-mo and 11-mo old WT and D257A

animals in several parameters, therefore, we occasionally report results obtained at both time

points.

Results for Specific Aim 1

Impaired mitochondrial bioenergetics in 11-month-old D257A mice

We have evaluated mitochondrial respiration in 3-month old and 11-month old WT and

D257A mice. 02 consumption by skeletal muscle mitochondria was almost identical between

WT and D257A mice at 3-mo of age for both state 4 (WT: 8.4 0.7 nmol/min/mg protein vs.

D257A: 8.2 1.2, p=0.9) and state 3 respiration (WT: 41.1 3.3 vs. D257A: 38.4 4.7, p=0.64)

(Fig A-3). This suggests that mtDNA mutations do not affect mitochondrial bioenergetics early

on in the D257A animal's life, and that the D257A phenotype is age-induced. At 11-months, 02

consumption during state 4, the resting state of the mitochondria, did not differ between

genotypes (WT: 12.7 1.3 vs. D257A: 11.9 0.95, p=0.31), which was not unexpected since 02









consumption during this state is usually minimal. However, at 11 months of age mutant

mitochondria displayed a marked decrease in oxygen consumption (-43 %) during state 3 (WT:

68.4 5.1 vs. D257A: 39 5.8, p=0.0006) (Fig 4-2), the phosphorylative state of the

mitochondria, which also led to a significantly lower respiratory control ratio (RCR: -43 %) for

the mutant mitochondria (WT: 5.7 0.49 vs. D257A: 3.27 0.39, p=0.0005). RCR is used as an

index of mitochondrial coupling and the significant decrease in the D257A mitochondria

suggests that there is significant uncoupling between oxidation and phosphorylation (Fig 4-2). It

is therefore evident that accumulation of mtDNA mutations may lead to mitochondrial

dysfunction associated with compromised state 3 respiration.

D257A mice display decreased content of ETC Complexes I, III, and IV that contain
mtDNA-encoded subunits

We measured the content of ETC complexes I, II, III, IV and F1 domain of ATPase in 11-

mo old WT and D257A skeletal muscle using blue native page. We found that the total contents

of complexes I (WT: 40050 2281 arbitrary units vs. D257A: 26100 2724, p=0.002), III

(WT: 50970 3673 vs. D257A: 31960 4925, p=0.0093), and IV (WT: 50900 4782 vs.

D257A: 25460 5532, p=0.0046), all of which contain subunits encoded by mtDNA, were

significantly reduced in D257A mice (Figs 4-3, 4-4), suggesting that complex formation in

D257A mice specifically those containing subunits encoded by mtDNA is abolished. In

contrast, the content of complex II (WT: 20710 4079 vs. D257A: 28610 7051, p=0.3513) and

Fl (WT: 19760 2831 vs. D257A: 18330 747.9, p=0.64), both of which contain only nuclear-

encoded subunits, was not different between genotypes (Figs 4-3, 4-4). The latter reinforces the

idea that the accumulation of mtDNA mutations directly impacts assembly of complexes that are

partly mitochondrial-encoded, while all nuclear-encoded complexes appear unaffected.









Electron transport chain complex specific activity remains unaffected by mtDNA
mutations in D257A mice

The activities of complex I and IV (partly mtDNA-encoded) appear greatly reduced in the

mutant mice (Fig 4-5) while for the all-nuclear-encoded complexes II, and F domain there are

no apparent differences between genotypes (Fig 4-5). However, when we normalized the activity

for each sample to the respective complex content we saw no differences between WT and

D257A mice for all complexes evaluated (Fig 4-6): Complex I (WT: 314.5 13.56 arbitrary

units vs. D257A: 349.1 + 28.8, p=0.29), complex II (WT: 313 + 118.9 vs. D257A: 163.8 + 26.3,

p=0.26), complex IV (WT: 364.7 19.6 vs. D257A: 440 100.5, p=0.49), Fl domain of ATPase

(WT: 435.1 96.5 vs. D257A: 384.3 18, p=0.62).When we take into account the complex

content and activity results (Figs 4-3, 4-4, 4-5, 4-6) as well, as the 02 consumption (Fig 4-2) and

the ATP content data (Fig 4-8), we can conclude that although the content of ETC complexes per

mitochondrion, or per amount of total mitochondrial protein is reduced, the activity of the

remaining complexes remains unaffected, at least at the time of the measurement which

represents a "snap shot" in the continuum of time. However, this still leaves mitochondria with

energy deficits which are well demonstrated in our experiments by greatly compromised

mitochondrial respiration and reductions in ATP content.

D257A mice show decreased content of both nuclear-encoded and mitochondrial-encoded
ETC subunits

Besides measuring the content of fully assembled and enzymatically active ETC

complexes, we further determined the content of selected individual subunits from each complex.

We evaluated the subunit NDUFA9 from complex I, which is nuclear-encoded, as well as the

subunit NDUFS3 from complex I which is mitochondrial-encoded. We also evaluated one

subunit from complex II (70 kDa) and 2 selected subunits from complex III, 29 kDa and 48 kDa,

all of which are nuclear-encoded. Last, we evaluated the COX1 subunit from complex IV which









is mitochondrial-encoded and is a part of the active redox center of this complex, and is thus,

essential for catalysis. We observed a significant down-regulation of protein expression in the

D257A mice compared to WT, for all subunits evaluated either nuclear- or mitochondrial-

encoded (NDUFA9-WT: 1.47 0.14 arbitrary units vs. D257A: 0.36 0.024, p<0.0001)

(NDUFS3-WT: 3.8 0.2 vs. D257A: 3.1 0.2, p=0.03) (Complex II 70kDa-WT: 0.8 0.025 vs.

D257A: 0.53 0.034, p<0.0001) (Complex III 48kDa-WT: 2.05 0.13 vs. D257A: 1.2 0.07,

p<0.0001) (COX1-WT: 1.44 0.12 vs. D257A: 0.53 0.05, p<0.0001) (Fig 4-7), with the

exception of the 29kDa subunit of complex III which was almost significantly affected in the

mutant mice (WT: 0.6 0.055 vs. D257A: 0.47 0.04, p=0.07) (Fig 4-7).

D257A mice display decreased ATP content

ATP content, determined at 11-mo of age was significantly lower in D257A mice

compared to WT (WT: 0.29 0.08 nmol/mg protein vs. D257A: 0.11 0.04, p=0.046) (Fig 4-8).

It is apparent that loss of ETC complex content (see Figs 4-3, 4-4) can have an impact on ATP

content. Therefore, if ETC complex content is reduced in D257A muscle per amount of total

mitochondrial protein, as we have observed (Figs 4-3, 4-4), it is only expected that ATP content

per amount of total mitochondrial protein would be reduced, as we also show, because there are

probably less ETC complexes per mitochondrion. ATP production at the same time point

remained unaffected by the accumulation of mtDNA mutations (WT: 142.3 19.65 nmol/mg

protein/min vs. D257A: 124.7 21.7, p=0.28) (Fig 4-8).

Mitochondrial membrane potential is significantly lower in D257A mice

We determined the effect of increased mtDNA mutational load on mitochondrial

membrane potential (Ax) in 13-mo old WT and D257A skeletal muscle. Membrane potential

was significantly lower in D257A mice during both state-4 (WT: 195.2 + 1.4 mV vs. D257A:

187.9 + 2.15 mV, p=0.017) (Fig 4-9) and state 3 (WT: 177.7 2.5 vs. D257A: 167.3 2.25,









p=0.01) (Fig 4-9). This drop in Ay is possibly conferred upon energy deficits in the

mitochondria due to the dysfunction of ETC complexes (see Figs 4-2, 4-3, 4-4, 4-7 and 4-8) and

can be the trigger for the mitochondrial-mediated apoptosis we detected in the mutant mice (see

Figs 4-16, 4-17, 4-18, 4-20).

Mitochondrial protein yield is reduced in skeletal muscle of D257A mice

We measured total mitochondrial protein yield in 13-mo old WT and D257A skeletal

muscle and found that mitochondrial yield is drastically reduced in D257A mice by 13-mo of age

(45.9% reduction compared to WT) compared to WT (WT: 4.3 0.14 mg of mitochondrial

protein/gram of muscle tissue vs. D257A: 2.35 0.2, p<0.0001) (Fig 4-10). Interestingly, this

may suggest that mitochondria are getting eliminated in D257A mice. We also compared this

content with a group of -10-11 mo old animals in order to observe whether it gets continuously

reduced as the D257A animals get older and closer to their mean lifespan. Indeed, at 11 months

we also observed a significant reduction (35% reduction) in the mitochondrial content but not to

the extend we saw at 13 months (WT: 4 0.14 vs. D257A: 2.6 0.06, p=0.0044) (Fig 4-10).

Combined the above results suggest that mitochondria are probably getting continuously

eliminated in skeletal muscle throughout the lifespan of D257A mice.

Skeletal muscle mitochondria from D257A mice produce significantly less ROS

The main tenet of the free radical theory of aging (70) is that aging is due to the progressive

accrual of ROS-inflicted damage, including mtDNA mutations, the accumulation of which has

been postulated to lead to a "vicious cycle" of further mitochondrial ROS generation and

mitochondrial dysfunction (5, 6). To test this hypothesis, we measured H202 produced by

skeletal muscle mitochondria of young and old (3-mo and 11-mo old) WT and D257A mice.

H202 production was measured during state 4 since ROS production is highest when electron

flow is low, while during state 3 ROS production is nearly negligible. Levels of H202 were not









significantly different between genotypes at the 3-mo time point (WT: 0.30 + 0.05 nmol H202/

min/mg protein vs. D257A: 0.26 0.06, p=0.6) (Fig A-4). Interestingly, at 11-mo of age, H202

production was significantly decreased (-36 %) in D257A mice (WT: 0.6 0.07 nmol H202/

min/mg protein vs. D257A: 0.4 0.05, p=0.01) (Fig 4-11), and coupled to the decreased state-3

respiration (Fig 4-2). The decreased H202 production by mutant mitochondria also led to the

calculation of a significantly lower free radical leak for the D257A mitochondria (WT: 2.6 +

0.3% vs. D257A: 1.8 0.3%, p=0.04) (Fig 4-11). These observations do not support the

"mitochondrial vicious-cycle" hypothesis of aging, but instead suggest that mtDNA mutational

load is causal to reduced mitochondrial function, as demonstrated by the marked decrease in

oxygen consumption and the significant mitochondrial uncoupling. However, the accumulation

of mutations does not induce an increase in mitochondrial ROS production. Similar results

regarding mitochondrial ROS production have recently been published using mouse embryonic

fibroblasts (MEFs) from D257A mutator mice (79) and this study also questioned the accuracy

of the mitochondrial "vicious cycle" theory.

D257A mitochondria produce less ROS in both main ROS generators of the ETC: Complex
I and Complex III

When we evaluated site specific ROS generation in 3-mo old mice we found no differences

between genotypes in ROS production at either complex I -representing total basal ROS

generation (Fig A-4: panels A) (WT: 0.3 0.05 nmol H202/ min/mg protein vs. D257A: 0.26 +

0.06, p=0.6) or Complex III (Reverse flux included-WT: 4.6 0.56 vs. D257A: 4.15 0.67,

p=0.6) (Reverse flux blocked-WT: 1.13 0.23 vs. D257A: 1.1 0.3, p=0.9) (Fig A-4: panels C,

D), or in the maximal capacity of these complexes to generate ROS (Fig A-4: panels B, E)

(Maximal complex I production WT: 1.6 0.19 vs. D257A: 1.75 0.18, p= 0.68) (Maximal

complex III production WT: 8.6 0.9 vs. D257A: 8.4 0.6, p= 0.84). As expected, the free









radical leak percent (Fig A-4: panel F) was also not different between WT and D257A mice

(WT: 2 0.45% vs. D257A: 1.75 0.4%, p=0.7). At 11-mo of age our results were consistent,

showing that, D257A mitochondria produce less ROS at complex III, either when reverse

electron flux is taken into account (WT: 6.3 0.47 nmol H202/ min/mg protein vs. D257A: 3.13

0.48, p=0.0002) (Fig 4-12: panel B) or when it's blocked (WT: 1.5 0.14 vs. D257A: 0.9

0.12, p=0.005) (Fig 4-12: panel C), and have reduced maximal capacity to generate ROS at both

complex I (WT: 2.9 0.23 vs. D257A: 1.2 0.19, p<0.0001) (Fig 4-12: panel A) and complex

III (WT: 11.9 + 1.2 vs. D257A: 6.75 0.88, p=0.003) (Fig 4-12: panel D) compared to WT.

Moreover, the fact that H202 production is decreased almost fourfold for both WT and D257A

mice when the reverse electron flux is blocked (when comparing Y axis values from Fig 4-12,

panels B and C) signifies that this reverse flow is a significant source of ROS produced by the

ETC. These observations combined provide further support to our previous results regarding

total basal ROS production by the mitochondria (see Fig 4-11), as well as, additional evidence

against the mitochondrial "vicious cycle" theory.

No difference in antioxidant enzyme mRNA expression between genotypes

We measured mRNA expression of Catalase and the mitochondrial-specific isoform of

SOD, MnSOD, in skeletal muscle of 11-mo old WT and D257A mice, via RT-PCR. We found

no difference in either Catalase (WT: 1.2 0.08 arbitrary units vs. D257A: 1.1 0.04, p=0.1)

(Fig 4-13) or MnSOD (WT: 0.8 0.01 arbitrary units vs. D257A: 0.7 0.07, p=0.3) (Fig 4-13)

between genotypes. This provides further support to the notion that mitochondria from D257A

mice actually produce less ROS and that the decrease we observed in H202 production was not

due to an adaptive up-regulation of antioxidant defenses in the mutant mice. In fact, in D257A

muscle, there was a strong trend toward decrease, especially, in Catalase mRNA expression. The









fact that mutant mice generate less H202 might explain the no-need for up-regulation of

antioxidant defenses compared to WT.

Mitochondrial DNA mutations cause aging phenotypes in the absence of increased
oxidative stress

In order to correlate our H202 results with further oxidative stress, we next examined a

marker of ROS-induced oxidative damage to DNA, by assessing the levels of 8-oxo-7,8-dihydro-

2'-deoxyguanosine (8-oxodGuo) in skeletal muscle mtDNA of 11-mo old WT and D257A mice,

using HPLC with electrochemical detection. We did not find any differences in the levels of

mtDNA oxidation between 11-mo old WT and D257A mice (WT: 51.4 6.3 8-oxodGuo/106

dGuo vs. D257A: 50.3 7.2, p=0.9) (Fig 4-14). Moreover, in published results, we also showed

no significant differences between WT and D257A skeletal muscle in F2-isoprostanes, a marker

of lipid peroxidation (13). Thus, an increased load of mtDNA mutations does not appear to be

associated with increased levels of oxidative damage to mtDNA (Fig 4-14), or elevated lipid

peroxidation in skeletal muscle. A recent publication provided further support to our outcomes

showing that protein carbonylation, and thus, oxidative damage to proteins was not significantly

different in mtDNA mutator mice compared to WT (79). Hence, despite increased mutational

load, mitochondria from D257A mice do not show increased oxidative stress.

Results for Specific Aim 2

D257A mice display significant skeletal muscle loss by 11-mo of age

We obtained data on 3-mo old and 11-mo old WT and D257A mice. At 3-mo-of age there

was no significant difference in skeletal muscle weight between WT and D257A mice (WT: 170

6 mg vs. D257A: 150 + 8, p=0.24) (Fig A-i). However, at 11-mo of age D257A mice

exhibited significant skeletal muscle loss in the gastrocnemius (-24 %) (WT: 160 6 mg vs.

D257A: 126 5, p=0.0004) (Fig 4-15), and in the quadriceps muscle (-19 %) (WT: 190 6 mg









vs. D257A: 0.156 0.007, p=0.0003) (Fig 4-15) compared to WT, which is indicative of

sarcopenia, since normally aged animals (30-mo WT) also showed similar degree of muscle loss

compared to young animals (5-mo WT) (WT: 145.7 9.3 mg vs. D257A: 109.9 6.6, p=0.0095)

(Fig 4-15).

Apoptosis in D257A skeletal muscle is evident by an increase in cytosolic mono- and oligo-
nucleosomes

We quantified apoptotic DNA fragmentation in skeletal muscle of 11-mo old WT and

D257A mice by measuring the amount of mono- and oligo-nucleosomes released in the cytosol,

using a quantitative "Cell Death" detection ELISA. These, are characteristic fragments of-180-

200 bp or multiples and are specific to apoptosis. We observed a significant release of these

fragments into the cytosol in the D257A muscle (WT: 0.11 0.006 OD/mg protein vs. D257A:

0.17 0.03, p=0.035) (Fig 4-16) indicating that apoptosis indeed occurs in these mice and is, at

least, partly responsible for the loss of skeletal muscle mass observed in these mice (Fig 4-15, 4-

16).

DNA laddering is evident in skeletal muscle of D257A mice

To further demonstrate and corroborate apoptosis in D257A skeletal muscle we performed

a standard measure of apoptosis: DNA laddering. This enables the detection and visualization of

nucleosomal ladders of -180-200 bp or multiples, characteristic of apoptosis. Prominent DNA

ladders are evident for the D257A mice while ladders are very minimal or non-existent for WT

mice (Fig 4-17). This small-scale DNA fragmentation further confirms that apoptosis is an

important mechanism of sarcopenia in the mutant mice.

Caspase-3 cleavage and activation is up-regulated in D257A mice and resembles caspse-3
activation during normal aging

We evaluated apoptosis in skeletal muscle by measuring the content of activated (cleaved)

caspase-3, by western blotting. Caspase-3 is the final effector caspase for many apoptotic









pathways and its cleavage at the C-terminal side of a specific aspartate residue is considered as

one of the hallmarks of apoptosis.

To determine if increased levels of cleaved caspase-3 is a feature of normal aging, we first

examined caspase-3 content in tissues of 5 mo-old and 30 mo-old WT mice (Fig 4-18) (13).

Cleaved caspase-3 levels significantly increased with normal aging in skeletal muscle of WT

mice by -32% (5-mo old: 43130 4704 arbitrary units vs. 30-mo old: 63620 4510, p=0.0085).

We further evaluated caspase-3 levels in skeletal muscle of 3-mo old and 11-mo old D257A and

WT mice. Levels of cleaved caspase-3 did not differ between WT and D257A mice at 3-mo of

age (WT: 26950 5802 vs. D257A: 21660 3924, p=0.46) (Fig A2), suggesting that the D257A

phenotype is age-induced. Similar to normal aging, cleaved caspase-3 levels were also

significantly elevated in D257A skeletal muscle by 11 months of age compared to controls (WT:

31580 1408 arbitrary units vs. D257A: 56780 + 8925, p=0.016) (Fig 4-18) (13), a time point at

which mutant animals also displayed significant loss of muscle mass. This suggests that

apoptosis, mediated by caspase-3 activation, is probably an important mechanism of skeletal

muscle loss in the mutant mice and also during normal aging.

Together, these findings suggest that normal aging, as well as, accelerated aging induced

by the accumulation of mtDNA mutations, are associated with the activation of a caspase-3

mediated apoptotic pathway in skeletal muscle. The observation of a similar response between

normal and accelerated aging constitutes the D257A an appropriate mouse model to study the

possible mechanisms of muscle wasting with age. Moreover, loss of critical, irreplaceable cells

through apoptosis may be a central mechanism of skeletal muscle loss associated with the

accumulation of mtDNA mutations during the aging process.









Results for Specific Aim 3

Cytochrome c release in the cytosol of D257A and WT skeletal muscle

We measured cytochrome c release in the cytosol by performing a western blot for

cytochrome c in the cytosolic fraction isolated from skeletal muscle of 13-mo old WT and

D257A mice. We did not detect significant differences between WT and D257A mice in

cytosolic cytochrome c content, although we expected that cytochrome c release in the cytosol of

D257A skeletal muscle would be significant (WT: 3.6 0.2 arbitrary units vs. 3.56 0.35,

p=0.87) (Fig 4-19). It is very possible that our cytosol was contaminated with mitochondrial

protein due to the mitochondrial isolation procedure. Basically, during mitochondrial isolation

although most of the mitochondria isolated are intact and fully functional, some may get

destroyed during homogenization, releasing many of the soluble proteins in the cytosol.

Unfortunately, once this occurs, even if we further purify the cytosol nothing changes in the case

of cytochrome c because it's a soluble protein.

Caspase-3 and caspase-9 activities are significantly higher in D257A mice: Evidence for
induction of the mitochondrial, caspase-dependent pathway of apoptosis

We measured caspase-3 activity in the cytosol of 11-mo old WT and D257A skeletal

muscle and found that it is significantly higher in the mutant mice (WT: 43 2.7 RFU/mg

protein vs. D257A: 57.7 1.97, p= 0.0003) (Fig 4-20). Similarly, caspase-9 activity showed the

same response: significant increase in D257A mice compared to WT (WT: 35.4 2 RFU/mg

protein vs. D257A: 45.3 1.7, p= 0.0014) (Fig 4-20). In addition, when we correlated caspase-3

activity with caspase-9 activity we found significant correlations for both WT (r = 0.97,

p<0.0001) and D257A mice (r = 0.8, p=0.0029) (Fig 4-21). These results provide proof for the

induction of the main mitochondrial mediated, caspase-dependent pathway of apoptosis since

activation of caspase-9 is evident in the mutant mice, which in turn leads to further cleavage and









activation of the final effector caspase, caspase-3 in this pathway (Fig 4-18, 4-20), which is

directly responsible for the downstream events (i.e. cleavage of endo-nucleases and DNA repair

enzymes) that lead to apoptosis.






































D257A mice at -13 months of age. Progeroid features including hair loss, graying
and kyphosis become apparent at ~9 months of age. (D) Kaplan-Meier survival
analysis of cohorts of WT (+/+), D257A heterozygous mice (D257A/+) and D257A

homozygous mice (D257A/D257A). At least 230 mice per genotype are represented
in the survival curves.





STATF A STATF 3 RCR


C

Q.
10-
E

E
o
Si 5-
0

E
C -


T


WT D257A


D257A


WT D257A


Figure 4- 2. Mitochondrial respiration is compromised in skeletal muscle of D257A mice. We
determined the effects of mtDNA mutations on 02 consumption of skeletal muscle
mitochondria obtained from 11-mo old WT and D257A mice. Oxygen consumption
was measured during state 4 (non-phosphorylative state and during state 3
(phosphorylative state). The respiratory control ratio (RCR), an index of
mitochondrial coupling, was calculated by dividing state 3 to state 4 respiration
values. Error bars represent SEM. *P < 0.5.


Y ~'L~


V B B V















WT WT D257A


III -

ATPase -


IVI







Fig. 4-3. D257A mice display decreased content of ETC Complexes I, III and IV that contain
mtDNA-encoded subunits. The total content of ETC complexes I, II, III, IV and the
Fl domain of the ATPase from skeletal muscle of 11-mo old WT and D257A mice
was determined using Blue Native Page electrophoresis followed by staining with
commassie blue stain. Proteins were separated according to molecular weight.
Representative blots are depicted above.















ETC Total Complex Content


Complex I content
50000-

40000-

30000-

20000-

10000-


WT D257A


Complex II content
40000-1


30000-


20000-


10000-


WT D257A


Complex III content
60000-

50000-

40000-

30000

20000-

10000

0
WT D257A


Complex IV content
60000-

50000-

40000-

30000-

20000-

10000-

0WT D257A
WT D257A


Complex V (FIATPase) content
250001


20000-

3 15000-

10000-

5000-

0-


WI UZbtA


Fig. 4-4. Statistical analysis of ETC complex I, II, III, IV and the Fl domain of the ATPase
content measured by Blue Native Page in skeletal muscle of 11-mo old WT and
D257A mice. Arbitrary units represent densitometry values normalized to total
protein loaded measured by the Bradford assay. Error bars represent SEM. *P < 0.5.



















II -





IV->





V->



Fl ATPase->


WT WT


Fig. 4-5. Electron transport chain complex activity in skeletal muscle of 11-mo old WT and
D257A mice. The activity of ETC complexes I, II, IV and the Fl domain of the
ATPase was determined using Blue Native Page electrophoresis followed by
enzymatic colorimetric reactions performed on the gels. Representative blots are
depicted above.


D257A











ETC Complex Specific Activity


Complex I activity


Complex II activity


-r


WT D257A


Complex IV activity
550-
500-
450-
400-
350-
300-
250-
200-
150-
100-
50-
0
WT D257A


WT D257A


Complex V (FIATPase) activity


WT D257A


Fig. 4-6. Statistical analysis of ETC complex activity in skeletal muscle of 11-mo old WT and
D257A mice. The activity of ETC complexes I, II, IV and the Fl domain of the
ATPase was determined using Blue Native Page electrophoresis followed by
enzymatic colorimetric reactions performed on the gels. Arbitrary units represent
activity densitometry values normalized to respective content densitometry values for
each sample. Error bars represent SEM.









WT


Cxl 39KDa

Cxl 30KDa

Cxll 70KDa

Cxlll 48KDa

Cxlll 29KDa

CxlV-COXI



AIF


Porin

Actin

Cxl-NDUFA9


WT D257A


4.5-
4.0-
3.5-

> 2.5-
S2.0-
* 2 1.5-
1.0-
0.5-
0.00


CxlI-48


D257A


Cxl-NDUFS3


WT D257A


CxlI-29


I i
-I



Fig. 4-7. D257A mice show decreased content of both nuclear-encoded and mitochondrial-
encoded ETC subunits. The content of selected nuclear- and mitochondrial-encoded
subunits from complexes I, II, III and IV, were evaluated by Western Blotting in 11-
mo old WT and D257A mice. Representative blots are depicted above. Results shown
above were normalized to porin. Error bars represent SEM. *P < 0.5. Cx: complex


WT D257A


CxlV-COX I


---r -


VVI ULOIA


VVI ULOIA


VVI ULOIA


-=o


I I I ma


I I













ATP content


ATP production


-r


D257A


Fig. 4-8. D257A mice display decreased ATP content. We determined the effects of mtDNA
mutations on ATP content and production in skeletal muscle mitochondria obtained
from 11-mo old WT and D257A mice. Error bars represent SEM. *P < 0.5.





Mitochondrial Membrane Potential


Glutamate/Malate
State 4
205-
195
185
> 175
E 165
155
145]
135
WT D257A


ADP
State 3


175-


145-


WT D257A


CCCP
Uncoupled state
140-
120
100
> 80-
E 60

40
20

WT D257A


Fig. 4-9. Mitochondrial membrane potential (Ay) drop in D257A mice. We determined the
effects of mtDNA mutations on Ay in skeletal muscle mitochondria obtained from
13-mo old WT and D257A mice. Changes in Ay were followed qualitatively by
monitoring the fluorescence of TMRM that accumulates in energized mitochondria.
Ay was measured during both state 4 (non-phosphorylative state) and during state 3
(phosphorylative state). Measurement of Ay after addition of CCCP served as a
control for TMRM binding. Error bars represent SEM. *P < 0.5.


0.01-











Mitochondrial Protein Yield

13 months 11 months
.E 4.5- 4 5-
o 4.0- o
3.5- CL 4-
.- 3.0- 2 ..

0 0
E 2.0- E
I 2-
S1.5-

o 0.5 o
E 0.0- E 0-
WT D257A WT D257A


Fig. 4-10. Mitochondrial yield is reduced in D257A skeletal muscle. We determined total
mitochondrial yield in 11- and 13-mo old WT and D257A mice by dividing the
mitochondrial protein content measured by the Bradford assay by the skeletal muscle
weight used each time to obtain the respective mitochondrial fractions. Error bars
represent SEM. *P < 0.5.



Basal Mitochondrial ROS Production

Pyruvate/Malate Free Radical Leak
S0.7 3
o 0.6
a 0.5 -







WT D257A WT D257A
E
S0.3
0
C4 0.2
I
S0.1
E
S0.0 0
WT D257A WT D257A


Fig. 4-11. D257A mitochondria produce less reactive oxygen species (ROS) during state 4. We
measured H202 production since it represents total basal mitochondrial ROS
generation. Skeletal muscle mitochondria were obtained from 11-mo old, WT and
D257A mice and supplemented with pyruvate/malate as substrate for oxidative
phosphorylation. Pyruvate/malate was used to study complex I ROS production
which also represents total mitochondrial ROS production. Free radical leak percent
(FRL%), an index of mitochondrial efficiency, was calculated by dividing the H202
value by twice the state 4 respiration value and the result was multiplied by 100 to
give a % final value. Error bars represent SEM. *P < 0.05.












Site Specific ROS Generation


Pyruvate/Malate + Rotenone
Maximal Complex I ROS production


WT D257A


Succinate + Rotenone
ROS generation at Complex III
Reverse electron flux blocked
1.75-
. 1.50- C
1.25-
1.00-
0.75-
0.50-
0.25-
0.00-
WT D257A


Succinate
ROS generation at Complex III
+ Reverse electron flux
7-

E 5-
B

r- 4-
E



c 3
0
2-

E

WT D257A


Succinate + Rotenone + Antimycin
Maximal Complex III ROS production


---


WT D257A


Fig. 4-12. D257A mitochondria produce less ROS in both main ROS generators: Complex I and
Complex III. Skeletal muscle mitochondria were obtained from 11-mo old, WT and
D257A mice. We used inhibitors of the ETC in order to study maximum rates of
H202 production from complexes I and III, since they represent the main sites of ROS
generation within the mitochondria. For complex I maximum rate (panel A) we used
rotenone added to pyruvate/malate supplemented mitochondria. For complex III
maximum rate (panel D) we used antimycin A plus rotenone, added to succinate
supplemented mitochondria. We also used mitochondria supplemented with succinate
alone in order to study complex III ROS production under near physiological
conditions (panel B). In addition, some of the assays with succinate as substrate were
performed in the presence of rotenone (panel C), in order to avoid the backwards flow
of electrons to Complex I. Error bars represent SEM. *P < 0.05.












D257A W


I I


Catalase

MnSOD

Actin


Catalase


MnSOD


WT D257A


--


D257A


Fig. 4-13. D257A mice show no difference in antioxidant enzyme mRNA expression. We
measured Catalase and MnSOD mRNA expression in 11-mo old WT and D257A
mice by RT-PCR. Arbitrary units represent specific mRNA densitometry values
normalized to actin mRNA densitometry values. Error bars represent SEM.


WT














mtDNA oxidation


-r T-


WT D257A


Fig. 4-14. Mitochondrial DNA oxidation in skeletal muscle of WT and D257A mice. We
examined a marker of ROS-induced oxidative damage to DNA, by assessing the
levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in skeletal muscle
mtDNA of 11-mo old WT and D257A mice, using HPLC with electrochemical
detection. We found no differences between 11-month old WT and D257A mice.
Error bars represent SEM.


Gastrocnemius weight
176-
160.
126,
100
765
60
26
01
WT D267A


Quadriceps weight
200- -


160

S100


WT D267A


B Gastrocnemius weight
176-
160-
126

E 100-
E 76

60
26
0
6-mo WT 30-mo WT


Quadriceps weight


160-


E100-

60-


6-mo WT 30-mo WT


Fig. 4-15. D257A mice display significant skeletal muscle loss by 11-mo of age compared to
age-matched WT (panel A) which resembles sarcopenia during normal aging (panel
B). Gastrocnemius and quadriceps muscles were extracted immediately following
sacrifice, rinsed in saline solution and weighed. Error bars represent SEM. *P < 0.05.










Cell death


0.00--


WT D257A


Fig. 4-16. Apoptosis evident in D257A muscle by increase in cytosolic mono- and oligo-
nucleosomes. Cytosolic fractions from 11-mo old WT and D257A skeletal muscle
were prepared. Apoptosis was quantified as the amount of mono- and oligo-
nucleosomes present in the cytosol, using a sandwich ELISA. Error bars represent
SEM. *P < 0.05.


WT


D257A


control
4


I


Fig. 4-17. DNA laddering evident in skeletal muscle of D257A mice. DNA from 13-mo old WT
and D257A mice was extracted and subjected to a DNA laddering-specific ligation
PCR. PCR products were electrophoresed through 1% agarose gels and visualized
under UV light for apoptosis-specific DNA ladders of 180-200bp pr multiples. Lane
1: 100bp molecular marker. Lanes 2-9: WT PCR products. Lanes 10-17: D257A PCR
products. Lane 18: Positive control. Lane 19: 500bp molecular marker.


I


















70000-
60000-
50000-
40000-
30000-
20000-
10000-



0 0


70000-
60000-
50000-
40000-
30000-
20000-
10000-
0-


,43


Blots for panel B

WT PG WT PG WT PG WT PG WT PG WT PG

Caspase-3


13-actin ___


Fig. 4-18. Caspase-3 activation in skeletal muscle of D257A mice resembles caspase-3 activation
during normal aging. Panel A: Cleaved (activated) caspase-3 content with normal
aging: Comparison of young (5-mo) vs old (30-mo) WT mice. Panel B: Comparison
of WT versus D257A cleaved caspase-3 levels at 11-mo of age. Skeletal muscle
cytosolic extracts from WT and D257A mice were subjected to SDS-polyacrylamide
gel electrophoresis and probed with a rabbit monoclonal antibody against cleaved
caspase-3. Representative blots are shown above. Arbitrary units represent caspase-3
densitometry values normalized to p-actin densitometry values. Error bars represent
SEM. *P < 0.05.

















Cytosolic Cytochrome C


-r


WT D257A


Fig. 4-19. Cytochrome c release in the cytosol of D257A and WT skeletal muscle. Skeletal
muscle cytosolic extracts from 13-mo old WT and D257A mice were subjected to
SDS-polyacrylamide gel electrophoresis and probed with a mouse monoclonal
antibody against cytochrome c. Arbitrary units represent cytochrome c densitometry
values normalized to tubulin densitometry values. Error bars represent SEM.


0-I-













Caspase-3 activity


WT D257A


Caspase-9 activity


WT D257A


Fig. 4-20. Caspase-3 and -9 activities are elevated in D257A muscle: Proof of activation of the
mitochondrial caspase-dependent pathway of apoptosis. Cytosolic fractions from 11-
mo old WT and D257A skeletal muscle were prepared. Caspase -3 and -9 activities
were measured using a fluorometric protease assay kit which is based on detection of
cleavage of the substrate DEVD-AFC or LEHD-AFC by caspase-3 and -9
respectively. Error bars represent SEM. *P < 0.05.


y= 0.7196x + 4.5332
r = 0.97


20 30 40 50 60


Caspase-3 activity


D257A


60

S55-

50-

S45

a 40-

35


y= 0.6949x+ 5.1651
r = 0.8


Caspase-3 activity


Fig. 4-21. Caspase-3 and caspase-9 activity Pearson correlations in WT and D257A mice:
Caspase-3 activity was correlated with caspase-9 activity in WT (panel A) and
D257A (panel B) mice. Pearson r values are shown on top right corner. Correlations
were significant for both genotypes. *P < 0.05.









CHAPTER 5
DISCUSSION

Overview of Principal Findings

The overall goal of this project was to determine "in vivo" whether mtDNA mutations,

known to accumulate with aging in skeletal muscle fibers, are causal to the demise of skeletal

muscle with age, the condition commonly termed sarcopenia. For this purpose mice having a

progeroid syndrome, due to a mutation in the exonuclease domain of POLy that led to an

increase in spontaneous mutation rates in mtDNA, were utilized. The experiments conducted

examined the impact of increased mtDNA mutational load on both mitochondrial function and

mitochondrial-induced apoptosis in skeletal muscle via three separate groups of experiments.

Specific aim 1 tested the following questions: (a) Do mtDNA mutations lead to

mitochondrial dysfunction? (b) If mitochondrial dysfunction is evident is it associated with an

increase in mitochondrial ROS production? (c) If mitochondrial ROS generation is elevated does

it lead to further oxidative damage in mtDNA? (d) What is the primary mechanism by which

mtDNA mutations induce mitochondrial dysfunction? Our results reveal that mtDNA mutations

induce mitochondrial dysfunction, apparent by compromised mitochondrial respiration during

state 3, decreased ATP content, and a significant drop in membrane potential during both state 3

and state 4. Importantly, this compromised mitochondrial function is not accompanied by

elevations in ROS production or further oxidative damage to mtDNA, which is in contrast to the

main premise of the "Vicious Cycle" theory of aging. In fact, it appears that in skeletal muscle,

the accumulation of mtDNA mutations is associated with a significant decrease in mitochondrial

ROS production which was coupled to the decrease in state 3 respiration. Moreover, the primary

cause of the mitochondrial dysfunction appears to be the abrogation of ETC complexes I, III and

IV, all of which contain mtDNA-encoded subunits. In addition, energy deficits due to the latter









are likely responsible for the drop in Ay we observed which in turn is likely responsible for the

induction of apoptosis intrinsic to the mitochondria.

Specific aim 2 tested the following questions: (a) Is apoptosis the mechanism responsible

for skeletal muscle loss in D257A mice? (b) If apoptosis is evident, is it caspase-dependent? Our

data indeed confirms that apoptosis is induced in skeletal muscle of D257A mice. Apoptosis was

evident by DNA laddering and increased release of mono-and oligo-nucleosomes in the cytosol.

Furthermore, apoptosis was caspase-dependent since significant increases in both the content and

activity of the final effector caspase, caspase-3, were observed.

Specific aim 3 tested the question: Is apoptosis mitochondrial-mediated? Although we

were not able to show differences in cytochrome c release in the cytosol between genotypes, we

detected a significant up-regulation of caspase-9 activity with further downstream activation of

caspase-3 (as the caspase-3-,caspase-9 correlations suggest on Fig 4-21), in D257A skeletal

muscle. This proves that the main, caspase-dependent mitochondrial pathway is activated in

D257A mice and is at least, partly responsible for the sarcopenia observed in these mice.

Hypothesis One: The Effect of mtDNA Mutations in Skeletal Muscle Mitochondrial
Function

The goal of this aim was to determine that mtDNA mutations are directly responsible for a

measurable deficiency in cellular oxidative phosphorylation activity and if this was proven to be

true, to identify the series of events that lead to mitochondrial dysfunction. Therefore, an

important aspect of this aim was to test the mitochondrial "Vicious Cycle" theory of aging, or in

other words to examine whether mtDNA mutations indeed lead to an enhanced ROS production,

which in turn gives rise to the rate of mtDNA damage and mutagenesis, thus causing a 'Vicious

Cycle' of exponentially increasing oxidative damage and dysfunction, which ultimately

culminates in death.









Mitochondrial DNA Mutations Cause Profound Deficiencies in Mitochondrial Function

Information on the specific contribution of mtDNA instability to human aging phenotypes

can be inferred through the analysis of disorders associated with increased mtDNA mutation or

deletion frequency. For example, in mitochondrial diseases, it is well demonstrated that mtDNA

deletions, when present at concentrations of 30% and greater in muscle tissue, can cause three

disorders, Kearns-Sayre syndrome (KSS), chronic progressive external opthalmoplegia (CPEO),

and Pearson's syndrome (PS) (20, 151, 152). The affected tissues show impaired electron

transport activity, ATP production, and mitochondrial protein synthesis and decreased

mitochondrial membrane potential (153-156). Furthermore, muscle biopsies from patients with

KSS or CPEO show ragged red fibers and cytochrome oxidase (COX) -negative fibers (157,

158). Tissues most affected by disorders associated with inherited mtDNA mutations are the

same tissues markedly affected by normal aging; these include the brain, heart, skeletal muscle,

kidney and the endocrine system (159). Because the most obvious consequence of mtDNA

mutations is an impairment of energy metabolism, most studies addressing aging effects have

focused on tissues that are post mitotic and display high energetic demands, such as the heart,

skeletal muscle, and the brain. Indeed, several studies have unambiguously demonstrated that

mtDNA base substitution mutations accumulate as a result of aging in a variety of tissues and

species, including rodents, rhesus monkeys, and humans.

An ongoing debate in the field relates to the issue of causality: are mtDNA mutations

merely markers of biological age, or do they lead to a decline in physiological function that

contributes to the aging process? Studies on sarcopenia in rodents and human samples have

helped to address this issue. Studies using laser capture microdissection to study the role of

mtDNA deletion mutations in single skeletal muscle fibers from sarcopenic rats have shown that

mtDNA deletions colocalize with electron transport chain dysfunction and fiber atrophy (7).









Interestingly, the mutations are largely clonal and absent from phenotypically normal regions

within individual muscle fibers (43). In a similar study of aged (69-82 years old) human muscle

biopsies, an association between a deficiency in the mitochondrially encoded cytochrome c

oxidase (COX) and clonally expanded base-substitution mutations and deletions in mtDNA was

shown (49).

Perhaps the strongest evidence that clonally expanded mtDNA mutations can be causal in

both age-related dysfunction and disease comes from recent studies of neurons present in the

substantial nigra region of the human brain. These dopamine-rich, pigmented neurons contain

very high levels of mtDNA deletions. Deleted mtDNA molecules are clonal in each neuron, can

accumulate, reaching up to 60% of the total mtDNA and are associated with oxidative

phosphorylation defects (160). Cytochrome c oxidase-deficient cells have also been shown to

increase with age in both hippocampal pyramidal neurons and choroid plexus epithelial cells

(161). Although these studies do not prove causality, they provide strong evidence in support of

the hypothesis that mtDNA deletions play a contributing role in age-related mitochondrial

dysfunction leading to aging phenotypes in post-mitotic tissues of mammals.

In order to test the in vivo effects of increased somatic mtDNA mutation accumulation,

Larsson's group was the first to report results on the D257A knock-in mice (the same mice used

in this project) showing that mtDNA mutations and deletions are responsible for a progressive

decline in respiratory function of mitochondrially encoded complexes, that was evident as early

as 12 weeks, resulting in decreased oxygen consumption and ATP production (14, 79). In

accordance with the aforementioned studies in this section, we also found profound decreases in

mitochondrial 02 consumption during state 3, the active state of the mitochondria when ATP is









produced (Fig 4-2). Moreover, ATP content was significantly lower in D257A mice, compared

to WT (Fig 4-8).

These findings clearly indicate that oxidative phosphorylation is compromised in skeletal

muscle of mutant mice and provide a causal role of mtDNA mutations specifically in skeletal

muscle mitochondrial dysfunction. In addition, since mitochondrial ETC enzyme activity

declines, decreases in ATP synthesis and state 3 respiration, and energy depletion are all well

documented in normal aging in various species and tissues, including human skeletal muscle (39,

40, 162-165) (see also more extensive background info on mitochondrial function with aging in

chapter 2), it can be deduced that mtDNA mutations may contribute to the sarcopenic phenotype

not only in D257A mice but also during normal aging.

Furthermore, D257A skeletal muscle mitochondria were uncoupled since respiratory

control ratios (state 3/state 4 02 consumption) in our experiments were less than 3.5 (Fig 4-2).

These defects in oxidative phosphorylation we have observed are likely the cause for the

disruption of mitochondrial membrane potential we have also detected in mitochondria from

mutant mice (Fig 4-9). In support of the latter, it has been shown that in mitochondrial diseases,

the accumulation of mtDNA deletions causes deficits in basic bioenergetic parameters including

mitochondrial membrane potential (156, 166, 167). For example, in Leber's hereditary optic

neuropathy (LHON), a late onset neurological disorder associated with specific mtDNA point

mutations, Battisti et al., showed that lymphocytes from patients with LHON treated with the

oxidizing agent dRib had significant depolarization of the mitochondrial membrane potential

compared to control cells and an increase in the percentage of apoptotic cells with respect to

controls (166). The authors concluded that their results confirmed the notion of a direct link

between complex I (commonly altered in patients with LHON) and changes in mitochondrial









membrane permeability. Furthermore, in cybrid cells incorporating two pathogenic

mitochondrial DNA point mutations, 3243A > G and 3302A > G in tRNALeu(UUR), it was

shown that the lowered mitochondrial membrane potentials exhibited by the cells led to a

disturbed intramitochondrial calcium homeostasis, which was postulated to be a major

pathomechanism in mitochondrial diseases, according to the authors (167). Lower mitochondrial

Ays have also been observed in normally aged mitochondria of rodents and in skin fibroblasts

from elderly human subjects (34, 168-171), and low Ays were found to correlate with reduced

ATP synthesis. As the levels of mtDNA mutations have also been shown to increase with age in

both humans and rodents (as has been extensively discussed in chapter 2), these observations

provide further support to the notion that mtDNA mutations are important culprits for tissue

dysfunction with age.

Mitochondrial DNA Mutations Cause Mitochondrial Dysfunction in the Absence of
Increased ROS Production or Oxidative Damage to mtDNA: Implications for the
Mitochondrial "Vicious Cycle" Theory of Aging

It has been thought that loss of mitochondrial function and increased mitochondrial ROS

production are important causal factors in aging. Every human cell contains hundreds of

mitochondria, and each mitochondrion has multiple copies of mitochondrial DNA (mtDNA).

Because the mitochondrial genome codes for 13 polypeptides constituting the respiratory

enzyme complexes required for normal functioning of the oxidative phosphorylation system,

somatic mutations in mtDNA may be directly involved in the mechanism by which ROS initiate

a vicious cycle and cause aging. The previously mentioned vicious cycle theory of oxidative

damage to mtDNA (172) holds that oxidative damage, or resultant mutation, of the mtDNA

causes the assembly of a defective respiratory chain, which in turn causes the production of more









ROS, and the cycle repeats, with ever increasing dysfunctions of the respiratory chain.

Eventually the cell dies (172).

Studies from aging humans and animals have shown good correlations between aging and

increased mitochondrial production of ROS and between mitochondrial function decline and

accumulation of mtDNA mutations (173). Certainly, oxidative stress could be playing a role in

the generation of mtDNA mutations in wild-type animals. The rate of mitochondrial ROS

production, extent of mtDNA (but not nuclear DNA), oxidative damage, and degree of

membrane fatty acid unsaturation (a determinant of vulnerability to lipid peroxidation) are all

inversely correlated with longevity across species (174-177). Mice expressing mitochondrion-

targeted catalase show reduced total DNA oxidative damage in skeletal muscle, fewer mtDNA

deletions, and extended mean and maximal lifespan (178), suggesting that mitochondrial

accumulation of oxidative damage can limit rodent lifespan.

The increased production of ROS as a consequence of a mtDNA mutation has been

demonstrated in some occasions, as discussed below. The presence of a specific mutation in

ATPase 6, a subunit of the FO portion of the ATP synthase, caused massive apoptosis in cultured

fibroblasts when glucose in the culture medium was replaced with galactose (179). Because both

the mitochondrial and cytosolic SOD activities were shown to be elevated, it was inferred that

superoxide production was increased, and it was proposed that it was the superoxide, rather than

the defect in oxidative phosphorylation, that directly caused the apoptosis. This hypothesis was

supported from an experiment in which a spin trap molecule, added to the medium, was able to

prevent apoptosis. The explanation for why this particular point mutation in mtDNA would cause

superoxide production is rather straightforward. The specific mutation inhibits the activity of the

ATP synthase (180), and, in coupled mitochondria, this inhibition would arrest respiration,









putting the mitochondria in "state 4." In this state, the electron carriers of the respiratory chain

are fully reduced, and superoxide production is maximal compared to almost negligible

superoxide formation in actively respiring (state 3) mitochondria (181, 182). Moreover, cells in

culture that have defects in complex I (the respiratory NADH dehydrogenase) produce higher

amounts of superoxide (183).

It should be noted that measurements of the level of one particular ROS may not provide

the complete picture of the relevant changes in the cell. For example, the amount of superoxide

at a given time is the net result of a balance between its formation and its degradation by

superoxide dismutase. In the study mentioned above (183) the more serious diseases resulting

from complex I deficiency were found to be associated with normal levels of superoxide and

greatly increased SOD activities, suggesting that the greater the superoxide production, the

greater the SOD activity. The data of Geromel et al. (179), mentioned previously, is also

consistent with the idea that SOD increases to compensate for increased superoxide production.

In our study, we measured the amount of H202 released by intact mitochondria in a surrounding

medium during state 4, as an index of total basal mitochondrial ROS production. By the addition

of SOD in the medium, we ensured that any superoxide remaining would be converted to H202.

Furthermore, H202 production in intact mitochondria is thought to be closer to a more

physiologic situation, while superoxide production is usually assayed in sub-mitochondrial

particles, since its half life is very short and it readily gets dismutated to H202 by SOD before it

exits the mitochondria (141). In contrast to the above studies, we and others have clearly

demonstrated that mitochondrial mutator mice do not have increased levels of oxidative stress

(13, 79, 80).









In this study we provide further support against the vicious cycle theory, showing that

specifically in skeletal muscle, mitochondrial ROS production is not only unchanged (as we

showed in the past for other tissues) but significantly decreased in mutant mice compared to WT

(Fig 4-11). These results suggest that ROS production is regulated in a tissue specific way and

does not necessarily play a role in the increased sensitivity to apoptosis. More importantly, since

we also found no up-regulation in either MnSOD or Catalase mRNA levels (Fig 4-13), we can

conclude that in contrast to previous studies mentioned above (179, 183), ROS levels are

decreased in mutant mice due to a lower ROS production and not due to a reactive up-regulation

in antioxidant defenses.

Respiratory enzyme complex I and the protonmotive Q cycle operating in complex III are

the major sites that generate ROS within the ETC (184). In order to evaluate whether D257A

mice produce different amounts of ROS compared to WT at these main generators, we used

specific complex inhibitors: rotenone in pyruvate/malate supplemented mitochondria, and

rotenone plus antimycin in succinate supplemented mitochondria, in order to assess maximal

ROS formation at complex I (Fig 4-12, panel A) and complex III (Fig 4-12, panel D)

respectively. Besides basal and maximal ROS production we also assessed the production from

complex III under normal conditions using succinate-supplemented mitochondria (Fig 4-12,

panels B and C). In every instance, we detected a decrease in the amount of ROS produced by

mutant mitochondria at complex I or complex III under physiological conditions, or when ETC

inhibitors were used for maximal ROS production at either complex. This reinforces the

hypothesis that mtDNA mutations are likely to induce mitochondrial dysfunction leading to

apoptosis in the absence of increased ROS, and that oxidative stress is not an obligate mediator

of aging phenotypes provoked by mitochondrial DNA mutations.









Furthermore, the ROS data fit very well with the lowered mitochondrial Ay data in D257A

mice, since increased ROS generation has been reported to occur at high mitochondrial Ays

while the opposite is also true (181, 185). For example, in 1973 Boveris and Chance have shown

that the protonophorous uncoupler of oxidative phosphorylation (CCCP) or ADP+Pi inhibit

H202 formation by mitochondria (185). Moreover, Skulachev and colleagues have shown that

the inhibition of H202 formation by the uncoupler malonate and ADP+Pi was proportional to the

Ay decrease by these compounds (181), and proposed that that a high proton motive force in

state 4 is potentially dangerous for the cell due to an increase in the probability of superoxide

formation (186). This is likely because at high Ays electron flow is not efficient and the chance

that electrons flow out of sequence thus leaking to form superoxide instead of reducing 02 to

H20 at the terminal cytochrome oxidase (complex IV) increases. Another hypothesis is that

activation of ROS production in state 4, when protonic potential is high and respiration rate is

limited by lack of ADP, is due to the fact that some transients of the respiratory chain electron

transport, capable of reducing 02 to superoxide, such as CoQH', become long-lived (181).

In agreement with the ROS data, we also found that mtDNA oxidative damage, measured

by 8-oxodG was not different between genotypes. This is the second time our group shows no

differences in oxidative damage in D257A mice. In 2005, we demonstrated that the amount of 8-

oxodG lesions in total liver DNA was not different compared to WT and 8-oxoG was actually

lower in liver RNA of mutant mice (13). In addition, mitochondrial protein carbonyl levels and

F2-isoprostanes were also unchanged compared to WT (13). The oxidative stress findings in

mitochondrial mutator mice were also confirmed by Larsson's group that showed no differences

in protein oxidation and no up-regulation of antioxidant enzymes in the heart and liver (79).

Furthermore, Zassenhaus and colleagues using mice with a heart-specific POLG mutation have









demonstrated no elevations in protein carbonyls, no differences in mtDNA 8-oxodG levels, no

up-regulation of antioxidant defense systems, normal ubiquitination levels and intact (not

oxidatively damaged) iron-sulfur centers in aconitase enzyme (80). The fact that we did not

detect an actual decrease in mtDNA 8-oxodG levels, like we did in H202 production in the

present study, may indicate that it is not only the ROS produced by the mitochondria that damage

mtDNA.

Taken all together, we do not postulate that chronic accumulation of ROS production and

oxidative stress are not important factors contributing to mtDNA damage and mutagenesis,

leading to aging and age-related phenotypes, such as sarcopenia. However, using the D257A

model, we do support the idea that, the mutagenesis partly due to chronic ROS insults to

mtDNA, does not lead to further increases in ROS production and oxidative stress and may not

be an important mediator of apoptosis. Hence, based on our results, we contradict and question

an important part of the mitochondrial vicious cycle theory (Fig 2-2) and we propose instead that

respiratory chain dysfunction per se is the primary inducer of the sarcopenic phenotype in

mtDNA mutator mice.

Studies on different transgenic mice further support the idea that increased mitochondrial

oxidative damage is not sufficient for accelerated aging. Mice with reduced levels of the

mitochondrial MnSOD enzyme (Sod2+ ) do not appear to age any faster than their wild-type

counterparts, despite harboring increased levels of oxidative damage to both nuclear and mtDNA

(187). Similarly, mice deficient for 8-oxoguanine DNA glycosylase (that repairs the vast

majority of 8-oxoguanine lesions) or 8-oxoGTPase (that prevents oxidized dGTP from being

incorporated into DNA) do not exhibit accelerated aging features (188-190). On the other hand,

mouse models such as the Anti mice exhibit elevated levels of ROS production (191) and









mitochondria treated with specific chemical electron transport chain (ETC) inhibitors can indeed

produce increased ROS levels (192). However, it should be noted that, inhibition of ETC

function in Anti mice or by chemical inhibitors may generate ROS because all mitochondria

show the same defect (e.g., lack of available ADP or blockage of electron flow at a specific point

in the ETC) (193). Upstream complexes can still function, resulting in electron stalling and

transfer to 02 to generate superoxide. By contrast, in the D257A mice, a variety of mutations is

present and multiple upstream complexes could be nonfunctional or be lacking subunits if

mitochondrial rRNA or tRNA mutations are numerous. Thus, electron flow through all the

complexes (except nucleus encoded complex II) may be impaired and reduced intermediates may

not be accumulating. In the case where mtDNA mutation levels are much lower, the presence of

many wild-type copies of mtDNA will mask the effects of specific respiratory mutations (193).

It is also important not to forget the observations of Bandy and Davison, the first

investigators to put forward a mechanistic elaboration of what later became known as the

mitochondrial 'vicious cycle' theory: while they showed that some mtDNA mutations may have

the same effect on the respiratory chain as small-molecule inhibitors of respiration, that is, to

stimulate ROS production, they also carefully noted that not all mutations stimulate superoxide

production (72). Specifically, they pointed out that mutations preventing the synthesis of

cytochrome b would actually abolish any superoxide production at complex III that normal

mitochondria might exhibit, because without cytochrome b in place, complex III cannot be

assembled (72). Later studies also reported that cells possessing large deletions, which

eliminated the genes for at least a couple of respiratory chain subunits, but also removed at least

one tRNA gene, would indisputably preclude assembly of both the enzyme complexes known to

be responsible for mitochondrial ROS production, complexes I and III (73-76).









Mitochondrial DNA Mutations Lead to Mitochondrial Dysfunction, Via Alterations of ETC
Complex Composition

One of the main goals of this specific aim was to characterize the D257A mice in terms of

skeletal muscle mitochondrial function and ROS production, and if dysfunction was evident to

try to identify how this dysfunction is induced. Since the mtDNA encodes a total of 13

polypeptides all-subunits of the complexes of the ETC, our hypothesis was that accumulation of

mutations will have a direct impact on transcription and translation of these genes leading to

miscoded, truncated and dysfunctional proteins, which in turn could preclude assembly of

functional complexes within the inner mitochondrial membrane. We therefore, went on to assess

the content and activity of the five ETC protein complexes. In order to determine whether

complete complex content corresponds with protein expression levels of individual subunits, we

further analyzed selected mitochondrial- or nuclear-encoded subunits from each complex.

Certainly, one can argue that mitochondrial dysfunction observed during normal aging is

not only due to mutations in mitochondrial genes leading to ETC complex misassembly. For

example, in WT old mice a compromised state 3 respiration, such as the one we observed in

D257A mice, could be due to several different factors. To name a few: (a) decrease in the

content and/or activity of respiratory complexes (due to the disruption in subunits encoded by

mitochondrial DNA) leading to impaired electron flux, (b) disruption of ADP phosphorylation

due to the decline in the activity of ATP Synthase (194), (c) impairment in the transport of ADP

in the mitochondria due to alterations in Adenine Nucleotide Translocase (ANT) due to

carbonylation or nitration (195), or (d) alterations in enzymes involved in TCA cycle or fatty

acid oxidation. In our mtDNA mutator mice a suboptimal concentration and/or activity of

respiratory complexes may be the primary cause of the observed mitochondrial dysfunction, and

(b), (c), (d) could be consequences of (a) rather than the cause.









Of course in D257A mitochondria it is almost certain that from the -1,500 nuclear-

encoded proteins that exist in the mitochondria at any given time, translation, interaction and/or

activity of many of these proteins are likely to be impacted by the high load of mtDNA

mutations. In this project we only focused on proteins that comprise the five ETC complexes

since we believe ETC complex dysfunction is directly and primarily affected by the high load of

mtDNA mutations. Furthermore, we are aware that aging and sarcopenia are complex processes,

that likely result from deregulation and interaction of multiple pathways. However, here, we only

tested one of these hypotheses, the role of mtNA mutations in sarcopenia which also appears to

be very relevant, specifically for skeletal muscle, since multiple papers show strong correlations

between the rate of somatic mtDNA mutations and skeletal muscle dysfunction.

Decreases in the content and/or activity of ETC complexes with age, and as a result of

accumulated mtDNA mutations, especially in skeletal muscle are well-documented in the

literature. Aiken's group has repeatedly demonstrated loss of COX (complex VI) staining

combined with hyperactive SDH staining in aged rat skeletal muscle cross-sections ( also known

as ragged red fibers) (7, 87, 102, 196). Interestingly, these abnormalities co-localized with clonal

intracellular expansions of unique somatically derived mtDNA deletion mutations. In the areas

of the fiber where the mutation abundance surpassed 90% of the total mitochondrial genomes,

the fibers lost COX activity and displayed abnormal morphology such as fiber splitting and

breakage, while normal areas of the same fiber contained only wild type mitochondrial genomes

and did not exhibit de-regulation of ETC complex activities (7, 196). Decreased activities of

complex I, III and IV with age were also reported in gastrocnemius muscle of mice, while the

nuclear-encoded complex II did not show significant changes with age (28). In the same study

the authors conducted a kinetic analysis for complex III and IV and indicated that Vmax for both









complexes decreased with age, which suggested a decrease in the total enzyme content (28).

Similarly, muscle biopsies from aged humans revealed that randomly deleted mtDNA appeared

mainly in the oldest subjects (beyond 80 years old), affecting up to 70% of mtDNA molecules

with the activities of partly mitochondrial-encoded complexes III and IV being lower in the aged

subjects (48) (see also chapter 2 for more extensive review on mtDNA mutations and ETC

abnormalities with age).

Our findings are in agreement with some of the findings mentioned above. We detected a

significant decrease in the content of complex I, III and IV, all of which contain subunits

encoded by mtDNA while the content of all nuclear-encoded complexes II and F domain of

ATPase showed no difference between genotypes (Figs 4-3, 4-4). This confirms our hypothesis

that indeed accumulation of mtDNA mutations directly impacts the assembly of ETC complexes

that are comprised of mtDNA encoded subunits and suggests that complex formation in D257A

mice is abolished. Interestingly, we did not detect significant differences between genotypes in

any of the complex activities (Fig 4-6), when each complex activity was normalized to the

respective complex content assessed by BN-page. Since per amount of mitochondrial protein

loaded on the BN-page we detected a lower ETC content in D257A mice but no differences in

activity, this suggests that although a significant amount of ETC complexes is lost the activity of

the remaining complexes in D257A muscle is for the most part normal. Based on these results, it

is reasonable to propose that per single D257A mitochondrion the amount to electron transport

chains assembled is probably significantly lower compared to a WT mitochondrion. Thus, even

if the activity of the remaining ETCs in mutant mitochondria are normal, the lower amount of

complexes still creates energy deficits in the mitochondria, leading to an overall decrease in ETC

activity in mitochondria and thus, in mutant skeletal muscle cells. It is also important to note that









even if maximum activity of isolated complexes is normal that does not exclude the possibility

that some of these complexes do not assemble into fully functional electrons transport chains,

especially in the case of mutant mitochondria.

Furthermore, a point that requires special attention regarding measurements of ETC

complex enzymatic activities is how the complex activities are expressed. In most cases complex

enzymatic activities are normalized just to total protein content used (usually expressed as

nmol/min/mg of protein) (162), or expressed as a ratio to nuclear-encoded citrate synthase

activity (48). In our case, we normalized the activity densitometry values to the respective

content densitometry values (content value was first normalized to total protein content loaded

per well). In this way we evaluated complex activity per unit of ETC complex content which

gives a more precise picture of what may be occurring. In most other cases the overall activity

per amount of mitochondrial protein is evaluated which may not always reflect decreases in the

actual activity of the individual complexes, but in many cases, decreases in the complex content.

In line with our assumptions, very recently Dubessay et al, reported significant decreases in the

activities of complex I, III and IV with age, expressed in nmol/min/mg, in drosophila

melanogaster (162). However, the authors clearly stated that these activity decreases may have

various causes, such as reduced concentrations of respiratory complexes in the inner

mitochondrial membrane or partial inactivation of the biological functions of the constituent

subunits of these complexes (162).

Moreover, we observed a significant down-regulation of protein expression in the D257A

muscle, for almost all of the ETC protein subunits evaluated either nuclear- or mitochondrial-

encoded (Fig 4-7). The reduced expression of mitochondrial-encoded subunits fits well with our

total complex content data: if expression of mtDNA-encoded subunits is abolished due to the









accumulation of mtDNA mutations that may impact their transcription or translation, it would be

expected that assembly of whole functional complexes may also be abolished, as we have

observed (see Figs 4-3, 4-4).

The reduction in the expression of nuclear-encoded subunits may be explained by the fact

that a reactive adaptation of the nucleus is occurring: if less ETC complexes are assembled due

to the miscoding of mtDNA-encoded subunits, expression of nuclear-encoded subunits would

also have to be reduced since there would be no need for the expression of extra subunits if more

fully functional electron transport chains are not created. However, the fact that, there is no

down-regulation in the content of all-nuclear encoded ETC complex II and F in mutant mice

(see Figs 4-3, 4-4), cannot be fully explained by these results and needs to be further

substantiated. It is possible that although there is a down-regulation of nuclear-encoded subunits

still these subunits more often combine to assemble functional complexes since it is less likely

that nuclear-encoded subunits would be truncated or have altered active sites, leading to loss of

activity and misassembly of a complex, etc., as it would be the case for mtDNA-encoded

subunits. However, this does not mean that functional nuclear-encoded complexes are

necessarily inserted in the inner membrane to assemble functional ETCs. In agreement with our

hypothesis of a reactive adaptation of the nucleus to the defects induced by mtDNA mutations,

Alemi et al. recently demonstrated that pathogenic mtDNA deletions in cells derived from KSS

and CPEO patients had a strong negative effect on nuclear-encoded mitochondrially targeted

genes (156). This was especially evident on Complex I transcripts, but also on Complex II and

Complex IV assembly genes, on Complex V, on several TCA cycle genes, and on components of

the mitochondrial ribosome (156). Based on their results, these authors also suggested that the

nucleus senses the irreversible depletion of mtDNA-encoded mitochondrial subunits and tRNAs,









and responds by down-regulating the interacting subunits that would normally form a functional

complex (156). They proposed that, the down-regulation of nuclear-encoded mitochondrial

ribosomal subunits, oxidative phosphorylation, and TCA cycle transcripts, possibly reinforces

the mitochondrial defect initiated by the deletions, and adds to the mitochondrial metabolic

defect in these patients (156).

Total Skeletal Muscle Mitochondrial Protein Yield Continuously Decreases as Time
Progresses in Mutant Mice

Several studies in the past, performed in skeletal muscle of rodents have reported decreases

in mitochondrial protein yield with aging (197, 198). To evaluate the overall oxidative capacity

of skeletal muscle of adult (6-mo) and elderly (24-mo) Fischer 344 rats, Hoppel and colleagues

determined the mitochondrial content. They measured the activity of two exclusively

mitochondrial enzymes, citrate synthase (CS) and succinate dehydrogenase (SDH), and they

used these data to calculate the mitochondrial content (198). They found that the activity of both

mitochondrial marker enzymes was significantly lower in skeletal muscle homogenates of 24-

mo-old compared with 6-mo-old adult animals (198). The average decrease for both CS and

SDH activities was 31%. In contrast to CS and SDH, no age-associated decrease was found in

lactate dehydrogenase activity, a cytosolic marker enzyme. The calculated mitochondrial content

was significantly lower in skeletal muscle of elderly rats with both mitochondrial marker

enzymes (25 and 20% based on CS and SDH, respectively). They concluded that aged skeletal

muscle has a significantly lower content of mitochondria in Fischer 344 rats. Moreover, the yield

of mitochondrial protein per gram wet weight of skeletal muscle was also less in elderly

compared with the adult animals, consistent with the lower mitochondrial content. Lower

skeletal muscle mitochondrial yield from elderly rats also has been reported by Beyer et al.









These authors reported a 35% decrease in mitochondrial protein yield from quadriceps femoris

of elderly Sprague-Dawley rats (197).

Consistent with the above findings we also found that in our accelerated-aging mice the

total mitochondrial protein yield, expressed as mg of mitochondrial protein per gram wet weight

of skeletal muscle, was significantly decreased compared to WT animals (Fig 4-10). By 11-mo

of age we detected a 35% reduction (Fig 4-10) which agrees with previous data in normally aged

skeletal muscle (197). Interestingly, at -13 months we saw a drastic, 46% reduction in the

protein yield (Fig 4-10) suggesting that mitochondrial content is continuously reduced in these

animals as they are approaching their mean lifespan which is -14 months. Although our data in

this area is limited to the only measurement of mitochondrial protein yield, it is tempting to

suggest that mitochondria in D257A mice are probably getting continuously eliminated. This

would make sense especially in the case where the accumulation of mtDNA mutations reach a

reported critical threshold (42, 51, 193, 196) before significant tissue dysfunction is observed.

And in the case of the post-mitotic skeletal muscle this threshold appears to be much later in the

D257A animal's lifespan compared to that of other tissues. For example, at 3-mo of age, the

aging phenotype for skeletal muscle is not evident in D257A mice, while in the case of rapidly

dividing cells in duodenum, thymus, and testes, we detected significant tissue dysfunction at the

same time point (13). Once the critical threshold of mtDNA mutations is reached, mitochondrial

dysfunction may ensue possibly leading to mitochondrial-mediated apoptosis and the elimination

of dysfunctional mitochondria. As the accumulation of mtDNA mutations was shown to be

exponential over time in several post mitotic tissues, with the most well-documented being

skeletal muscle (51, 73, 193, 199, 200), we also see that the mitochondrial protein yield in

D257A skeletal muscle further decreases over time.









In line with the above, our BN-page results, showing that mitochondrial complex content is

decreased and suggesting that less ETC complexes may exist per mutant mitochondrion, also

point to the direction that indeed elimination of these mitochondria is a very likely hypothesis,

since these mitochondria would not be able to keep up with the energy demands of the cell.

Mitochondrial elimination in skeletal muscle would lead to compromised oxidative capacity and

tissue dysfunction. On the other hand, one can argue that accumulation of dysfunctional

mitochondria and inhibition of mitochondrial autophagy may be the cause for tissue dysfunction.

However, based on the fact that mitochondrial protein yield is profoundly decreased in the

D257A mice we don not believe this is the case. Nevertheless, our hypothesis remains to be

further explored and confirmed.

If indeed mitochondria from D257A muscle are eliminated this creates an important

question as to the mechanism responsible for their elimination (Fig 5-1). Would dysfunctional

mitochondria in D257A muscle trigger an autophagic response? How is mitochondrial

biogenesis impacted by the accumulation of dysfunctional mitochondria? Very recently

Cortopassi's group showed that specific cell types with pathogenic mtDNA deletions derived

from KSS and CPEO patients had a significant induction of the ATG12 transcript (156). The

ATG12 transcript encodes the first and most important product in the mammalian autophagy

cascade (201, 202) and was induced in the microarray data from fibroblasts, lymphoblasts, and

myoblasts from KSS patients, and in NT2 neural cells bearing deletions. To determine whether

the induction of ATG12 was a specific consequence of mtDNA deletions, they quantified

ATG12 transcript levels in 143B osteosarcoma cybrid fusion controls (i.e., cell lines that had

gone through the process of cybridization but with normal mtDNA), in osteosarcoma cybrids

harboring deletions, and in osteosarcoma cell lines lacking the mitochondrial genome. The









ATG12 transcript levels were highly correlated with the presence of deletions, were significantly

higher in cells bearing deletions, and highest in cells lacking mtDNA (156). Consistent with the

hypothesis of an induction of autophagy in mutant cells, they also observed the induction of

several SNARE/vesicular transcripts, proteins that are also essential for the process of autophagy

(203, 204). The authors concluded that the induction of autophagic transcripts is a specific

consequence of mtDNA deletions (156). Based on the above findings, activation of autophagy

could also be a plausible mechanism mediating mitochondrial removal in D257A mice.

Regarding mitochondrial biogenesis, it has been shown that the expression of nuclear

genes encoding the transcription factors TFAM, TFB 1, TFB2 and DmTTF, which are essential

for the maintenance and expression of mtDNA, are decreased in old and dysfunctional

mitochondria (162). It is possible that attenuation in mitochondrial biogenesis in conjunction

with an up-regulation of autophagy may be occurring in skeletal muscle of mutant mice, and are

responsible for the robust declines in mitochondrial yield. Although the mitochondrial protein

yield responses in normal aging and accelerated aging in D257A mice appear to be similar, the

respective mechanisms responsible for the decreases in mitochondrial protein yields remain to be

determined.

Hypothesis Two: the Effect of mtDNA Mutations on Skeletal Muscle Apoptosis

The goal of this aim was to demonstrate that mitochondrial dysfunction observed in aim #

1 ultimately culminates in apoptosis, and to prove that the sarcopenia observed in D257A mice

was due to apoptosis. In this way we could show a direct causal relationship between the

accumulation of mtDNA mutations and skeletal muscle loss through apoptosis and since the

D257A model is an aging model we could extrapolate our results to normal aging and deduce

that: a) Accumulation of mtDNA mutations with age is an important culprit for tissue









dysfunction, in this case, skeletal muscle loss and b) Apoptosis is a central mechanism

responsible for the age-induced sarcopenia.

Apoptosis is a programmed process of cell death that has a tightly regulated initiation and

execution. In Greek, apoptosis means "dropping off' of petals or leaves from plants or trees. The

phrase had a medical meaning to the Greeks over two thousand years ago. Hippocrates (460-370

BC) used the term to describe "the falling off of the bones" and Galen extended its meaning to

"the dropping of the scabs". A re-introduction of the term for medical use occurred in 1972

when Kerr, Wyllie, and Currie deduced that there was a specific controlled mechanism of cell

death distinct from uncontrolled necrotic death (205). They noticed a characteristic, identical

sequence of events in many different types of cells and published their observations in a seminal

1972 paper that coined the phrase "apoptosis" and was largely ignored for fifteen years (205)!

The concept that death is essential for life according to Wyllie went "against twentieth century

philosophy".

Apoptosis with Aging

To date, evidence has been accumulating to suggest that de-regulation of apoptosis may

contribute to age-associated changes such as progressive decline of physiologic function and

significant increases in the incidence of cancer and degenerative diseases (206). Progressive cell

loss mediated by apoptosis is linked to many age-related disorders. Moreover, many studies have

demonstrated that apoptosis is up-regulated during aging in various post-mitotic cells such as

those of the central nervous system, cardiomyocytes, and skeletal muscle fibers (206-210). For

example, the loss of neurons through apoptosis is closely associated with functional impairments

such as dementia and motor neuron disability in neurodegenerative diseases such as Alzheimer

disease, amyotrophic lateral sclerosis, and Parkinson disease (211). The aging process that

occurs in the heart is characterized in animals and humans by a loss of cardiomyocytes and









reactive hypertrophy of the remaining cells, which ultimately results in impairment of cardiac

function in advanced age (206). In skeletal muscle, there is increasing evidence indicating that

deregulation of apoptosis plays a key role in the pathophysiology of skeletal muscle cell loss.

Indeed, accelerated skeletal muscle apoptosis has been well documented to occur with aging

(105, 207).

In accordance with the above published reports we found a significant skeletal muscle loss

in WT aging mice compared to young WT counterparts (Fig 4-15). Sarcopenia in normally aged

mice was also associated with up-regulation in cleaved caspase-3 content (Fig 4-18), suggesting

that the apoptotic program is activated in skeletal muscle of old animals. Cell loss in these tissues

can cause functional deterioration, thereby leading to aging. These observations suggest that

aging enhances apoptosis under physiologic conditions and increases the susceptibility to

apoptosis triggered by challenges.

Mitochondrial DNA Mutations and Apoptosis

Aging-associated accumulation of oxidative damage to macromolecules in mitochondria

results in mitochondrial dysfunction. Oxidative damage to mtDNA induced by ROS is probably

a major source of mitochondrial genomic instability since much of this damage can be mutagenic

(212). Indeed, MtDNA mutations are gradually accumulated and the activity/efficiency of energy

metabolism declines in aging tissue cells that often exhibit a higher susceptibility to apoptosis

(213, 214). This instability of mtDNA, leading to respiratory dysfunction and apoptosis, is

thought to be one of the most important factors in aging (212). Pathogenic A3243G and A8344G

mutations as well as the 4977-bp deletion in mtDNA render human cells more susceptible to

apoptosis stimuli such as UV irradiation (215, 216). In addition, studies on mice with a knockout

of the mitochondrial transcription factor showed that defects in the respiratory chain are

associated with massive apoptosis of affected cells (217). It is conceivable that impairment of









mitochondrial ATP production and the resulting energy depletion can lead to apoptosis (212).

Therefore, aging-induced inadequate supply of energy from mitochondria may contribute to an

increased susceptibility of aging human and animal cells to apoptosis.

Several laboratories have addressed the question of whether apoptosis is a part of the

pathogenic mechanisms associated with mtDNA deletions and point mutations. In support of this

hypothesis, human cells bearing mutations causing Leber's hereditary optic neuropathy, an

inherited mtDNA disease, are sensitized to Fas-induced apoptosis (218). Furthermore, TUNEL

positive staining was observed in up to 75% or more of the muscle fibers in patients with

mitochondrial encephalomyopathy, carrying a high percentage (>40%) of a mtDNA deletion

(219). In patients carrying high proportions (>70%) of the A32443G MELAS mutation in the

mitochondrial tRNALeu (UUR) gene, or the A8344G MERRF mutation in the mitochondrial

tRNALys gene, 25-75% of the muscle fibers exhibited TUNEL-positive nuclei. It appears that

the apoptotic program is initiated in muscle fibers of patients carrying high proportions of

mutations affecting mitochondrial tRNAs. Recently, Zassenhaus and colleagues proposed an

intriguing mechanism whereby mtDNA mutations would generate a pool of misfolded

mitochondrial proteins, some small proportion of which might have the conformation necessary

to bind to Bax or Bak and thereby activate apoptosis or perhaps bind to cyclophilin D and inhibit

its chaperone function (220). This hypothesis could explain how heteroplasmic mtDNA

mutations could elicit a cell-death response in the presence of many wild-type copies of mtDNA.

Very recently, Aiken and colleagues demonstrated that aged rat muscle fibers possessed

segmental, clonal intracellular expansions of unique somatically derived mtDNA deletion

mutations (196). In the areas where less than wild type genomes were detected the fibers

displayed ETC abnormalities and abnormal morphology such as fiber splitting, atrophy, and









breakage (196). Deletion mutation accumulation was linked to these aberrant morphologies with

more severe cellular pathologies resulting from higher deletion mutation abundance. In addition,

in fiber regions distant from the ETC abnormalities with normal morphology, only wild type

genomes were detected, and mtDNA deletion mutations were undetectable (196).

In summary, these measurements corroborate previous studies of the same group (7, 43,

87), and indicate that age-induced mtDNA deletion mutations expand within individual muscle

fibers, eliciting fiber dysfunction and atrophy. Our study is in agreement with those findings

showing that mtDNA mutations lead to ETC abnormalities and skeletal muscle atrophy, and

extends the conclusions by showing that the skeletal muscle atrophy observed in the D257A

mice is actually due to apoptosis (Figs 4-16, 4-17, 4-18, 4-20). Therefore, the mitochondrial

mutator mice suggest that activation of apoptosis is important for the induction of the aging

phenotype in skeletal muscle. While previous studies demonstrated an increased susceptibility of

cells with pathogenic mtDNA mutations to apoptotic stimuli, or showed a correlative

relationship between mtDNA mutations and apoptosis in mitochondrial diseases, we are showing

a direct causal relationship between the accumulation of somatic mtDNA mutations in vivo and

apoptosis in skeletal muscle. Hence, loss of myonuclear domain through apoptosis, possibly

leading to the loss of irreplaceable skeletal muscle fibers, appears to be a central mechanism of

sarcopenia associated with the accumulation of mtDNA mutations.

Disruption of Mitochondrial Membrane Potential and Role for Apoptosis

During mitochondrial-mediated apoptosis, the release of cytochrome c from the

mitochondrial intermembrane space induces the assembly of the apoptosome that is required for

activating downstream caspases. However, the actual mechanism of its release is still debatable.

In particular, the relation between mitochondrial physiology and the release of cytochrome c and

other apoptogenic factors from mitochondria is not clear (221). It is conceivable that the ETC









abnormalities we have detected in the D257A model, leading to energy depletion is likely the

cause for the drop in membrane potential we observed in D257A mice (Fig 4-9). Previous studies

have described the relationship between mitochondrial membrane potential and apoptosis

showing that a reduction in Ay leads to matrix condensation and exposure of cytochrome c to the

intermembrane space, facilitating cytochrome c release and cell death following an apoptotic

insult (221).

Changes in the Ay have been originally postulated to be early, obligate events in the

apoptotic signaling pathway (222, 223). Multiple lines of research demonstrate that the nuclear

features of apoptosis are preceded by changes in mitochondrial structure and Ay in some

regimes of induction of apoptosis. Rat embryo cells induced to undergo apoptosis by the SV40

large T antigen, show a lowered Ay and a decrease in mitochondrial respiration and translation,

which is detectable early in the apoptotic process (224). Human peripheral blood mononuclear

cells treated with dexamethasone show a reduced uptake of the mitochondrial AY determining

fluorochrome, 3,3'dihexyloxacarbocyanine iodide before the appearance of any morphological

signs of apoptosis (223). The separation of these cells prior to dexamethasone treatment into

populations with high Ay and low Ay revealed that cells with a lowered AY undergo

spontaneous apoptosis after a short term in culture at 37 o C (223). Also, dexamethasone induces


early mitochondrial effects in thymocytes undergoing apoptosis, which show both an early

decrease in Ay as determined by another Ay fluorochrome, 5,5,6,6-tetrachloro-1,1',3,3'-

tetraethyl benzimidazolylcarbocyanine iodide (JC-1), and altered mitochondrial structure as

demonstrated by electron microscopy (225). Furthermore, cell death in the Dictyostelium

discoideum, a single-celled slime mold involves early disruption of Ay that precedes









phosphatidylserine exposure, nuclear shrinkage, DNA fragmentation and the release of AIF

(226), suggesting the evolutionary conservation between unicellular and multicellular organisms.

In contrast to the above results, po cells, (devoid of mitochondrial DNA) which typically

have only 40-60% of the Ay of their parental cell line, can also undergo apoptosis in response to

range of agents with similar kinetics as the parental cells. These include Ca2+ and atractyloside

(227), staurosporine (228), anti-Fas antibodies (229), TNFa plus cycloheximide (227), or

didemnin B (230). As there seems to be no acceleration in the apoptotic process, even though the

Ay in these po cells is already significantly decreased compared to their parental cell line(230),

the notion that lowering Ay will predispose cells for apoptosis cannot be generalized (223).

Based on our outcomes, we suggest that it is the drop in Ay observed in skeletal muscle

mitochondria from mutant mice that leads to the leakage of pro-apoptotic proteins into the

cytosol and triggers apoptosis, although other mechanisms not investigated in the present project

(e.g. Bax-Bak pores in the mitochondrial outer membrane) could also be additionally responsible

for this induction, and thus, cannot be excluded.

Apoptosis is Evident in Skeletal Muscle of D257A Mice

Apoptosis in our model was evident by an increased release of mono- and oligo-

nuclesomal fragments into the cytosol (Fig 4-16). Cells undergoing apoptosis can release mono-

or oligo-nucleosomes comprising DNA fragments and histones from their nuclei into the

cytoplasm or even into the extracellular compartment and this process is very characteristic to

apoptosis (231, 232).

Moreover, degradation of chromosomal DNA is one of the biochemical hallmarks of

apoptosis: Late in the apoptotic process, caspase-activated endogenous endonucleases cleave

chromosomal DNA between the nucleosomes, generating a series of DNA fragments with









multiples of 180 to 220 bases (233-235) that form a ladder when the extracted DNA is separated

by gel electrophoresis and stained by ethidium bromide. We have detected prominent DNA

ladders in D257A mice while DNA ladders were almost not detectable in WT mice (Fig 4-17),

which further confirms that apoptosis is the mechanism responsible for the sarcopenia observed

in D257A mice.

In addition, protein cleavage by caspases, the central executioners of the apoptotic

pathway, accounts for the distinctive cytoplasmic and structural changes seen in apoptotic cells.

Cleavage and activation of the effector caspase-3 during apoptosis has been very well

documented in the scientific literature (119, 236-239). Here, we also show that the DNA

fragmentation we detected in D257A muscle is caspase-3-mediated since we demonstrated up-

regulation in both the content and the activity of caspase-3 (Figs 4-18, 4-20). As mentioned

previously, caspase-3 is activated by proteolytic cleavage at the C-terminal side of a specific

aspartate residue. In figure 4-18 we show the content of the large (17/19 kDa) activated fragment

of caspase-3 resulting from cleavage adjacent to Asp 175. Furthermore, disruption of

mitochondrial membrane potential leading to activation of caspase-3 and subsequent apoptosis,

has been well documented (240-242), and this is also in agreement with our findings showing a

drop in Ay, caspase-3 activation and subsequent apoptotic DNA fragmentation. More

importantly, we showed that apoptosis through caspase-3 activation is also an important

mechanism for skeletal muscle loss during normal aging (Fig 4-18), providing further support to

the usefulness of our model to study mechanisms of sarcopenia in aging skeletal muscle.

Hypothesis Three: Identify the Specific Apoptotic Signaling Pathway Responsible for
Sarcopenia in D257A Mice

The goal of this aim was to demonstrate that the apoptosis observed in mutator mice is

intrinsic to the mitochondria. In this way, we intended to show that the mitochondrial-mediated









pathway is the pathway responsible for the apoptosis induced by increased mtDNA mutational

load, and make the inference that mitochondrial-mediated apoptosis is an important mechanism

for the age-associated skeletal muscle loss.

Studies have suggested that age-related apoptosis and/or necrosis in response to energy

depletion may occur through activation of the mitochondria-mediated signaling pathway (41,

212, 213, 221). Izyumov et al. has shown that in HeLa cells, complete inhibition of oxidative

phosphorylation by oligomycin, myxothiazol or FCCP (trifluoromethoxycarbonylcyanide

Phenylhydrazone) combined with partial inhibition of glycolysis by 2-deoxyglucose resulted in a

steady threefold decrease in the intracellular ATP level (41). In 48 h after a transient (3 h) [ATP]

lowering followed by recovery of the ATP level, the majority of the cells had committed suicide

by means of mitochondrial-mediated apoptosis. Apoptosis was accompanied by Bax

translocation to mitochondria, cytochrome c release into cytosol, caspase activation, and

reorganization and decomposition of chromatin (41). Similarly, it has been shown that, in

mitochondria isolated from healthy cells, matrix condensation can be induced by either depletion

of oxidizable substrates or by protonophores that dissipate the membrane potential (221). Matrix

remodeling to the condensed state results in cristae unfolding and exposes cytochrome c to the

intermembrane space facilitating its release from the mitochondria and the induction of apoptosis

(221).

In accordance with the above studies, we showed that disruption of oxidative phosphorylation

and a drop in the ATP content in the D257A skeletal muscle (Figs 4-2, 4-8) indeed leads to

mitochondrial-mediated apoptosis evident by up-regulation in the activity of the initiator

caspase-9 which mediated the downstream cleavage and activation of caspase-3 (Figs 4-18, 4-20,

4-21). The significant positive correlation between caspase-3 and caspase-9 (Fig 4-21) adds









further support to the notion that the up-regulation of caspase-9, and thus the activation of the

caspase-dependent mitochondrial pathway, is indeed responsible for the activation of caspase-3.

In addition, disruption of mitochondrial membrane potential followed by caspase-9 activation

and downstream caspase-3 activation has also been previously demonstrated (240). Although we

were unable to show differences in cytochrome c release into the cytosol most probably due to

contamination of our cytosolic fraction with ruptured mitochondria which can occur during the

mitochondrial isolation procedure we would expect that cytochrome c release is indeed the case

in D257A animals, since activation of caspase-9 can only occur after formation of the

apoptosome, which requires cytochrome c release in the cytosol.

Furthermore, studies exist to support a critical role of mtDNA mutations in apoptosis

intrinsic to the mitochondria (82, 243). Zassenhaus's group studied mice that express a

proofreading-deficient POLy specifically in the heart, and develop cardiac mtDNA mutations, in

order to determine whether low frequency mitochondrial mtDNA mutations are pathogenic.

They found that sporadic myocytic death occurred in all regions of the heart, due to apoptosis as

assessed by histological analysis and TUNEL staining (82). While in their model they showed

that mitochondrial respiratory function, ultrastructure, and number remained normal, they also

pointed out that cytochrome c was released from mitochondria and concluded that mtDNA

mutations are pathogenic, and seem to trigger apoptosis through the mitochondrial pathway (82).

In another study and in order to confirm whether apoptotic processes are truly related to

muscle fiber degeneration in mitochondrial encephalomyopathies, Ikezoe et al. evaluated

apoptosis in muscle fibers from patients with chronic progressive external ophthalmoplegia

(CPEO; associated with a mtDNA deletion), MELAS, or MERRF (243). The criterion for

selecting the patients for this study was that >5% of the muscle fibers were "ragged red fibers,"









(RRFs) i.e., fibers with the characteristic subsarcolemmal accumulation of mitochondria found in

mitochondrial diseases with deficient mitochondrial protein synthesis (usually RRFs show loss

of COX activity with concomitant hyperactivation of SDH activity). The proportion of mtDNA

carrying the relevant mutation was unknown. However, markers of mitochondrial-mediated

apoptosis appeared to be upregulated in RRFs: Bax and Apaf-1 expression and cytochrome c

release from mitochondria were seen in RRFs (243). Caspase-3 activation was also confirmed in

RRFs of MELAS, CPEO and MERRF, but not in control muscles (243).

It is therefore evident from the current literature that a rise in mtDNA mutations can lead to

apoptosis mediated by the mitochondria. The present dissertation study confirms results from

previous studies and ties these studies together showing that an induced in vivo rise in somatic

mtDNA mutations results in ETC abnormalities, such as profound decreases in complex I and

COX content, and compromised oxidative phosphorylation, which in turn lead to energy

depletion, loss of mitochondrial membrane potential and induction of apoptosis mediated by the

mitochondria.

Proposed Mechanism for the Skeletal Muscle Loss Induced by High Load of Somatic
mtDNA Mutations

Based on our outcomes, we describe below a hypothetical mechanism of how somatic

mtDNA mutations can lead to apoptosis, responsible for the sarcopenia observed in D257A

skeletal muscle (Figure 5-1).

From the blue native page results we observed that the content of mitochondrial ETC

complexes I, III and IV is significantly lower in D257A mice (Figs 4-3, 4-4) while their activities

remain unaffected (Figs 4-5, 4-6). We have also determined that total mitochondrial content per

gram of skeletal muscle tissue in D257A mice is -35% lower by 11 months, and almost half

(-46% lower) compared to that of WT by 13-mo of age (Fig 4-13), which suggests that









mitochondria in D257A mice are getting continuously eliminated. The decreased ETC complex

content per total protein loaded may suggests that assembly of ETC complexes, specifically

those containing mtDNA-encoded subunits, is abolished. We can still detect normal levels of all-

nuclear-encoded complexes, such as, complex II. However, if formation of partly mitochondrial-

encoded complexes is abrogated that means that possibly fewer fully functional electron

transport chains exist per skeletal muscle mitochondrion in D257A mice. Thus, even if levels of

all-nuclear encoded complexes are not different compared to WT it is most possible that they

may just accumulate in mitochondria without being inserted in the mitochondrial inner

membrane to form fully functional ETCs. If fewer ETCs exists per mitochondrion this would

still leave the mitochondrion at energy deficit even though the activity of the remaining ETCs is

normal. Our results fit very well this hypothesis since we indeed show impairment of

mitochondrial oxygen consumption at state 3 (Fig 4-2), as well as, decreased ATP content (Fig

4-8) in mitochondria isolated from D257A mice. We believe that these energy deficits due to the

assembly of fewer ETCs in mutant mitochondria lead to the disturbance in the mitochondrial

membrane potential we have observed (Fig 4-9). A decrease in membrane potential in turn, may

be directly responsible for the rupture of the mitochondrial outer membrane and the release of

cytochrome c and other pro-apoptotic proteins from the inter-membrane space into the cytosol,

which triggers apoptosis. Release of cytochrome c will lead to the formation of the apoptosome

causing activation of caspase-9 and downstream activation of the final effector caspase, caspase-

3, which will be responsible for carrying out the proteolytic events that lead to DNA

fragmentation. In accordance with the above we demonstrated increased skeletal muscle

apoptosis in D257A mice (Figs 4-16, 4-17, 4-18, 4-20) which was indeed intrinsic to the

mitochondria, since activation of caspase-9 and caspase-3 was observed (Figs 4-18, 4-20). We









believe that the main, mitochondrial caspase-dependent pathway is a central pathway responsible

for sarcopenia associated with the accumulation of somatic mtDNA mutations and, it may be

also as critical for the skeletal muscle loss associated with normal aging. A mechanistic series of

events is depicted in Figure 5-1. Last, and as previously mentioned, based on our total

mitochondrial protein yield findings, it appears that mitochondria in skeletal muscle fibers are

getting eliminated. Although this provides support to our hypotheses, since we would expect that

mutant mitochondria with energy deficits and disrupted membrane potentials would eventually

get destroyed, it also poses the important question as to how their distraction is mediated. What

is the mechanism of their elimination? These are important questions that await answers.

Synopsis

This project utilized the D257A knock in mouse, as an "in vivo" model of increased

spontaneous mutation rates in mtDNA in order to elucidate the role of mtDNA mutations in

sarcopenia. This mouse contained a mutation that resulted in the functional disruption of the

exonuclease domain of mouse mitochondrial DNA polymerase y, leading to the abolishment of

its proofreading function without significantly affecting the polymerase activity (13, 14, 148).

Three separate but interrelated hypotheses were tested. Major findings include the

following: (a) mtDNA mutations in skeletal muscle lead to compromised mitochondrial

bioenergetics, evident by profound decreases in mitochondrial 02 consumption, ATP content,

and a significant drop in Ay. (b) The accumulation of mtDNA mutations in skeletal muscle of

D257A mice leads to a significant decrease in the content of ETC complexes I, III, and IV, all of

which contain mtDNA-encoded subunits. This finding represents the primary mechanism

responsible for the impaired mitochondrial bioenergetics observed and the disturbance in the Ay,

since elimination of ETC complexes from the inner mitochondrial membrane are likely to leave









mitochondria and cells in energy deficits. (c) Importantly, our observations, thus far, do not

support the idea that mtDNA mutations contribute to increased mitochondrial ROS production

and further oxidative damage to mtDNA, in contrast to the main tenet of the free radical theory

of aging (4-6, 19, 71). Instead, it is evident that mtDNA mutations can induce mitochondrial

dysfunction in the absence of increased ROS production, and this finding has been also

demonstrated by other groups (79). (d) Up-regulation of apoptosis in D257A mice is evident by

DNA laddering, increased release of mono- and oligo-nucleosomes in the cytosol, and increases

in cleaved cas-3 content and activity. The apoptosis data when combined with the significant loss

of muscle mass in 11-mo-old D257A mice suggest that loss of irreplaceable, post-mitotic cells

through apoptosis may be a central mechanism of sarcopenia induced by the accumulation of

mtDNA mutations. (e) Involvement of the main mitochondrial caspase-dependent pathway is

apparent by the up-regulation of caspase-9 activity resulting in downstream activation of the

final effector caspase-3 in D257A mice. The drop in mitochondrial membrane potential is likely

the trigger for mitochondrial-mediated apoptosis in the D257A mice. A mechanistic series of

events is depicted in Figure 5-1.

The results of these experiments provide a unique contribution to the existing research,

utilizing the first "in vivo" mammalian system to examine the role of mtDNA mutations in

skeletal muscle aging. In contrast to previous correlative studies, these new outcomes establish a

direct link between the accumulation of mtDNA mutations and sarcopenia. Importantly, this

work is also the first to demonstrate that, specifically in skeletal muscle, mtDNA mutations do

not lead to increases in mitochondrial ROS production, introducing a break in the mitochondrial

"Vicious Cycle" theory.









Conclusions

Concurrent with the age-dependent loss of muscle fibers, multiple mtDNA mutations

accumulate over time in many tissues and species (11, 73, 200, 244, 245). MtDNA mutations and

deletions were initially considered to be at low abundance (<0.1%) when calculated against the

total mitochondria pool in tissue homogenates (246, 247). When, however, discrete numbers of

muscle fibers were analyzed, the abundance of mtDNA mutations was found to be inversely

proportional to the number of cells analyzed (248). In situ hybridization studies demonstrated

that mtDNA deletion mutations were not distributed homogeneously throughout a tissue, but

amplified focally within a subset of individual cells, appearing as a segmental pattern along the

length of muscle fibers and as a mosaic distribution between cells (75, 158, 249-253). This

provides a mechanism for significant tissue dysfunction induced by mtDNA mutations, the focal

accumulation of which may cause significant impairment of mitochondrial function in individual

cells in spite of low overall levels of mitochondrial DNA mutations in muscle (49).

The hypothesis that aging is due in part to mtDNA damage and associated mutations (5, 6)

was based on the observations that mtDNA is located in the organelle that generates most

cellular ROS, that mtDNA is relatively unprotected from ROS damage due to a lack of histones,

and also that mtDNA repair may be limited. It is important to note that aging and aging -

associated phenotypes, such as sarcopenia, are complex processes that are likely to have

multifactorial causes. Mitochondrial DNA mutations can arise directly from errors during DNA

replication (193). Oxidative stress may also generate mtDNA mutations as well as damaged

proteins that might be able to directly signal apoptosis through a misfolded protein response

(193, 220). Respiratory deficiency could contribute to apoptotic signaling or be directly

responsible for some aspects of tissue dysfunction (193) The limited and sometimes

contradictory evidence available concerning the capacity of pathogenic mtDNA mutations to









start and support the development of the apoptotic process and the role of the production of ROS

in this phenomenon makes it difficult to reach general conclusions.

The still limited understanding of the pathogenic mechanisms of many of the disease-

causing mutations and of all the factors capable of promoting and controlling the various

apoptotic pathways adds greatly to the complexity of the problem. Last, because cells may have

hundreds of mitochondria, and each carries multiple copies of mtDNA, the contribution of

mtDNA mutations and deletions to normal aging and aging phenotypes, remains a controversial

issue. It is clear, however, that progress in these areas will lead to a better understanding of the

resources available to the cell for compensating and possibly reversing the process leading to cell

death, with potential implications for the therapy of sarcopenia, as well as degenerative diseases

associated with mtDNA mutations.

Future Directions

Since the D257A mouse model represents a relatively new model to study aging, there is a

lot of work left to be done.

A significant finding of this study was that the total mitochondrial protein content per gram

of skeletal muscle tissue is getting continually decreased in D257A mice, being half that in WT

by 13-mo of age. This, points out to the fact that mitochondria are probably getting continuously

eliminated in these mice which very nicely agrees with the fact that current examined

mitochondria exhibit loss of ETC complexes and disruptions in membrane potential. However, it

also poses a question as to the mechanism responsible for the elimination of mitochondria. Is it

autophagy that is responsible for the decrease in mitochondrial content or maybe a decrease in

mitochondrial biogenesis, or maybe a combination of both?

In this project we evaluated what would be directly impacted by the increased mtDNA

mutational load, assessing mostly proteins of the ETC, however, future research is needed to









determine the adaptive responses of nuclear genes to the changes in mitochondrial-encoded

genes or in other words, how mtDNA mutations affect nuclear-encoded genes in the

mitochondria, especially the ones involved in the Krebs cycle, ATP production, as well as, genes

involved in the regulation of the permeability transition pore opening.

Moreover, more work is required to clarify the role of Bcl-2 family proteins such as Bax,

Bad, Bak, and Bcl-2 in apoptosis induced in the D257A model and the formation of pores in the

outer membrane that also lead to mitochondrial-mediated apoptosis.

Importantly, future work should also focus on answering the very interesting question of

why skeletal muscle mitochondria with accumulated mtDNA mutations produce significantly

less ROS, which is in contrast to the mitochondrial "Vicious Cycle" theory. We provided the

first indications /observations to that, suggesting that, it is the abrogation of ETC complexes I

and III, the main generators of ROS within the ETC, that lead to a decrease in ROS production in

the mutant mice. The drop in mitochondrial Ay can also be a potential mechanism explaining the

decrease in ROS production but this may also be linked back to the energy deficits due to the

decrease in the content of ETC complexes, specifically those containing mtDNA-encoded

subunits. The exact mechanism for this significant decline in ROS generation is far from being

completely understood and warrants additional research.

Ultimately, testing the effect of reduced mtDNA mutation accumulation on lifespan and

aging phenotypes, including sarcopenia, will provide the strongest support of a causal

relationship between mtDNA mutations and aging.










D257A
Mitochondrion


Energy deficits 4 02 Consumption
in mitochondria 4, ATP Content



Muscle Fiber


??? Mechanism J


tMitochondrial /Mitochondrial
Autophagy?? ( Biogenesis??


Apoptosis









Fig 5-1. Proposed mechanism for the skeletal muscle loss induced by high load of somatic
mtDNA mutations. Abolishment of ETC complexes in D257A mice leads to
assembly of less functional electron transport chains per mutant mitochondrion. This
can create energy deficits leading to mitochondrial dysfunction, evident by severely
compromised mitochondrial respiration and reduced ATP content in D257A muscle.
Ultimately, this dysfunction results in significant drop in mitochondrial membrane
potential and release of cytochrome c from the intermembrane space into the cytosol.
Cytochrome c in the cytosol results in apoptosome formation, activation of caspase-9
and downstream activation of caspase-3 which ultimately results in apoptotic DNA
fragmentation. Apoptosis appears to be a central mechanism of skeletal muscle loss in
D257A mice. Moreover, the observation of reduced mitochondrial yield in D257A
skeletal muscle suggests that mitochondria are eliminated. The mechanism for their
elimination still remains to be determined although up-regulation of autophagy,
down-regulation of mitochondrial biogenesis or both are likely mechanisms.











APPENDIX: ADDITIONAL FIGURES





3-mo-old 11-mo-old


I-
E






I-


WT D257A


0.175-
0.150-
0.125-
0.100-
0.075-
0.050-
0.025-
0.000-


WT D257A


Fig A-1. Skeletal muscle mass gastrocnemiuss) in 3-mo old (N=8 per group), and 11-mo old
(N=11 per group), WT and D257A mice. Error bars represent SEM. *P < 0.05.


3-mo-old


70000-
60000-
50000-
40000-
30000-
20000-
100001
0"


tb'


35000'
30000,
25000,
20000,
15000'
10000.
5000.
0.


70000-
60000-
50000-
40000-
30000-
20000-
10000-
0-


11-mo-old

-F


Fig A-2. Caspase-3 activation in gastrocnemius muscle. (A): Caspase-3 content with normal
aging: Comparison of young (5-mo) versus old (30-mo) WT mice. (B): Comparison
of WT versus D257A caspase-3 levels at 3 months of age. (C): Comparison of WT
versus D257A caspase-3 levels at 11 months of age. Cytosolic extracts from WT and
D257A mice of the indicated ages were subjected to SDS-polyacrylamide gel
electrophoresis and probed with a rabbit monoclonal antibody against cleaved
caspase-3. Error bars represent SEM. N=7 per group. *P < 0.05.


0.175-
0.150-
0.125-
0.100-
0.075-
0.050-
0.025-
0.000-


cYn

) E
"E

0o


~ ~2P~
6'


~



















Mitochondrial Respiration in 3-mo Old Mice


State 4 State 3 RCR
.S 10.0- 45 6-
ST 40-
o o 5-
0. 7.5- .
CD Do 30 4
C 5.0- C 3-
.... 20-
E E
N N 15- 2-
O 2.5- O 10

E E
C 0.0 0 0
WT D257A WT D257A WT D257A





Fig A-3. Mitochondrial respiration in skeletal muscle of 3-mo old mice. We determined the
effects of mtDNA mutations on 02 consumption of skeletal muscle mitochondria
obtained from 3-mo old (N=8 per group) WT and D257A mice. The respiratory
control ratio (RCR), an index of mitochondrial coupling, was calculated by dividing
state 3 to state 4 respiration values. Error bars represent SEM.















ROS Production in 3-mo Old Mice

B C


Pyruvate/Malate
Basal mitochondrial ROS production


UZ~I/


Succinate + Rotenone
ROS generation at Complex III
Reverse electron flux blocked
= 1.5,

0.



E
I 0

0C 0.^
WT D257A


Pyruvate/Malate + Rotenone
Maximal Complex I ROS Production


VV I


Succinate + Rotenone + Antimycin
Maximal Complex III ROS production
= 10.0.
0.

S 7.0
cE

0 2.
-6
E
C 0.11


Succinate
ROS generation at Complex III
+ Reverse electron flux


UZrd/


Free Radical Leak


Fig A-4. Reactive oxygen species production during state 4 in isolated mitochondria from 3-mo
old mice. Skeletal muscle mitochondria were obtained from 3-mo old (N=8 per
group) WT and D257A mice. We measured H202 production in mitochondria
supplemented with pyruvate/malate (panel A) since it represents total basal
mitochondrial ROS generation. We also used inhibitors of the ETC in order to study
maximum rates of H202 production from complexes I and III, since they represent the
main sites of ROS generation within the mitochondria. For complex I maximum rate
(panel B) we used rotenone added to pyruvate/malate supplemented mitochondria.
For complex III maximum rate (panel E) we used antimycin A plus rotenone, added
to succinate supplemented mitochondria. We also used mitochondria supplemented
with succinate alone in order to study complex III ROS production under near
physiological conditions (panel C). In addition, some of the assays with succinate as
substrate were performed in the presence of rotenone (panel D), in order to avoid the
backwards flow of electrons to Complex I. Free radical leak percent (FRL%), an
index of mitochondrial efficiency (panel F), was calculated by dividing the H202
value by twice the state 4 respiration value and the result was multiplied by 100 to
give a % final value. Error bars represent SEM.











Young- 3-mo old


WT
Female Male


Cxl39KDa |j I

Cxl30KDa IL._ -I
Cxll70KDa

CxII48KDa
Cxlll29KDa
CxlV-COXI

AIF
A.F 04W

Porin

Actin


D257A
Female Male











-
S-~ -lri


WT
Female Male


D257A
Female Male






-1 -





-I -


ETC complex subunit content 3 mo old mice


Cxl-NDUFAS


0.751


1.5-

1.0-

0.5-


Cxlll-48


Cxl-NDUFS3


4 1.5-
* = 0
10-

0.5-


Cxlll-29


WT D257A


D257A


Fig. A-5. Protein expression of nuclear-encoded and mitochondrial-encoded ETC subunits in
skeletal muscle of 3-mo old and 11-mo old WT and D257A mice. The content of
selected nuclear- and mitochondrial-encoded subunits from complexes I, II, III and
IV, as well as, AIF were evaluated by Western Blotting. Representative blots are
depicted above. Results shown above were normalized to porin. Error bars represent
SEM. *P < 0.5.


CxlV-COXI


AIF- 3 mo old mice
1.25,1


>,
* '5
I
^B


WT D257A


0.00*


WT D257A


Old-11-mo old


I- ~ '




I_-~
I II


I, I








IC~



--


I ~I


I







~I


0.0-L









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BIOGRAPHICAL SKETCH

Asimina Hiona was born in Ioannina, Greece. She attained her bachelor's degree from

Aristotelian University of Thessaloniki with major in exercise physiology. Following

graduation, she moved to the US where she obtained a master's degree from Queen College, NY

in exercise physiology, with main focus in clinical exercise physiology. While in New York she

worked for two years as an exercise physiologist in "Plus One Holding Inc." Finally, deciding to

focus her career in basic science, Asimina moved to University of Florida in 2001, to pursue a

Ph.D degree. The main focus of her doctoral research is the contribution of mtDNA mutations in

skeletal muscle aging, specifically in sarcopenia. Asimina has been a co-author in several peer-

reviewed publications. In 2004 she was awarded an American Heart Association Fellowship and

in 2005 she was awarded the Leighton E. Cluff award in aging research. She was awarded her

Ph.D in summer 2007, with major in biochemistry and molecular biology.





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1 THE ROLE OF MITOCHONDRIAL DNA MUTATI ONS IN SARCOPENIA: IMPLICATIONS FOR THE MITOCHONDRIAL VICIOUS CYCLE THEORY AND APOPTOSIS By ASIMINA HIONA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Asimina Hiona

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3 To my family Thank you for your unwavering love and support. In the memory of my deceased grandfather La mbros who I cherished so much Thank you for the great childhood memories.

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4 ACKNOWLEDGMENTS This work would not have been possible without the support and guidance of several important people. First, I thank my family for their love and support duri ng these difficult years. Their support has not been only moral but also fi nancial. Without their help it would have been impossible for me to complete a Ph.D especially I also thank my parents for teaching me some very important values in life: determination, persisten ce and how to always rely on my abilities and be an honest and fair person. Without their love and support I never could have come this far. From my colleagues I first and foremost thank Dr. Barry Drew for his scientific guidance during my first years in the lab a nd his friendship. I appreciate ever ything that he taught me and more importantly, his ability to make the lab such an enjoyable environment to work in. He is a best friend of mine for life. I also thank Stephane Servais for his guidance and great mentorship regarding my project and my fu ture career. I thank Alberto Sanz from Spain for all his contributions to my thesis and all the laughs we ha d during my thesis studies. I want to especially thank Rizwan Kalani, an outst anding undergraduate student in our lab and current medical student for always helping me dur ing big studies, whenever I need ed him. A special thanks to Alex Samhan Arias and Miguel Garcia from Spain that have been so helpful to me during their short stay in our lab. They made me laugh a lo t and became great friends. Moreover, I thank Suma Kendhayia and Evelyn Kowenhoven for their help in the lab. I would also like to thank my advisor Christiaan Leeuwenburgh for all the opp ortunities I had in the lab during my Ph.D. From University of Wisconsin, Madison, I espe cially thank Dr. Toma s Prolla. Without his help, guidance and mentoring I wouldnt have the thesis project I have, and great career opportunities. I also thank Dr. Greg Kujoth, a post-doc in Dr. Prollas lab for constructing the

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5 transgenic mice I used for my project, and for al ways being so helpful w ith providing me with transgenic mice for my studies and answering my multiple questions. Importantly, I thank my committee members drs Meyer, Kilberg and Kaushal, for being such great, insightful, and helpful committee memb ers. I would also like to thank them for their mentoring and for being so helpful to me duri ng my applications for post-doc positions. They really made my journey to the final defense much easier. Individually, I thank first and foremost Dr. Meyer for tremendously helping me with my future career. I think that words do not do justice to how much I appreciat e his help. I thank Dr. Kilberg for being so supportive of my transfer to the IDP, which I beli eve was one of the best things out of my Ph.D, since I enjoyed my classes so much and the extra knowledge, and for all his help regarding my job applications. Last, but not least, I thank Dr. Ka ushal for all his help with my future career and for believing in me. If I had to choose anyone of the three to be my advisor I would do it in a heartbeat but it would be a very hard choice. Finally, and most importantly, I want to tha nk two very important people I met while in Gainesville: Giorgos Leonis, w hos been an amazing friend all th ese years and Joel French, for loving me and for being the most supportive boyfriend a girl could ever have.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION AND HYPOTHESES..............................................................................14 Specific Aim 1. Determine the Effect of mtDNA Mutations in Skeletal Muscle Mitochondrial Function.......................................................................................................15 Hypothesis 1................................................................................................................... .16 Specific Aim 2. Determine Whether Increased Load of mtDNA Mutations Leads to Apoptosis in Skeletal Muscle..............................................................................................17 Hypothesis 2................................................................................................................... .17 Specific Aim 3. Identify the Specific Apopt otic Signaling Pathway Responsible for Sarcopenia in D257A Mice.................................................................................................17 Hypothesis 3................................................................................................................... .18 2 BACKGROUND AND SIGNIFICANCE..............................................................................19 Introduction................................................................................................................... ..........19 Age-Related Changes in Mitochondrial Function..................................................................20 Electron Transport Chain Abnormalities a nd Mitochondrial DNA Mutations in Aging.......21 Suggested Molecular Mechanisms for th e Propagation of mtDNA Mutations, and Potential Reasons for the Greater Occu rrence of mtDNA Mutations Compared to Nuclear DNA, with Age......................................................................................................24 Mitochondrial DNA Damage and the Mitochondri al Vicious Cycle Theory of Aging.......26 Challenging the Mitocho ndrial Vicious Cycle Theory of Aging.........................................28 Direct Evidence for a Causal Role of mtDNA Mutations in Aging: D257A Mice................29 Mitochondrial Dysfunction, Apoptosis and Skeletal Muscle Aging/Sarcopenia...................30 Mitochondrial-Mediated Pa thways of Apoptosis...................................................................34 3 MATERIALS AND METHODS...........................................................................................41 Experimental Design............................................................................................................ ..41 General Procedures............................................................................................................. ....41 Animals........................................................................................................................ ....41 Mitochondrial and Cytosolic Isolation............................................................................42 Specific Methods............................................................................................................... .....43 Specific Aim 1. Effect of mtDNA Mutations on Skel etal Muscle Mitochondrial Function.......................................................................................................................43 Rationale...................................................................................................................44

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7 Experimental approach.............................................................................................45 Mitochondrial H2O2 generation................................................................................45 Mitochondrial respiration.........................................................................................47 ATP content and production....................................................................................47 Mitochondrial membrane potential..........................................................................48 Blue native page (BN-page) for dete rmination of content and enzymatic activity of respiratory complexes..........................................................................49 Determination of protein content of selected mitochondrialand nuclearencoded subunits from ETC complexes I, II, III and IV......................................50 Determination of mitochondrial protein yield..........................................................51 Determination of MnSOD and Catalase mRNA expression by RT-PCR................52 Oxidative damage to mtDNA...................................................................................52 Specific Aim 2. mtDNA Mutations and Apoptosis in Skeletal Muscle..........................53 Rationale...................................................................................................................54 Experimental approach.............................................................................................54 Determination of cleaved caspase-3 content............................................................54 Enzymatic measurement of caspase 3 activity.........................................................55 Determination of cytosolic monoand oligonucleosomes.......................................56 DNA laddering.........................................................................................................56 Specific Aim 3. Identification of th e Specific Apoptotic Signaling Pathway Responsible for Skeletal Muscle Loss in D257A Mice...............................................57 Rationale...................................................................................................................57 Experimental approach.............................................................................................58 Determination of cytochrome c content by Western Blotting..................................58 Enzymatic measurement of caspase-9 activity.........................................................59 Statistical Analyses........................................................................................................... ......59 4 RESULTS........................................................................................................................ .......61 Mouse Characterization Data from Dr. Prol las Lab: Generation and Phenotype of D257A Mice..................................................................................................................... ...61 Data from Our Lab.............................................................................................................. ....62 Results for Specific Aim 1..............................................................................................62 Impaired mitochondrial bioenerget ics in 11-month-old D257A mice.....................62 D257A mice display decreased content of ETC Complexes I, III, and IV that contain mtDNA-encoded subunits........................................................................63 Electron transport chain complex specific activity remains unaffected by mtDNA mutations in D257A mice.......................................................................64 D257A mice show decreased cont ent of both nuclear-encoded and mitochondrial-encoded ETC subunits..................................................................64 D257A mice display decreased ATP content...........................................................65 Mitochondrial membrane potential is significantly lower in D257A mice..............65 Mitochondrial protein yield is reduced in skeletal muscle of D257A mice.............66 Skeletal muscle mitochondria from D257A mice produce significantly less ROS.......................................................................................................................66 D257A mitochondria produce less ROS in both main ROS generators of the ETC: Complex I and Complex III........................................................................67

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8 No difference in antioxidant enzyme mRNA expression between genotypes.........68 Mitochondrial DNA mutations cause ag ing phenotypes in the absence of increased oxidative stress.....................................................................................69 Results for Specific Aim 2..............................................................................................69 D257A mice display significant skel etal muscle loss by 11-mo of age...................69 Apoptosis in D257A skeletal muscle is evident by an increase in cytosolic monoand oligo-nucleosomes..............................................................................70 DNA laddering is evident in skel etal muscle of D257A mice.................................70 Caspase-3 cleavage and activation is up-regulated in D257A mice and resembles caspse-3 activation during normal aging.............................................70 Results for Specific Aim 3..............................................................................................72 Cytochrome c release in the cytosol of D257A and WT skeletal muscle................72 Caspase-3 and caspase-9 activities are significantly higher in D257A mice: Evidence for induction of the mitochondr ial, caspase-dependent pathway of apoptosis...............................................................................................................72 5 DISCUSSION..................................................................................................................... ....89 Overview of Principal Findings..............................................................................................89 Hypothesis One: The Effect of mtDNA Muta tions in Skeletal Muscle Mitochondrial Function....................................................................................................................... .......90 Mitochondrial DNA Mutations Cause Prof ound Deficiencies in Mitochondrial Function.......................................................................................................................91 Mitochondrial DNA Mutations Cause Mitoc hondrial Dysfunction in the Absence of Increased ROS Production or Oxidative Dama ge to mtDNA: Implications for the Mitochondrial Vicious Cy cle Theory of Aging.......................................................94 Mitochondrial DNA Mutations Lead to M itochondrial Dysfuncti on, Via Alterations of ETC Complex Composition..................................................................................101 Total Skeletal Muscle M itochondrial Protein Yield Continuously Decreases as Time Progresses in Mutant Mice...............................................................................106 Hypothesis Two: the Effect of mtDNA Mu tations on Skeletal Muscle Apoptosis..............109 Apoptosis with Aging....................................................................................................110 Mitochondrial DNA Mutations and Apoptosis.............................................................111 Disruption of Mitochondrial Membrane Potential and Role for Apoptosis..................113 Apoptosis is Evident in Skel etal Muscle of D257A Mice.............................................115 Hypothesis Three: Identify the Specific A poptotic Signaling Pathway Responsible for Sarcopenia in D257A Mice...............................................................................................116 Proposed Mechanism for the Skeletal Muscle Loss Induced by High Load of Somatic mtDNA Mutations.............................................................................................................119 Synopsis....................................................................................................................... .........121 Conclusions.................................................................................................................... .......123 Future Directions.............................................................................................................. ....124 APPENDIX: ADDITIONAL FIGURES.....................................................................................128 LIST OF REFERENCES.............................................................................................................132

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9 BIOGRAPHICAL SKETCH.......................................................................................................153

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10 LIST OF FIGURES Figure page 2-1 Contributions of the mitochondrial and nuc lear DNA to protein subunits of the complexes of the ETC.......................................................................................................39 2-2 The mitochondrial vicious cycle theory..........................................................................39 2-3 Mitochondrial-mediated apoptosis.....................................................................................40 3-1 Experimental design and summary of the pa rameters measured in specific aims 1, 2 and 3.......................................................................................................................... ........60 4-1 D257A mice display a premature aging phenotype...........................................................74 4-2 Mitochondrial respiratio n is compromised in skeletal muscle of D257A mice.................74 4-3 D257A mice display decreased content of ETC Complexes I, III and IV that contain mtDNA-encoded subunits.................................................................................................75 4-4 Statistical analysis of ETC complex I, II, III, IV and the F1 domain of the ATPase content measured by Blue Native Page in skeletal muscle of 11-mo old WT and D257A mice..................................................................................................................... ..76 4-5 Electron transport chain complex activity in skeletal muscle of 11-mo old WT and D257A mice..................................................................................................................... ..77 4-6 Statistical analysis of ETC complex activity in skeletal muscle of 11-mo old WT and D257A mice..................................................................................................................... ..78 4-7 D257A mice show decreased content of both nuclear-encoded and mitochondrialencoded ETC subunits.......................................................................................................79 4-8 D257A mice display decreased ATP content....................................................................80 4-9 Mitochondrial membrane potential ( ) drop in D257A mice.........................................80 4-10 Mitochondrial yield is reduced in D257A skeletal muscle................................................81 4-11 D257A mitochondria produce less reactiv e oxygen species (ROS) during state 4...........81 4-12 D257A mitochondria produce less ROS in both main ROS generators: Complex I and Complex III................................................................................................................ .82 4-13 D257A mice show no difference in an tioxidant enzyme mRNA expression....................83 4-14 Mitochondrial DNA oxidation in skelet al muscle of WT and D257A mice.....................84

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11 4-15 D257A mice display signif icant skeletal muscle loss by 11-mo of age compared to age-matched WT................................................................................................................84 4-16 Apoptosis evident in D257A muscle by increase in cytosolic monoand oligonucleosomes.................................................................................................................... ...85 4-17 DNA laddering evident in skeletal muscle of D257A mice..............................................85 4-18 Caspase-3 activation in skeletal musc le of D257A mice resembles caspase-3 activation during normal aging..........................................................................................86 4-19 Cytochrome c release in the cytoso l of D257A and WT skeletal muscle..........................87 4-20 Caspase-3 and -9 activities are elevated in D257A muscle: Proof of activation of the mitochondrial caspase-dependent pathway of apoptosis...................................................88 4-21 Caspase-3 and caspase-9 activity Pears on correlations in WT and D257A mice..............88 5-1 Proposed mechanism for the skeletal musc le loss induced by high load of somatic mtDNA mutations............................................................................................................127 A-1 Skeletal muscle mass (gastrocnemius) in 3-mo old (N=8 per group), and 11-mo old (N=11 per group), WT and D257A mice.........................................................................128 A-2 Caspase-3 activation in gastrocnemius muscle................................................................128 A-3 Mitochondrial respir ation in skeletal muscle of 3-mo old mice......................................129 A-4 Reactive oxygen species production during st ate 4 in isolated mitochondria from 3mo old mice.................................................................................................................... ..130 A-5 Protein expression of nuclear-encoded and mitochondrial-encoded ETC subunits in skeletal muscle of 3-mo old a nd 11-mo old WT and D257A mice.................................131

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SIMINA HIONA Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF MITOCHONDRIAL DNA MUTATIONS IN SARCOPENIA: IMPLICATIONS FOR THE MITOCHONDRIAL VICIOUS CYCLE THEORY AND APOPTOSIS 12 By AAugust 2007 Chair: Christiaan Leeuwenburgh Major: Medical Sciences--Biochemistry and Molecular Biology Aging results in a progressive loss of skel etal muscle, a condition termed sarcopenia which can have significant effects on physical function and quality of life as aging commences. At the cellular level, the aging process can activate stress-associat ed signal transduction pathways that result in mitochondrial dysfunction and apoptosis. Because the mitochondrion contains its own DNA, a centr al role for mitochondrial DNA (m tDNA) mutations in mammalian aging has been postulated. In fact, mtDNA mutations have been shown to accumulate with aging in skeletal muscle fibers of various species The purpose of my disse rtation project was to determine whether mtDNA mutations are causal to sarcopenia. The central hypothesis tested was that mutations in mitochondrial DNA known to be associated with aging in many post mitotic tissues, play a causal role in skeletal muscle loss, possibly by inducing mitochondrial dysfunction, leading to the activation of a m itochondrial-mediated apoptotic program. In order to demonstrate a causal relationship between mtDNA mutations and skeletal muscle loss with age, we used a transgenic mouse model that expresses a proofreading-deficient versi on of the mitochondrial DNA polymerase gamma (PolgD257A), resulting in increased spontaneous mutation rates in mtDNA. The causal role of mtDNA mutations in mammalian aging is supported in this mouse

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13 model by the observation that mice with the PolgD257A (D257A ) phenotype develop several aging phenotypes among which, is skeletal muscle loss. We specifically hypothesized that the accumu lation of mtDNA muta tions in skeletal muscle will lead to compromised mitochondrial bioenergetics. We found that D257A mice have decreased protein content of co mplexes I, III and IV, all of which contain subunits encoded by mitochondrial DNA, compared to wild type (WT) mice at 11-mo of age. Mitochondrial dysfunction was also evident in D257A mice by decreased mitochondri al oxygen consumption, lower membrane potential during both state 3 (pho sphorylative state) and state 4 (resting state), and lower ATP content. However, this dysfunc tion was not accompanied by an increase in mitochondrial reactive oxygen sp ecies (ROS) production or oxida tive damage. In fact, we detected a decrease in the rate of H2O2 production by intact D 257A mitochondria and no difference in mtDNA oxidative modification m easured by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), compared to WT. This is in contrast to the mitochondrial Vic ious Cycle theory of aging which suggests that mtD NA mutations may lead to mito chondrial dysfunction via further increases in mitochondrial ROS production. We further hypothesized that mitochondrial dysfunction will result in mitochondrial-mediated apoptosis, which would be responsible for the loss of skeletal muscle mass we have observe d in D257A mice. We detected DNA laddering and an increase in the amount of cytosolic monoand oligo-nucleosomes in D257A mice compared to WT, indicative of apoptosis. Concurrently, we demonstrated increased activity of both, the initiator caspase-9, and the effector caspase-3, as well as an increase in cleaved (activated) caspase-3 content. This suggests that apoptosis in mutant mice is mitochondrial-mediated, and is conferred upon mitochon drial dysfunction. Thus, mutations in mtDNA play a causal role in sarcopenia, through enhancing apoptosis induced by mitoch ondrial dysfunction.

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14 CHAPTER 1 INTRODUCTION AND HYPOTHESES Aging individuals lose muscle mass at a rate of 1-2% per year past the age of 50 (1, 2). This age-related muscle atrophy, termed sarcopenia, is associated with muscle weakness and can have significant effects on an i ndividuals health and quality of life. Sarcopenia affects a growing population, occurring in 10-25% of individuals under the age of 70 and in more than 40% of the elderly over the age of 80 (1, 2). The annual co st of treating sarcopeni a is greater than the amount spent due to osteoporosis, yet little effort is made to increase public awareness to prevent sarcopenia (3). Thus, sarcopenia and the subseque nt loss of physical f unction is a significant public health problem. Mitochondria are the main source of cellular ATP and play a cen tral role in a variety of cellular processes, including fatty acid -oxidation, calcium signali ng, reactive oxygen species (ROS) generation and apoptosis. The aging process can introduce a variety of stressors that result in the collapse of mitochondr ial function, causing apoptot ic cell death. Because the mitochondrion contains its own ~16-kilobase circ ular DNA, that is also intron-less and histoneless, and close to the main ROS generator in the cell: the electron transport chain (ETC), a central role for mitochondrial DNA (mtDNA) muta tions in aging has been postulated (4-6). Indeed, mtDNA mutations have been shown to accumulate with aging in several tissues of various species (7-12), in cluding skeletal muscle. The central hypothesis tested in my research project is that mutations in mitochondrial DNA, known to be associated with aging in ma ny post mitotic tissues, pl ay a causal role in sarcopenia, possibly through e nhancing apoptosis mediated by mitochondrial dysfunction. Previous studies have provided strong experi mental support for an association between mitochondrial DNA mutations and tissue dysfunction, particularly in long -lived post-mitotic

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15 cells such as cardiomyocytes, skeletal muscle fibers and neurons (7, 9-12). However, such association studies can provide only correlative data. In order to determine whether mtDNA mutations underlie the aging process, we used a genetically engineered mouse model that expresses a proofreading-defi cient version of the mitochondrial DNA polymerase gamma (PolgD257A), resulting in increased sp ontaneous mutation rates in mitochondrial DNA. Previously (13), we have characterized accelerated agi ng in D257A mice and found that these mice exhibited various age-related phenotypes including thymic involution, loss of bone mass, cardiac dysfunction and skeletal muscle loss. In mitochondr ia from the tissues examined we showed that mtDNA mutations do not lead to increases in ROS production or oxidative stre ss, contrary to the free radical theory of aging. Importantly, in most tissues exam ined, including skeletal muscle, we demonstrated increased levels of cleaved (activated) caspase-3, wh ich is indicative of apoptosis (13). Hence, the accumulation of mt DNA mutations may be associated with the induction of apoptosis irrespectiv e of elevations in ROS produc tion and oxidative stress in mitochondria with age. Th e D257A mouse provides an in vivo model to study the mechanisms of apoptosis in skeletal muscle with age, speci fically, the contribution of mtDNA mutations. Our hypotheses have implications for both the basic bi ology of aging and clinical approaches to agerelated diseases of skeletal muscle, such as sarcopenia. Specific Aim 1. Determine the Effect of mtDNA Mutations in Skeletal Muscle Mitochondrial Function We, as well as, others have shown that mice with a mitochondrial mutator phenotype develop several aging phenotypes (13, 14). We f ound that D257A mice have significant skeletal muscle loss by 11-mo of age compared to WT, wh ich is indicative of sarcopenia. However, our findings thus far, also indicate that despite increased mutational load, mitochondria from D257A mice do not show increased levels of oxidative stress in all currently examined tissues (13).

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16 These observations do not support the idea that mtDNA mutations contribut e to increased ROS production and oxidative stress in mitochondria with age, placing the mitochondrial vicious cycle theory of aging in questi on. In this aim we determined whether mtDNA mutations lead to mitochondrial dysfunction and further investigat ed the mechanism by which mutations induce mitochondrial dysfunction. Since these mice have elevated levels of mtDNA mutations, we expected that the structure a nd/or function of proteins enco ded by mitochondrial DNA would be affected, ultimately affecting mitochondrial bioenergetics, and leading to mitochondrial dysfunction. The concentration, and enzymatic activity of respiratory complexes I, II, III, IV, and F1F0 ATPase, mitochondrial respiration, basal oxidant production, ATP content and production, mitochondrial membrane potential and oxida tive damage to mtDNA were determined. Hypothesis 1 We hypothesized that the accumulation of mtDNA mutations would lead to mitochondrial dysfunction, due to alterations in the content and/or activity of th e respiratory complexes I, III, IV and F1F0 ATPase, which contain subunits encoded by mtDNA, leading to compromised ETC activity in skeletal muscle of 11-mo-old D257A mice compared to WT. Decrease in ETC activity would lead to a more extensive decrement in stat e 3 respiration, since this is the active state of the mitochondria when electron flux is highest We expected that the decrease in state 3 respiration would be associated with reduced ATP content and production in skeletal muscle mitochondria, ultimately leading to mitochondr ial dysfunction. We further hypothesized that mitochondrial dysfunction would not be associated with increases in basal ROS production in the D257A mice, compared to WT, and specifically, with increases in ROS production at the main ROS generators of the ETC, Complex I and Complex III. Rather, mitochondrial dysfunction would lead to loss of mitochondrial membrane potential and greater susceptibility of mitochondria to apoptosis.

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17 Specific Aim 2. Determine Whether Increase d Load of mtDNA Mutations Leads to Apoptosis in Skeletal Muscle We have already demonstrated that D257A mice lose skeletal muscle and exhibit significantly greater content of cleaved (activated) caspase-3 in skeletal muscle by 11-mo of age, compared to WT mice (13). However, additional m easures are needed in order to determine that apoptosis is indeed a central mechanism responsible for skeletal muscle loss associated with the accumulation of mtDNA mutations. In this aim, we wanted to corroborate and more extensively investigate apoptosis in skeletal muscle by pe rforming specific apoptotic measures. We further measured caspase-3 activity, the content of monoand oligonucleosomes re leased in the cytosol following apoptotic DNA fragmentation, and DNA laddering, evident on agarose gel during apoptotic nucleosomal fragmentation. Hypothesis 2 We hypothesized that the levels of apoptosis would be elevated in skeletal muscle of D257A mice compared to WT by 11-mo of age, which may explain the decline in skeletal muscle mass we observed in D257A animals. Specific Aim 3. Identify the Specific Apop totic Signaling Pathway Responsible for Sarcopenia in D257A Mice The significant skeletal musc le loss in conjunction with th e elevated cleaved caspase-3 levels in the D257A animals suggest that loss of critical, irreplaceable cells through apoptosis may be a central mechanism of tissue dysfunctio n associated with the accumulation of mtDNA mutations. In this aim we determined the pathwa y of skeletal muscle apoptosis in the D257A mice. We speculated that the si gnaling pathway responsible for induction of apoptosis in D257A skeletal muscle has to be intrinsic to the mito chondria since loss of musc le mass in these animals is conferred upon accumula tion of mtDNA mutations. Moreover, the mitochondrial pathway may encompass both caspase-dependent and independent induction. Since the caspase-

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18 independent path still remains to be elucidated, we assessed the activation of key proteins from the main mitochondrial-mediated, caspase-dependent pathway to ev aluate whether this path is activated in response to the in crease in mtDNA mutational load. Hypothesis 3 We hypothesized that mitochondrial dysfunction in D257A mice would ultimately lead to mitochondrial outer membrane permeability and efflux of cytochrome c into the cytosol. Cytochrome c release would instigate formation of the apoptosome, leading to cleavage and activation of the initiator cas pase-9. Caspase-9 would furthe r cleave and activate the final effector caspase-3 which is respon sible for carrying out the actual pr oteolytic events that result in cellular breakdown, leading to mito chondrial-mediated apoptosis.

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19 CHAPTER 2 BACKGROUND AND SIGNIFICANCE Introduction Over the past two decades, increasing ev idence suggests that mitochondrial dysfunction may play a causal role in the aging process. Th e essential role of mito chondria in cellular ATP production, the generation of reactive oxygen specie s (ROS), and the induction of apoptosis suggest a number of mechanisms for mitochondria l pathology. There is now strong evidence that age induces alterations in the mitochondrial geno me that lead to def ects in mitochondrial function, especially in tissues w ith high energy requirements such as the heart, liver, brain and skeletal muscle (15-18). It was proposed that duri ng an individuals life span, ROS, by-products of oxidative metabolism, accumulate and alter cell components (19). Mitochondria, one of the primary sources of ROS, are particularly affected, leading to changes in their structure as well as in the genetic information of mtDNA. Th e observed alterations of mtDNA include oxidative damage to DNA bases, point mutations and large scale de letions or duplications. MtDNA mutations are known to have deleterious effects on oxidative phosphorylation, especially in patients with mitochondrial diseases (20, 21), and tissues that rely heavily on oxidative phosphorylation are expected to be more affected. We chose skelet al muscle as the focus tissue in this project because this tissue is highly dependent on oxi dative phosphorylation and suffers marked agerelated degeneration (sarcopenia). The background information presented in this section first focuses on the changes that are induced in mitochondrial function as a resu lt of aging, and the cellular impact of mtDNA mutations, by providing direct evidence for a ca usal role of mtDNA mutations in the aging process. This is followed by an explanation of how mitochondrial dysfunction can lead to

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20 apoptosis initiated by the mitochondria, and an examination of the sp ecific pathways and functions of the molecules involved in mitoc hondrial-mediated apoptosis Although much of the evidence suggests an important role for mtDNA mutations in aging, this evidence was largely correlative until 2004 when Trifunovic et al. publ ished the first results on the POLG mouse model (14). We propose that this mouse model (a lso used in the present study), with altered mitochondrial mutation rates, represents a valuable tool to critically assess in vivo the role of mtDNA mutations on sarcopenia. Age-Related Changes in Mitochondrial Function The mitochondrial genome is a double-stranded, circular DNA molecule of 16,569 bp, lacking histones and compactly organized (i.e ., no introns) that, apart from a 1.1 kbp non-coding D-loop, encodes for 13 protein co mponents of the ETC (Fig 2-1) 22 tRNAs and 2 rRNAs. There are approximately 2 to 10 copies of the mitocho ndrial genome per mitoc hondrion (22) and 10s to 100s of mitochondria per cell, dependi ng on the cells energy requirements (23). Biochemical analyses of ET C complex activities pe rformed in tissue homogenates from humans, rhesus monkeys and rodents have, in genera l, identified age-associated decreases in the activities of complexes I and IV. Those tissues in which robust bi ochemical declines have been repeatedly detected are the highly oxidative, non-replicating tissues such as skeletal muscle, heart and brain (16, 24-31). Commonly used markers for mitochond rial ETC abnormalities include the loss of cytochrome c oxidase (COX) activity and the concomit ant increase in succinate dehydrogenase (SDH) activity (SDH hyperreactive regions, also known as ragged red fibers (RRF)). In situ histochemical studies of human and m onkey myocardial tissue have focused on detection of cells deficient in COX activity (32, 33); in these studies, the number of cardiomyocytes displaying defects in the ETC COX enzyme was found to increase with age in humans from 2-3 defects/cm2 in the second and third decades, to 50 defects/cm2 in the fifth to ninth

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21 decades (32). Recent cytochemical-immunocytochemical studies of oxidative phosphorylation enzymes in monkeys (1025 years of age) showed complex III, complex IV and complex V defects in skeletal muscles, diaphragm, myocardium and extraocular muscles of 25-year-old animals. These defects were randomly distributed and not asso ciated with a loss of complex II, which is all nuclear encoded (33). Furthermore, in rats, aged mitochondria exhib it lower mitochondrial membrane potential (34), reduced cardiolipin leve ls (35-37), and a decrease in the activity of carnitine acetyltransferase, a key mitochondrial enzyme for fuel u tilization (38). In general, the main feature of these age-related alterations in post-mitotic tissues is the development of a shift in activity ratios among different complexes, such that it would tend to hinder the ability of mitochondria to effectively tran sfer electrons down the respir atory chain and thus, adversely affect oxidative phosphorylation. In accordance with the above, energy depletion in the mitochondria during aging is also evident; Our laboratory recently found that mitochondrial ATP content and production in gastrocn emius muscle from aged rats significantly decreased, although H2O2 production and mtDNA 8-oxodGuo levels were unchanged compared to young animals (39). This decline observed in skeletal muscle may be a factor in the process of sarcopenia, which increases in incidence with advancing age (39). Consistent with our findings, a decline in human skeletal muscle mitoc hondrial ATP production with adva ncing age has recently been observed (40). Eventually, energy depletion could impair important cellula r functions including damage repair/removal mechanisms, and also trigger apoptosis (41) Electron Transport Chain Abnormalities a nd Mitochondrial DNA Mutations in Aging There is growing evidence that the accumula tion of mitochondrial mutations and deletions, associated with aging, result in tissue dysfunction. For example, in the rat, the levels of a particular deletion in mtDNA ( 4834 deletion) in the dorsal root ganglion were about 300-fold higher in old compared to young rats. The a bundance of this particular mtDNA deletion in

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22 dorsal root ganglia from individual rats correlated strongly with th eir decline in function (42). In normal aging, impaired respiratory function and oxi dative phosphorylation in muscle fibers is becoming increasingly evident (39, 40), and point-m utations and deletions in mtDNA have been found to correlate with this re duced capacity (43-45). Cao et al ., showed that fibers from the femoris muscle of 38month-old rats, with el ectron transport system abnormalities, also had large mtDNA deletions (4.4.7 kb), whereas normal ETC fiber regions had wild-type mtDNA. Deletions occurred at the major arc of the mtDNA spanning the origin of replication, and were clonal within the fibers, with different deletions between the fibers (43) Similarly, Wanagat et al, have demonstrated an age-related increase in skeletal muscle fibers that display ETC abnormalities, and that, mtDNA deletion muta tions, co-localize with segmental ETC abnormalities (7). Specifically, th ey showed that the proportion of ETC abnormal fibers that displayed the RRF phenotype (i.e., loss of COX activity with concomita nt hyperactivation of SDH activity) increased significan tly with age, and there were no ETC abnormal fibers with the RRF phenotype observed in the 5-month-old musc les, whereas 42% of the total ETS abnormal fibers in the 38-month-old animals displayed th e RRF phenotype. They further detected shorter than wild type genomes in al l of the RRFs, while mtDNA deletion mutations were not detected in ETS normal fibers from the same sections Multiple microdissections along the same RRF amplified identically sized products, supporting th e clonal nature of the mtDNA deletion (7). Moreover, human studies also provide evidence for an increase in mtDNA mutations with aging, and a correlation between mtDNA mutations and the occurrence of skeletal muscle abnormalities with advancing age (44-46). Michik awa et al., showed that human fibroblast mtDNA from normal old individuals, revealed high c opy point mutations at specific positions in the control region for replication, which was not evident in young s ubjects (47). Furthermore, in

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23 longitudinal studies, one or more mutations appeared in an indivi dual only at an advanced age. Most strikingly, a T414G transversion was found, in a generally high proportion (up to 50%) of mtDNA molecules, in 8 of 14 indi viduals above 65 years of age ( 57 percent) but was absent in 13 younger individuals (47). Wang et al., showed that muscle-specific mutations accumulate with aging in critical human mtDN A control sites for replication; specifically, they demonstrated that most of 26 individuals 53 to 92 years old, without a known hist ory of neuromuscular disease, exhibited an accumul ation of two new point mutations, i.e., A189G and T408A, at mtDNA replication control sites in muscle which were absent or marginally present in 19 young individuals. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years of age (T408A), or were present only in thr ee subjects in very low amounts (A189G)(12). The investigators suggested that the striking tissue specificity of the muscle mtDNA mutations detected, and their mapping at critical site s for mtDNA replication, strongly point to the involvement of a specific mutagenic machiner y and to the functional relevance of these mutations. Latest experimental evidence also suggests that randomly deleted mtDNA appears mainly in skeletal muscle of healthy old subjects (be yond 80 years old), affecting up to 70% of mtDNA molecules, and coincides with a decrease in the activities of complexes III and IV of the ETC, which contain subunits encoded by mtDNA (Fig 21) (48). Most importantly, high levels of clonally expanded mtDNA point muta tions were identified in cy tochrome c oxidase deficient muscle fibers, from old individuals without muscle disease, but in none of the normal fibers (49). Immunohistochemical experiments showed that the majority of the cytochrome c oxidase deficient muscle fibers expressed reduced levels of subunit II of cytochrome c oxidase, which is encoded by mitochondrial DNA, whereas there was normal or increased expression of subunit IV

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24 of cytochrome c oxidase, which is encoded by nuclear DNA (49). The au thors concluded that mtDNA point mutations are associated with cy tochrome c oxidase deficient muscle fiber segments in aging, the focal accumulation of which may cause significant impairment of mitochondrial function in individu al cells in spite of low ove rall levels of mitochondrial DNA mutations in muscle (49). Indeed, although onl y a few cells develop COX deficiency, the resultant cellular dysfunction might have substantial effects, especially if the cell is part of a complex networke.g., the central nervous system (50) It is therefore, like ly that, in skeletal muscle of aged individuals, normal mtDNA devoid of deletions or point mutations may represent a minority of the total mtDNA pool. There is also evidence that the rate of mitochondrial mutagenesis is faster in mice than humans per unit time (51), a necessary condition if mitochondrial mutations are causally linked to ag ing. When taken as a whole, these studies provide compelling evidence for an important role of mitochondrial DNA mutations in aging. Suggested Molecular Mechanisms for the Propagation of mtDNA Mutations, and Potential Reasons for the Greater Occurrence of mt DNA Mutations Compared to Nuclear DNA, with Age The cellular and physiological ramifications of mtDNA disruption have been first made clear from studies of a broad class of ne uromuscular disorders known as mitochondrial myopathies and encephalomyopathies (reviewed by Wallace, 1999; DiMauro, 1993) (52, 53). These diseases are, clin ically and biochemically, a diverse gr oup of disorders affecting primarily those tissues having the highest energy demands: br ain, skeletal muscle and heart. The mtDNA abnormalities associated with thes e disorders were shown to range from point mutations (21, 54) to large mtDNA deletions in the mitoc hondrial encephalomyopathies (53, 55). The mtDNA abnormalities found in mitochondrial myopathies have been linked to many oxidative defects in cells, with a very common defect being the RRF phenotype which is also common in skeletal muscle fibers of aged individuals.

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25 Mathematical models suggest that the same basic cellular mechanisms are responsible for the amplification of mutant mtDNA in agi ng and in mtDNA diseases (50); Using an in silico model of mitochondrial genetic pr ocesses within indivi dual non-dividing cel ls, which was based on a contemporary understanding of relaxed replication of mtDNA (unlike nuclear DNA, which replicates once during th e cell cycle, mitochondrial DNA is degraded and replaced continuously, even in non-dividing cells such as skeletal muscle fibers and central neurons), Chinnery et al. introduced a copy-error (mut ation) rate, and meas ured the proportion of individual simulated cells over time (50). With this approach they were able to show that, even for very rare somatic mutations, relaxed replic ation leads to random dr ift of the amount of mutant mtDNA within the cell. This powerful mech anism alone leads to th e clonal expansion of mutant mtDNA during the lifetime of a person, a nd the accumulation of COX-negative cells at a similar rate to that seen in vivo (50). They furt her showed that random genetic drift was also the mechanism for the clonal expa nsion of mutant mtDNA in pr ogressive mtDNA disease. The mechanism proposed in this study is interesting, yet the actual process of propagation of somatic mtDNA mutations remains to be determined. Several other hypotheses regarding the propaga tion of mtDNA mutations and thus, of the mutant mitochondria, have also been suggested. De Grey suggested in 19 97 that it is precisely the loss of superoxide production that gives mutant mitochondria and their DNA a selective advantage and drives their clona l expansion: in this surviva l of the slowest hypothesis, mitochondrial turnover by autophagy is driven by self-inflicted free radical damage to the mitochondrial membranes, so a mutant mitochondr ion is less suicidal and is more often replicated simply because it is more long-lived (56). Another hypothesis, known as the crippled

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26 mitochondrion hypothesis, states that, the internal biochemi stry of mutant mitochondria somehow stimulates them to replicate (57). Furthermore, the greater occu rrence of point-mutations a nd deletions observed in mtDNA compared to nuclear DNA could be due to: a) a gr eater mtDNA exposure to re actants, b) the lack of protective histones, and c) to a less advanced DNA repair system (58-60). In fact, mitochondria are believed to enti rely lack nucleotide excision re pair (NER), which constitutes a major nuclear defense system acting on various nDNA lesions including pyrimidine dimmers (58, 59). Mitochondria also have other discrepancie s and may also have less advanced mismatch repair (MMR) (59). Like crosslinks betw een DNA bases (such as thymine dimmers), DNA protein crosslinks, or bulky DNAadducts can cause a stall duri ng mtDNA replication which can induce DNA double-strand brakes (61, 62), contributi ng to the occurrence of mtDNA deletions with aging, and as previous re search suggests, it is likely fo r mtDNA containing deletions, to acquire a replicative advantage ove r longer wild type mtDNA (43). Mitochondrial DNA Damage and the Mitoch ondrial Vicious Cycle Theory of Aging Despite the fact that in animal cells mtDNA co mprises only 1% of genetic material, several lines of evidence suggest that its contribution to cellular physiol ogy could be much greater than would be expected from its size alone (63). For instance, (i) it mutates at higher rates than nuclear DNA, which may be a consequence of its close proximity to the ETC (electron transfer chain); (ii) it encodes either polypeptides of ETC or components required for their synthesis and, therefore, any coding mutations in mtDNA will affect the ETC as a whole; this could affect both the assembly and function of the products of num erous nuclear genes in ETC complexes; (iii) defects in the ETC can have pleiotropic effects b ecause they affect cellular energetics as a whole (63).

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27 Mitochondria have been shown to accumulate high levels of lipophilic carcinogens such as polycyclic aromatic hydrocarbons (64, 65) which can preferentially damage mtDNA (66). Other mutagenic chemicals also have been shown to preferentially target mtDNA (64, 67-69). Therefore, it is conceivable that life-long exposure to certain envi ronmental toxins could result in a preferential accumulation of mtDNA damage and accelerate aging. However, by far, the predominant kind of insult to which mtDNA is e xposed is oxidative damage. The susceptibility of the mitochondrial genome to oxidative DNA damage may be due to a number of factors including: 1) its close proximity to the ETC, whose complexes I and III are believed to be the predominant sites of ROS production inside the cell, 2) lack of protective histones and, 3) the compactness of its genetic inform ation is such that damage at any point in the genome will likely occur in a gene. The phenotypic implications of mtDNA mutations are dependent on which gene product is disrupted and one might predict that da mage may occur in those complexes to which the mitochondrial genome makes th e greatest contributions. The free radical theory of aging first put forw ard by Harman (4, 5, 70, 71) states that it is the mitochondrial production of RO S, such as superoxide and H2O2, and the resulting accumulation of damage to macromolecules that causes aging. Cumulative damage to biological macromolecules was proposed to overwhelm th e capacity of biological systems to repair themselves, resulting in an inevitable functiona l decline (63). The mitochondrial vicious cycle theory of aging can be considered as an extensio n and refinement of the free radical theory (63). Its major premise is that mtDNA mutations accumulate progressively during li fe, as a side effect of respiration, and are directly responsible for a measurable de ficiency in cellular oxidative phosphorylation activity, leading to an enhanced ROS production (63). In turn, increased ROS production results in an increased rate of mt DNA damage and mutagenesis, thus causing a

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28 vicious cycle of exponentially increasing oxidative damage and dysfunction, which ultimately culminates in death (Fig 2-2) (63). Challenging the Mitochondrial Vic ious Cycle Theory of Aging Bandy and Davison were the first to put forwar d a mechanistic elaboration of what later became known as the mitochondrial vicious cycle theory (72). Wh ile they showed that mtDNA mutations may have the same effect on the respir atory chain as small-molecule inhibitors of respiration, that is, to stimulate the one-elect ron reduction of molecular oxygen to superoxide (therefore, increasing ROS production), they also carefully noted that not all mutations stimulate superoxide production. Specifically, they pointed out that a mutati on preventing the synthesis of cytochrome b would actually abolish any superoxide production at complex III that normal mitochondria might exhibit, because without cytochrome b in place, complex III cannot be assembled (72). Later studies repo rted that respiration-deficient cells of several tissues possessed mutations that would indisputab ly preclude assembly of both the enzyme complexes known to be responsible for mitochondrial ROS production, co mplexes I and III (73-76). These mutations were large deletions, which eliminated the gene s for at least a couple of respiratory chain subunits, but also removed at least one tRNA gene There is no redundancy of tRNA genes in the mtDNA, so the loss of any such gene abolishes the synthesis of all 13 mtDNA-encoded proteins (77, 78). These findings are highly re levant to the plausibility of the vicious cycle theory in normal aging. Recently, there is an increasing body of eviden ce challenging the vicious cycle theory of aging (63, 78-80), and our results provide direct proof against the theory (13) (Figs 4-11, 4-12, 414). In our specific aim #1 we tested the hypothe sis that the accumulati on of mtDNA mutations indeed leads to impaired synthesis of mtDNA-en coded ETC subunits and loss of activity of the mitochondrial complexes, which could explain the decrease in O2 consumption in conjunction

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29 with the decrease in H2O2 production we have shown in the D257A skeletal muscle mitochondria (Figs 4-2, 4-11, 4-12). Direct Evidence for a Causal Role of mt DNA Mutations in Aging: D257A Mice As already discussed, there is an ever growing body of research that supports an important role for mtDNA mutations in aging by providing experimental support for an association between mtDNA mutations, apoptosis and tissue dys function, particularly in long-lived post mitotic cells such as cardiomyocytes and skeletal muscle fibers (7, 81). However, most of the studies to date are correlative in nature. In pa rticular, until recently, it has been unclear whether mtDNA mutations are simply associat ed with aging in va rious tissues, or if they actually cause alterations in tissue function. In 2004, Trifunovic et al. published th e first experimental evidence providing a causative link betw een mtDNA mutations and mammalian aging (14). They showed that homozygous knock-in mice that express a pr oof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polym erase, develop a mtDNA mutator phenotype with a threefold to fivefold increase in the le vels of point mutations as well as increased amounts of deleted mtDNA. This increase in so matic mtDNA mutations was associated with reduced lifespan and premature onset of agingrelated phenotypes such as alopecia, kyphosis, osteoporosis, and heart enlargement. A year later we corroborated Trifunovics findings regarding the impact of mtDNA mu tations in aging, using mice w ith the same mutation, and we also showed that the accumulation of mtDNA mutations was not associ ated with increased levels of oxidative stress or a defect in cellular pr oliferation, but was correlated with the induction of apoptotic markers, suggesting that accumu lation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging (13). Moreover, Zassenhaus and colleagues studied mice that express a proofreading-deficient POL specifically in the heart, and develop cardiac mtDNA mutations, in order to determine

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30 whether low frequency mitochondrial mtDNA mu tations are pathogenic. They found that sporadic myocytic death occurred in all regions of the heart, due to apoptosis as assessed by histological analysis and TUN EL staining (82). They also pointed out that cytochrome c was released from mitochondria and concluded that mtDNA mutations are pat hogenic, and seem to trigger apoptosis through the m itochondrial pathway (82). The use of D257A mice in the present investigation allowe d us to elucidate in vivo, the contribution of mtDNA mutations to the aging phenotype in skeletal muscle, and their role in apoptosis. Mitochondrial Dysfunction, Apoptosis a nd Skeletal Muscle Aging/Sarcopenia An age-related loss of muscle mass and function occurs in skeletal mu scle of a variety of mammalian species; this process is referred to as sarcopenia, and is reflected by 25% to 35% decreases in the cross-sectional ar ea of several limb muscles due to muscle fiber atrophy and loss (83). The public health ramificatio ns of this large decline are evid ent in the clinical presentation, which includes decreased mobility and respirat ory function. These declines have significant effects on individual health and qua lity of life, affecting the ab ility of elderly people to live independently. In humans, specific skeletal muscles may unde rgo a ~40% decline in muscle mass between the ages of 20 and 80 years (84). What is more, a 25% decrease in cross-sectional area of vastus lateralis (VL) (the most studied muscle in the context of sarcop enia) is consistently seen in comparisons of 70to 75-y ear-olds with 20to 30-year-olds (85, 86 ). Large declines also occur in the number of fibers in the VL. Progressive muscle wasti ng has also been demonstrated in murine and nonhuman primates. These sarcopen ic changes are evidenced by a significant reduction in muscle cross sectional area, muscle mass loss and fiber number loss over time. In the Fischer 344 x Brown Norway (FBN) hybrid rat, the difference between the rectus femoris muscles of 18and 38-month-old animals is stri king. Muscle cross sectio nal area is reduced by

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31 30% in the older animals and the muscle com position is more heterogeneous including an increase in fibrotic tissue (87). A significan t reduction in muscle mass (45%) is observed between 18and 36/38-months of age, as well as, a significant (27%) loss of muscle fibers (87). Another study in male FxBNF1 rats found that atrophy occurs from 9 to 31 months of age in the soleus (13%), EDL (15%), plantaris (22%), and gastrocnemius (25%) (88). In C57BL/6 mice (the same background strain used in th is study) skeletal muscle mass al so decreases with aging with percent atrophy reported ranging from 15% to more than 30% (89). When we compared aged WT C57BL/6 mice (30-mo) to young WT (5-mo old) animals we also found significant muscle atrophy (Fig 4-15). Although the specific characteris tics of sarcopenia in murine depend on the strain, gender, muscle and age groups studied, th e relative magnitude of muscle at rophy in old animals resembles that of old persons (90). We have observed a simila r degree of atrophy in th e skeletal muscle of 11mo-old D257A mice (24% atrophy in gastrocnemius and 19% atr ophy in quadriceps) that very closely resembles sarcopenia during aging (Fig 4-15). Although the molecular events responsible for sarcopenia are unknown, the muscle mass loss is due to both fiber atrophy (84, 91, 92) and fiber loss (84, 93, 94), and proposed mechanisms for fiber loss include mtDNA mutations and altered apoptotic signaling (87). It is also important to point out that skel etal muscle does not possess the high repair capacities that occur in more mitotically active tissues, which makes it more susceptible to age-induced deterioration; altho ugh satellite cells are capable of replacing lost muscle fibers, both the percentage of satellite cells and their prolif erative capacities decrease with aging (95). Therefore, skeletal muscle degenerates w ith aging in both huma ns and murine, and may represent an important target fo r age-related mitochondrial dysfunction.

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32 The consequences on skeletal muscle of mt DNA disruption, ranging fr om point mutations to large deletions, are clear from neuromuscu lar disorders known as mitochondrial myopathies (96). The mtDNA abnormalities associated with these disorders cause ETC dysfunctions in muscle fibers (97). Moreover, mutations aff ecting the mitochondrial genome can increase the susceptibility of cells to apoptos is (98). Several prominent exampl es include Friedreichs Ataxia, a neurodegenerative disease in which mtDNA mutation sensitizes the cells to undergo apoptosis (99, 100) and Leigh syndrome, the most common ne urodegenerative disorder affecting oxidative phosphorylation in children, in wh ich mtDNA mutations also incr ease mitochondrial-mediated apoptosis (101). In the normally aged skeletal mu scle of rats it was clear ly shown that segmental mitochondrial abnormalities coloca lize with mtDNA deletion mutations (7). Importantly, these muscle fibers harboring mitochondrial abnormali ties displayed a striki ng decrease in cross sectional area indicative of at rophy, and fiber splitting, strongly suggesting a causal role for ageassociated mitochondrial DNA deletion mutations and mitochondrial dysfunction in sarcopenia (7). The same group also showed that the vast us lateralis muscle, wh ich undergoes a high degree of sarcopenia, exhibited more ETS abnormalities a nd associated fiber loss than the soleus and adductor longus muscles, which are more resistan t to sarcopenia, suggesti ng a direct association between ETS abnormalities and fiber loss (102) Moreover, Prolla and co-workers have demonstrated that aging of specifi c organs, including skeletal muscle is associated with specific patterns of transcriptional alterations that serve as molecular biomarkers to indicate mitochondrial dysfunction in the aging process (103). There is evidence indicating th at deregulation of apoptosis plays a key role in the pathophysiology of skeletal muscle cell loss. Ind eed, accelerated skeletal muscle apoptosis has been documented to occur with aging (104, 105). Our laboratory has previously shown that in

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33 the gastrocnemius muscle of old Fischer-344 rats, apoptosis is significantly elevated compared to young rats and this also coincided with a significa nt increase in the levels of cleaved caspase-3 (105). These findings also agree with the elevated caspase-3 levels we have detected in the gastrocnemius of aged, 30-mo old WT mice, a nd in the gastrocnemiu s of 11-mo old D257A mice, a time-point when the sarcopenic phenotype is evident in these mice (13) (Fig 4-18). Despite a very likely role of apoptosis in sa rcopenia, there are only sparse reports on the occurrence of skeletal muscle cell apoptosis in humans with normal aging. In 1999, Strasser et al. showed that, in humans, an age-dependent increase in apoptosis of the stri ated muscle fibers of the rhabdosphincter led to a dramatic decrease in the number of striated mu scle cells (106); in a 5-week-old neonate, 87.6% of the rhabdosphincter consisted of striat ed muscle cells, in striking contrast with only 34.2% in a 91-ye ar-old subject. To our knowledge this was the first report on the role of apoptosis in human skeletal muscle atrophy with age. Results from a very recent study in humans indicate that apoptosis appears to be a contributing pathway to skeletal muscle wasting in healthy older adults compared to healthy young adults (107). This was marked by significant increases in TUNEL positive cells stained for DNA fr agmentation (older adults showing an increase of 87% over young adu lts) in the vastus lateralis (107). Several investigations have implicated th e mitochondria as key mediators involved in sarcopenia. Cortopassi and ot hers (100, 108, 109) suggested th at mitochondrial dysfunction could induce the mitochondrial permeability transiti on pore (PTP), the release of cytochrome c and eventually initiation of a poptosis. Furthermore, Fitts et al (110) showed increases in glycolysis and glycogen utilization during cont ractile activity in aged rats, suggesting an increased reliance on energy production from glycol ytic processes possibly as a consequence of an age-related mitochondrial dys function. Importantly, in the wh ite gastrocnemius of Fischer-

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34 344 rats, it was recently demonstr ated that aging significantly increased DNA fragmentation and cleaved caspase-3 content and this also coincided with a 35.4% lower mean fiber crosssectional area in the old sedentary rats versus the young sedentary controls (111). Additionally, proapoptotic Bax was increased in the old rats compared to young, while anti-apoptotic Bcl-2 protein expression declined in both white gastrocnemius and sole us muscles (111), suggesting that mitochondria may be a target for skeletal muscle aging and alte rations in mitochondrial apoptotic regulatory proteins may be responsib le for the observed age-induced muscle fiber atrophy. Moreover, Leeuwenburgh et al., showed that the soleus muscle weight and cross sectional area from 32-mo-old rats were 24% an d 26% lower, respectively, compared to 6-moold animals, and in the old rats there was a six-fold higher incidence of total TUNEL-positive nuclei compared to young rats (112). Interest ingly, Endonuclease G translocation from the mitochondria to the nucleus also occurred in ol d, but not in young animals, implicating the mitochondrial mediated apoptosis in the lo ss of skeletal muscle mass with age. In summary, our review of the literature s uggests that: a) aging is undoubtedly associated with loss of skeletal muscle mass, b) aging is associated with mito chondrial abnormalities in skeletal muscle of several species, including humans, and c) age-asso ciated mtDNA mutations, leading to mitochondrial dysfuncti on, may be important contributors to sarcopenia. Furthermore, mitochondrial-mediated apoptosis appears to be a likely mechanism for sarcopenia, but this still remains to be substantiated. Mitochondrial-Mediated Pathways of Apoptosis. Apoptosis is a cell suicide program that is highly c onserved among species. Under physiological conditions, apoptosis is essential for embryonic development, tissue homeostasis and removal of cells whose persistence would be detrimental to the or ganism (e.g. self-reactive immune cells, neoplastic cells, vi rus-infected cells) (113). Apoptosis is up-regulated in many

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35 tissues with age. Accelerated apoptosis in mito tic tissues during aging, such as liver and white blood cells, is most likely beneficial as it may serve, respectively, to prevent age-associated tumorigenesis and to maintain overall contro l of immunocompetent cells. However, excessive apoptosis in post mitotic cells, su ch as skeletal muscle fibers and cardiomyocytes may lead to a decline in function and the development of pa thological conditions, such as sarcopenia and cardiac dysfunction, since these types of cells have a limited ability to regenerate. Apoptosis is executed via act ivation of specific signaling pathways which are tightly regulated. Hence, particular mo rphological, biochemical, and molecu lar events occur, such as DNA fragmentation, nuclear condens ation, and formation of apoptot ic bodies, which are then engulfed by macrophages or neighboring cells without initiating an inflammatory response (60). Recent evidence has implicated mitochondria as key regulators of apoptosis (114-116). Although other apoptotic pathways exist, this proposal focuses exclusively on the main mitochondrial-mediated pathway, since mtDNA muta tions are expected to affect mitochondrial function and possibly mitochondrial outer membrane permeability (MOMP). MOMP can lead to 1) release of molecules implicat ed in the activation of caspase s that orchestrate downstream events associated with apoptosis, 2) release of molecules involved in caspase-independent cell death, and 3) loss of mitochondrial functions esse ntial for cell survival. The key step in the initiation of mitochondrial-mediated apoptosis is the release of pro-apoptotic proteins from the mitochondrial inter-membrane space into the cytosol. Mitochondrial-mediated apoptosis entails both caspase-dependent and caspase-independent modes (Fig 2-3). Caspases normally exist in an inactivated state, called procaspases, in the cytoplasm but can be activated by dimerization or proteolytic cleavage. Cytochrome c release from the mitochondrial intermembrane space, in the caspase-dependent pathway, is one of the

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36 most intensively studied pathways of apoptosis. Upon receiving a death-inducing signal, there is a disruption of the mitochondrial inner transmembrane potential, wh ich results in the opening of the mitochondrial permeability transition pore (PTP ), that involves components of the outer mitochondrial membrane (VDAC, Bax and Bcl2), inner mitochondrial membrane (ANTadenine nucleotide translocase), and matrix (c yclophilin D). PTP opening results in loss of membrane potential, osmotic swelling of the mitochondrial matrix, r upture of the outer membrane, and the release of cytochrome c and other apoptogenic factors from the intermembrane space (117, 118). A second model proposes that at least some of the pro-apoptotic Bcl-2 proteins (e.g Bax and Bak) are able to form tetrameric outer me mbrane channels that could also mediate the release of apoptogenic factors fr om the inter-membrane space, w ithout the involvement of inner mitochondrial membrane components (119). Cytochrome c in the cytosol combines w ith procaspase-9 and (Apoptotic Protease Activating Factor 1) Apaf-1, which is constituti vely expressed in the cytoplasm, and in the presence of ATP (which is required for the i nduction of apoptosis) forms the apoptosome. This complex cleaves off the pro-enzyme of caspase-9 into the active form. This allows the molecule to change conformation, and bind to another clea ved caspase-9 precursor, forming a homodimer. Caspase-9 is autocatalytic, thus it activates other caspase-9 molecules by cleaving off their Nterminal prodomain. This is known as the "Caspa se Cascade." Caspase-9 also activates caspase3, which is not autocatalytic, by cleavage at the Cterminal side of a spec ific aspartate residue. Activation of the final apoptosis-effector, casp ase-3, which carries out the actual proteolytic events that result in cellular br eakdown will irreversibly commit th e cell to suicide (120) (Fig 23).

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37 Alternatively, mitochondria ca n release apoptosis inducing factor (AIF) and endonuclease G (EndoG), which have been suggested to func tion in a caspase-independent fashion (121-124), as both AIF and EndoG induce apoptotic changes in purified nuclei, even in the presence of caspase inhibitors (125, 126). U pon release from the mitochondria, both mediators translocate to the nucleus and may lead to large-scale DNA fragmentation and peripheral chromatin condensation (Fig 2-3), but not oligonucleosomal DNA laddering. In vitro, AIF appears to be an essential mitochondrial pathway for cell death sin ce caspase inhibitors block only 40-50% of cell death (127). Moreover, genetic analyses in C. elegans indicate that AIF cooperates with EndoG to participate in the regulation of cell death (128), however, it is unclear whether the same occurs in mammalian cells. Recent data also reveal an important contradiction to the idea that AIF functions in a strictly caspase -independent manner (128, 129). Arnoult et al. showed that the mitochondrial release of AIF, occurring in HeLa and Jurkat cell lines treated with general apoptosis inducers, such as stauro sporine or actinomycin D, is suppr essed (or at least delayed) by caspase inhibitors. The authors suggested that AIF would be re leased only after cytochrome c release, subsequent to apoptos ome-mediated caspase activation (129). However, it has not been elucidated whether and how AI F can induce DNA fragmentation si nce it has no reported intrinsic DNAse activity. Since all mammalian nucleated cells have the ability to undergo apoptosis (130), several apoptotic regulatory mechanisms have evolved, some inhibiting mitochondrial release of proapoptotic proteins and others pr eventing caspase activati on in the cytosol (131) One of these, the Bcl-2 family of proteins consists of both pro(Bax) and anti-apopt otic (Bcl-2) proteins that are structurally related, and act to either prev ent or promote the release of cytochrome c in the cytosol (132). It appears that the relative ratios of these proteins influences whether a cell lives or

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38 dies. In the aged rat heart, the Bcl-2/Bax ra tio has been shown to decrease while cytosolic cytochrome c rises, indicating that the heart becomes mo re sensitive to apoptotic stimuli (133). A very recent study showed that in the white gastrocnemius, the Bax/Bcl-2 ratio increased by 98% with age and this increase was associated wi th a dramatic increase in cleaved caspase-3 and in histone-associated DNA fragmentation (111) Furthermore, endogenous inhibitors of apoptosis proteins (IAPs), in itially discovered in baculoviruses, also exist in mammalian cells (134-136). Am ongst them, the X-linked IAP (XIAP) is regarded as the most potent suppr essor of mammalian cell death. At least one explanation for the vers atile suppression of cell death exhibi ted by this protein resides in its ability to bind directly to, and inhibit, caspases in the cytosol (137). Specifically, the BIR2 region of XIAP is a potent and specific inhibitor of caspa se-3, whereas the BIR3 domain is specific for caspase-9 (137). Apoptosis mediated by the mitochondria ap pears to be the most likely pathway responsible for skeletal muscle loss in th e D257A mice, since the observed mitochondrial dysfunction (Figs 4-2, 4-9) can be a trigger for apoptosis. In our sp ecific aim # 3 we investigated the impact of mtDNA mutations in the induction of the main caspase-dependent mitochondrialmediated pathway of apoptosis.

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39 Figure 2-1. Contributions of the mitochondrial and nuclear DNA to protein subunits of the complexes of the ETC. Depicted are the 5 enzymatic complexes of the mitochondrial ETC embedded in the inner mitochondrial membrane. The number of subunits from each complex encoded from mtDNA and nuc lear DNA are also shown. Note that complex II is all nuclear encoded while co mplex IV has the great est contribution of mtDNA-encoded subunits. Adapted from Wallace, 1997. Figure 2-2. The mitochondrial vicious cycle theory. H +Inter membrane space MatrixNADH FADH 2 H + H + H + ADP + P i ADP ATP TRANSLOCATOR CYT C H 2 O 1 / 2 O 2 2 H + H + ATP 1 2e -Q Q 2 3 4 MtDNA Nuclear 7 0 1 3 2 324 91010 A A p p o o p p t t o o s s i i s s E E v v e e n n h h i i g g h h e e r r i i n n R R O O S S p p r r o o d d u u c c t t i i o o n n E E T T C C a a c c t t i i v v i i t t y y M M i i t t o o d d y y s s f f u u n n c c t t i i o o n n m m t t D D N N A A m m u u t t a a t t i i o o n n s s m m t t D D N N A A d d a a m m a a g g e e O O x x i i d d a a t t i i v v e e s s t t r r e e s s s s m m t t D D N N A A

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40 Figure 2-3. Mitochondrial-mediated apoptosis Scheme was adapted from Cell Signaling Technology Inc. and modified. 2 2 1 1 P P P T T T P P P

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41 CHAPTER 3 MATERIALS AND METHODS Experimental Design In recent published results we performed experiments at different time points of the D257A animals lifespan in order to determine the point at which the D257A phenotype deviates significantly from the WT phenotype (13). For post mitotic tissues, such as skeletal muscle and heart, we detected a phenotype that resembles nor mal aging at ~ 9-10 mo of age in D257A mice, while at 3-mo of age there was no difference in phenotype between WT and D257A for the parameters measured, which indicates that the phenotype is age-related and is not due to developmental defects. To ensure the selection of an ideal time-point for the present dissertation project, in our pilot study we performed the sa me experiments at two different time points: 3 months and 11 months, and our re sults corroborated our previous findings regarding the change in the phenotype. Indeed, we did not detect a ny differences in skeletal muscle mass, oxygen consumption, ROS production, free radical leak or caspase-3 levels, between WT and D257A mice at 3 months of age (Appendix-a, Figs A-1, A-2, A-3, A-4). Therefore, in our design, we selected a time point between 11-13-mo old for our experiments in order to ensure that we will get the age-related phenotype. Fo r all three specific aims the e xperimental groups are identical. We used and compared two groups: 11-13 mo-old WT (n = 11) versus 11-13 mo-old D257A (n = 11) mice (Fig 3-1). General Procedures Animals C57BL/6 strain WT and D257A male and female mice were obtained at ~11 months of age from University of Wisconsin, Madison, from th e lab of our collaborato r Dr. Tom Prolla. The animals were housed in quarantines, in the anim al care facility located at the Progress Park

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42 (Specific-pathogen free and accredited facility). The fac ility is climatea nd light-controlled. After one week of acclimation in the facility the animals were sacrificed by rapid cervical dislocation followed by extraction of the gastro cnemious and quadriceps muscles and immediate isolation of mitochondria for the measurements of mitochondrial resp iratory and functional parameters. Cervical dislocation was chosen in or der to avoid the influenc e of other anesthetics (e.g. volatile gases) on some of the paramete rs to be measured, primarily mitochondrial functional parameters (basal m itochondrial respiration, and RO S production) (138, 139). Four animals a day were sacrificed. The number of animals used per group is n 11 (Fig 3-1). This number was determined via a power analysis based on detecting a 50% difference, using previous data from our laboratory. The power was set at 0.90 and al pha level at p<0.05. Mitochondrial and Cytosolic Isolation Mitochondrial and cytosolic protein frac tions were isolated using differential centrifugation. Immediately after sacrifice, skeletal muscle (both gastrocnemious and quadriceps muscles were mixed) was removed, cleaned, and weighed. Skeletal muscle was finely minced into small pieces and homogenized in (1:5 wt/v ol) ice-cold isolation buffer containing 0.21 M mannitol, 0.07 M sucrose, 0.005 Hepes, 0.001 M EDTA 0.2% fatty acid free BSA, pH 7.4, using a Potter-Elvehjem glass homogenizer. The homogenate was centrifuged at 1,000 g for 10 min. After the first spin, the pellets containing nuclei we re frozen for future analysis. The supernatant was then centrifuged at 14,000 g for 20 min. The supe rnatant (crude cytosol) was stored at -80 C and the mitochondrial pellet wa s re-suspended in isolation buffer without BSA and was centrifuged again at 14,000 g for 10 min. The final mitochondrial pellet was re-suspended in 350 l of isolation buffer without BSA, and was used immediately for the measurements of mitochondrial H2O2 production, oxygen consumption and ATP content and production. All

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43 centrifugation steps were carried out at 4C. Protein concentrati ons were determined using the Bradford method (140). Specific Methods Specific Aim 1. Effect of mtDNA Mutations on Skeletal Muscle Mitochondrial Function In this specific aim we investigated the impact of the accumulation of mtDNA mutations on mitochondrial bioenerge tics, specifically, on O2 consumption, ATP content and production, and ROS generation. Using specific complex i nhibitors we also determined maximum ROS production at the main ROS-generating sites with in the ETC, complex I and complex III. In our preliminary studies we had measured mitoc hondrial oxygen consumption and our results match our hypothesis: D257A mitochondria show co mpromised respiration during state 3, and uncoupling between oxidation and phosphoryl ation (Fig 4-2). Interestingly, H2O2 production and free radical leak from mutant m itochondria was lower compared to that of WT (Fig 4-11, 4-12). As previously mentioned, this finding doesnt support the mitochondrial vicious cycle theory of aging, suggesting that mtDNA mutations ma y lead to mitochondrial dysfunction without increases in ROS production. In this aim we are providing additional evid ence that oxidative stress levels are not elevated in response to high mt DNA mutational rate, by demonstra ting that 8-oxodGuo levels in skeletal muscle mtDNA were not significantly different between WT and D257A mice (Fig 414). In this aim we further determined whethe r the mitochondrial dysfunction we have observed in the mutant mice leads to loss of membrane potential thus making the mutant mitochondria more susceptible to apoptosis. We examined the possible mechanism by which this dysfunction is induced by assessing the cont ent and activity of ETC respirator y complexes I, III, IV and F1F0 ATPase, which contain subunits encoded by the mitochondrial genome, and compare them to complex II which is all nuclear-encoded.

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44 Rationale Part of the focus in this aim was to determine whether mtDNA mutations induce mitochondrial dysfunction. We have evaluated se veral mitochondrial func tional parameters and we determined that indeed mitochondrial dysfunction is evident by the reduction in mitochondrial respiration at stat e 3, and the higher degree of unc oupling we have observed in the D257A mice. As previously mentioned, D257A mice accu mulate mutations due to the mutated exonuclease domain of POL which is devoid of proof-readi ng activity. We expected that mutated gene-encoding areas of the mitochondrial genome will have a direct effect on protein transcription and/or translati on. Therefore, we anticipated th at the proteins encoded by the mitochondrial genome will be directly affected. Depending on the type of mutations introduced and the location at which they occur, the pr oteins encoded by mtDNA may be truncated, or completely absent, may partially or totally lo se activity. We hypothesize d that the end result would be that the content and/or activity of the proteins encoded by mtDNA would be compromised in the mutant mice. Since the prot eins encoded by the mitochondria (total of 13 proteins) are all protei ns of the electron transport chain (ETC), we expected that the mtDNA mutations would have a direct impact on mitochondrial ETC activity and hence, on mitochondrial function. Differently stated, we hy pothesized that the redu ction in the content and/or activity of ETC proteins would be the primary mechanism of the observed mitochondrial dysfunction associated with mtDNA mutations. We evaluated the total concentration and maximum activity of the respiratory complexes I, III, IV and F1F0 ATPase. These are the ETC complexes that contain subunits encoded by mtDNA. We also evaluated the content of selected individual subunits from these complexes that are either nuclearor mitochondrial-encoded.

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45 These measures helped us understand and expl ain how the mitochondrial dysfunction we have observed in the D257A mice is induced. Furthermore, the measurement of mitochondrial me mbrane potential was critical to help us understand why mutant mitochondria produce le ss ROS (evident by the decrease in H2O2 generation), and explain the induc tion of apoptosis in the muta nt mice. Importantly, loss of membrane potential represents a link between mitochondrial dysfunction and apoptosis and hence, a link between aim #1 and aims #2, #3 (see also Fig 3-1). Experimental approach Mitochondrial H2O2 generation The rate of mitochondrial H2O2 production was assayed in fre shly isolated mitochondria by a highly sensitive fluorometric method accordi ng to Barja (141), and adapted to a microplate reader. H2O2 generation was monitored by measur ing the increase in fluorescence (excitation at 312 nm, emission at 420 nm) due to the oxi dation of homova nillic acid by H2O2 in the presence of horseradish peroxidase like it is shown in the reaction below: The assay was performed in incubation buffer (145-mM KCl, 30-m Hepes, 5-mM KH2PO4, 3-mM MgCl2, 0.1-mM EGTA 0.1% BSA, pH 7.4) at 37 C, and the reaction conditions were: 0.25 mg of mito chondrial protein per mL, 6 U/mL of horseradish peroxidase, 0.1-mM homovanillic acid and 50-U/mL of su peroxide dismutase (SOD). The reaction was started by the addition of 2.5 mM pyruvate/2.5 mM malate or 5 mM succinate as substrates.

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46 Pyruvate/malate was used to study complex I RO S production, and succinate was used to study complex III ROS production (for details also se e Barja, 1999) (142). We also used inhibitors of the ETC in order to stud y maximum rates of H2O2 production from complexe s I and III, since they represent the main sites of ROS generation (esp ecially complex I) within the mitochondria. For complex I maximum rate we used 2 M rotenone added to pyr uvate/malate supplemented mitochondria. For complex II I maximum rate we used 2 M antimycin A plus 2 M rotenone, added to succinate supple mented mitochondria. In addition, some of the as says with succinate as substrate were performed in the presence of 2 M rotenone alone, in order to avoid the backwards flow of electrons to Complex I. After 15 min of incubation at 37 C, the reaction was stopped and the samples were transferred on ice and a stop solution (0.1-M glycine, 25-mM EDTA, pH 12) was added. Known amounts of H2O2 generated in parallel by glucose oxidase, with glucose as substrate, were used as standards. Sin ce the SOD added in ex cess converts all the O2 produced (if any) to H2O2, the measurement represents the total (O2 plus H2O2) rate of mitochondrial ROS production. All samples were run in duplicate. H2O2 production and O2 consumption were measured in parallel in the same muscle mitochondria under similar experimental conditions. This allowed the calculation of the fraction of electr ons out of sequence which reduce O2 to ROS at the respiratory chain (the percent free radical leak or FRL%) instead of reaching cytochrome oxidase to reduce O2 to water. Since two electrons are needed to reduce 1 mole of O2 to H2O2 whereas four electrons are transferred in the re duction of 1 mole of O2 to water, the percent free radical leak can be calculated as the rate of H2O2 production divided by two times the rate of O2 consumption, and the result is multiplied by 100.

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47 Mitochondrial respiration Mitochondrial oxygen consumption was measur ed at 37C by polarography, with a Clarktype oxygen electrode (Oxytherm Hansatech, Norfolk, UK) under the same conditions used (same mitochondria, buffer composition and substrate concentrations) for H2O2 production measurements: incubation buffer (145 mM KCl, 30 mM Hepes, 5 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, pH 7.4) with 0.25 mg of mitoc hondrial protein per ml and 2.5 mM pyruvate/2.5 mM malate as substrates. The a ssay was performed in the absence (State 4-resting state) and in the presence (State 3-ph osphorylating state) of 500 M ADP. Clark-type elec trode without (State 4) and with (State 3) saturant ADP allows ca lculation of the respirat ory control ratio (RCR) (State 3/State 4 oxygen consumption) as an indi cator of the degree of coupling and metabolic activity of the mitoc hondrial preparations. ATP content and production Mitochondria isolated from skeletal muscle were used immediately after isolation to determine mitochondrial ATP content and produc tion, following the method of Drew (143). This bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. Upon addi tion, ATP combines with luciferi n to form luciferyl adenylate and inorganic pyrophosphate (PPi) on the surface of the luciferase enzyme as shown in reaction 1 below: Reaction 1 Luciferin + ATP Luciferase Luciferyl Adenylate + PPi While bound to the enzyme, luciferyl adenylate combines with O2 to form oxyluciferin and AMP through a series of enzymatic redox reactions. As oxyluciferin and AMP are released from the enzymes surface, a quantum yield of light is emitted in proportion to the ATP concentration as shown in reaction 2 : Reaction 2 Luciferyl Adenylate + O2 Oxyluciferin + AMP + hv

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48 The light emission ( h v) can then be recorded and quantified using a chemiluminometer. ATP content methodology was modi fied from a method of Mol ecular Probes (A-22066, Eugene, OR). Chemicals used are D -luciferin, luciferase (40 L of a 5 mg/mL solution in 25 mM Trisacetase, pH 7.8, 0.2 M ammonium sulfate, 15% (v/v ) glycerol and 30% (v/v) ethylene glycol), dithiothreitol (DTT), adenosine 5 -triphosphate (ATP), and a Reac tion Buffer (10 mL of 500 mM Tricine buffer, pH 7.8, 100 mM MgSO4, 2mM EDTA and 2 mM sodium azide). The reagents and reaction mixture were combined according to the protocol by Molecular Probes. In order to determine ATP content, freshly isolated mito chondria were added to a cuvette containing reaction buffer, D -luciferin, luciferase and DTT. In addition, 2.5 mM pyruvate and 2.5 mM malate were added to the reaction mixture, as substrates for oxidative phosphorylation. Immediately after the ATP content measurem ents, 2.5 mM ADP was added to the cuvette containing the reaction mixture and mitochondria in order to determine the rate of ATP production. A blank cuvette contai ning no sample, only reaction mixture, was assayed to account for background luminescence, and known concen trations of ATP standards were used to establish a standard curve. The values for ATP c ontent and rate of production were normalized to total mitochondrial protein concen tration. All mitochondrial sample s were assayed in duplicate, and an average of these results was used to calc ulate final ATP content and rate of production. Mitochondrial membrane potential Mitochondrial membrane potential changes in isolated skeletal muscle mitochondria were followed qualitatively by monitoring the fluor escence of tetramethyl rhodamine methyl ester (TMRM, Molecular Probes, Eugene, OR), a catio nic lipid-soluble probe that accumulates in energized mitochondria. The method of Scaduto (144) was followed without modification. Briefly, mitochondria (0.25 mg/ml) were incubated at 37 C in a medium composed of 135 mM

PAGE 49

49 KCl, 20 mM MOPS, 5 mM K2HPO4, and 5 mM MgCl2 at pH 7.00. The experiment was initiated by the addition of mitochondria to th e medium, also containing 0.33 mM TMRM and either 5mM succinate or 5mM glutamate + 2.5mM ma late in order to record membrane potential during state 4. Fluorescence at 546 and 573 nm excitation was monitored using an emission wavelength of 590 nm. This was followed by the addition of ADP (0.17 mM) to record membrane potential during state 3. Addition of 0. 5 mM CCCP followed to serve as a control for TMRM binding. An increase in fluorescence repres ents de-quenching of TMRM when the probe is released into the medium upon mitochondrial depolarization. Blue native page (BN-page) for determinat ion of content and enzymatic activity of respiratory complexes For determination of the content of the ETC complexes we followed the protocol as described by Schagger et al. with some modifica tion (145). Skeletal muscle was homogenized in buffer 1 containing 20 mM MOPS, 440 mM sacch arose, 1 mM EDTA and 0.5 mM PMSF, pH 7.2 at 4C. The homogenates were centrifuge d at 20,000 g for 20 min. The pellet was resuspended in 80 l of buffer containing 1 M aminocapro ic acid, 50 mM Bis-tris and 0.5 mM PMSF, pH 7.0. The membranes were then solubilized by the addiction of 30 l ndodecylmaltoside (10 %, prepared fresh). Mitoch ondrial suspensions were incubated on ice for 30 minutes with vortex mixing every 5 min, followed by ultracentrifugation for 25 min at 100,000 g (Beckman, Optima LE-80K). The supernat ant, containing all the solubilized mitochondrial membrane proteins was used for the BN-page. 7 l of 5% w/v coomassie brilliant blue G-250 in aminocaproic acid (1M) were added to 100 l of supernatant. Samples were then stored on ice for no more than 30 min prior to gel loading. For electrophoresis, a 3-12 % gradient gel with 4% of stacker was used. The anode buffer was comprised of 50 mM Bis-Tris, pH 7.0. The cathode buffer was comprised of 50 mM tricin e, 15 mM Bis tris, and coomassie brilliant

PAGE 50

50 blue G-250 (0.02% w/v), pH 7.0. Samples were electrophoresed at 90 V for 20 min, and thereafter at 170 V for 2 h, at 4C. Immediately after electrophor esis gels were incubated in coomassie brilliant blue G-250 solution (0.1% co omassie in 10% acetic acid and 40% methanol) for 1h, followed by incubation in de-staining so lution (10% acetic acid, 40% methanol) for 2h. After de-staining gels were photographed and an alyzed using the Alpha Innotech FluorChem SP imaging system. Densitometry values were normalized to total protein loaded per well, measured by the Bradford assay. For determination of enzymatic activity, enzyma tic colorimetric reactions were performed on the BN-PAGE. Gels were incubated overnight at room temperature with the following solutions: Complex I: 2mM TrisHCl, pH 7.4, 0.1 mg/ml NADH, and 2.5 mg/ml NTB (nitrotetrazolium blue). Complex II: 4.5 mM EDTA, 10mM KCN, 0.2mM phenazine methasulfate, 84mM succinic acid and 50mM NTB in 1.5mM phosphate buffer, pH 7.4. Complex IV: 5 mg 3:30-Diamidobenzidine te trahydrochloride (DAB) dissolved in 9ml phosphate buffer (0.05 M, pH 7.4), 1ml catalase (20 g/ml), 10 mg cytochrome c, and 750 mg sucrose. Complex V: 35mM Tris, 270mM glycine, 14mM MgSO4, 0.2% Pb(NO3)2, and 8mM ATP, pH 7.8. Gels were then washed in distilled water and p hotographed immediately. Densitometry values for activity were normalized to respective content densitometry values. Determination of protein content of selected mitochondrialand nuc lear-encoded subunits from ETC complexes I, II, III and IV Skeletal muscle tissue was immersed and ri nsed in cold homogenization buffer: 50mN Tris-HCl pH 7.4, 1% Tween 20 (Amersham Biosciences), 0.25% sodium deoxycholate, 150 mM NaCl, 1mM disodium ethylenediaminet etraacetate dehydrate (EDTA), 1mM Diethylenetriaminepenta-acetic acid (DTPAC), 1 M 2,6-di-tert-butyl-4-methylphenol (BHT), and 1.5% Protease Inhibitor Mix (Amersham Biosciences). This was followed by homogenization in

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51 25 ml homogenization buffer/g of skeletal muscle with a Potter-Elvehjem type homogenizer system (Glas-Col, Terre Haute, IN). The homoge nate was then centrifuged at 500 x g for 5 min at 4C yielding a pellet corresponding to crude nuclear fraction. Prot ein concentration was determined by the Bradford method using BioRad reagent and BSA as standard. Homogenates were immediately frozen at C until further analysis. The protein content of skeletal muscle mitochondrial respiratory chain complexes wa s estimated using western blot analysis. Immunodetection was performed using specific antibodies for the 39KDa (NDUF A9) and 30KDa (NDUFS3) subunit of complex I (1 :1000 and 1:1000, re spectively), 70KDa su bunit (Flavoprotein) of complex II (1:500), 48KDa (CORE 2) and 29 KDa (Rieske iron-sulfur protein) subunits of complex III (1:1000 and 1:1000, re spectively), and COXI subunit of complex IV (1:1000) (ref. A21344, A21343, A1114 2, A11143, A21346 and A6403, respectively; Molecular Probes). An antibody to porin (1 :5000, A31855, Mo lecular Probes) or beta-actin (1:5000, AB20272, Abcam), as a loading control for tota l mitochondrial mass or total protein content, was also used. Appropriate peroxidase-coupled secondary an tibodies and chemiluminescence HRP substrate (Millipore, MA, USA) were used for primary antibody detection. Signal quantification and recording was performed with a ChemiDoc equipmen t (Bio-Rad Laboratories, Inc., Barcelona, Spain). Protein concentration was determined by the Bradfo rd method. Data were expressed as Arbitrary Units. Determination of mitochondrial protein yield In order to determine mitochondrial yield, we first determined the total protein concentration in each mitochondrial extract by the Bradford assay. We then multiplied each concentration value by the total volume of each mitochondrial extract. Last, we divided this product by the skeletal muscle we ight used each time to obtai n the respective mitochondrial extract. In this way we were able to norma lize and express the total mitochondrial content per gram of skeletal muscle tissue.

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52 Determination of MnSOD and Cata lase mRNA expression by RT-PCR To extract RNA skeletal muscles (1/10 weight /reagent volume) were homogenized in 1mL of Trizol reagent and the Tr izol protocol for RNA isola tion was followed. Briefly, after homogenization samples were centrifuged at 12,000 x g for 10 min in order to remove insoluble material. To the clear supernatant, 0.2mL of chloroform was added and the supernatant was centrifuged at 12,000 x g for 15 min. This separate s the mixture into 3 phases: a red organic phase, an interphase and a colorless upper aque ous phase containing RNA. To the aqueous phase 0.6 mL of isopropanol was added and the mixt ure was centrifuged again at 12,000 x g for 10 min. The precipitated RNA was washed with 75% ethanol, centrifuged again at 7,500 x g for 5 min and the resulting RNA pellets were dried for 5-10 minutes under a vacuum. 1 g of isolated RNA was reverse transcribed (Eppen dorf RT plus PCR kit) using o ligo (dT) primer, as described by the manufacturer's instructions. PCR was performed on 3l aliquots from each cDNA reaction, using primer sets for detecting MnSOD (5 -GGTGGCCTTGAGCGGGGACTTG-3 5 GGTGGGTGGGGAGGTAGGGAGGAT-3 sense and antisense, respectively) and Catalase (5 ATGGCCTCCGAGATCTTTTCAATG-3 5 -GAGCGCGGTAGGGACAGTTCAC-3 sense and antisense, respectively). The sizes of th e amplification products were 611 bp for MnSOD and 366 bp for Catalase. Conditions for PCR reactions were for MnSOD: 94 C for 30 sec, 58 C for 30 sec, and 72 C for 30 sec and for Catalase: 94 C for 30 sec, 57.7 C for 30 sec, and 72 C for 30 sec. PCR amplification was conducted for 29 cycles for both MnSOD and Catalase. RTPCR products were analyzed by agarose gel electr ophoresis and digital imaging of the ethidium bromide-stained gel, using the Alpha Innotech FluorChem SP imaging system. Oxidative damage to mtDNA Mitochondrial DNA oxidation was measured according to Sanz et al.(146), with modification. Briefly, mitochondrial DNA, free of nDNA, were isolated by the method of

PAGE 53

53 Latorre et al. (1986), adapted to mammals (As uncion et al., 1996). After isolation mtDNA was completely dissolved in 85 L of 30 M DFOM, DNA was digested wi th 4 U of Nuclease P1 (dissolved in 300 mM sodium acetate, 0.2 mM Zn Cl2, pH 5.3), and 5 U of alkaline phosphatase during 60 min at 50C. After filtering, samples we re put into an autosampler vial for HPLC-ECUV analysis. 8-oxodG and dG were measured by HPLC with online electrochemical and ultraviolet detection respectively. For analysis, the nucleoside mixture was injected into two Delta-Pak (150x3.9mm id, 5 m) C-18 reversed-phase columns (Waters, Milford, MA). 8oxodG was detected with an electrochemical de tector (Coulochem III, ESA Inc, Chelmsford, MA, USA) with a PEEK filter protected 5011A an alytical cell (ESA, 5 nA, screen electrode: +205 mV analytical electrode: +275), and dG was measured with a Spectra SYSTEM UV1000 detector (Thermo Electron Corp., San Jose, CA USA) set at 290 nm. Chromatograms were recorded using EZChrome Elite (Scientific So ftware INC., Pleasant on, CA, USA). Calibration curves for dG and 8-oxodG were constructed by in jection of each standard 3-4 times. The HPLC buffer consisted of 9% v/v methanol and 50 mM sodium acetate, set to pH 5.3, with acetic acid filtered through a CN 0.2 m filter from Nalgene Nunc (Rochester, NY, USA). Specific Aim 2. mtDNA Mutations and Apoptosis in Skeletal Muscle In out pilot study we had already measured cleaved (activated) cas pase-3 content in skeletal muscle of 3-mo and 11-mo old WT and D257A mice. Proteolytic activation of caspase-3 is a key event in the execution of apoptosis, marking the point at which the cell is committed to die. We have shown significant elevation in cleav ed caspase-3 levels by 11-mo of age in skeletal muscle of D257A mice compared to WT, which was not evident in D257A mice at 3-mo-of age, a time-point when the D257A phenotype is also not evident (Figs 4-18, A-2). We also have found that 30-mo-old WT mice ha ve significant muscle atrophy in concert with elevated

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54 caspase-3 levels compared to young, 5-mo-old mice (Figs 4-15, 4-18) (13). Therefore, the elevation in cleaved caspase-3 le vels, coupled to a significant sk eletal muscle loss at 11-mo of age (Figs 4-15, 4-18), suggests th at D257A muscle becomes sarcopenic (13). Together, these findings suggest that caspase-3-mediated apoptosi s may be one of the main pathways responsible for the decline in skeletal muscle loss associat ed with mtDNA mutations in the accelerated aging D257A mice and the same m echanism may also be responsible during normal aging. Rationale Although we showed increased leve ls of one marker of apopto sis (caspase-3), additional measures are needed in order to corroborate th at apoptosis is indeed a central mechanism responsible for skeletal muscle loss in the D257A mice. Therefore, in this aim, we wanted to further investigate apoptosis in skeletal mu scle, by conducting specific apoptotic measures. Besides the content of cleaved caspase-3 we fu rther evaluated caspase-3 activity. Furthermore, we used a quantitative ELISA to measure the am ount of monoand oligo-nucleosomes released in the cytosol after apoptotic DNA fragmentation. Last, we isolated DNA from skeletal muscle, performed a DNA laddering-specific ligation PCR in order to amp lify apoptotic fragments, and subjected PCR products to electrophoresis through agarose gel in order to detect oligonucleosomal fragmentation evident by the fo rmation of specific ladders of ~180-200 bps or multiples in the gel. Experimental approach Determination of cleaved caspase-3 content The active form of caspase-3, cleaved caspa se-3, was quantified by Western blotting. Activation of caspase-3 requires proteolytic proc essing of its inactive zymogen into activated fragments. The specific antibody used detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resu lting from cleavage adjacent to Asp175. Proteins were separated

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55 using 15% PAGEr Gold pre-cast Tris-glycine gels (Cambrex, USA) under denaturing conditions, and then transferred to PVDF membranes (0.2 m, Trans-Blot Transfer Medium, Bio-Rad Laboratories, CA USA) Protein concentration was determined using the Bradford assay, and samples were subsequently normalized so that the protein content among samples is identical. Subsequently, 20 l of sample were loaded to each well. HL-60 cells induced with etoposide were also loaded in a well as an appr opriate positive control. Membranes were blocked for 1.5 hrs using a blocking solution containi ng TBS and 5% milk. Membranes were then incubated overnight in the 5% blocking solu tion containing the monoclonal primary antibody caspase-3 (Cell Signaling, Beverly, MA, USA) in a dilution of 1:500. The following day membranes were incubated for 1 h at room temp erature with IgG horsera dish peroxidase-linked whole secondary antibody (1:1000, Amersham Biosciences UK Ltd, Amersham, UK). Specific protein bands were visualized using ECL reagent (Amersham, UK) The resulting Western blots were analyzed using the Alpha Innotech FluorChem SP imaging system. Specific protein bands were further normalized to b-actin bands. Valu es were expressed as arbitrary units after normalizing and expressing samples as % of a control sample that was included in all membranes. Enzymatic measurement of caspase 3 activity Caspase-3 activity was measured using a fl uorometric protease assay kit: (Caspase3/CPP32, Biovision, Mountain View, CA, USA) according to manufacturers instructions. Briefly, the assay is based on detection of cl eavage of the substrate DEVD-AFC (AFC: 7-amino4-trifluoromethyl coumarin) by caspas e-3. DEVD-AFC emits blue light ( max = 400 nm); upon cleavage of the substrate by caspase-3, fr ee AFC emits a yellow-green fluorescence ( max = 505

PAGE 56

56 nm), which can be quantified using a fluorescen ce microplate reader. Samples were run in triplicate and values were expressed as raw fluorescence units per mg of cytosolic protein. Determination of cytosolic monoand oligonucleosomes Endogenous endonucleases activated during a poptosis cleave double-stranded DNA in the linker region between nucleosomes to generate monoand oligonucleosomes of ~180 bp or multiples. Apoptotic DNA fragmentation was quan tified in skeletal muscle by measuring the amount of cytosolic monoand oligonucleosomes using a Cell Death detection ELISA (Roche Molecular Biochemicals, Germany). The assay is based on the quantit ative sandwich-enzymeimmunoassay-principle. Briefl y, wells were coated with a monoclonal anti-histone antibody. Nucleosomes in the sample bound to the an tibody followed by the addition of anti-DNAperoxidase, which reacted with the DNA associated with the histones. The amount of peroxidase retained in the immunocomplex was determined spectrophotometrically w ith ABTS (2.2-azinodi-[3-ethylbenzthiazoline sulfonate ]) as a substrate. All samples were run in triplicate and the means were expressed as arbitrary OD units normalized to milligrams of cytosolic protein, with sample protein concentrations determined by the Bradford method. DNA laddering To enable detection of nucleosomal ladders in apoptotic cells, the DNA ladder assay was performed. Skeletal muscle was homogenized in 1 mL DNAzol (Molecular Research Center Inc., Cincinnati, OH). Proteina se K (Qiagen, Valencia, CA) was added to the homogenates, which, after a 3 h incubation period, were centr ifuged (10,000 g for 10 min at 4C) and the supernatants were precipitated and washed with 100% and 75% ethanol, respectively. After digestion with RNase A, DNA samples were s ubjected to a DNA ladder-specific ligation PCR, following the manufacturers prot ocol (Maxim Biotech, CA). Brie fly, isolated DNA is subjected to an overnight ligation r eaction using de-phosphorylated adaptors (12-mer: 5-

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57 AGTCGACACGTG-3, 27-mer: 5-GACGTCG ACGTCGTACACGTGTCGACT-3) that are ligated to the ends of DNA fragments generate d during apoptosis, using T4 DNA ligase. In mammalian cells, such fragments generally have 5-phosphorylated blunt ends and 3-OH ends, thus only the 27-mer is ligated to the DNA fragments. When the mixture is heated to 55 C, the 12-mer is released. Next, the 5 protruding ends of the molecules are filled by Taq polymerase. The 27-mer then serves as a primer for PCR in which the fragments with adaptors on both ends are amplified. Conditions for PCR reactions were 72 C for 10 min, 94 C for 1min, 94 C for 1 min, and 70 C for 2 min. PCR amplification was conducted for 30 cycles. PCR products were electrophoresed through 1% ag arose gels containing 0.5 g/mL ethidium bromide at 80 V for 1 h, and were examined under UV light for the pres ence of apoptosis-specific nucleosomal ladders. Specific Aim 3. Identification of the Specific Apoptotic Sign aling Pathway Responsible for Skeletal Muscle Loss in D257A Mice In this aim we hypothesized that mitochondr ial dysfunction in D257A mutator mice would lead to mitochondrial outer membrane pe rmeability and leakage of cytochrome c and other proapoptotic factors into th e cytosol. Cytochrome c release from the mitochondria may subsequently activate the caspase-dependent m itochondrial-mediated pathway of apoptosis, leading to the activation of caspase-9 and downstream cleavage and activation of caspase-3 that we have observed in these mice (Figs 4-18, 4-20). Rationale The demonstration that the effector caspase-3 is cl eaved and thus, activated, in D257A mice does not provide proof of activation of a mitochondrial-mediated pathway of apoptosis, since other apoptotic pathways, such as, recept or-mediated (extrinsic pathway), and ER-stressmediated pathways may also lead to the activat ion of the final effect or caspase-3. Since the accumulation of mtDNA mutations was expect ed to cause changes in mitochondrial

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58 bioenergetics, ultimately leading to mitochondr ial dysfunction, we hypothesized that the pathway of apoptosis would be intrinsic to the mito chondria. The D257A mouse model allowed us to elucidate the relevant mitochondr ial pro-apoptotic proteins that are activated in response to mtDNA mutations. Apoptosis originating from these pathwa ys has been strongly implicated to be causal in the aging process and is also highly relevant to ma ny clinical conditions in humans that are associated w ith mtDNA mutations (147). Since the specific functions of AIF and EndoG (caspase-independent pathway) in mitochondrial function, as well as, in apoptosis-initiated by the mitochondria remain to be substantiated, we evaluated the levels of key regulators of the main caspase-dependent, mitochondrial-mediated pathway of apoptosis: Cytochrome c and the initiator caspase-9. We further correlated caspase9 activity levels with caspase-3 activity levels (Fig 4-21) in order to demons trate that activation of caspase -9 indeed leads to downstream activation of caspase-3 and apoptosis. Experimental approach Determination of cytochrome c content by Western Blotting For quantification of cytochrome c content by Western blot analys is, proteins were separated using 15% PAGEr Gold pre-cast Tris-glycine gels (Cambrex, USA) under denaturing conditions, and then transferre d to PVDF membranes (0.2 m, Trans-Blot Transfer Medium, Bio-Rad Laboratories, CA USA). Prot ein concentration was determined using the Bradford assay, and was subsequently normalized so that the protein content among samples is identical. Subsequently, 20 l of sample were loaded to each well. 5 l of purified human heart mitochondria were also loaded in a well as an appropriate positive co ntrol. Membranes were blocked for 1.5 hrs using a blocking solution containing TBS and 5% milk. Membranes were then incubated overnight in the 5% blocking solution containing the cytochrome c monoclonal

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59 primary antibody at a dilution of 1:1000. The fo llowing day membranes were incubated for 1 h at room temperature with IgG horseradish pe roxidase-linked whole secondary antibody (1:1000, Amersham Biosciences UK Ltd, Amersham, UK). Sp ecific protein bands were visualized using ECL plus reagent (Amersham Pharmacia Biot ech, UK). The resulting Western blots were analyzed using the Alpha Innotech FluorChem SP imaging system. Specific protein bands were further normalized to tubulin. Values were expr essed as arbitrary units after normalizing and expressing samples as % of a control sample that was included in all membranes. Enzymatic measurement of caspase-9 activity Caspase -9 activity was measured using a fl uorometric protease assay kit: (Caspase9/Mch6, Biovision, Mountain View, CA, USA) according to manufacturers instructions. Briefly, the assay is based on detection of cl eavage of the substrate LEHD-AFC (AFC: 7-amino4-trifluoromethyl Coumar in) by caspase-9. LEHD-AFC emits blue light ( max = 400 nm); upon cleavage of the substrate by caspase-9, fr ee AFC emits a yellow-green fluorescence ( max = 505 nm), which can be quantified using a fluorescence microtiter plate reader. Samples were run in triplicate and values were expressed as raw fluorescence units per mg of cytosolic protein. Statistical Analyses All results are expressed as means SEM and the means obtained were used for independent t tests. Statistical analyses were ca rried out using the Graph-Pad Prism 4.0 statistical analysis program (San Diego, CA, USA). Statistical significance was set at P<0.05.

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60 Figure 3-1. Experimental design and summary of the parameters measur ed in specific aims 1, 2 and 3. Mitochondrial pathway of apoptosis Caspase-dependent C C C y y y t t t o o o c c c h h h r r r o o o m m m e e e c c c c c c o o o n n n t t t e e e n n n t t t C C C a a a s s s p p p a a a s s s e e e 9 9 9 aa a c c c t t t i i i v v v i i i t t t y y y Mitochondrial ROS production/ MtDNA oxidative damage T T T o o o t t t a a a l l l H H H2 2 2O O O2 2 2 p p p r r r o o o d d d u u u c c c t t t i i i o o o n n n C C C o o o m m m p p p l l l e e e x x x I I I H H H2 2 2O O O2 2 2 C C C o o o m m m p p p l l l e e e x x x I I I I I I I I I H H H2 2 2O O O2 2 2 8 8 8 o o o x x x o o o d d d G G G 1 1 1 1 1 1 3 3 m m o o o o l l d d W W T T n n = = 1 1 1 1 Apoptosis measures C C C l l l e e e a a a v v v e e e d d d C C C a a a s s s p p p a a a s s s e e e 3 3 3 C C C a a a s s s p p p a a a s s s e e e 3 3 3 a a a c c c t t t i i i v vv i i i t t t y y y D D D N N N A A A f f f r r r a a a g g g m m m e e e n n n t t t a a a t t t i i i o o o n n n D D D N N N A A A l l l a a a d d d d d d e e e rr r i i i n n n g g g Mitochondrial functional parameters S S S t t t a a a t t t e e e 3 3 3 O O O2 2 2 c c c o o o n n n s s s u u u m m m p p p t t t i i i o o o n n n S S S t t t a a a t t t e e e 4 4 4 O O O2 2 2 c c c o o o n n n s s s u u u m m m p p p t t t i i i o o o n n n A A A T T T P P P c c c o o o n n n t t t e e e n n n t t t A A A T T T P P P p p p r r r o o o d d d u uu c c c t t t i i i o o o n n n M M M e e e m m m b b b r r r a a a n n n e e e p p p o o o t t t e e e n n n t t t i i i a a a l l l Concentration/acti vity of respiratory complexes C C C o o o m m m p p p l l l e e e x x x I I I C C C o o o m m m p p p l l l e e e x x x I I I I I I C C C o o o m m m p p p l l l e e e x x x I I I I I I I I I C C C o o o m m m p p p l l l e e e x x x I I I V V V F F F 1 1 1 A A A T T T P P P a a a s s s e e e 1 1 1 1 1 1 3 3 m m o o o o l l d d D D 2 2 5 5 7 7 A A n n = = 1 1 1 1 S S S k k k e e e l l l e e e t t t a a a l l l m m m u u u s s s c c c l l l e e e e e e x x x t t t r r r a a a c c c t t t i i i o o o n n n / / / s s s u u u b b b f f f r r r a a a c c c t t t i i i o o o n n n a a a t t t i i i o o o n n n S S p p e e c c i i f f i i c c A A i i m m 1 1 S S p p e e c c i i f f i i c c A A i i m m 2 2 S S p p e e c c i i f f i i c c A A i i m m 3 3 L L i i n n k k b b e e t t w w e e e e n n m m i i t t o o c c h h o o n n d d r r i i a a l l d d y y s s f f u u n n c c t t i i o o n n a a n n d d a a p p o o p p t t o o s s i i s s

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61 CHAPTER 4 RESULTS Mouse Characterization Data from Dr. Prolla s Lab: Generation and Phenotype of D257A Mice In order to elucidate the role of mtDNA muta tions in skeletal muscle loss, observed with age, we will use a knock in mouse model (PolgD257A) with increased spontaneous mutation rates in mtDNA. In brief, these mice contain a mu tation that results in a functional disruption of the exonuclease domain of mous e mitochondrial DNA polymerase POLG. Based on yeast, site directed mutagenesis st udies, our collaborators constructed a mutation that corresponds to the D230 substitution in yeast (D257 in mice), which was the substitution that elicited the str ongest mutator phenotype in vivo among the substitutions tested (148). This residue, D257, is conserved in all POLG proteins identified to date and is involved in dNMP and divalent ion binding, playing an essential role in the catalytic activity of the 3-5 exonuclease (149, 150). This mutation completely abolishes POLG exonuclease activity in yeast and mice, but has no significant effect on polymerase activity (148). The mouse POLG locus, PolgA, was cloned a nd gene targeting in embryonic stem (ES) cells was used to introduce an AC CT two-base substitution that corresponds to positions 1054 and 1055 of the exonuclease-encoding domain (see supporting data on Science online). This mutation results in a critical residue subs titution in the conserved exonuclease domain of POLG, impairing its proofreading ability (14). Seven correctly targeted ES cell clones were expanded and the cells were inject ed into blastocysts derived fr om B6 female mice. Injected blastocysts were implanted in pseudo-pregnant fe males for generation of chimeric mice. Several chimeric mice were identified as determined by coat color. Of these, six chimeras, representing four different ES cell clone lines, resu lted in germline transmission of the PolgD257A allele when

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62 mated to B6 females. Germline transmission of the mutation produced PolgAD257A/+ mice, which were then intercrossed to generate homozygous PolgAD257A/D257A mice. Mice carrying one copy of the PolgD257A allele are healthy and fertile, and are co ntinuously used to generate homozygote PolgD257A mice. Young homozygous PolgD257A mice, which are devoid of WT Polg protein, were indistinguishable from wild-type littermates, however, long-term follo w-up revealed a striking premature aging phenotype beginning at ~ 9 months of age (13). Phenotypes are age-related and consisted of: hair loss, loss of bone mass, h earing loss, kyphosis, skelet al muscle loss, and cardiac dysfunction (Fig 4-1). The mutant mice ha ve a significantly re duced life span (for D257A mice, maximum survival 460 days, median survival 416 days; for wild-type littermates, maximum and median survival >850 days (Fig 4-1) (13). Data from Our Lab In our pilot study we had obtained data on both 3-mo and 11-m o old WT and D257A animals in several parameters, therefore, we occasionally report results obtained at both time points. Results for Specific Aim 1 Impaired mitochondrial bioenerget ics in 11-month-old D257A mice We have evaluated mitochondrial respirati on in 3-month old and 11-month old WT and D257A mice. O2 consumption by skeletal muscle mito chondria was almost identical between WT and D257A mice at 3-mo of age for both st ate 4 (WT: 8.4 0.7 nmol/min/mg protein vs. D257A: 8.2 1.2, p=0.9) and state 3 respirat ion (WT: 41.1 3.3 vs. D257A: 38.4 4.7, p=0.64) (Fig A-3). This suggests that mtDNA mutations do not affect mitochondrial bioenergetics early on in the D257A animals life, and that the D257A phenotype is age-induced. At 11-months, O2 consumption during state 4, the resting state of the mitochondria, did not differ between genotypes (WT: 12.7 1.3 vs. D257A:11.9 0.95, p= 0.31), which was not unexpected since O2

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63 consumption during this state is usually mini mal. However, at 11 months of age mutant mitochondria displayed a marked decrease in oxygen consumption (-43 %) during state 3 (WT: 68.4 5.1 vs. D257A: 39 5.8, p=0.0006) (Fig 42), the phosphoryla tive state of the mitochondria, which also led to a significantly lower respiratory control ratio (RCR: -43 %) for the mutant mitochondria (WT: 5.7 0.49 vs. D257A: 3.27 0.39, p=0.0005). RCR is used as an index of mitochondrial coupling and the sign ificant decrease in the D257A mitochondria suggests that there is significant uncoupling be tween oxidation and phosphoryl ation (Fig 4-2). It is therefore evident that accumulation of mtDNA mutations may lead to mitochondrial dysfunction associated with compromised state 3 respiration. D257A mice display decreased content of ETC Complexes I, III, and IV that contain mtDNA-encoded subunits We measured the content of ETC complexes I, II, III, IV and F1 domain of ATPase in 11mo old WT and D257A skeletal mu scle using blue native page. We found that the total contents of complexes I (WT: 40050 2281 arbitrar y units vs. D257A: 26100 2724, p=0.002), III (WT: 50970 3673 vs. D257A: 31960 4925, p=0.0093), and IV (WT: 50900 4782 vs. D257A: 25460 5532, p=0.0046), all of which cont ain subunits encoded by mtDNA, were significantly reduced in D257A mi ce (Figs 4-3, 4-4), suggesting that complex formation in D257A mice specifically t hose containing subunits encode d by mtDNA is abolished. In contrast, the content of complex II (W T: 20710 4079 vs. D257A: 28610 7051, p=0.3513) and F1 (WT: 19760 2831 vs. D257A: 18330 747.9, p=0.64), both of which contain only nuclearencoded subunits, was not different between genot ypes (Figs 4-3, 4-4). The latter reinforces the idea that the accumulation of mtDNA mutations dire ctly impacts assembly of complexes that are partly mitochondrial-encoded, while all nuc lear-encoded complexes appear unaffected.

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64 Electron transport chain complex specifi c activity remains unaffected by mtDNA mutations in D257A mice The activities of complex I and IV (partly mt DNA-encoded) appear greatly reduced in the mutant mice (Fig 4-5) while for the all-nuclea r-encoded complexes II, and F1 domain there are no apparent differences between genotypes (Fig 4-5). However, when we normalized the activity for each sample to the respective complex c ontent we saw no differences between WT and D257A mice for all complexes evaluated (Fig 4-6): Complex I (WT: 314.5 13.56 arbitrary units vs. D257A: 349.1 28.8, p=0.29), complex II (WT: 313 118.9 vs. D257A: 163.8 26.3, p=0.26), complex IV (WT: 364.7 19.6 vs. D257A : 440 100.5, p=0.49), F1 domain of ATPase (WT: 435.1 96.5 vs. D257A: 384.3 18, p=0.62).When we take into account the complex content and activity results (Figs 4-3, 4-4, 4-5, 4-6) as well, as the O2 consumption (Fig 4-2) and the ATP content data (Fig 4-8), we can conclude that although the conten t of ETC complexes per mitochondrion, or per amount of total mitochondr ial protein is reduced, the activity of the remaining complexes remains unaffected, at l east at the time of the measurement which represents a snap shot in the continuum of time. However, this still leaves mitochondria with energy deficits which are well demonstrated in our experiments by greatly compromised mitochondrial respiration and reductions in ATP content. D257A mice show decreased content of both nuclear-encoded and mitochondrial-encoded ETC subunits Besides measuring the content of fully assembled and enzymatically active ETC complexes, we further determined the content of selected individual subunits from each complex. We evaluated the subunit NDUFA9 from complex I, which is nuclear-encoded, as well as the subunit NDUFS3 from complex I which is mito chondrial-encoded. We also evaluated one subunit from complex II (70 kDa) and 2 selected subunits from complex III, 29 kDa and 48 kDa, all of which are nuclear-encoded. Last, we eval uated the COX1 subunit from complex IV which

PAGE 65

65 is mitochondrial-encoded and is a part of the ac tive redox center of this complex, and is thus, essential for catalysis. We obser ved a significant down-regulation of protein expression in the D257A mice compared to WT, for all subunits evaluated either nuclearor mitochondrialencoded (NDUFA9-WT: 1.47 0.14 arbitr ary units vs. D257A: 0.36 0.024, p<0.0001) (NDUFS3-WT: 3.8 0.2 vs. D257A: 3.1 0.2, p=0.03) (Complex II 70kDa-WT: 0.8 0.025 vs. D257A: 0.53 0.034, p<0.0001) (Complex III 48kDa-WT: 2.05 0.13 vs. D257A: 1.2 0.07, p<0.0001) (COX1-WT: 1.44 0.12 vs. D257A: 0.53 0.05, p<0.0001) (Fig 4-7), with the exception of the 29kDa subunit of complex III whic h was almost significantly affected in the mutant mice (WT: 0.6 0.055 vs. D257A: 0.47 0.04, p=0.07) (Fig 4-7). D257A mice display decreased ATP content ATP content, determined at 11-mo of ag e was significantly lower in D257A mice compared to WT (WT: 0.29 0.08 nmol/mg pr otein vs. D257A: 0.11 0.04, p=0.046) (Fig 4-8). It is apparent that loss of ETC complex content (see Figs 4-3, 44) can have an impact on ATP content. Therefore, if ETC complex content is reduced in D257A muscle per amount of total mitochondrial protein, as we have observed (Figs 43, 4-4), it is only exp ected that ATP content per amount of total mitochondrial protein would be reduced, as we also show, because there are probably less ETC complexes per mitochondri on. ATP production at the same time point remained unaffected by the accumulation of mtDNA mutations (WT: 142.3 19.65 nmol/mg protein/min vs. D257A: 124.7 21.7, p=0.28) (Fig 4-8). Mitochondrial membrane potential is significantly lower in D257A mice We determined the effect of increase d mtDNA mutational load on mitochondrial membrane potential ( ) in 13-mo old WT and D257A skeletal muscle. Membrane potential was significantly lower in D257A mice duri ng both state-4 (WT: 195.2 1.4 mV vs. D257A: 187.9 2.15 mV, p=0.017) (Fig 4-9) and st ate 3 (WT: 177.7 2.5 vs. D257A: 167.3 2.25,

PAGE 66

66 p=0.01) (Fig 4-9). This drop in is possibly conferred upo n energy deficits in the mitochondria due to the dysfuncti on of ETC complexes (see Figs 4-2, 4-3, 4-4, 4-7 and 4-8) and can be the trigger for the mitoc hondrial-mediated apoptosis we detected in the mutant mice (see Figs 4-16, 4-17, 4-18, 4-20). Mitochondrial protein yield is reduced in skeletal muscle of D257A mice We measured total mitochondrial protein yi eld in 13-mo old WT and D257A skeletal muscle and found that mitochondrial yield is dras tically reduced in D257A mice by 13-mo of age (45.9% reduction compared to WT) compared to WT (WT: 4.3 0.14 mg of mitochondrial protein/gram of muscle tissue vs. D257A: 2.35 0.2, p<0.0001) (Fig 4-10). Interestingly, this may suggest that mitochondria are getting elimin ated in D257A mice. We also compared this content with a group of ~10-11 mo old animals in order to observe whether it gets continuously reduced as the D257A animals get older and closer to their mean lifespan. Indeed, at 11 months we also observed a significant re duction (35% reduction) in the mitochondrial content but not to the extend we saw at 13 months (WT: 4 0.14 vs. D257A: 2.6 0.06, p=0.0044) (Fig 4-10). Combined the above results suggest that mito chondria are probably getting continuously eliminated in skeletal muscle thr oughout the lifespan of D257A mice. Skeletal muscle mitochondr ia from D257A mice produce significantly less ROS The main tenet of the free radical theory of aging (70) is that aging is due to the progressive accrual of ROS-inflicted damage, including mtDNA mutations, the accumulation of which has been postulated to lead to a "vicious cycle" of further mitochondrial ROS generation and mitochondrial dysfunction (5, 6) To test this hypothesis, we measured H2O2 produced by skeletal muscle mitochondria of young and old (3-mo and 11-mo old) WT and D257A mice. H2O2 production was measured during state 4 sin ce ROS production is highest when electron flow is low, while during state 3 ROS produc tion is nearly negligible. Levels of H2O2 were not

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67 significantly different between genotypes at the 3mo time point (WT: 0.30 0.05 nmol H2O2/ min/mg protein vs. D257A: 0.26 0.06, p=0.6) (Fig A-4). Interestingly, at 11-mo of age, H2O2 production was significantly decreased (36 %) in D257A mice (WT: 0.6 0.07 nmol H2O2/ min/mg protein vs. D257A: 0.4 0.05, p=0.01) (Fig 4-11), and coupled to the decreased state-3 respiration (Fig 4-2). The decreased H2O2 production by mutant mitochondria also led to the calculation of a significantly lower free radical leak for th e D257A mitochondria (WT: 2.6 0.3% vs. D257A: 1.8 0.3%, p=0.04) (Fig 4-11 ). These observations do not support the mitochondrial vicious-cycle hypothesis of agin g, but instead suggest that mtDNA mutational load is causal to reduced mitochondrial functi on, as demonstrated by the marked decrease in oxygen consumption and the significant mitoc hondrial uncoupling. However, the accumulation of mutations does not induce an in crease in mitochondrial ROS production. Similar results regarding mitochondrial ROS production have recently been published using mouse embryonic fibroblasts (MEFs) from D257A mutator mice (7 9) and this study also questioned the accuracy of the mitochondrial vicious cycle theory. D257A mitochondria produce less ROS in both main ROS generators of the ETC: Complex I and Complex III When we evaluated site speci fic ROS generation in 3-mo old mice we found no differences between genotypes in ROS production at either complex I -represen ting total basal ROS generation (Fig A-4: panels A) (WT: 0.3 0.05 nmol H2O2/ min/mg protein vs. D257A: 0.26 0.06, p=0.6) or Complex III (Reverse flux included-WT: 4.6 0.56 vs. D257A: 4.15 0.67, p=0.6) (Reverse flux blocked-WT: 1.13 0.23 vs. D257A: 1.1 0.3, p=0.9) (Fig A-4: panels C, D), or in the maximal capacity of these complexe s to generate ROS (Fig A-4: panels B, E) (Maximal complex I production WT: 1.6 0.19 vs. D257A: 1.75 0.18, p= 0.68) (Maximal complex III production WT: 8.6 0.9 vs. D257A: 8.4 0.6, p= 0.84). As expected, the free

PAGE 68

68 radical leak percent (Fig A-4: panel F) was also not different between WT and D257A mice (WT: 2 0.45% vs. D257A: 1.75 0.4%, p=0.7). At 11-mo of age our results were consistent, showing that, D257A mitochondria produce less ROS at complex III, either when reverse electron flux is taken into account (WT: 6.3 0.47 nmol H2O2/ min/mg protein vs. D257A: 3.13 0.48, p=0.0002) (Fig 4-12: panel B) or when its blocked (WT: 1.5 0.14 vs. D257A: 0.9 0.12, p=0.005) (Fig 4-12: panel C), and have reduced maximal capacity to generate ROS at both complex I (WT: 2.9 0.23 vs. D257A: 1.2 0.19, p< 0.0001) (Fig 4-12: panel A) and complex III (WT: 11.9 1.2 vs. D257A: 6.75 0.88, p=0.003) (F ig 4-12: panel D) compared to WT. Moreover, the fact that H2O2 production is decreased almost f ourfold for both WT and D257A mice when the reverse electron flux is blocked (when comparing Y axis values from Fig 4-12, panels B and C) signifies that this reverse flow is a significa nt source of ROS produced by the ETC. These observations combined provide furthe r support to our previous results regarding total basal ROS production by the mitochondria (s ee Fig 4-11), as well as, additional evidence against the mitochondrial vicious cycle theory. No difference in antioxidant enzyme mRNA expression between genotypes We measured mRNA expression of Catalase and the mitochondrial-specific isoform of SOD, MnSOD, in skeletal muscle of 11-mo old WT and D257A mice, via RT-PCR. We found no difference in either Catalase (WT: 1.2 0.08 arbitrary units vs. D257A: 1.1 0.04, p=0.1) (Fig 4-13) or MnSOD (WT: 0.8 0.01 arbitrar y units vs. D257A: 0.7 0.07, p=0.3) (Fig 4-13) between genotypes. This provides further support to the notion that m itochondria from D257A mice actually produce less ROS and that the decrease we observed in H2O2 production was not due to an adaptive up-regulation of antioxidant de fenses in the mutant mice. In fact, in D257A muscle, there was a strong trend toward decrease, especially, in Catalase mRNA expression. The

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69 fact that mutant mice generate less H2O2 might explain the no-n eed for up-regulation of antioxidant defenses compared to WT. Mitochondrial DNA mutations cause aging phenotypes in the absence of increased oxidative stress In order to correlate our H2O2 results with further oxidative stress, we next examined a marker of ROS-induced oxidative damage to DNA, by assessing the levels of 8-oxo-7,8-dihydro2'-deoxyguanosine (8-oxodGuo) in skeletal muscle mtDNA of 11-mo old WT and D257A mice, using HPLC with electrochemical detection. We did not find any differences in the levels of mtDNA oxidation between 11-mo old WT a nd D257A mice (WT: 51.4 6.3 8-oxodGuo/106 dGuo vs. D257A: 50.3 7.2, p=0.9) (Fig 4-14). Moreove r, in published results, we also showed no significant differences between WT and D257A skeletal muscle in F2-isoprostanes, a marker of lipid peroxidation (13). Thus, an increased lo ad of mtDNA mutations does not appear to be associated with increased levels of oxidative damage to mtDNA (Fig 4-14), or elevated lipid peroxidation in skeletal muscle. A recent publica tion provided further support to our outcomes showing that protein carbonylati on, and thus, oxidative damage to proteins was not significantly different in mtDNA mutator mi ce compared to WT (79). Hence, despite in creased mutational load, mitochondria from D257A mice do not show increased oxidative stress. Results for Specific Aim 2 D257A mice display significant skelet al muscle loss by 11-mo of age We obtained data on 3-mo old and 11-mo old WT and D257A mice. At 3-mo-of age there was no significant difference in skeletal muscle weight between WT and D257A mice (WT: 170 6 mg vs. D257A: 150 8, p=0.24) (Fig A-1) However, at 11-mo of age D257A mice exhibited significant skeletal muscle loss in the gastrocnemius (-24 %) (WT: 160 6 mg vs. D257A: 126 5, p=0.0004) (Fig 4-15), and in th e quadriceps muscle (-19 %) (WT: 190 6 mg

PAGE 70

70 vs. D257A: 0.156 0.007, p=0.0003) (Fig 4-15) comp ared to WT, which is indicative of sarcopenia, since normally aged animals (30-mo WT) also showed similar degree of muscle loss compared to young animals (5-mo WT) (WT: 145.7 9.3 mg vs. D257A: 109.9 6.6, p=0.0095) (Fig 4-15). Apoptosis in D257A skeletal muscle is eviden t by an increase in cytosolic monoand oligonucleosomes We quantified apoptotic DNA fragmentation in skeletal muscle of 11-mo old WT and D257A mice by measuring the amount of monoa nd oligo-nucleosomes rele ased in the cytosol, using a quantitative Cel l Death detection ELISA. These, ar e characteristic fragments of ~180200 bp or multiples and are specific to apoptosis We observed a significant release of these fragments into the cytosol in the D257A muscle (WT: 0.11 0.006 OD/mg protein vs. D257A: 0.17 0.03, p=0.035) (Fig 4-16) indicati ng that apoptosis indeed occu rs in these mice and is, at least, partly responsible for th e loss of skeletal muscle mass obs erved in these mice (Fig 4-15, 416). DNA laddering is evident in skeletal muscle of D257A mice To further demonstrate and corroborate apoptosi s in D257A skeletal muscle we performed a standard measure of apoptosis : DNA laddering. This enables the detection and visualization of nucleosomal ladders of ~180-200 bp or multiples, characteristic of apoptosis. Prominent DNA ladders are evident for the D257A mice while ladders are very mi nimal or non-existent for WT mice (Fig 4-17). This small-scale DNA fragmenta tion further confirms that apoptosis is an important mechanism of sarcopenia in the mutant mice. Caspase-3 cleavage and activation is up-regulat ed in D257A mice and resembles caspse-3 activation during normal aging We evaluated apoptosis in skeletal muscle by measuring the content of activated (cleaved) caspase-3, by western blotting. Caspase-3 is th e final effector caspase for many apoptotic

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71 pathways and its cleavage at the C-terminal side of a specific aspartate re sidue is considered as one of the hallmarks of apoptosis. To determine if increased levels of cleaved ca spase-3 is a feature of normal aging, we first examined caspase-3 content in tissues of 5 mo -old and 30 mo-old WT mice (Fig 4-18) (13). Cleaved caspase-3 levels significantly increased with normal aging in sk eletal muscle of WT mice by ~32% (5-mo old: 43130 4704 arbitr ary units vs. 30-mo old: 63620 4510, p=0.0085). We further evaluated caspase-3 levels in skelet al muscle of 3-mo old and 11-mo old D257A and WT mice. Levels of cleaved caspase-3 did not differ between WT and D257A mice at 3-mo of age (WT: 26950 5802 vs. D257A: 21660 3924, p=0.46) (Fig A2), suggesting that the D257A phenotype is age-induced. Similar to normal ag ing, cleaved caspase-3 levels were also significantly elevated in D257A skeletal muscle by 11 months of age compared to controls (WT: 31580 1408 arbitrary units vs. D257A: 56780 8925, p= 0.016) (Fig 4-18) (13), a time point at which mutant animals also displayed significan t loss of muscle mass. This suggests that apoptosis, mediated by caspase-3 activation, is probably an important mechanism of skeletal muscle loss in the mutant mice and also during normal aging. Together, these findings suggest that normal aging, as well as, accelerated aging induced by the accumulation of mtDNA mutations, are associ ated with the activation of a caspase-3 mediated apoptotic pathway in skeletal muscle The observation of a si milar response between normal and accelerated aging constitutes the D2 57A an appropriate mouse model to study the possible mechanisms of muscle wasting with age. Moreover, loss of critic al, irreplaceable cells through apoptosis may be a central mechanism of skeletal muscle loss associated with the accumulation of mtDNA mutations during the aging process.

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72 Results for Specific Aim 3 Cytochrome c release in the cytosol of D257A and WT skeletal muscle We measured cytochrome c release in the cytosol by performing a western blot for cytochrome c in the cytosolic fraction isolated from skeletal muscle of 13-mo old WT and D257A mice. We did not detect significant di fferences between WT and D257A mice in cytosolic cytochrome c content, although we expect ed that cytochrome c re lease in the cytosol of D257A skeletal muscle would be signifi cant (WT: 3.6 0.2 arbitr ary units vs. 3.56 0.35, p=0.87) (Fig 4-19). It is very possible that ou r cytosol was contaminat ed with mitochondrial protein due to the mitochondrial isolation pro cedure. Basically, during mitochondrial isolation although most of the mitochondria isolated are intact and fully func tional, some may get destroyed during homogenization, releasing many of the soluble proteins in the cytosol. Unfortunately, once this occurs, even if we furthe r purify the cytosol nothing changes in the case of cytochrome c because its a soluble protein. Caspase-3 and caspase-9 activities are signif icantly higher in D257A mice: Evidence for induction of the mitochondrial, casp ase-dependent pathway of apoptosis We measured caspase-3 activity in the cyto sol of 11-mo old WT and D257A skeletal muscle and found that it is significantly highe r in the mutant mice (WT: 43 2.7 RFU/mg protein vs. D257A: 57.7 1.97, p= 0.0003) (Fig 4-20) Similarly, caspase-9 activity showed the same response: significant increase in D257A mice compared to WT (WT: 35.4 2 RFU/mg protein vs. D257A: 45.3 1.7, p= 0.0014) (Fig 4-20). In addition, when we correlated caspase-3 activity with caspase-9 activ ity we found significant correlations for both WT (r = 0.97, p<0.0001) and D257A mice (r = 0.8, p=0.0029) (Fig 4-21 ). These results provide proof for the induction of the main mitochondrial mediated, cas pase-dependent pathwa y of apoptosis since activation of caspase-9 is evident in the mutant mice, which in turn leads to further cleavage and

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73 activation of the final effector caspase, caspase -3 in this pathway (Fig 4-18, 4-20), which is directly responsible for the downstream events (i.e. cleavage of endo-nucleases and DNA repair enzymes) that lead to apoptosis.

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74 Figure 4 -1. D257A mice display a premature ag ing phenotype. Shown are (A) WT and (B, C ) D257A mice at ~13 months of age. Progero id features includi ng hair loss, graying and kyphosis become apparent at ~9 months of age. (D) Kaplan-Meier survival analysis of cohorts of WT (+/+), D 257A heterozygous mice (D257A/+) and D257A homozygous mice (D257A/D257A). At least 230 mice per genotype are represented in the survival curves. Figure 42. Mitochondrial respir ation is compromised in skelet al muscle of D257A mice. We determined the effects of mtDNA mutations on O2 consumption of skeletal muscle mitochondria obtained from 11-mo old WT and D257A mice. Oxygen consumption was measured during state 4 (non-phos phorylative state a nd during state 3 (phosphorylative state). The respirator y control ratio (RCR), an index of mitochondrial coupling, was calculated by di viding state 3 to st ate 4 respiration values. Error bars represent SEM. *P < 0.5. STATE 4 WT D257A 0 5 10 15nmol O2/min/mg protein STATE 3 WT D257A 0 25 50 75*nmol O2/min/mg protein RCR WT D257A 0 1 2 3 4 5 6 7*

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75 Fig. 4-3. D257A mice display decreased content of ETC Complexes I, III and IV that contain mtDNA-encoded subunits. The total content of ETC complexes I, II, III, IV and the F1 domain of the ATPase from skeletal muscle of 11-mo old WT and D257A mice was determined using Blue Native Page el ectrophoresis followed by staining with commassie blue stain. Proteins were sepa rated according to molecular weight. Representative blots are depicted above. WT WT D257A I III ATPase IV II

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76 Fig. 4-4. Statistical analysis of ETC complex I, II, III, IV and the F1 domain of the ATPase content measured by Blue Native Page in skeletal muscle of 11-mo old WT and D257A mice. Arbitrary units represent dens itometry values normalized to total protein loaded measured by the Bradford a ssay. Error bars represent SEM. *P < 0.5. Complex I content WT D257A 0 10000 20000 30000 40000 50000*Arbitrary units Complex II content WT D257A 0 10000 20000 30000 40000Arbitrary units Complex III content WT D257A 0 10000 20000 30000 40000 50000 60000*Arbitrary units Complex IV content WT D257A 0 10000 20000 30000 40000 50000 60000*Arbitrary units Complex V (F1ATPase) content WT D257A 0 5000 10000 15000 20000 25000Arbitrary unitsETC Total Complex Content

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77 Fig. 4-5. Electron transport chain complex activity in skeletal muscle of 11-mo old WT and D257A mice. The activity of ETC complexes I, II, IV and the F1 domain of the ATPase was determined using Blue Native Page electrophoresis followed by enzymatic colorimetric reactions performed on the gels. Representative blots are depicted above. WT WT D257A I II IV V F1 ATPase

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78 Fig. 4-6. Statistical analysis of ETC complex activity in skeletal muscle of 11-mo old WT and D257A mice. The activity of ETC complexes I, II, IV and the F1 domain of the ATPase was determined using Blue Native Page electrophoresis followed by enzymatic colorimetric reactions performed on the gels. Arbitrary units represent activity densitometry values normalized to re spective content densitometry values for each sample. Error bars represent SEM. Complex I activity WT D257A 0 50 100 150 200 250 300 350 400Arbitrary units Complex II activity WT D257A 0 50 100 150 200 250 300 350 400 450Arbitrary units Complex IV activity WT D257A 0 50 100 150 200 250 300 350 400 450 500 550Arbitrary units Complex V (F1ATPase) activity WT D257A 0 50 100 150 200 250 300 350 400 450 500 550Arbitrary unitsETC Complex Specific Activity

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79 Fig. 4-7. D257A mice show decreased content of both nuclear-encode d and mitochondrialencoded ETC subunits. The content of sel ected nuclearand mitochondrial-encoded subunits from complexes I, II, III and IV were evaluated by We stern Blotting in 11mo old WT and D257A mice. Representative blots are depicted above. Results shown above were normalized to porin. Error ba rs represent SEM. *P < 0.5. Cx: complex CxI 39KDa CxI 30KDa CxII 70KDa CxIII 48KDa CxIII 29KDa CxIV-COXI AIF Porin Actin WT D257A CxI-NDUFA9 WT D257A 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75*Arbitrary units CxI-NDUFS3 WT D257A 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5*Arbitrary units Cx II WT D257A 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9*Arbitrary units CxIII-48 WT D257A 0.0 0.5 1.0 1.5 2.0 2.5*Arbitrary units CxIII-29 WT D257A 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7Arbitrary units CxIV-COX I WT D257A 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75*Arbitrary units

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80 Fig. 4-8. D257A mice display decreased ATP cont ent. We determined the effects of mtDNA mutations on ATP content and production in skeletal muscle mitochondria obtained from 11-mo old WT and D257A mice. Error bars represent SEM. *P < 0.5. Fig. 4-9. Mitochondrial me mbrane potential ( ) drop in D257A mice. We determined the effects of mtDNA mutations on in skeletal muscle mitochondria obtained from 13-mo old WT and D257A mice. Changes in were followed qualitatively by monitoring the fluorescence of TMRM that accumulates in energized mitochondria. was measured during both state 4 (non-phos phorylative state) and during state 3 (phosphorylative state). Measurement of after addition of CCCP served as a control for TMRM binding. Error bars represent SEM. *P < 0.5. ATP content WT D257A 0.0 0.1 0.2 0.3 0.4nmol/mg of protein ATP production WT D257A 0 25 50 75 100 125 150 175nmol/mg of protein/min* Glutamate/Malate State 4 WT D257A 135 145 155 165 175 185 195 205*mV ADP State 3 WT D257A 135 145 155 165 175 185*mV CCCP Uncoupled state WT D257A 0 20 40 60 80 100 120 140mVMitochondrial Membrane Potential

PAGE 81

81 Fig. 4-10. Mitochondrial yield is reduced in D257A skeletal muscle. We determined total mitochondrial yield in 11and 13-mo old WT and D257A mice by dividing the mitochondrial protein content measured by the Bradford assay by the skeletal muscle weight used each time to obtain the resp ective mitochondrial fractions. Error bars represent SEM. *P < 0.5. Fig. 4-11. D257A mitochondria produce less reac tive oxygen species (ROS) during state 4. We measured H2O2 production since it represents total basal mitochondrial ROS generation. Skeletal muscle mitochondria were obtained from 11-mo old, WT and D257A mice and supplemented with pyruva te/malate as substrate for oxidative phosphorylation. Pyruvate/malate was used to study complex I ROS production which also represents total mitochondria l ROS production. Free radical leak percent (FRL%), an index of mitochondrial effici ency, was calculated by dividing the H2O2 value by twice the state 4 respiration va lue and the result was multiplied by 100 to give a % final value. Error bars represent SEM. *P < 0.05. WT D257A 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5*13 monthsmg of mitochondrial protein per gram of tissue 11 months WT D257A 0 1 2 3 4 5*mg of mitochondrial protein per gram of tissueMitochondrial Protein Yield Pyruvate/Malate WT D257A 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 *nmol H2O2/min/mg protein Free Radical Leak WT D257A 0 1 2 3*%FRLBasal Mitochondrial ROS Production

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82 Fig. 4-12. D257A mitochondria produce less ROS in both main ROS generators: Complex I and Complex III. Skeletal muscle mitochondria were obtained from 11-mo old, WT and D257A mice. We used inhib itors of the ETC in order to study maximum rates of H2O2 production from complexes I and III, since they represent the main sites of ROS generation within the mitochond ria. For complex I maximum rate (panel A) we used rotenone added to pyruvate/malate suppl emented mitochondria. For complex III maximum rate (panel D) we used antimy cin A plus rotenone, added to succinate supplemented mitochondria. We also used mitochondria supplemented with succinate alone in order to study complex III ROS production under near physiological conditions (panel B). In addition, some of th e assays with succinate as substrate were performed in the presence of ro tenone (panel C), in order to avoid the backwards flow of electrons to Complex I. Erro r bars represent SEM. *P < 0.05. Pyruvate/Malate + Rotenone Maximal Complex I ROS production WT D257A 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5*nmol H2O2/min/mg prot Succinate ROS generation at Complex III + Reverse electron flux WT D257A 0 1 2 3 4 5 6 7*nmol H2O2/min/mg prot Succinate + Rotenone ROS generation at Complex III Reverse electron flux blocked WT D257A 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75*nmol H2O2/min/mg prot Succinate + Rotenone + Antimycin Maximal Complex III ROS production WT D257A 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14*nmol H2O2/min/mg protSite Specific ROS GenerationA B CD

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83 Fig. 4-13. D257A mice show no difference in antioxidant enzyme mRNA expression. We measured Catalase and MnSOD mRNA e xpression in 11-mo old WT and D257A mice by RT-PCR. Arbitrary units represent specific mRNA densitometry values normalized to actin mRNA densitometry va lues. Error bars represent SEM. D D 2 2 5 5 7 7 A A W W T T C C a a t t a a l l a a s s e e M M n n S S O O D D A A c c t t i i n n Catalase WT D257A 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4Arbitrary units MnSOD WT D257A 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0Arbitrary units

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84 Fig. 4-14. Mitochondrial DNA oxida tion in skeletal muscle of WT and D257A mice. We examined a marker of ROS-induced oxida tive damage to DNA, by assessing the levels of 8-oxo-7,8-dihydro-2'-deoxyguanos ine (8-oxodGuo) in skeletal muscle mtDNA of 11-mo old WT and D257A mice, using HPLC with electrochemical detection. We found no differences betw een 11-month old WT and D257A mice. Error bars represent SEM. Fig. 4-15. D257A mice display significant skeletal muscle loss by 11-mo of age compared to age-matched WT (panel A) which resemb les sarcopenia during normal aging (panel B). Gastrocnemius and quadriceps muscle s were extracted immediately following sacrifice, rinsed in saline solution and we ighed. Error bars represent SEM. *P < 0.05. Gastrocnemius weight WT D257A 0 25 50 75 100 125 150 175mg Quadriceps weight WT D257A 0 50 100 150 200mg* Gastrocnemius weight 5-mo WT 30-mo WT 0 25 50 75 100 125 150 175mg Quadriceps weight 5-mo WT 30-mo WT 0 50 100 150 200mg* A B mtDNA oxidation WT D257A 0 10 20 30 40 50 608-oxodGuo/106 dGuo

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85 Fig. 4-16. Apoptosis evident in D257A muscle by increase in cytosolic monoand oligonucleosomes. Cytosolic fractions from 11mo old WT and D257A skeletal muscle were prepared. Apoptosis was quantifie d as the amount of monoand oligonucleosomes present in the cytosol, usi ng a sandwich ELISA. Error bars represent SEM. *P < 0.05. Fig. 4-17. DNA laddering evident in skeletal musc le of D257A mice. DNA from 13-mo old WT and D257A mice was extracted and subject ed to a DNA laddering-specific ligation PCR. PCR products were electrophoresed th rough 1% agarose gels and visualized under UV light for apoptosis-specific DNA la dders of ~180-200bp pr multiples. Lane 1: 100bp molecular marker. Lanes 2-9: WT PCR products. Lanes 10-17: D257A PCR products. Lane 18: Positive contro l. Lane 19: 500bp molecular marker. + + c c o o n n t t r r o o l l D D 2 2 5 5 7 7 A A W W T T Cell death WT D257A 0.00 0.05 0.10 0.15 0.20*OD /mg protein

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86 Fig. 4-18. Caspase-3 activation in skeletal muscle of D257A mice resembles caspase-3 activation during normal aging. Panel A: Cleaved (ac tivated) caspase-3 content with normal aging: Comparison of young (5-mo) vs ol d (30-mo) WT mice. Panel B: Comparison of WT versus D257A cleaved caspase-3 leve ls at 11-mo of age. Skeletal muscle cytosolic extracts from WT and D257A mi ce were subjected to SDS-polyacrylamide gel electrophoresis and probed with a rabbit monoclonal antibody against cleaved caspase-3. Representative bl ots are shown above. Arbitrar y units represent caspase-3 densitometry values normalized to -actin densitometry valu es. Error bars represent SEM. *P < 0.05. 5-mo-WT 30-mo-WT 0 10000 20000 30000 40000 50000 60000 70000Cleaved Caspase-3 Arbitrary units W T D2 5 7A 0 10000 20000 30000 40000 50000 60000 70000*A B*C C a a s s p p a a s s e e 3 3 WT PG WT PG WT PG WT PG WT PG WT PG a a c c t t i i n n Blots for panel B

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87 Fig. 4-19. Cytochrome c release in the cytosol of D257A and WT skeletal muscle. Skeletal muscle cytosolic extracts from 13-mo ol d WT and D257A mice were subjected to SDS-polyacrylamide gel electrophoresis and probed with a mouse monoclonal antibody against cytochrome c. Arbitrary un its represent cytoch rome c densitometry values normalized to tubulin densitometr y values. Error bars represent SEM. Cytosolic Cytochrome C WT D257A 0 1 2 3 4Arbitrary units

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88 Fig. 4-20. Caspase-3 and -9 activities are elevated in D257A muscle: Proof of activation of the mitochondrial caspase-dependent pathway of apoptosis. Cytosolic fractions from 11mo old WT and D257A skeletal muscle were prepared. Caspase -3 and -9 activities were measured using a fluorometric protease assay kit which is based on detection of cleavage of the substrate DEVD-AFC or LEHD-AFC by caspase-3 and -9 respectively. Error bars represent SEM. *P < 0.05. Fig. 4-21. Caspase-3 and caspase -9 activity Pearson correlati ons in WT and D257A mice: Caspase-3 activity was correlated with caspase-9 activity in WT (panel A) and D257A (panel B) mice. Pearson r values are shown on top right corner. Correlations were significant for both genotypes. *P < 0.05. Caspase-3 activity WT D257A 0 10 20 30 40 50 60*RFU/mg protein Caspase-9 activity WT D257A 0 10 20 30 40 50*RFU/mg protein D257Ay = 0.6949x + 5.1651 r = 0.835 40 45 50 55 60 45556575 Caspase-3 activityCaspase-9 activity B WTy = 0.7196x + 4.5332 r = 0.97 20 25 30 35 40 45 50 55 203040506070Caspase-3 activityCaspase-9 activity A

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89 CHAPTER 5 DISCUSSION Overview of Principal Findings The overall goal of this project was to determine in vivo whether mtDNA mutations, known to accumulate with aging in skeletal muscle fibers, are causal to the demise of skeletal muscle with age, the condition commonly termed sarcopenia. For this purpose mice having a progeroid syndrome, due to a mutation in the exonucleas e domain of POL that led to an increase in spontaneous mutation rates in mt DNA, were utilized. The experiments conducted examined the impact of increased mtDNA muta tional load on both mitochondrial function and mitochondrial-induced apoptosis in skeletal muscle via three separate grou ps of experiments. Specific aim 1 tested the following questi ons: (a) Do mtDNA mutations lead to mitochondrial dysfunction? (b) If mitochondrial dysfunction is eviden t is it associated with an increase in mitochondrial ROS production? (c) If mitochondrial RO S generation is elevated does it lead to further oxidative damage in mtDNA? (d) What is the primary mechanism by which mtDNA mutations induce mitochondrial dysfunction? Our results reveal that mtDNA mutations induce mitochondrial dysfunction, apparent by compromised mito chondrial respiration during state 3, decreased ATP content, and a significant drop in membrane potential during both state 3 and state 4. Importantly, this compromised mitochondrial function is not accompanied by elevations in ROS production or further oxidative damage to mtDNA, which is in contrast to the main premise of the Vicious Cycle theory of ag ing. In fact, it appears th at in skeletal muscle, the accumulation of mtDNA mutations is associat ed with a significant de crease in mitochondrial ROS production which was coupled to the decrease in state 3 respiration. Moreover, the primary cause of the mitochondrial dysfunction appears to be the abrogation of ETC complexes I, III and IV, all of which contain mtDNA-encoded subunits. In addition, energy defic its due to the latter

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90 are likely responsib le for the drop in we observed which in turn is likely responsible for the induction of apoptosis intr insic to the mitochondria. Specific aim 2 tested the following questions: (a) Is apoptosis the mechanism responsible for skeletal muscle loss in D257A mice? (b) If apoptosis is eviden t, is it caspase-dependent? Our data indeed confirms that apoptosis is induced in skeletal muscle of D257A mice. Apoptosis was evident by DNA laddering and increased release of mono-and oligo-nucleosomes in the cytosol. Furthermore, apoptosis was caspase-dependent sin ce significant increases in both the content and activity of the final effector cas pase, caspase-3, were observed. Specific aim 3 tested the question: Is a poptosis mitochondrial-mediated? Although we were not able to show differences in cytochrome c release in the cytosol between genotypes, we detected a significant up-regulati on of caspase-9 activity with fu rther downstream activation of caspase-3 (as the caspase-3-,caspase-9 correlatio ns suggest on Fig 4-21), in D257A skeletal muscle. This proves that the main, caspase-dep endent mitochondrial path way is activated in D257A mice and is at least, partly responsible fo r the sarcopenia observed in these mice. Hypothesis One: The Effect of mtDNA Muta tions in Skeletal Muscle Mitochondrial Function The goal of this aim was to determine that mt DNA mutations are direc tly responsible for a measurable deficiency in cellula r oxidative phosphorylati on activity and if this was proven to be true, to identify the series of events that lead to mitochondr ial dysfunction. Therefore, an important aspect of this aim was to test the mitochondrial Vicious Cycle theory of aging, or in other words to examine whether mtDNA mutations indeed lead to an enhanced ROS production, which in turn gives rise to the rate of mtDNA da mage and mutagenesis, thus causing a Vicious Cycle of exponentially incr easing oxidative damage and dysfunction, which ultimately culminates in death.

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91 Mitochondrial DNA Mutations Cause Profound De ficiencies in Mito chondrial Function Information on the specific c ontribution of mtDNA instabil ity to human aging phenotypes can be inferred through the analysis of disorder s associated with incr eased mtDNA mutation or deletion frequency. For example, in mitochondrial di seases, it is well de monstrated that mtDNA deletions, when present at concen trations of 30% and greater in muscle tissue, can cause three disorders, Kearns-Sayre syndrome (KSS), chroni c progressive external opthalmoplegia (CPEO), and Pearson's syndrome (PS) (20, 151, 152). The affected tissues show impaired electron transport activity, ATP production, and mitoc hondrial protein synt hesis and decreased mitochondrial membrane potential (153-156). Furthe rmore, muscle biopsies from patients with KSS or CPEO show ragged red fibers and cyto chrome oxidase (COX) -negative fibers (157, 158). Tissues most affected by disorders associ ated with inherited mtDNA mutations are the same tissues markedly affected by normal aging; these include the brain, h eart, skeletal muscle, kidney and the endocrine system (159). Becau se the most obvious consequence of mtDNA mutations is an impairment of energy metabolism, most studies addressi ng aging effects have focused on tissues that are post mitotic and displa y high energetic demands, such as the heart, skeletal muscle, and the brain. Indeed, several studies have unambiguously demonstrated that mtDNA base substitution mutations accumulate as a result of aging in a variety of tissues and species, including rodents, rh esus monkeys, and humans. An ongoing debate in the field relates to th e issue of causality: are mtDNA mutations merely markers of biological ag e, or do they lead to a declin e in physiological function that contributes to the aging proces s? Studies on sarcopenia in ro dents and human samples have helped to address this issue. Studies using laser capture microd issection to st udy the role of mtDNA deletion mutations in single skeletal muscle fibers from sarcopenic rats have shown that mtDNA deletions colocalize with electron tran sport chain dysfunction and fiber atrophy (7).

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92 Interestingly, the mutations are largely clonal and absent from phenotypically normal regions within individual muscle fibers (43). In a similar study of aged (69 years old) human muscle biopsies, an association between a deficiency in the mitochondrially encoded cytochrome c oxidase (COX) and clonally expanded base-subs titution mutations and deletions in mtDNA was shown (49). Perhaps the strongest evidence that clonally expanded mtDNA mutations can be causal in both age-related dysfunction and disease comes fr om recent studies of neurons present in the substantia nigra region of the human brain. These dopamine-rich, pigmented neurons contain very high levels of mtDNA deletions. Deleted mtDNA molecules are clonal in each neuron, can accumulate, reaching up to 60% of the total mtDNA and are associated with oxidative phosphorylation defects (160). Cytochrome c oxidase deficient cells have also been shown to increase with age in both hippocampal pyramidal neurons and choroid plexus epithelial cells (161). Although these studies do not prove causalit y, they provide strong evidence in support of the hypothesis that mtDNA deletions play a co ntributing role in ag e-related mitochondrial dysfunction leading to aging phenotypes in post-mitotic tissues of mammals. In order to test the in vivo effects of increased somatic mtDNA mutation accumulation, Larssons group was the first to report results on the D257A knockin mice (the same mice used in this project) showing that mtDNA mutations and deletions are respons ible for a progressive decline in respiratory function of mitochondrially encoded complexes, that was evident as early as 12 weeks, resulting in d ecreased oxygen consumption a nd ATP production (14, 79). In accordance with the aforementioned studies in this section, we also found profound decreases in mitochondrial O2 consumption during state 3, the active st ate of the mitochondria when ATP is

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93 produced (Fig 4-2). Moreover, ATP content was significantly lower in D257A mice, compared to WT (Fig 4-8). These findings clearly indicate that oxidative phosphorylation is compromised in skeletal muscle of mutant mice and provide a causal role of mtDNA mutations specifically in skeletal muscle mitochondrial dysfunction. In additi on, since mitochondrial ETC enzyme activity declines, decreases in ATP synthesis and state 3 respiration, and energy depletion are all well documented in normal aging in various species a nd tissues, including human skeletal muscle (39, 40, 162-165) (see also more extensive background info on mitochondrial function with aging in chapter 2), it can be deduced that mtDNA mutati ons may contribute to the sarcopenic phenotype not only in D257A mice but also during normal aging. Furthermore, D257A skeletal muscle mito chondria were uncoupled since respiratory control ratios (state 3/state 4 O2 consumption) in our experiment s were less than 3.5 (Fig 4-2). These defects in oxidative phosphorylation we ha ve observed are likely the cause for the disruption of mitochondrial membrane potential we have also detected in mitochondria from mutant mice (Fig 4-9). In support of the latter, it has been shown that in mitochondrial diseases, the accumulation of mtDNA deletions causes deficits in basic bioenergetic parameters including mitochondrial membrane potential (156, 166, 167). Fo r example, in Lebers hereditary optic neuropathy (LHON), a late onset neurological disorder associat ed with specific mtDNA point mutations, Battisti et al., showed that lymphoc ytes from patients with LHON treated with the oxidizing agent dRib had signifi cant depolarization of the mito chondrial membrane potential compared to control cells and an increase in the percentage of apoptotic cells with respect to controls (166). The authors concluded that thei r results confirmed the notion of a direct link between complex I (commonly a ltered in patients with LHON) and changes in mitochondrial

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94 membrane permeability. Furthermore, in c ybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A > G and 3302A > G in tRNALeu(UUR), it was shown that the lowered mitochondrial membrane potentials exhibited by the cells led to a disturbed intramitochondrial calcium homeosta sis, which was postulated to be a major pathomechanism in mitochondrial di seases, according to the aut hors (167). Lower mitochondrial s have also been observed in normally aged m itochondria of rodents and in skin fibroblasts from elderly human subj ects (34, 168-171), and low s were found to correlate with reduced ATP synthesis. As the levels of mtDNA mutations have also been shown to increase with age in both humans and rodents (as has been extensivel y discussed in chapter 2), these observations provide further support to the notion that mtDNA mutations are important culprits for tissue dysfunction with age. Mitochondrial DNA Mutations Cause Mitocho ndrial Dysfunction in the Absence of Increased ROS Production or Oxidative Da mage to mtDNA: Implications for the Mitochondrial Vicious Cycle Theory of Aging It has been thought that loss of mitochondr ial function and increas ed mitochondrial ROS production are important causal factors in ag ing. Every human cell contains hundreds of mitochondria, and each mitochondrion has multiple copies of mitochondrial DNA (mtDNA). Because the mitochondrial genome codes for 13 polypeptides constituting the respiratory enzyme complexes required for normal functioni ng of the oxidative phosphorylation system, somatic mutations in mtDNA may be directly in volved in the mechanism by which ROS initiate a vicious cycle and cause aging. The previously mentioned vicious cycle theory of oxidative damage to mtDNA (172) holds that oxidative damage, or resultant mutation, of the mtDNA causes the assembly of a defectiv e respiratory chain, which in tu rn causes the production of more

PAGE 95

95 ROS, and the cycle repeats, with ever incr easing dysfunctions of the respiratory chain. Eventually the cell dies (172). Studies from aging humans and animals have shown good correlations between aging and increased mitochondrial production of ROS and between mitochondrial function decline and accumulation of mtDNA mutations (173). Certainly, oxidative stress coul d be playing a role in the generation of mtDNA mutations in wild-type animals. The rate of mitochondrial ROS production, extent of mtDNA (but not nuclear DNA), oxidative damage, and degree of membrane fatty acid unsaturation (a determinant of vulnerability to lipid peroxidation) are all inversely correlated with longevity across species (174-177). Mice expressing mitochondriontargeted catalase show reduced total DNA oxida tive damage in skeletal muscle, fewer mtDNA deletions, and extended mean and maximal lif espan (178), suggesting that mitochondrial accumulation of oxidative damage can limit rodent lifespan. The increased production of ROS as a c onsequence of a mtDNA mutation has been demonstrated in some occasions, as discussed below. The presence of a specific mutation in ATPase 6, a subunit of the F0 portion of the ATP synthase, caused massive apoptosis in cultured fibroblasts when glucose in the culture medium was replaced with galactose (179). Because both the mitochondrial and cytosolic S OD activities were shown to be elevated, it was inferred that superoxide production was increase d, and it was proposed that it wa s the superoxide, rather than the defect in oxidative phosphor ylation, that directly caused the apoptosis. This hypothesis was supported from an experiment in wh ich a spin trap molecule, adde d to the medium, was able to prevent apoptosis. The explanati on for why this particular poin t mutation in mtDNA would cause superoxide production is rather st raightforward. The specific mutation inhibits the activity of the ATP synthase (180), and, in coupled mitochondri a, this inhibition would arrest respiration,

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96 putting the mitochondria in state 4. In this stat e, the electron carriers of the respiratory chain are fully reduced, and superoxi de production is maximal comp ared to almost negligible superoxide formation in actively respiring (state 3) mitochondria (181, 182). Moreover, cells in culture that have defects in complex I (t he respiratory NADH dehydr ogenase) produce higher amounts of superoxide (183). It should be noted that measurements of the level of one particular ROS may not provide the complete picture of the relevant changes in the cell. For example, the amount of superoxide at a given time is the net result of a bala nce between its formation and its degradation by superoxide dismutase. In the study mentioned a bove (183) the more serious diseases resulting from complex I deficiency were found to be asso ciated with normal leve ls of superoxide and greatly increased SOD activities, suggesting that the greater the superoxide production, the greater the SOD activity. The data of Geromel et al. (179), mentioned previously, is also consistent with the idea that SOD increases to compensate for increased superoxide production. In our study, we measured the amount of H2O2 released by intact mitochondria in a surrounding medium during state 4, as an index of total basal mitochondrial ROS production. By the addition of SOD in the medium, we ensured that any s uperoxide remaining would be converted to H2O2. Furthermore, H2O2 production in intact mitochondria is thought to be closer to a more physiologic situation, while superoxide producti on is usually assayed in sub-mitochondrial particles, since its half life is very sh ort and it readily gets dismutated to H2O2 by SOD before it exits the mitochondria (141). In contrast to th e above studies, we and others have clearly demonstrated that mitochondrial mutator mice do not have increased levels of oxidative stress (13, 79, 80).

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97 In this study we provide furt her support against the viciou s cycle theory, showing that specifically in skeletal muscle, mitochondria l ROS production is not only unchanged (as we showed in the past for other tissu es) but significantly de creased in mutant mice compared to WT (Fig 4-11). These results suggest that ROS production is regulated in a tissue specific way and does not necessarily play a role in the increase d sensitivity to apoptosis. More importantly, since we also found no up-regulation in either MnSOD or Catalase mRNA levels (Fig 4-13), we can conclude that in contrast to previous studies mentioned above (179, 183), ROS levels are decreased in mutant mice due to a lower ROS production and not due to a reactive up-regulation in antioxidant defenses. Respiratory enzyme complex I and the prot onmotive Q cycle operating in complex III are the major sites that generate ROS within the ETC (184). In order to evaluate whether D257A mice produce different amounts of ROS compared to WT at these main generators, we used specific complex inhibitors: rotenone in pyr uvate/malate supplemented mitochondria, and rotenone plus antimycin in succinate supplemente d mitochondria, in order to assess maximal ROS formation at complex I (Fig 4-12, pa nel A) and complex III (Fig 4-12, panel D) respectively. Besides basal and maximal ROS production we also assessed the production from complex III under normal conditions using succin ate-supplemented mitochondria (Fig 4-12, panels B and C). In every instance, we detect ed a decrease in the amount of ROS produced by mutant mitochondria at complex I or complex III under physiological conditions, or when ETC inhibitors were used for maximal ROS producti on at either complex. This reinforces the hypothesis that mtDNA mutations are likely to induce mitochondrial dys function leading to apoptosis in the absence of incr eased ROS, and that oxidative stre ss is not an obligate mediator of aging phenotypes provoked by mitochondrial DNA mutations.

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98 Furthermore, the ROS data fit very well with the lowered mitochondrial data in D257A mice, since increased ROS generation has b een reported to occur at high mitochondrial s while the opposite is also true (181, 185). For example, in 1973 Boveris and Chance have shown that the protonophorous uncoupler of oxidative phosphorylati on (CCCP) or ADP+Pi inhibit H2O2 formation by mitochondria (185). Moreover, Skulachev and colleagues have shown that the inhibition of H2O2 formation by the uncoupler malonate and ADP+Pi was proportional to the decrease by these compounds (181), and propos ed that that a high proton motive force in state 4 is potentially dangerous for the cell due to an increase in the probability of superoxide formation (186). This is likely because at high s electron flow is not efficient and the chance that electrons flow out of seque nce thus leaking to form s uperoxide instead of reducing O2 to H2O at the terminal cytochrome oxidase (complex IV) increases. Another hypothesis is that activation of ROS production in st ate 4, when protonic potential is high and respiration rate is limited by lack of ADP, is due to the fact that some transients of the respiratory chain electron transport, capable of reducing O2 to superoxide, such as CoQH, become long-lived (181). In agreement with the ROS data, we also found that mtDNA oxidati ve damage, measured by 8-oxodG was not different between genotypes. This is the second time our group shows no differences in oxidative damage in D257A mice. In 2005, we demonstrated that the amount of 8oxodG lesions in total liver DNA was not differe nt compared to WT and 8-oxoG was actually lower in liver RNA of mutant mice (13). In a ddition, mitochondrial protein carbonyl levels and F2-isoprostanes were also unchanged compared to WT (13). The oxidative stress findings in mitochondrial mutator mice were also confirmed by Larssons group that showed no differences in protein oxidation and no up-re gulation of antioxidant enzymes in the he art and liver (79). Furthermore, Zassenhaus and colleagues using mi ce with a heart-specific POLG mutation have

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99 demonstrated no elevations in protein carbonyl s, no differences in mtDNA 8-oxodG levels, no up-regulation of antioxidant defense systems, normal ubiquitination leve ls and intact (not oxidatively damaged) iron-sulfur centers in aconitase enzyme ( 80). The fact that we did not detect an actual decrease in mtD NA 8-oxodG levels, like we did in H2O2 production in the present study, may indicate that it is not only the ROS produced by the mitochondria that damage mtDNA. Taken all together, we do not postulate that chronic accumulation of ROS production and oxidative stress are not important factors co ntributing to mtDNA damage and mutagenesis, leading to aging and age-related phenotypes, su ch as sarcopenia. However, using the D257A model, we do support the idea that, the mutagene sis partly due to chronic ROS insults to mtDNA, does not lead to further increases in ROS production and oxidative stress and may not be an important mediator of apoptosis. Hence, based on our results, we contradict and question an important part of the mitochondrial vicious cycl e theory (Fig 2-2) and we propose instead that respiratory chain dysfunction per se is the primary inducer of the sarcopenic phenotype in mtDNA mutator mice. Studies on different transgenic mice further s upport the idea that in creased mitochondrial oxidative damage is not sufficient for accelera ted aging. Mice with reduced levels of the mitochondrial MnSOD enzyme (Sod2+/) do not appear to age any fa ster than their wild-type counterparts, despite harboring increased levels of oxidative damage to both nuclear and mtDNA (187). Similarly, mice deficient for 8-oxoguanine DNA glycosylase (tha t repairs the vast majority of 8-oxoguanine lesions) or 8-oxoGTPas e (that prevents oxidi zed dGTP from being incorporated into DNA) do not exhibit accelerated aging features (188-190). On the other hand, mouse models such as the Ant1/ mice exhibit elevated levels of ROS production (191) and

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100 mitochondria treated with specific chemical electr on transport chain (ETC) inhibitors can indeed produce increased ROS levels (192). However, it should be noted that, inhibition of ETC function in Ant1/ mice or by chemical inhibitors may generate ROS because all mitochondria show the same defect (e.g., lack of available ADP or blockage of electron flow at a specific point in the ETC) (193). Upstream complexes can st ill function, resulting in electron stalling and transfer to O2 to generate superoxide. By contrast, in the D257A mice, a variety of mutations is present and multiple upstream complexes coul d be nonfunctional or be lacking subunits if mitochondrial rRNA or tRNA mutations are numerous. Thus, electron flow through all the complexes (except nucleus encoded complex II) may be impaired and reduced intermediates may not be accumulating. In the case where mtDNA mutati on levels are much lower, the presence of many wild-type copies of mtDNA will mask the e ffects of specific respiratory mutations (193). It is also important not to forget the observations of Bandy and Davison, the first investigators to put forward a mechanistic ela boration of what later became known as the mitochondrial vicious cycle theory: while they showed that some mtDNA mutations may have the same effect on the respiratory chain as smallmolecule inhibitors of respiration, that is, to stimulate ROS production, they also carefully noted that not all mu tations stimulate superoxide production (72). Specifically, they pointed out that mutations preven ting the synthesis of cytochrome b would actually abolish any superoxide production at complex III that normal mitochondria might exhibit, because without cytochrome b in place, complex III cannot be assembled (72). Later studies also reported that cells possessing large deletions, which eliminated the genes for at least a couple of resp iratory chain subunits, but also removed at least one tRNA gene, would indisputab ly preclude assembly of both the enzyme complexes known to be responsible for mitochondrial ROS production, complexes I and III (73-76).

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101 Mitochondrial DNA Mutations Lead to Mitocho ndrial Dysfunction, Via Alterations of ETC Complex Composition One of the main goals of this specific aim wa s to characterize the D257A mice in terms of skeletal muscle mitochondrial function and RO S production, and if dysfunction was evident to try to identify how this dysfunction is i nduced. Since the mtDNA encodes a total of 13 polypeptides all-subunits of the complexes of the ETC, our hypothesis was that accumulation of mutations will have a direct impact on transcription and translation of these genes leading to miscoded, truncated and dysfunctional proteins, which in turn could preclude assembly of functional complexes within the inner mitochondria l membrane. We therefore, went on to assess the content and activity of the five ETC protei n complexes. In order to determine whether complete complex content corresponds with protei n expression levels of individual subunits, we further analyzed selected mitochondrialor nuclear-encoded subunits from each complex. Certainly, one can argue that mitochondrial dysfunction observed during normal aging is not only due to mutations in mitochondrial ge nes leading to ETC complex misassembly. For example, in WT old mice a compromised state 3 respiration, such as the one we observed in D257A mice, could be due to several different factors. To name a fe w: (a) decrease in the content and/or activity of resp iratory complexes (due to the disruption in s ubunits encoded by mitochondrial DNA) leading to impaired electr on flux, (b) disruption of ADP phosphorylation due to the decline in th e activity of ATP Synthase (194), (c) impairment in the transport of ADP in the mitochondria due to alterations in Adenine Nucleotide Translocase (ANT) due to carbonylation or nitration (195), or (d) alterations in enzymes involved in TCA cycle or fatty acid oxidation. In our mtDNA mutator mice a subop timal concentration and/or activity of respiratory complexes may be the primary cause of the observed mitochondrial dysfunction, and (b), (c), (d) could be consequences of (a) rather than the cause.

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102 Of course in D257A mitochondria it is al most certain that from the ~1,500 nuclearencoded proteins that exist in the mitochondria at any given time, translation, interaction and/or activity of many of these prot eins are likely to be impacted by the high load of mtDNA mutations. In this project we only focused on pr oteins that comprise the five ETC complexes since we believe ETC complex dysfunction is direc tly and primarily affected by the high load of mtDNA mutations. Furthermore, we are aware that aging and sarcopenia are complex processes, that likely result from deregulation and interactio n of multiple pathways. However, here, we only tested one of these hypotheses, th e role of mtNA muta tions in sarcopenia which also appears to be very relevant, specifically for skeletal musc le, since multiple papers show strong correlations between the rate of somatic mtDNA mutations and skeletal muscle dysfunction. Decreases in the content and/or activity of ETC complexes wi th age, and as a result of accumulated mtDNA mutations, especially in skel etal muscle are well-documented in the literature. Aikens group has re peatedly demonstrated loss of COX (complex VI) staining combined with hyperactive SDH staining in aged ra t skeletal muscle cros s-sections ( also known as ragged red fibers) (7, 87, 102, 196). Interestingl y, these abnormalities co-l ocalized with clonal intracellular expansions of unique somatically derived mtDNA deletion mutations. In the areas of the fiber where the mutation abundance surp assed 90% of the total mitochondrial genomes, the fibers lost COX activity and displayed abno rmal morphology such as fiber splitting and breakage, while normal areas of the same fiber contained only wild type mitochondrial genomes and did not exhibit de-regulati on of ETC complex activities ( 7, 196). Decreased activities of complex I, III and IV with age were also report ed in gastrocnemius muscle of mice, while the nuclear-encoded complex II did not show significan t changes with age (28). In the same study the authors conducted a kinetic analysis for complex III and IV and indicated that Vmax for both

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103 complexes decreased with age, which suggested a decrease in the tota l enzyme content (28). Similarly, muscle biopsies from aged humans re vealed that randomly deleted mtDNA appeared mainly in the oldest subjects (beyond 80 years old), affecting up to 70% of mtDNA molecules with the activities of partly mitochondrial-encoded complexes III a nd IV being lower in the aged subjects (48) (see also chapter 2 for more extensive review on mtDNA mutations and ETC abnormalities with age). Our findings are in agreement with some of the findings mentioned above. We detected a significant decrease in the content of complex I, III and IV, all of which contain subunits encoded by mtDNA while the content of all nuc lear-encoded complexes II and F1 domain of ATPase showed no difference between genotypes (Figs 4-3, 4-4). This confirms our hypothesis that indeed accumulation of mt DNA mutations directly impacts the assembly of ETC complexes that are comprised of mtDNA encoded subunits a nd suggests that complex formation in D257A mice is abolished Interestingly, we did not detect signi ficant differences between genotypes in any of the complex activities (Fig 4-6), when each complex activity was normalized to the respective complex content asse ssed by BN-page. Since per am ount of mitochondrial protein loaded on the BN-page we detected a lower ETC content in D257A mice but no differences in activity, this suggests that alt hough a significant amount of ETC complexes is lost the activity of the remaining complexes in D257A muscle is for the most part normal. Based on these results, it is reasonable to propose that per single D257A mitochondrion the amount to electron transport chains assembled is probably significantly lower compared to a WT mitochondrion. Thus, even if the activity of the remaining ETCs in mutant mitochondria are normal, the lower amount of complexes still creates energy deficits in the m itochondria, leading to an overall decrease in ETC activity in mitochondria and thus, in mutant skeletal muscle cells It is also important to note that

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104 even if maximum activity of isolated complexes is normal that does not exclude the possibility that some of these complexes do not assemble in to fully functional electrons transport chains, especially in the case of mutant mitochondria. Furthermore, a point that requires special attention regarding m easurements of ETC complex enzymatic activities is how the complex activities are expressed. In most cases complex enzymatic activities are normalized just to tota l protein content used (usually expressed as nmol/min/mg of protein) (162), or expressed as a ratio to nuclear-enc oded citrate synthase activity (48). In our case, we normalized the activity densitometry values to the respective content densitometry values (content value was first normalized to total protein content loaded per well). In this way we evaluated complex activity per unit of ETC complex content which gives a more precise picture of what may be occurring. In most other cases the overall activity per amount of mitochondrial protein is evaluated which may not always reflect decreases in the actual activity of the individual co mplexes, but in many cases, decreases in the complex content. In line with our assumptions, ve ry recently Dubessay et al, repor ted significant decreases in the activities of complex I, III and IV with age, expressed in nmol/min/mg, in drosophila melanogaster (162). However, the authors clearly stated that these activity decreases may have various causes, such as reduced concentrati ons of respiratory complexes in the inner mitochondrial membrane or partial inactivation of the biological functions of the constituent subunits of these complexes (162). Moreover, we observed a signifi cant down-regulation of protein expression in the D257A muscle, for almost all of the ETC protein subun its evaluated either nuclearor mitochondrialencoded (Fig 4-7). The reduced expression of mi tochondrial-encoded subun its fits well with our total complex content data: if expression of mtDNA-encoded subun its is abolished due to the

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105 accumulation of mtDNA mutations that may impact th eir transcription or translation, it would be expected that assembly of whole functional co mplexes may also be abolished, as we have observed (see Figs 4-3, 4-4). The reduction in the expressi on of nuclear-encoded subunits ma y be explained by the fact that a reactive adaptatio n of the nucleus is occurring: if less ETC complexes are assembled due to the miscoding of mtDNA-encoded subunits, expression of nuclear-e ncoded subunits would also have to be reduced since th ere would be no need for the expression of extra subunits if more fully functional electron transpor t chains are not created. However, the fact that, there is no down-regulation in the content of all-nuclear encoded ETC complex II and F1 in mutant mice (see Figs 4-3, 4-4), cannot be fully explaine d by these results and needs to be further substantiated. It is possible th at although there is a down-regulat ion of nuclear-encoded subunits still these subunits more often combine to asse mble functional complexes since it is less likely that nuclear-encoded subunits woul d be truncated or have altered active sites, leading to loss of activity and misassembly of a complex, etc., as it would be the case for mtDNA-encoded subunits. However, this does not mean that functional nuclear-encoded complexes are necessarily inserted in the inner membrane to assemble functional ETCs. In agreement with our hypothesis of a reactive adaptation of the nucleus to the defects induced by mtDNA mutations, Alemi et al. recently demonstrated that pathogen ic mtDNA deletions in cells derived from KSS and CPEO patients had a strong negative effect on nuclear-encoded mitochondrially targeted genes (156). This was especially evident on Comp lex I transcripts, but also on Complex II and Complex IV assembly genes, on Complex V, on several TCA cycle genes, and on components of the mitochondrial ribosome (156). Based on their re sults, these authors al so suggested that the nucleus senses the irreversible depletion of mtDNA-encoded mitochondrial subunits and tRNAs,

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106 and responds by down-regulating th e interacting subunits that w ould normally form a functional complex (156). They proposed that, the down-regulati on of nuclear-encod ed mitochondrial ribosomal subunits, oxidative pho sphorylation, and TCA cycle tran scripts, possibly reinforces the mitochondrial defect initia ted by the deletions, and adds to the mitochondrial metabolic defect in these patients (156). Total Skeletal Muscle Mitochondrial Prot ein Yield Continuously Decreases as Time Progresses in Mutant Mice Several studies in the past, perf ormed in skeletal muscle of rodents have reported decreases in mitochondrial protein yield with aging (197, 1 98). To evaluate the ove rall oxidative capacity of skeletal muscle of adult (6-mo) and elde rly (24-mo) Fischer 344 ra ts, Hoppel and colleagues determined the mitochondrial content. They measured the activity of two exclusively mitochondrial enzymes, citrate synthase (CS) and succinate dehydrogenase (SDH), and they used these data to calculate th e mitochondrial content (198). They found that the activity of both mitochondrial marker enzymes was significantly lo wer in skeletal muscle homogenates of 24mo-old compared with 6-mo-old adult animals (198). The average decrease for both CS and SDH activities was 31%. In contra st to CS and SDH, no age-asso ciated decrease was found in lactate dehydrogenase activity, a cytosolic marker enzyme. The calculated mitochondrial content was significantly lower in skeletal muscle of elderly rats with bot h mitochondrial marker enzymes (25 and 20% based on CS and SDH, respec tively). They concluded that aged skeletal muscle has a significantly lower content of mito chondria in Fischer 344 ra ts. Moreover, the yield of mitochondrial protein per gram wet weight of skeletal muscle was also less in elderly compared with the adult animals, consistent with the lower mitochondrial content. Lower skeletal muscle mitochondrial yield from elderly rats also has been reported by Beyer et al.

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107 These authors reported a 35% decrease in mito chondrial protein yield fr om quadriceps femoris of elderly Sprague-Dawley rats (197). Consistent with the above findings we also found that in our accelerated-aging mice the total mitochondrial protein yield, expressed as mg of mitochondrial protein per gram wet weight of skeletal muscle, was significan tly decreased compared to WT animals (Fig 4-10). By 11-mo of age we detected a 35% reducti on (Fig 4-10) which agrees with previous data in normally aged skeletal muscle (197). Intere stingly, at ~13 months we saw a drastic, 46% reduction in the protein yield (Fig 4-10) suggesting that mitochondr ial content is continuo usly reduced in these animals as they are approaching their mean lif espan which is ~14 months. Although our data in this area is limited to the only measurement of mitochondrial protein yi eld, it is tempting to suggest that mitochondria in D257A mice are pr obably getting continuously eliminated. This would make sense especially in the case wh ere the accumulation of mt DNA mutations reach a reported critical threshold (42, 51, 193, 196) before significant tissue dys function is observed. And in the case of the post-mitotic skeletal muscle this threshold appears to be much later in the D257A animals lifespan compared to that of ot her tissues. For example, at 3-mo of age, the aging phenotype for skeletal muscle is not eviden t in D257A mice, while in the case of rapidly dividing cells in duodenum, thymus, and testes, we detected significant tissue dysfunction at the same time point (13). Once the critical thresh old of mtDNA mutations is reached, mitochondrial dysfunction may ensue possibly lead ing to mitochondrial-mediated apoptosis and the elimination of dysfunctional mitochondria. As the accumula tion of mtDNA mutations was shown to be exponential over time in several post mitotic tis sues, with the most well-documented being skeletal muscle (51, 73, 193, 199, 200), we also s ee that the mitochondrial protein yield in D257A skeletal muscle furt her decreases over time.

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108 In line with the above, our BN-page results, showing that mitochondrial complex content is decreased and suggesting that less ETC comple xes may exist per mutant mitochondrion, also point to the direction that indeed elimination of these mitochondria is a very likely hypothesis, since these mitochondria would not be able to keep up with the energy demands of the cell. Mitochondrial elimination in skel etal muscle would lead to comp romised oxidative capacity and tissue dysfunction. On the other hand, one can argue that accumulation of dysfunctional mitochondria and inhibition of mitochondrial aut ophagy may be the cause for tissue dysfunction. However, based on the fact that mitochondrial protein yield is prof oundly decreased in the D257A mice we don not believe th is is the case. Nevertheless our hypothesis remains to be further explored and confirmed. If indeed mitochondria from D257A muscle are eliminated this creates an important question as to the mechanism responsible for th eir elimination (Fig 51). Would dysfunctional mitochondria in D257A muscle trigger an au tophagic response? How is mitochondrial biogenesis impacted by the accumulation of dys functional mitochondria? Very recently Cortopassis group showed that specific cell types with pathog enic mtDNA deletions derived from KSS and CPEO patients had a significant i nduction of the ATG12 transcript (156). The ATG12 transcript encodes the first and most important product in the mammalian autophagy cascade (201, 202) and was induced in the microarray data from fibroblasts, lymphoblasts, and myoblasts from KSS patients, and in NT2 neural cells bearing de letions. To determine whether the induction of ATG12 was a specific conse quence of mtDNA deletions, they quantified ATG12 transcript levels in 143B osteosarcoma c ybrid fusion controls (i .e., cell lines that had gone through the process of c ybridization but with normal mt DNA), in osteosarcoma cybrids harboring deletions, and in os teosarcoma cell lines lacking the mitochondrial genome. The

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109 ATG12 transcript levels were hi ghly correlated with th e presence of deletions, were significantly higher in cells bearing deletions, and highest in cells lacking mtDNA (156). Consistent with the hypothesis of an induction of autophagy in mutant cells, they also observed the induction of several SNARE/vesicular transcript s, proteins that are also esse ntial for the process of autophagy (203, 204). The authors concluded that the induction of autopha gic transcripts is a specific consequence of mtDNA deletions (156). Based on the above fi ndings, activation of autophagy could also be a plausible mechanism media ting mitochondrial removal in D257A mice. Regarding mitochondrial biogenesis, it has be en shown that the expression of nuclear genes encoding the transcription factors TFAM, TFB1, TFB2 and DmTTF, which are essential for the maintenance and expression of mtD NA, are decreased in old and dysfunctional mitochondria (162). It is possible that attenuati on in mitochondrial biogenesis in conjunction with an up-regulation of autophagy may be occurring in skeletal muscle of mutant mice, and are responsible for the robust decl ines in mitochondrial yield. Al though the mitochondrial protein yield responses in normal aging and accelerated ag ing in D257A mice appear to be similar, the respective mechanisms responsible for the decrease s in mitochondrial protein yields remain to be determined. Hypothesis Two: the Effect of mtDNA Mu tations on Skeletal Muscle Apoptosis The goal of this aim was to demonstrate that mitochondrial dysfunction observed in aim # 1 ultimately culminates in apoptosis, and to pr ove that the sarcopenia observed in D257A mice was due to apoptosis. In this way we could show a direct causal relationship between the accumulation of mtDNA mutations and skeletal muscle loss through apoptosis and since the D257A model is an aging model we could extrapolate our results to normal aging and deduce that: a) Accumulation of mtDNA mutations wi th age is an important culprit for tissue

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110 dysfunction, in this case, skeletal muscle loss and b) Apoptosis is a central mechanism responsible for the age-induced sarcopenia. Apoptosis is a programmed process of cell deat h that has a tightly regulated initiation and execution. In Greek, apoptosis means dropping off of petals or leaves from plants or trees. The phrase had a medical meaning to the Greeks over two thousand years ago. Hippocrates (460-370 BC) used the term to describe the falling o ff of the bones and Galen extended its meaning to the dropping of the scabs. A re-introduction of the term for medical use occurred in 1972 when Kerr, Wyllie, and Currie deduced that there was a specific controlled mechanism of cell death distinct from uncontrolled necrotic death (205). They noticed a ch aracteristic, identical sequence of events in many differe nt types of cells and published th eir observations in a seminal 1972 paper that coined the phras e apoptosis and was largely i gnored for fifteen years (205)! The concept that death is essential for life accord ing to Wyllie went against twentieth century philosophy. Apoptosis with Aging To date, evidence has been accumulating to su ggest that de-regulation of apoptosis may contribute to age-associated ch anges such as progressive decl ine of physiologic function and significant increases in the incide nce of cancer and degenerative di seases (206). Progressive cell loss mediated by apoptosis is linked to many agerelated disorders. Moreover, many studies have demonstrated that apoptosis is up-regulated during agi ng in various post-mitotic cells such as those of the central nervous sy stem, cardiomyocytes, and skeletal muscle fibers (206-210). For example, the loss of neurons through apoptosis is closely associated with functional impairments such as dementia and motor neuron disability in neurodegenerative diseases such as Alzheimer disease, amyotrophic late ral sclerosis, and Parkinson dis ease (211). The aging process that occurs in the heart is characterized in anim als and humans by a loss of cardiomyocytes and

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111 reactive hypertrophy of the remaining cells, which ultimately results in impairment of cardiac function in advanced age (206). In skeletal musc le, there is increasing evidence indicating that deregulation of apoptosis plays a key role in the pathophysiology of skeletal muscle cell loss. Indeed, accelerated skeletal muscle apoptosis has been well documented to occur with aging (105, 207). In accordance with the above published reports we found a significant skeletal muscle loss in WT aging mice compared to young WT counterpa rts (Fig 4-15). Sarcopeni a in normally aged mice was also associated with up-regulation in cleaved caspase-3 conten t (Fig 4-18), suggesting that the apoptotic program is activated in skeletal muscle of old animals. Cell loss in these tissues can cause functional deterioration, thereby lead ing to aging. These obser vations suggest that aging enhances apoptosis under physiologic cond itions and increases the susceptibility to apoptosis triggered by challenges. Mitochondrial DNA Mutations and Apoptosis Aging-associated accumulation of oxidative damage to macromolecules in mitochondria results in mitochondrial dysfunction. Oxidativ e damage to mtDNA induced by ROS is probably a major source of mitochondrial geno mic instability since much of this damage can be mutagenic (212). Indeed, MtDNA mutations are gradually accumulated and the ac tivity/efficiency of energy metabolism declines in aging tissue cells that often exhibit a higher susceptibility to apoptosis (213, 214). This instability of mtDNA, leading to respiratory dysfunction and apoptosis, is thought to be one of the most important factor s in aging (212). Pathogenic A3243G and A8344G mutations as well as the 4977-bp deletion in mt DNA render human cells more susceptible to apoptosis stimuli such as UV irradiation (215, 216) In addition, studies on mice with a knockout of the mitochondrial transcripti on factor showed that defects in the respiratory chain are associated with massive apoptosis of affected cells (217). It is conceivable that impairment of

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112 mitochondrial ATP production and the resulting en ergy depletion can lead to apoptosis (212). Therefore, aging-induced inade quate supply of energy from mitochondria may contribute to an increased susceptibility of aging hum an and animal cells to apoptosis. Several laboratories have addr essed the question of whether apoptosis is a part of the pathogenic mechanisms associated with mtDNA de letions and point mutati ons. In support of this hypothesis, human cells bearing mutations causing Lebers heredi tary optic neuropathy, an inherited mtDNA disease, are sensitized to Fa s-induced apoptosis (218) Furthermore, TUNEL positive staining was observed in up to 75% or mo re of the muscle fibers in patients with mitochondrial encephalomyopathy, carrying a hi gh percentage (>40%) of a mtDNA deletion (219). In patients carrying hi gh proportions (>70%) of the A32443G MELAS mutation in the mitochondrial tRNALeu (UUR) gene, or the A8344G MERRF mutation in the mitochondrial tRNALys gene, 25% of the muscle fibers exhi bited TUNEL-positive nuclei. It appears that the apoptotic program is initiated in muscle fibers of patients carry ing high proportions of mutations affecting mitochondrial tRNAs. Recently, Zassenhaus and colleagues proposed an intriguing mechanism whereby mtDNA mutati ons would generate a pool of misfolded mitochondrial proteins, some sm all proportion of which might have the conformation necessary to bind to Bax or Bak and thereby activate apoptos is or perhaps bind to cyclophilin D and inhibit its chaperone function (220). This hypothesis could explai n how heteroplasmic mtDNA mutations could elicit a cell-death response in th e presence of many wild-t ype copies of mtDNA. Very recently, Aiken and colleagues demonstrated that aged rat muscle fibers possessed segmental, clonal intracellular expansions of unique somatically derived mtDNA deletion mutations (196). In the areas where less than w ild type genomes were detected the fibers displayed ETC abnormalities and abnormal mor phology such as fiber splitting, atrophy, and

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113 breakage (196). Deletion mutation accumulation wa s linked to these aberrant morphologies with more severe cellular pathologies resulting from higher deletion mutation abundance. In addition, in fiber regions distant from the ETC abnor malities with normal morphology, only wild type genomes were detected, and mtDNA dele tion mutations were undetectable (196). In summary, these measurements corroborat e previous studies of the same group (7, 43, 87), and indicate that age-induced mtDNA deletion mutations expand within individual muscle fibers, eliciting fiber dysfuncti on and atrophy. Our study is in ag reement with those findings showing that mtDNA mutations lead to ETC abnormalities and skeletal muscle atrophy, and extends the conclusions by showing that the sk eletal muscle atrophy observed in the D257A mice is actually due to apoptosis (Figs 4-16, 417, 4-18, 4-20). Therefore, the mitochondrial mutator mice suggest that activa tion of apoptosis is important for the induction of the aging phenotype in skeletal muscle. While previous stud ies demonstrated an increased susceptibility of cells with pathogenic mtDNA mutations to apopt otic stimuli, or showed a correlative relationship between mtDNA mutations and apoptosi s in mitochondrial diseases, we are showing a direct causal relationship between th e accumulation of somatic mtDNA mutations in vivo and apoptosis in skeletal muscle Hence, loss of myonuclear domain through apoptosis, possibly leading to the loss of irreplaceable skeletal muscle fibers, appears to be a central mechanism of sarcopenia associated with the accumulation of mtDNA mutations. Disruption of Mitochondrial Membrane Potential and Role for Apoptosis During mitochondrial-mediated apoptosis, the release of cytochrome c from the mitochondrial intermembrane space induces the asse mbly of the apoptosome that is required for activating downstream caspases. However, the actual mechanism of its release is still debatable. In particular, the relation be tween mitochondrial physiology and th e release of cytochrome c and other apoptogenic factors from mitochondria is not clear (221). It is conceivable that the ETC

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114 abnormalities we have detected in the D257A mode l, leading to energy depletion is likely the cause for the drop in membrane potential we obser ved in D257A mice (Fig 4-9). Previous studies have described the relationship between mito chondrial membrane potential and apoptosis showing that a reduction in leads to matrix condensation and exposure of cytochrome c to the intermembrane space, facilitating cytochrome c release and cell death following an apoptotic insult (221). Changes in the have been originally postulated to be early, obligate events in the apoptotic signaling pathway (222, 223). Multiple lines of research demonstrate that the nuclear features of apoptosis are preceded by changes in mitochondrial structure and in some regimes of induction of apoptosis. Rat embryo cells induced to undergo apoptosis by the SV40 large T antigen, show a lowered and a decrease in mitochondria l respiration and translation, which is detectable early in the apoptotic process (224). Hu man peripheral blood mononuclear cells treated with dexamethasone show a reduced uptake of the mitochondrial determining fluorochrome, 3,3dihexyloxacar bocyanine iodide before the appearance of any morphological signs of apoptosis (223). The se paration of these cells prior to dexamethasone treatment into populations with high and low revealed that cells with a lowered undergo spontaneous apoptosis after a short term in culture at 37 C (223). Also, dexamethasone induces early mitochondrial effects in thymocytes unde rgoing apoptosis, which show both an early decrease in as determined by another fluorochrome, 5,5,6,6-tetrachloro-1,1,3,3tetraethyl benzimidazolylcarbocy anine iodide (JC-1), and alte red mitochondrial structure as demonstrated by electron microscopy (225). Furthermore, cell deat h in the Dictyostelium discoideum, a single-celled slime mold involves early disruption of that precedes

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115 phosphatidylserine exposure, nuclear shrinkage DNA fragmentation and the release of AIF (226), suggesting the evolutionary conservation between unicellular and multicellular organisms. In contrast to the above results, 0 cells, (devoid of mitoc hondrial DNA) which typically have only 40% of the of their parental cell line, can also undergo a poptosis in response to range of agents with similar kinetics as the parental cells. These include Ca2+ and atractyloside (227), staurosporine (228), an ti-Fas antibodies (229), TNF plus cycloheximide (227), or didemnin B (230). As there seems to be no accele ration in the apoptotic process, even though the in these 0 cells is already significan tly decreased compared to their parental cell line(230), the notion that lowering will predispose cells for apoptos is cannot be generalized (223). Based on our outcomes, we sugge st that it is the drop in observed in skeletal muscle mitochondria from mutant mice that leads to th e leakage of pro-apopto tic proteins into the cytosol and triggers apoptosis, although other mechanisms not inve stigated in the present project (e.g. Bax-Bak pores in the mitochondrial outer memb rane) could also be additionally responsible for this induction, and thus, cannot be excluded. Apoptosis is Evident in Skel etal Muscle of D257A Mice Apoptosis in our model was evident by an increased release of monoand oligonuclesomal fragments into the cy tosol (Fig 4-16). Ce lls undergoing apoptosis can release monoor oligo-nucleosomes comprising DNA fragments and histones from their nuclei into the cytoplasm or even into the extracellular compartm ent and this process is very characteristic to apoptosis (231, 232). Moreover, degradation of chromosomal DNA is one of the biochemical hallmarks of apoptosis: Late in the apoptotic process, cas pase-activated endogenous endonucleases cleave chromosomal DNA between the nucleosomes, generating a series of DNA fragments with

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116 multiples of 180 to 220 bases (233-235) that form a ladder when the extracted DNA is separated by gel electrophoresis and staine d by ethidium bromide. We have detected prominent DNA ladders in D257A mice while DNA la dders were almost not detect able in WT mice (Fig 4-17), which further confirms that apoptosis is the m echanism responsible for the sarcopenia observed in D257A mice. In addition, protein cleavage by caspases, th e central executioners of the apoptotic pathway, accounts for the distinctiv e cytoplasmic and structural cha nges seen in apoptotic cells. Cleavage and activation of the effector caspa se-3 during apoptosis has been very well documented in the scientific literature ( 119, 236-239). Here, we also show that the DNA fragmentation we detected in D257A muscle is caspase-3-mediated since we demonstrated upregulation in both the co ntent and the activity of caspase-3 (Figs 4-18, 4-20). As mentioned previously, caspase-3 is activated by proteolytic cleavage at the C-terminal side of a specific aspartate residue. In figure 4-18 we show the cont ent of the large (17/19 kDa) activated fragment of caspase-3 resulting from cleavage adj acent to Asp175. Furthermore, disruption of mitochondrial membrane potential leading to activ ation of caspase-3 and subsequent apoptosis, has been well documented (240-242), and this is also in agreem ent with our findings showing a drop in caspase-3 activation and subsequent apoptotic DNA fragmentation. More importantly, we showed that apoptosis through caspase-3 activation is also an important mechanism for skeletal muscle loss during normal aging (Fig 4-18), provid ing further support to the usefulness of our model to study mechanisms of sarcopenia in aging skeletal muscle. Hypothesis Three: Identify the Specific Ap optotic Signaling Pathway Responsible for Sarcopenia in D257A Mice The goal of this aim was to demonstrate that the apoptosis observed in mutator mice is intrinsic to the mitochondria. In this way, we in tended to show that the mitochondrial-mediated

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117 pathway is the pathway responsible for the apoptosis induced by in creased mtDNA mutational load, and make the inference that mitochondrialmediated apoptosis is an important mechanism for the age-associated skeletal muscle loss. Studies have suggested that ag e-related apoptosis and/or necr osis in response to energy depletion may occur through activ ation of the mitochondria-medi ated signaling pathway (41, 212, 213, 221). Izyumov et al. has shown that in He La cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP (trifluoromethoxycarbonylcyanide Phenylhydrazone) combined with partial inhibition of glycolysis by 2-deoxyglucose resulted in a steady threefold decrease in the intracellular ATP le vel (41). In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells had committed suicide by means of mitochondrial-mediated apopt osis. Apoptosis was accompanied by Bax translocation to mitochondria, cytochrome c re lease into cytosol, caspase activation, and reorganization and decom position of chromatin (41) Similarly, it has been shown that, in mitochondria isolated from healthy cells, matrix condensation can be induced by either depletion of oxidizable substrates or by pr otonophores that dissipate the me mbrane potential (221). Matrix remodeling to the condensed state results in cr istae unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria and the induction of apoptosis (221). In accordance with the above studies, we showed that disruption of oxidative phosphorylation and a drop in the ATP content in the D257A skel etal muscle (Figs 4-2, 4-8) indeed leads to mitochondrial-mediated apoptosis evident by up-r egulation in the activ ity of the initiator caspase-9 which mediated the downstream cleavag e and activation of caspase-3 (Figs 4-18, 4-20, 4-21). The significant positive correlation between caspase-3 and caspase-9 (Fig 4-21) adds

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118 further support to the no tion that the up-re gulation of caspase-9, and t hus the activation of the caspase-dependent mitochondrial pa thway, is indeed responsible fo r the activation of caspase-3. In addition, disruption of mito chondrial membrane potential fo llowed by caspase-9 activation and downstream caspase-3 activation has also b een previously demonstrated (240). Although we were unable to show differences in cytochrome c release into the cytosol most probably due to contamination of our cytosolic fraction with r uptured mitochondria which can occur during the mitochondrial isolation procedure we would expect that cytochrome c releas e is indeed the case in D257A animals, since activ ation of caspase-9 can only oc cur after formation of the apoptosome, which requires cytochro me c release in the cytosol. Furthermore, studies exist to support a cri tical role of mtDNA mutations in apoptosis intrinsic to the mitochondria (82, 243). Za ssenhauss group studied mice that express a proofreading-deficient POL specifically in the heart, and de velop cardiac mtDNA mutations, in order to determine whether low frequency m itochondrial mtDNA mutations are pathogenic. They found that sporadic myocytic death occurred in all regions of the hear t, due to apoptosis as assessed by histological analysis and TUNEL staining (82). While in their model they showed that mitochondrial respiratory f unction, ultrastructure, and number remained normal, they also pointed out that cytochrome c was released from mitochondr ia and concluded that mtDNA mutations are pathogenic, and seem to trigger apoptosis through the mitochondrial pathway (82). In another study and in order to confirm whet her apoptotic processes are truly related to muscle fiber degeneration in mitochondrial en cephalomyopathies, Ikezoe et al. evaluated apoptosis in muscle fibers from patients with chronic progressive ex ternal ophthalmoplegia (CPEO; associated with a mtDNA deletion), MELAS, or MERRF (243). The criterion for selecting the patients for this study was that 5% of the muscle fibers were ragged red fibers,

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119 (RRFs) i.e., fibers with the ch aracteristic subsarcolemmal accu mulation of mitochondria found in mitochondrial diseases with deficient mitochondr ial protein synthesis (usually RRFs show loss of COX activity with concomitant hyperactivat ion of SDH activity). The proportion of mtDNA carrying the relevant mutation was unknown. Ho wever, markers of mitochondrial-mediated apoptosis appeared to be upre gulated in RRFs: Bax and Apaf-1 expression and cytochrome c release from mitochondria were seen in RRFs (243). Caspase-3 act ivation was also confirmed in RRFs of MELAS, CPEO and MERRF, but not in control muscles (243). It is therefore evident from th e current literature that a rise in mtDNA mutations can lead to apoptosis mediated by the mitochondria. The pres ent dissertation study confirms results from previous studies and ties these studies together showing that an induced in vivo rise in somatic mtDNA mutations results in ETC abnormalities, su ch as profound decreases in complex I and COX content, and compromised oxidative phosp horylation, which in turn lead to energy depletion, loss of mitoc hondrial membrane potential and induc tion of apoptosis mediated by the mitochondria. Proposed Mechanism for the Skeletal Muscle Loss Induced by High Load of Somatic mtDNA Mutations Based on our outcomes, we describe belo w a hypothetical mechanism of how somatic mtDNA mutations can lead to apoptosis, respons ible for the sarcopenia observed in D257A skeletal muscle (Figure 5-1). From the blue native page results we obser ved that the content of mitochondrial ETC complexes I, III and IV is significantly lower in D257A mice (Figs 4-3, 4-4) while their activities remain unaffected (Figs 4-5, 4-6). We have also determined that total mitochondrial content per gram of skeletal muscle tissue in D257A mice is ~35% lower by 11 months, and almost half (~46% lower) compared to that of WT by 13mo of age (Fig 4-13), which suggests that

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120 mitochondria in D257A mice are getting continu ously eliminated. The decreased ETC complex content per total protein loaded may suggests th at assembly of ETC complexes, specifically those containing mtDNA-encoded subunits, is abolishe d. We can still detect normal levels of allnuclear-encoded complexes, such as, complex II. However, if formation of partly mitochondrialencoded complexes is abrogated that means that possibly fewer fully functional electron transport chains exist per skeletal muscle mitoch ondrion in D257A mice. Thus, even if levels of all-nuclear encoded complexes are not different compared to WT it is most possible that they may just accumulate in mitochondria without being inserted in th e mitochondrial inner membrane to form fully functional ETCs. If fewer ETCs exists per mitochondrion this would still leave the mitochondrion at energy deficit even though the activity of the remaining ETCs is normal. Our results fit very well this hypothe sis since we indeed show impairment of mitochondrial oxygen consumption at state 3 (Fig 42), as well as, decreased ATP content (Fig 4-8) in mitochondria isolated fr om D257A mice. We believe that these energy deficits due to the assembly of fewer ETCs in mutant mitochondria lead to the disturbanc e in the mitochondrial membrane potential we have obser ved (Fig 4-9). A decrease in me mbrane potential in turn, may be directly responsible for the rupture of the mitochondrial outer membra ne and the release of cytochrome c and other pro-apoptotic pr oteins from the inter-membrane space into the cytosol, which triggers apoptosis. Release of cytochrome c will lead to the formation of the apoptosome causing activation of caspase-9 and downstream activ ation of the final effector caspase, caspase3, which will be responsible for carrying out the proteolytic even ts that lead to DNA fragmentation. In accordance with the above we demonstrated increased skeletal muscle apoptosis in D257A mice (Figs 4-16, 4-17, 418, 4-20) which was indeed intrinsic to the mitochondria, since activation of caspase-9 a nd caspase-3 was observe d (Figs 4-18, 4-20). We

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121 believe that the main, mitochondr ial caspase-dependent pathway is a central pathway responsible for sarcopenia associated with the accumula tion of somatic mtDNA mutations and, it may be also as critical for the skeletal muscle loss associated with norm al aging. A mechanistic series of events is depicted in Figure 5-1. Last, a nd as previously mentioned, based on our total mitochondrial protein yield findings it appears that mitochondria in skeletal muscle fibers are getting eliminated. Although this provides support to our hypotheses, since we would expect that mutant mitochondria with energy deficits and di srupted membrane potenti als would eventually get destroyed, it also poses the im portant question as to how their distraction is mediated. What is the mechanism of their elimination? These are important questions that await answers. Synopsis This project utilized the D257A knock in mouse, as an in vivo model of increased spontaneous mutation rates in mtDNA in order to elucidate the role of mtDNA mutations in sarcopenia. This mouse containe d a mutation that resulted in the functional disruption of the exonuclease domain of mouse mitochondrial DNA polymerase leading to the abolishment of its proofreading function without significantly affecting the polymerase activity (13, 14, 148). Three separate but interrel ated hypotheses were tested. Major findings include the following: (a) mtDNA mutations in skeletal muscle lead to compromised mitochondrial bioenergetics, evident by prof ound decreases in mitochondrial O2 consumption, ATP content, and a significant drop in (b) The accumulation of mtDNA mu tations in skeletal muscle of D257A mice leads to a significant decrease in the content of ETC complexes I, III, and IV, all of which contain mtDNA-encoded subunits. This finding represents the primary mechanism responsible for the impaired m itochondrial bioenergetics observe d and the disturbance in the since elimination of ETC complexes from the in ner mitochondrial membrane are likely to leave

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122 mitochondria and cells in energy deficits. (c) Importantly, our observatio ns, thus far, do not support the idea that mtDNA mutations contribu te to increased mitochondrial ROS production and further oxidative damage to mtDNA, in contrast to the main tenet of the free radical theory of aging (4-6, 19, 71). Instead, it is evident that mtDNA mutations can induce mitochondrial dysfunction in the absence of increased ROS production, and this finding has been also demonstrated by other groups (79). (d) Up-regula tion of apoptosis in D 257A mice is evident by DNA laddering, increased release of monoand oli go-nucleosomes in the cytosol, and increases in cleaved cas-3 content and activity. The apoptosis data when combined with the significant loss of muscle mass in 11-mo-old D257A mice suggest that loss of irreplaceable, post-mitotic cells through apoptosis may be a central mechanism of sarcopenia induced by the accumulation of mtDNA mutations. (e) Involvement of the main mitochondrial caspase-d ependent pathway is apparent by the up-regulation of caspase-9 activ ity resulting in downstr eam activation of the final effector caspase-3 in D257A mice. The drop in mitochondrial membrane potential is likely the trigger for mitochondrial-medi ated apoptosis in th e D257A mice. A mechanistic series of events is depicted in Figure 5-1. The results of these experiments provide a unique contribution to the existing research, utilizing the first in vivo mammalian system to examine the role of mtDNA mutations in skeletal muscle aging. In contrast to previous correlative studie s, these new outcomes establish a direct link between the accumulation of mtDNA mutations and sarcopenia. Importantly, this work is also the first to demonstrate that, sp ecifically in skeletal muscle, mtDNA mutations do not lead to increases in mitochondrial ROS pr oduction, introducing a break in the mitochondrial Vicious Cycle theory.

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123 Conclusions Concurrent with the age-dependent loss of muscle fibers, multiple mtDNA mutations accumulate over time in many tissues and species (11, 73, 200, 244, 245). MtDNA mutations and deletions were initially consid ered to be at low abundance ( < 0.1%) when calculated against the total mitochondria pool in tissue homogenates (246, 247). When, however, discrete numbers of muscle fibers were analyzed, the abundance of mtDNA mutations was found to be inversely proportional to the number of cells analyzed (248). In situ hybridization studies demonstrated that mtDNA deletion mutations were not dist ributed homogeneously th roughout a tissue, but amplified focally within a subset of individual cells, appearing as a segmen tal pattern along the length of muscle fibers and as a mosaic distribution between cells (75, 158, 249-253). This provides a mechanism for significant tissue dys function induced by mtDNA mutations, the focal accumulation of which may cause significant impair ment of mitochondrial function in individual cells in spite of low overa ll levels of mitochondrial DNA mutations in muscle (49). The hypothesis that aging is due in part to mt DNA damage and associat ed mutations (5, 6) was based on the observations that mtDNA is lo cated in the organelle that generates most cellular ROS, that mtDNA is relatively unprotected from ROS damage due to a lack of histones, and also that mtDNA repair may be limited. It is important to note that aging and aging associated phenotypes, such as sarcopenia, ar e complex processes that are likely to have multifactorial causes. Mitochondri al DNA mutations can arise di rectly from errors during DNA replication (193). Oxidative stress may also ge nerate mtDNA mutations as well as damaged proteins that might be able to directly signal apopt osis through a misfolded protein response (193, 220). Respiratory deficiency could contribute to apoptotic signaling or be directly responsible for some aspect s of tissue dysfunction (193) The limited and sometimes contradictory evidence available concerning th e capacity of pathogenic mtDNA mutations to

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124 start and support the development of the apoptotic process and th e role of the production of ROS in this phenomenon makes it difficult to reach general conclusions. The still limited understanding of the pathoge nic mechanisms of many of the diseasecausing mutations and of all the factors capab le of promoting and controlling the various apoptotic pathways adds greatly to the complexity of the problem. Last, because cells may have hundreds of mitochondria, and each carries multi ple copies of mtDNA, the contribution of mtDNA mutations and deletions to normal aging and aging phenotypes, remains a controversial issue. It is clear, however, that progress in thes e areas will lead to a be tter understanding of the resources available to the cell for compensating a nd possibly reversing the process leading to cell death, with potential implications for the therapy of sarcopenia, as well as degenerative diseases associated with mtDNA mutations. Future Directions Since the D257A mouse model represents a rela tively new model to study aging, there is a lot of work left to be done. A significant finding of this study was that the total mitochondrial protein content per gram of skeletal muscle tissue is ge tting continually d ecreased in D257A mice, be ing half that in WT by 13-mo of age. This, points out to the fact that mitochondria are proba bly getting continuously eliminated in these mice which very nicely ag rees with the fact that current examined mitochondria exhibit loss of ETC complexes and disruptions in membrane potential. However, it also poses a question as to the mechanism respons ible for the elimination of mitochondria. Is it autophagy that is responsible for the decrease in mitochondrial content or maybe a decrease in mitochondrial biogenesis, or maybe a combination of both? In this project we evaluated what would be directly impacted by the increased mtDNA mutational load, assessing mostly proteins of the ETC, however, fu ture research is needed to

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125 determine the adaptive responses of nuclear genes to the changes in mitochondrial-encoded genes or in other words, how mtDNA muta tions affect nuclear-encoded genes in the mitochondria, especially the ones involved in th e Krebs cycle, ATP production, as well as, genes involved in the regulation of the permeability transition pore opening. Moreover, more work is required to clarify the role of Bcl-2 family proteins such as Bax, Bad, Bak, and Bcl-2 in apoptosis induced in the D257A model and the formation of pores in the outer membrane that also lead to mitochondrial-mediated apoptosis. Importantly, future work should also focus on answering the very in teresting question of why skeletal muscle mitochondria with accu mulated mtDNA mutations produce significantly less ROS, which is in contrast to the mitoc hondrial Vicious Cycle th eory. We provided the first indications /observations to that, suggesting that it is the abrogati on of ETC complexes I and III, the main generators of ROS within the ETC, that lead to a decrease in ROS production in the mutant mice. The drop in mitochondrial can also be a potential mechanism explaining the decrease in ROS production but this may also be linked back to th e energy deficits due to the decrease in the content of ETC complexes, specifically those containing mtDNA-encoded subunits. The exact mechanism for this significant decline in ROS generation is far from being completely understood and warra nts additional research. Ultimately, testing the effect of reduced mtDNA mutation accumulation on lifespan and aging phenotypes, including sa rcopenia, will provide the strongest support of a causal relationship between mtDNA mutations and aging.

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126 Muscle Fiber I I I I I I I I I I V V I I I I I I I I I I I I I I I I I I V V ADP + Pi ADP + Pi ATP ATPATP Synthase ATP Synthase H+ H+ H+H+H+ H+ H+ H+ H+H+H+ H+ H+H+H+ H+ NADH H2O 2eH2O FADH2 Matrix K K r r e e b b s s C C y y c c l l e e 2H++ 1/2O2 2H++ 1/2O2 2eMatrix Energy deficits in mitochondria O2 Consumption ATP Content Cyt c Apaf-1 ATPCas-3 Cas-9 D257A Mitochondrion ??? Mechanism A p o p tosis Mitochondrial Autophagy?? Mitochondrial Biogenesis?? (+)

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127 Fig 5-1. Proposed mechanism for the skeletal muscle loss induced by high load of somatic mtDNA mutations. Abolishment of ETC complexes in D257A mice leads to assembly of less functional electron transport chains pe r mutant mitochondrion. This can create energy deficits leading to mitochondrial dysf unction, evident by severely compromised mitochondrial respiration a nd reduced ATP content in D257A muscle. Ultimately, this dysfunction results in si gnificant drop in mitochondrial membrane potential and release of cytochrome c from the intermembrane space into the cytosol. Cytochrome c in the cytosol results in a poptosome formation, activation of caspase-9 and downstream activation of caspase-3 wh ich ultimately results in apoptotic DNA fragmentation. Apoptosis appears to be a centr al mechanism of skeletal muscle loss in D257A mice. Moreover, the observation of reduced mitochondrial yield in D257A skeletal muscle suggests that mitochondria are eliminated. The mechanism for their elimination still remains to be dete rmined although up-regulation of autophagy, down-regulation of mitochondrial biogene sis or both are likely mechanisms.

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128 APPENDIX: ADDITIONAL FIGURES Fig A-1. Skeletal muscle mass (gastrocnemius) in 3-mo old (N=8 per group), and 11-mo old (N=11 per group), WT and D257A mice. Error bars represent SEM. *P < 0.05. Fig A-2. Caspase-3 activation in gastrocnemius muscle. (A ): Caspase-3 content with normal aging: Comparison of young (5-mo) versus old (30-mo) WT mice. (B): Comparison of WT versus D257A caspase-3 levels at 3 months of age. (C): Comparison of WT versus D257A caspase-3 levels at 11 months of age. Cytosolic extracts from WT and D257A mice of the indicat ed ages were subjected to SDS-polyacrylamide gel electrophoresis and probed with a rabb it monoclonal antibody against cleaved caspase-3. Error bars represent SEM. N=7 per group. *P < 0.05. 3-mo-old WT D257A 0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175Gastroc Weight (grams) 11-mo-old WT D257A 0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175Gastroc Weight (grams)* WT 5 -mo-WT 30 moW T 0 10000 20000 30000 40000 50000 60000 70000Cleaved Caspase-3 (OD/mm2) 3-mo-old WT D2 57A 0 5000 10000 15000 20000 25000 30000 35000 11-mo-old WT D2 57A 0 10000 20000 30000 40000 50000 60000 70000*A BC*

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129 Fig A-3. Mitochondrial respiration in skeletal muscle of 3-mo old mice. We determined the effects of mtDNA mutations on O2 consumption of skeletal muscle mitochondria obtained from 3-mo old (N=8 per group) WT and D257A mice. The respiratory control ratio (RCR), an inde x of mitochondrial coupling, was calculated by dividing state 3 to state 4 respiration va lues. Error bars represent SEM. State 4 WT D257A 0.0 2.5 5.0 7.5 10.0nmol O2/min/mg protein State 3 WT D257A 0 5 10 15 20 25 30 35 40 45nmol O2/min/mg protein RCR WT D257A 0 1 2 3 4 5 6Mitochondrial Respiration in 3-mo Old Mice

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130 Fig A-4. Reactive oxygen species production during st ate 4 in isolated mitochondria from 3-mo old mice. Skeletal muscle mitochondria were obtained from 3-mo old (N=8 per group) WT and D257A mice. We measured H2O2 production in mitochondria supplemented with pyruvate/malate (panel A) since it represents total basal mitochondrial ROS generation. We also used inhibitors of the ETC in order to study maximum rates of H2O2 production from complexes I and III, since they represent the main sites of ROS generation within the mitochondria. For complex I maximum rate (panel B) we used rotenone added to pyruvate/malate supplemented mitochondria. For complex III maximum rate (panel E) we used antimycin A plus rotenone, added to succinate supplemented mitochondria. We also used mitochondria supplemented with succinate alone in order to study complex III ROS production under near physiological conditions (panel C). In additi on, some of the assays with succinate as substrate were performed in the presence of rotenone (panel D), in order to avoid the backwards flow of electrons to Complex I. Free radical leak percent (FRL%), an index of mitochondrial efficiency (panel F), was calculated by dividing the H2O2 value by twice the state 4 respiration va lue and the result was multiplied by 100 to give a % final value. Error bars represent SEM. Pyruvate/Malate Basal mitochondrial ROS production WT D257A 0.0 0.1 0.2 0.3 0.4nmol H2O2/min/mg protein Pyruvate/Malate + Rotenone Maximal Complex I ROS Production WT D257A 0.0 0.5 1.0 1.5 2.0nmol H2O2/min/mg protein Succinate ROS generation at Complex III + Reverse electron flux WT D257A 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5nmol H2O2/min/mg protein Succinate + Rotenone ROS generation at Complex III Reverse electron flux blocked WT D257A 0.0 0.5 1.0 1.5nmol H2O2/min/mg protein Succinate + Rotenone + Antimycin Maximal Complex III ROS production WT D257A 0.0 2.5 5.0 7.5 10.0nmol H2O2/min/mg protein Free Radical Leak WT D257A 0.0 0.5 1.0 1.5 2.0 2.5%FRLROS Production in 3-mo Old MiceA B C D E F

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131 Fig. A-5. Protein expression of nuclear-encode d and mitochondrial-encoded ETC subunits in skeletal muscle of 3-mo old and 11-mo old WT and D257A mice. The content of selected nuclearand mitochondrial-encoded subunits from complexes I, II, III and IV, as well as, AIF were evaluated by Western Blotting. Representative blots are depicted above. Results shown above were normalized to porin. Error bars represent SEM. *P < 0.5. CxI-NDUFA9 WT D257A 0.00 0.25 0.50 0.75*Arbitrary units CxI-NDUFS3 WT D257A 0.0 0.5 1.0 1.5 2.0*Arbitrary units CxII WT D257A 0.0 0.5 1.0 1.5 2.0*Arbitrary units CxIII-48 WT D257A 0.0 0.5 1.0 1.5 2.0 2.5*Arbitrary units CxIII-29 WT D257A 0.0 0.5 1.0 1.5 2.0 2.5*Arbitrary units CxIV-COXI WT D257A 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25*Arbitrary unitsETC complex subunit content 3 mo old mice WT D257A 0.00 0.25 0.50 0.75 1.00 1.25*AIF3 mo old miceArbitrary units WT D257A Female Male Female Male WT D257A Female Male Female Male CxI39KDa CxI30KDa CxII70KDa CxIII48KDa CxIII29KDa CxIV-COXI AIF Porin Actin Old 11 moold Young3-mo old

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153 BIOGRAPHICAL SKETCH Asimina Hiona was born in Ioannina, Greece. She attained her bachelors degree from Aristotelian University of Thessaloniki with major in exercise physiology. Following graduation, she moved to the US where she obtain ed a masters degree from Queen College, NY in exercise physiology, with main focus in clinical exercise p hysiology. While in New York she worked for two years as an exercise physiologist in Plus One Holding Inc. Finally, deciding to focus her career in basic science, Asimina move d to University of Florida in 2001, to pursue a Ph.D degree. The main focus of her doctoral research is the c ontribution of mtDNA mutations in skeletal muscle aging, specifically in sarcopenia. Asimina has been a co-author in several peerreviewed publications. In 2004 sh e was awarded an American He art Association Fellowship and in 2005 she was awarded the Leighton E. Cluff aw ard in aging research. She was awarded her Ph.D in summer 2007, with major in biochemistry and molecular biology.