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Role of Autophagy in Neuronal Injury Models of the Central Nervous System

Permanent Link: http://ufdc.ufl.edu/UFE0022094/00001

Material Information

Title: Role of Autophagy in Neuronal Injury Models of the Central Nervous System
Physical Description: 1 online resource (92 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: apoptosis, autophagic, autophagy, brain, excitotoxicity
Neuroscience (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: Autophagy is an intracellular phenonmenon which is induced under conditions of stress. It is characterized by the presence of double membrane vesicles in the cytoplasm, autophagosomes. Autophagosomes sequester cytoplasmic organelles and finally fuse with the lysosomes. The lysosomal hydrolases subsequently breakdown the sequestered organelles in the vesicles into its constituent amino acids. The amino acids are then recycled into the protein machinery of the cell to sustain cell survival. Autophagy has been reported to aid cell survival under conditions of stress such as nutrient starvation or cell injury. Prolonged autophagy has also been known to result in autophagic cell death. While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Excitotoxicity has been documented as one of the major biochemical pathways leading to cell death, we examined the role of autophagy induction and prolonged autophagy in our neuronal cell culture model. Excitotoxicity, a form of acute stress due to excessive release of glutamate, was mimicked by exposing primary rat cerebellar granule neurons to the excitotoxin N-methyl-d-aspartate (NMDA). Our results demonstrated that excitotoxic NMDA exposure induced autophagosome formation in both the cell bodies and the neurites in as early as 3 h post-treatment, as evidenced by autophagy protein marker LC3 immunostaining, beclin-1 immunoblotting and fluorescent labeling with the monodansylcadaverine (MDC) dye. We also observed the increased levels of the autophagy proteins Atg8 (LC3) and beclin-1 (Atg6) in our animal model of controlled cortical impact. Prolonged exposure of the cultures to NMDA (12-24 h) however, produced abnormal and aggregated autophagosomes. Co-treatment of neurons with autophagy inhibitor 3-methyl adenine (3-MA) and NMDA reduced the levels of autophagosome-associated form of LC3 (LC3-II) and suppressed NMDA-induced autophagosome formation. Importantly, NMDA-mediated neuronal death was also robustly suppressed by 3-MA. Biochemical analysis furthermore showed that the neuroprotective effects of 3MA were likely mediated through suppression of NMDA-induced caspase-3 activity and oxidative stress. We also observed significant increases in the levels of the processed form of LC3 (LC3-II) and the beclin-1/bcl-2 ratio in the ipsilateral cortex of rats subjected to controlled cortical impact at various time points, after injury. Both of these changes are indicative of autophagy-enabling events following brain trauma. We thus conclude that autophagy and possibly autophagic cell death might play a role following the manifestation of brain trauma and may be neuroprotective when exploited initially but when prolonged results in autophagic cell death. Collectively, our data strongly suggest that autophagy induction and later autophagic cell death might be a significant component of either neuroprotection or later neuronal death following brain trauma.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Wang, Kevin K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022094:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022094/00001

Material Information

Title: Role of Autophagy in Neuronal Injury Models of the Central Nervous System
Physical Description: 1 online resource (92 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: apoptosis, autophagic, autophagy, brain, excitotoxicity
Neuroscience (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: Autophagy is an intracellular phenonmenon which is induced under conditions of stress. It is characterized by the presence of double membrane vesicles in the cytoplasm, autophagosomes. Autophagosomes sequester cytoplasmic organelles and finally fuse with the lysosomes. The lysosomal hydrolases subsequently breakdown the sequestered organelles in the vesicles into its constituent amino acids. The amino acids are then recycled into the protein machinery of the cell to sustain cell survival. Autophagy has been reported to aid cell survival under conditions of stress such as nutrient starvation or cell injury. Prolonged autophagy has also been known to result in autophagic cell death. While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Excitotoxicity has been documented as one of the major biochemical pathways leading to cell death, we examined the role of autophagy induction and prolonged autophagy in our neuronal cell culture model. Excitotoxicity, a form of acute stress due to excessive release of glutamate, was mimicked by exposing primary rat cerebellar granule neurons to the excitotoxin N-methyl-d-aspartate (NMDA). Our results demonstrated that excitotoxic NMDA exposure induced autophagosome formation in both the cell bodies and the neurites in as early as 3 h post-treatment, as evidenced by autophagy protein marker LC3 immunostaining, beclin-1 immunoblotting and fluorescent labeling with the monodansylcadaverine (MDC) dye. We also observed the increased levels of the autophagy proteins Atg8 (LC3) and beclin-1 (Atg6) in our animal model of controlled cortical impact. Prolonged exposure of the cultures to NMDA (12-24 h) however, produced abnormal and aggregated autophagosomes. Co-treatment of neurons with autophagy inhibitor 3-methyl adenine (3-MA) and NMDA reduced the levels of autophagosome-associated form of LC3 (LC3-II) and suppressed NMDA-induced autophagosome formation. Importantly, NMDA-mediated neuronal death was also robustly suppressed by 3-MA. Biochemical analysis furthermore showed that the neuroprotective effects of 3MA were likely mediated through suppression of NMDA-induced caspase-3 activity and oxidative stress. We also observed significant increases in the levels of the processed form of LC3 (LC3-II) and the beclin-1/bcl-2 ratio in the ipsilateral cortex of rats subjected to controlled cortical impact at various time points, after injury. Both of these changes are indicative of autophagy-enabling events following brain trauma. We thus conclude that autophagy and possibly autophagic cell death might play a role following the manifestation of brain trauma and may be neuroprotective when exploited initially but when prolonged results in autophagic cell death. Collectively, our data strongly suggest that autophagy induction and later autophagic cell death might be a significant component of either neuroprotection or later neuronal death following brain trauma.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Wang, Kevin K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022094:00001


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1 ROLE OF AUTOPHAGY IN NEURONAL INJURY MODELS OF THE CENTRAL NERVOUS SYSTEM By SHANKAR SADASIVAN 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 2008

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2 2008 Shankar Sadasivan

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3 Om Sai Ram To my beloved parents for their encouragement, vision and blessings during my academic career

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4 ACKNOWLEDGMENTS First and forem ost I would like to express my h eartfelt gratitude to my beloved parents without whom none of this work would have been possi ble. Their constant encouragement and vision was the guiding light during my academic progress. I want to thank my brother and his family for their loving support during the co urse of my education. I also wi sh to thank my grandparents, my cousin and aunt in Minneapolis my uncle and aunt in India, my parents-in-law and the rest of my family for their support and patience during my research work. Lastly, I would like to thank my wife for her everlasting love, encour agement, support and patience during the final phases of my Ph.D. work. I wish to thank my mentor Dr. Kevin K Wang, for giving me the opportunity and provide resources to pursue research in the field of autophagy. It was hi s guidance that helped me face the challenges meted out to me in my research project and come out successful at the end. I would like thank him for the fina ncial support in the form of Gr aduate Research Assistantship, his patience, understanding, friends hip and his confidence in me a nd my abilities. I would also like to thank my committee members Drs. Ronald Hayes, Lucia Notterpek, William Dunn Jr. and John Petito for their valuable insights, encour agement and intellectual and emotional guidance during the course of my project. I would like to thank Ms. Betty J Streetman and Susan Gardner for their assistance with the graduate student paperwork. I would like to express my grat itude to all the members of th e Wang and Hayes lab. I would in particular like to thank Drs. Stephen Larner an d Firas Kobeissy for th eir assistance and support in my research work. I am grateful to Veronika Akle, Erin Golden, Regi na Wolper, Drs. Andrew Ottens, Ming Chen Liu, Wenrong Zheng and Zhiqun Zhang, Barbara OSteen, Rebecca Ellis, Brian Fuller for their guidance and friendship. I also want to acknowledge my friends Mukur and

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5 Saumya Gupta, Ankit and Sunaina Dhawan, Am y Chen, Grace Ha, Arseima Del Valle Pinero, Jason Weinstein, Cara Weinstein, Gin Fu Ch en, Mayling Chen, Kathy Laughlin, Gretchen Lopez, Roslyn Frank, Joseph Brown, Amber Shatzer, Tolga Barker and Amanda Dubose for their valued friendship and support du ring my years in the Ph.D. program. Finally, I would like to thank the Almighty fo r giving me the patience and bestowing his blessings on me.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF FIGURES.........................................................................................................................8ABSTRACT...................................................................................................................................10CHAPTER 1 LITERATURE REVIEW.......................................................................................................13Autophagy: A General Overview........................................................................................... 13Classification of Autophagy...................................................................................................14Autophagosome Formation.................................................................................................... 15Different Forms of Cell Death................................................................................................ 16Traumatic Brain Injury......................................................................................................... ..19Autophagy in Brain Injury and Homeostasis..........................................................................202 MATERIALS AND METHODS........................................................................................... 25Animal Treatment...................................................................................................................25Gel Electrophoresis and Electrotransfer................................................................................. 25Immunoblot Analysis and Antibodies....................................................................................26Cell Culture and Amino Acid Starvation Treatment.............................................................. 26Primary Cultures and Treatments........................................................................................... 27MTT Assay.............................................................................................................................27Lactate Dehydrogenase Release Assa y (LDH assay) of Cell Death...................................... 28MDC Labeling of Autophagosomes and Nuclear Morphology Using Hoechst 33258 Staining....................................................................................................................... ........28Immunocytochemistry............................................................................................................ 29Caspase-3 Activity Assay.......................................................................................................30Materials.................................................................................................................................30Chemicals and Antibodies......................................................................................................31Experimental Paradigm of Traumatic Brain Injury................................................................ 31Tissue Collection Post Traumatic Brain Injury Procedures................................................... 32Statistical Analysis........................................................................................................... .......333 AMINO ACID STARVATION INDUCED AU TOPHAGIC CELL DEATH IN PC-12 CELLS: EVIDENCE FOR ACTIVATION OF CASPASE3 BUT NOT CALPAIN-1........ 34Introduction................................................................................................................... ..........34Results.....................................................................................................................................35Autophagy Is Induced under Amino Acid Starvation Conditions................................... 35Prolonged Amino Acid Starvation In duces Cell Death and Evidence of II-spectrin Breakdown...................................................................................................................36

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7 Activation of Caspase-3 and not Calpains in Am ino Acid Starvation Mediated Autophagy....................................................................................................................37Prolonged Amino Acid Starvation Induced Nuclear Morphology and Autophagosome Abnormalities................................................................................... 39Amino Acid Starvation Mediated Cell Death is Suppressed by Autophagy Inhibitors but not Caspase or Calpain Inhibitors......................................................... 39Discussion...............................................................................................................................40Conclusion..............................................................................................................................444 ACUTE NMDA TOXICITY IN RAT CE REBELLAR GRANULE NEURONS IS ACCOMPANIED BY AUTOPHAGY AN D 3-METHYLADENINE-SENSITIVE LATE ONSET AUTOPHAGIC CELL DEATH.................................................................... 52Introduction................................................................................................................... ..........52Results.....................................................................................................................................53Acute NMDA Exposure Induces Autophagy in Cerebellar Granule Neurons in Culture..........................................................................................................................53Autophagy Protein Marker Beclin-1 is Up-Regulated Following Early Phase NMDA Exposure.........................................................................................................54Autophagy Inhibitor 3-Methyladenine (3-MA) Effectively Suppresses NMDAInduced Autophagy......................................................................................................55Cell Death in NMDA-Treated Neurons was Alleviated by 3-MA.................................. 55NMDA-Induced Caspase-3 Activation is Suppressed by 3-MA.....................................56NMDA Mediated Protein Nitration in Cerebe llar Neurons is Also Attenuated by 3MA...............................................................................................................................57Discussion...............................................................................................................................58Conclusion..............................................................................................................................615 CHANGES IN AUTOPHAGY PROTEINS IN A RAT MODEL OF CONTROLLED CORTICAL IMPACT BRAIN INJURY ................................................................................ 72Introduction................................................................................................................... ..........72Results.....................................................................................................................................73Autophagy Induction Increases After Brain Injury in the Cortex................................... 73Beclin-1 Levels are Increased Following Brain Injury................................................... 73Immunoblot Detection of Beclin-1/B cl-2 Ratio After Brain Injury................................ 73Discussion...............................................................................................................................74Conclusion..............................................................................................................................766 CONCLUSION..................................................................................................................... ..80Summary and Scientific Applications.................................................................................... 80Future Directions....................................................................................................................82LIST OF REFERENCES...............................................................................................................83BIOGRAPHICAL SKETCH.........................................................................................................92

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8 LIST OF FIGURES Figure page 1-1 Illustration of the sequence of events in the induction of auto phagy and the for mation of autophagosomes in eukaryotic cells..............................................................................231-2 Autophagy proteins (Atg) involved in the formation of autophagosomes........................ 243-1 Autophagosome staining with MDC and LC3 after amino acid starvation of PC-12 cells....................................................................................................................................453-2 Amino Acid starvation induces cell death......................................................................... 463-3 Evidence for the lack of calpains under starvation conditions in EBSS............................473-4 Evidence for caspase-3 activation u nder amino acid starvation conditions...................... 493-5 Activation of cathepsin B following starvation................................................................. 493-6 Hoechst 33258 nuclear and MDC staini ng of cells under different treatment conditions...........................................................................................................................503-7 Effects of caspase, calpain and auto phagy inhibitors on AA starvation induced autophagic cell death and SBDP120 formation................................................................. 514-1 NMDA excitotoxicity results in the induction of LC3-positive autophagosomes in rat cerebellar granule neurons.................................................................................................634-2 NMDA exposure induces the formation of MDC-positive autophagosomes in cerebellar granule neurons.................................................................................................644-3 NMDA exposure of neurons results in an increase in the beclin-1 levels in vitro............ 654-4 Autophagy inhibitor 3-MA suppresses LC3-II formation................................................. 664-5 NMDA-induced autophagosome fo rmation is inhibited by 3-MA.................................... 674-6 Autophagy inhibitor 3-MA protects neurons against NMDA excitotoxcity.....................684-7 NMDA-induced caspase-3 activation is suppressed by 3-MA..........................................694-8 Protein nitration in cer ebellar granule neurons following NMDA-treatment is alleviated by 3-MA............................................................................................................704-9 Schematic representation of the involvement of autophagy and autophagic cell death in neurons following excitotoxic NMDA challenge..........................................................715-1 Increased levels of MAP-LC3-II are obser ved following controlled cortical impact....... 77

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9 5-2 Beclin-1 levels increase following cortical injury. ............................................................ 785-3 Increases in the ratio of beclin-1/bcl-2 indi cate autophagy induction............................... 79

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10 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 AUTOPHAGY IN NEURONAL INJURY MODELS OF THE CENTRAL NERVOUS SYSTEM By Shankar Sadasivan May 2008 Chair: Kevin Wang Major: Medical Sciences-Neuroscience Autophagy is an intracellular phenonmenon which is induced under conditions of stress. It is characterized by the presence of double membra ne vesicles in the cytoplasm, autophagosomes. Autophagosomes sequester cytoplasmic organelles and finally fuse with the lysosomes. The lysosomal hydrolases subsequently breakdown the seque stered organelles in the vesicles into its constituent amino acids. The amino acids are then recycled into the protein machinery of the cell to sustain cell survival. Autophagy has been repo rted to aid cell surviv al under conditions of stress such as nutrient starvation or cell inju ry. Prolonged autophagy has also been known to result in autophagic cell death. While the apoptotic and necrotic cell death pathways have been well studied, there lacks a co mprehensive understanding of th e molecular events involving autophagic cell death. We examined the potential roles of the apoptosislinked caspase-3 and the necrosis/apoptosis-linked calpa in-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was obser ved as early as three hours following amino acid withdrawal. Cell death, meas ured by lactate dehydr ogenase (LDH) and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu m bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred followi ng AA starvation was significantly alleviated by

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11 treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensit ive autophagic cell death due to AA starvation in PC-12 cells is mechanisti cally and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Excitotoxicity has been documented as one of the major biochemical pathways leading to cell death, we examined the role of aut ophagy induction and prolonged autophagy in our neuronal cell culture model. Excito toxicity, a form of acute stre ss due to excessive release of glutamate, was mimicked by exposing primary rat cerebellar granule neurons to the excitotoxin N-methyl-d-aspartate (NMDA). Our results demonstrated that excitotoxic NMDA exposure induced autophagosome formation in both the cell bod ies and the neurites in as early as 3 h posttreatment, as evidenced by autophagy protein marker LC3 immunostaining, beclin-1 immunoblotting and fluorescent labeling with the monodansylcadaverine (MDC) dye. We also observed the increased levels of the autophagy pr oteins Atg8 (LC3) and b eclin-1 (Atg6) in our animal model of controlled co rtical impact. Prolonged exposure of the cultures to NMDA (12-24 h) however, produced abnormal and aggregated autophagosomes. Co-treatment of neurons with autophagy inhibitor 3-methyl ad enine (3-MA) and NMDA reduced the levels of autophagosomeassociated form of LC3 (LC3-II) and suppre ssed NMDA-induced autophagosome formation. Importantly, NMDA-mediated neuronal death was also robustly suppressed by 3-MA. Biochemical analysis furthermore showed that the neuroprotective effects of 3MA were likely mediated through suppression of NMDA-induced caspase-3 activity and oxidative stress. We also observed significant increases in the levels of the processed form of LC3 (LC3-II) and the beclin-1/bcl-2 ratio in the ipsilateral cortex of rats subjected to contro lled cortical impact at various time points, after injur y. Both of these changes are indicative of autophagy-enabling

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12 events following brain trauma. We thus conclude that autophagy and possibly autophagic cell death might play a role following the manifestat ion of brain trauma and may be neuroprotective when exploited initially but wh en prolonged results in autophag ic cell death. Collectively, our data strongly suggest that autophagy inducti on and later autophagic cell death might be a significant component of either ne uroprotection or later neuronal death following brain trauma.

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13 CHAPTER 1 LITERATURE REVIEW Autophagy: A General Overview Autophagy is an intracellular phenom enon th at is activated un der conditions of intracellular and extracellular moda lities of stress such as endoplasmic (ER) stress or nutrient deprivation/starvation. This phenomenon is char acterized by the presence of double membrane cytosolic vacuoles called autophagosomes. The autophagosomes have been documented to engulf and sequester cytosolic organelles under cond itions of stress and ultimately fuse with the lysosomes. The lysosomal hydrolases then breakdown the organelles and recycle the proteins and amino acids into the cell machinery for its survival in nutrient compromised conditions (Dunn, 1990a, b; Klionsky, 2004; Levine a nd Klionsky, 2004; Meijer and Codogno, 2004). Autophagy has been documented to be a highly conserved phenomenon from yeast to mammals. A common unified nomenclature has been proposed to name the prot eins involved in the process of autophagy. The genes involve d in mammalian autophagy are denoted as ATG-genes while the proteins are depicted as atg-proteins (Klionsky et al., 2003). Al so a very recent report suggests and outlines the guidelines for the interp retation and the use of assays used to study autophagy in higher eukaryotes (Klionsky et al., 2008). Thus the studies in atuophagy are gaining momentum and becoming more structured. Though autophagy has been documented to assi st cell survival, prolonged periods of autophagy induction have been recently suggested to be responsible for another type of cell death called autophagic cell death (t ype II) (Clarke, 1990; Locksh in and Zakeri, 2004b). Autophagic cell death is considered as a programmed cell death as its execution involves a sequential activation of a number of enzymes and autophagy pr oteins. Growth factor or nutrient deprivation mediated autophagic cell death has been documente d in neuronal cell culture systems (Xue et al.,

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14 1999; Larsen and Sulzer, 2002). Autophagic cell deat h, a conserved integral pathway involved in mammalian cell development (Levine and Kli onsky, 2004) is also activated under certain pathological conditions such as the neurodegenerative diseases (S tefanis et al., 2001). Also, the activity of autophagosomes during prolonged peri ods of autophagy induction might end in the sequesteration and degradation of seminal cell or ganelles such as the mitochondria resulting in cell death. Classification of Autophagy Autophagy is classified into 3 types: a) m acroautophagy, b) microautophagy and c) chaperone-mediated autophagy. Macroautophagy: It is the main route for bulk protein degradation under conditions of stress and st arvation. Macroautophagy is a multi-step process and involves the formation of a double membrane d vesicular structure called the autophagosome and is presumably derived from the endoplasmic reticulum (ER) (Dunn, 1990a). Autophagosomes engulf cytoplasmic components incl uding whole organelles and transport them to the lysosomes. Once it reaches the lysosome the outer membrane of the autophagosome fuses with the membrane of the lysosome and matures into a structure known as the autophagolysosome. The vesicle transported into the lysosome delivers its contents following the disintegration of its membra ne. The vesicular contents are then broken down into amino acids and recycled into the protein machinery of the cell to sustain survival under nutrient deprivation conditions (Fig. 11). Recently, mammalian proteins involved in the process of macroautophagy have been identified and unified under a single nomenclature. Some of the proteins well known include Atg8, Atg6, At g7, Atg12 and Atg5 (Klionsky et al., 2003) demonstrates the processes involved in m acroautphagy (Klionsky and Emr, 2000). Since macroautophagy is the predominant form observe d, it is often referred to as autophagy.

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15 Microautophagy: In this process internalization of the proteins is made directly through the lysosomal membrane by invagination of the me mbrane at different locations forming a multivesicular body. It is non-selective and has an inherently basic activation level in the cell. Thus microautophagy seems to be activat ed even under normal conditions unlike macroautophagy (Larsen and Sulzer, 2002). Chaperone-mediated autophagy: This process is re stricted to eliminati on of proteins that possess an amino acid sequence biochemically rela ted to the pentapeptide Lys-Phe-Glu-Arg-Gln (KFERQ) during conditions of pr olonged starvation (Chiang and Dice, 1988; Dice et al., 1990). The proteins are tagged by the he at shock cognate (hsc-73) protein of 73 kDa and binds to a lysosome membrane receptor LAMP-2a which faci litates the entry of the complex into the lysosome for degradation purposes (Terlecky and Dice, 1993; Cuervo et al., 1994; Cuervo and Dice, 1996). Autophagosome Formation Autophagy is characterized by the form ation of autophagosomes. Formation of autophagosomes is a well regulated process invol ving a number of proteins, some of which is mentioned above. One of the main proteins which control the formation of these double membraned vesicles is the mammalian target of rapamycin (mTOR). The TOR proteins are assigned to a protein family termed the phosphati dylinositol kinase relate d kinases (PIKKs) and function as Ser/Thr protein ki nases (Hunter, 1995; Hoekstra, 1997; Raught et al., 2001). This protein is sensitive to the nutrient levels in the cell and is a regulator of autophagy. Under normal/healthy conditions, mTOR is hyperphosphorylated and exerts an inhibitory influence on the activation of the auto phagy proteins. Under starvation conditi ons, this inhibitory influence is lifted and the phosphorylation state of the Atg13 changes (hypophosphorylated) which then

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16 effectively binds to the Atg1 pr otein and initiates a cascading set of events resulting in the induction of autophagy. Autophagosome formation, once initiated afte r nucleation from the endoplasmic reticulum (ER), undergoes a series of steps towards its ma turation. The process is similar to the ubiquitinproteosome pathway. One of the autophagosome pr oteins, Atg12, is conjugated to Atg7 (E1-like) and then to Atg10 (E2-like) forming thioester in termediates through its COOH-terminal glycine. Finally, Atg12 conjugates to Atg5 via an internal ly sine residue in the latter. Atg16 then binds to this conjugate non-covalently and dimerizes wi th another Atg12-Atg5-Atg16 complex to form a complex required for autophagosome formation. Recen t reports illustrate the co-localization of the Atg12-Atg5 protein complex with LC3 (mamma lian orthologue of Atg8) (Mizushima et al., 2001). Figure 1-2 underlines the different At g proteins involved in the autophagosome elongation following cell stress conditions such as nutrient deprivation or anoxia (Gozuacik and Kimchi, 2004). Different Forms of Cell Death Cell death is a highly regulated event in euka ryotic cells that can be both essential and detrim ental in different cicumstances (Nels on and White, 2004). According to literature, cell death has been classified into th ree types: a) apoptotic (Type I) b) autophagic (Type II) and c) necrotic/oncotic (Type III) (Baehr ecke, 2003; Liu et al., 2004). A r ecent report adds to this list with the introduction of a new fo rm of cell death called necropt osis (Degterev et al., 2005). Although apoptotic and autophagic cell death are examples of programmed cell death, oncotic cell death is uncontrolled and involve s an inflammatory response. Apoptotic cell death is characterized by cell shrinkage and blebbing, nuclear fragmentation and no inflammation. Activation of caspases such as caspase-3 and caspase-9 has long been considered the hallmark for apoptosis. Classical apoptotic process does not involve inflammation

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17 and caspase activation is late (Lockshin and Zakeri, 2004a). Apoptosis has generally been considered the core biochemical pathway of programmed cell death and is conserved from nematodes to mammals (Metzstein et al., 1998 ; Green, 2005). With the recent advances in understanding the apoptotic pathway, it can be said that cell death due to apoptosis depends on the cell type and the death stimulus and that morphological features may not always corelate with the biochemical features (S tefanis, 2005). The apoptotic path way can be divided into three biochemical pathways activating different effect ors leading to cell death. The intrinsic pathway involves the release of factors su ch as cytochrome c from the mitochondria and recruitment of caspase-9 to the apoptosome (a large protein co mplex in the cytosol) where it is activated through homophilic interaction and dimerization before activating caspases downstream such as caspase-3, finally resulting in cel l death (Degterev et al., 2003). The second pathway essentially involves th e death receptor wherein the extracellular ligands bind to the death receptor. This binding results in the formation of a death receptor protein complex, which recruits cas pase-8 to the membrane. Caspase-8 then activates the effector caspases directly, leading to cel l death. Though these pathways are activated differently, there is still some crosstalk that occurs between these pathways (D egterev et al., 2003). Apoptotic cell death has recently been shown to occur under ER stress conditions. Studies have shown that ER stress is cap able of indirectly activating both the extrinsic and the intrinsic pathways of apoptosis mediating cell death w ith caspase-12 being the primary caspase involved (Rao et al., 2004). Excess endoplasmic stress can be induced by the accumulation of unfolded or misfolded proteins which subsequently activ ates multiple caspase-associated pathways. Although autophagy has been primarily thought to be a cell survival mechanism under conditions of stress and disruption of homeostasis, recent evidence suggests that it might play a

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18 role in programmed cell death is gaining mo mentum (Baehrecke, 2005) Autophagic cell death has been observed in the non-feeding metamorphosi s stages in insects and amphibians, such as D.melanogaster and Xenopus laevis. This form of programmed cell death is involved in eliminating the cells not required for the organi sm. The presence of autophagosomes in the dying cells represents one of the morphological manife stations in type II ce ll death. The cells also manifest caspase activation towards the later stages in the cell death process. The phosphatidylinositol 3kinase (PI3K)/Akt signa ling pathway is an important regulator of autophagy and is involved in th e maintenance of homeostasis in the cell (Hay and Sonenberg, 2004). Inhibition of different clas ses of PI3K results in varying effects on autophagy. Inhibition of class1 family of PI3K results in activation of autophagy while inhibition of class III family of PI3K inhibits autophagy (Baeh recke, 2005). Though there is m ounting evidence suggesting autophagic cell death as a form of cell death, ther e is still divided opinion about its existence. There seems to be considerable interactions in the autophagic and apoptotic pathways and recent studies suggest autophagy may play a role in apoptosis either by preceding and eventually initiating it or by delaying its ons et (Canu et al., 2005) or both pathways might be mutually exclusive (Gozuacik and Kimchi, 2004). Oncosis or necrotic cell death is the most drastic set of events that can occur in programmed cell death. It refers to process by which a cell ru ptures following swelling and releases its intracellular com ponents into the cytoplasm. It is normally a result of a harsh mechanical or chemical insult to the cell that di srupts its homeostasis lead ing to the release of a variety of factors that assist in the cell death process. Massive inflammation results which either helps in the healing or elimination of the damage d tissue. Necrotic cell death can also substitute in cases where the apoptotic process is nonfunctional (Proskuryakov et al., 2003). Recent

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19 reports have revealed the therapeutic benefits of this form of cell death in trea ting cancer (Zong et al., 2004). Traumatic Brain Injury Traum atic brain injury (TBI) has been one of the major health problems affecting young male adults between the ages of 15 to 24 and elde rly people of both sexes over 75 yeas of age in the U.S.A. TBI is defined as acquired brain injury due to sudden mechanical trauma to the head region that causes brain damage (http://www.ninds.nih.gov/disorders/tbi/detail_tbi.htm) (Pineda et al., 2004b). The financial burden associated w ith TBI grosses to more than $56 billion a year with more than 5 million Americans alive today who have had a TBI resulting in a permanent damage to the brain. Survivors of TBI often suffer cognitive deficits and also changes in the behavior and communicative abil ities. Some patients are al so susceptible to secondary complication such as epilepsy, Alzheimers dise ase, Parkinsons dise ase and post-traumatic dementia. Traumatic brain injury (TBI) is a complex neur ological disorder that involves a sequence of events leading to cell death in the brain. Cell death in TBI has been attributed to the primary mechanical injury followed by a cascade of protea se activation leading to biochemical secondary injury. Trauma to the head can result in damage to the underlying soft ti ssue. Majority of brain contusions result in seizures and depending on the severity of the injury, the underlying brain tissue and meninges can suffer shear damage due to skull fractures. Brain trauma also results in damage to the vascular system and can re sult in hematomas whic h may, depending on its location have a detrimental effect on neuronal surv ival. The secondary biochemical injury occurs days to weeks after the primary mechanical injury. This occurs in the form of a variety of insults such as neurotoxic ischemia, proteolysis, oxidati ve stress and inflammatory cytokines mediated by activation of host immune system (microglia and lymphocytes) (McIntosh et al., 1998; Wang

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20 et al., 2004; Ottens et al., 2006; Wieloch and Nikolich, 2006). Another major player is the disruption in Ca+2 ion homeostasis following injury. Following injury there is an increase in the intracellular calcium concentration which result s in the activation of many proteases one of which is the cysteine prot ease calpain. Increases in Ca+2 levels also induce excessive release of the excitatory amino acid glutamat e via activation of the glutamate receptor leading to glutamate excitotoxicity. These biochemical events have th e potential to lead to prolonged depolarization accompanied with ATP depletion, compromise in the axonal integrity and also an increase in intracranial pressure (Kupina et al., 2003; Yi and Hazell, 2006). The well documented proteolytic events following TBI ha s been cell death resulting from the activation of calpains and caspases resulting in neuronal cell death in a mu lti-faceted manner (Raghupathi, 2004). Oncotic cell death is sudden and more dras tic, occurring due to the mechanical injury to the brain tissue and is more immediate. Apoptotic cell death foll owing brain trauma resulting from activation of caspases is a more delayed and secondary bioc hemical response to the primary mechanical injury. The evidence for activation of calpains and caspases protease has been well studied employing the cytoskeletal protein II-spectrin. TBI studies have demonstrated II-spectrin to undergo proteolysis by both calpains and caspases is II-spectrin (Pineda et al., 2004a; Czogalla and Sikorski, 2005). Thus, understanding the molecular and cellular mechanisms following TBI would help develop better therap ies to arrest the neuronal and f unctional loss associated with the injury. Autophagy in Brain Injury and Homeostasis Recent stud ies have demonstrated an integral ro le for autophagy in maintaining the homeostasis and development of the mammalian brain. Separate studies have shown the importance of the autophagy protein Atg5 in the development of th e central nervous syst em (Hara et al., 2006; Komatsu et al., 2006). The presence of autophagic vacuoles has been observed in dystrophic

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21 neurons and implicated to be either benefici al or detrimental in the pathogenesis of neurodegenerative diseases such as Alzheimers and Parkinsons diseas e (Webb et al., 2003; Zhu et al., 2003; Li et al., 2007; Nixon, 2007; Sarkar et al., 2007b) Autophagy has also been demonstrated to be beneficial in the clearance of mutant aggr egates in polyglutamine disease states (Williams et al., 2006; Floto et al., 2007; Sarkar et al., 2007b; King et al., 2008). Excitotoxicity resulting from excess release of the neurotransmitter, glutamate has been one of the major factors contributing to the pathology afte r brain injury (Ankarcr ona et al., 1995; Ferrer et al., 1995; Portera-Cailliau et al ., 1997). Excessive stimulation of the glutamate receptor in the spinal cord motor neurons and the organotypic hippocampal slice cultures has shown the induction of autophagosome formation and subs equent neuronal death. The presence of autophagosomes evidenced in the neurons made the authors speculate if ex citotoxicity-associated autophagy induction results in auto phagic cell death (Borsello et al., 2003; Tarabal et al., 2005). (Wang et al., 2006) demonstrated the induction of autophagy in Lurcher mice, a genetically engineered animal model of excitotoxicity, shows the accumulation of autophagosomes at the distal ends of the axons sugges ting a breakdown in the retrograde transport of materials in the neuron. Studies have also demonstrated the i nduction of autophagy after mechanical or biochemical insult in neurons in the brains of experimental animals. Beclin-1 (Atg6), LC3 (Atg8), Atg7 and Atg5 have been the well documen ted autophagy proteins in studies associated with brain injury. (Diskin et al., 2005) demons trated the up regulation of one of the key autophagy proteins beclin-1 (Atg6) n ear the site of injury in their rat model of closed head injury. Beclin-1 is a bcl-2 interacting protein that has be en documented to be an important player in the induction of autophagy. Bcl-2, the an ti-apoptotic protein has been s hown to interact with beclin1 via the BH-3 domain on beclin-1. Bcl-2 exerts a controlling effect on the activity of beclin-1.

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22 Under normal homeostatic conditions, beclin-1 is bound to bcl-2 and hence not available to induce autophagy. Following a disruption in the cell homeostasis due to stress conditions or insults, beclin-1 interaction with bcl-2 is w eakened and beclin-1 now becomes available to induce autophagy. Beclin-1, thus is available to complex with the Class III PI3K and the Vps34 protein to signal and activate autophagy protei ns downstream. This inte raction between these proteins is of utmost significance as it gove rns the switch between inducing autophagy or apoptosis (Pattingre et al., 2005; Pattingre and Levine, 2006; Er lich et al., 2007; Feng et al., 2007; Maiuri et al., 2007b). Elevated levels of beclin-1 was observ ed at early stages post-injury and maintained in the neurons for at least 3 weeks after injury. High levels of beclin-1 were also observed in astrocytes starting at 3 days following injury. A follow up study by the same group showed that the increased levels in beclin-1 were observed in neurons in response to the injury. They speculated that the overexpression of beclin-1 protein at th e site of injury can enhance autophagy induction as a mechanism to discard inju red cells and reduce the extent of damage to cells from the injured components (Erlich et al ., 2006). Recently autophagy assosciated neuronal death has also been studied in neuronal injury models. (Uchiyam a et al., 2008) demonstrated that hypoxia/ischemia brain injury in the neonatal brain results in en ergy failure, oxidative stress and unbalanced ion fluxes inducing elevated levels of autophagy in the brain neurons. Their results demonstrated caspase-dependent and independe nt cell death in the hippocampal pyramidal neurons with the accumulation of LC3-PE positive autophagosomes after ischemic brain insult. Significant neuroprotection of the pyramidal neur ons was observed in mice deficient in ATG7, a gene involved in the regulation of autophagy, im plying autophagic cell death (Koike et al., 2008). Autophagy has also been demonstrated in th e brains of rodent models by the presence and accumulation of autophagy protein LC3 (Atg8) follo wing traumatic brain inju ry (Liu et al., 2007;

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23 Clark et al., 2008). Studies exploring the role for autophagy in brain trauma are still in its infancy. Further studies have to be conducted to elucidate the role of autophagy and autophagic cell death after brain injury. Figure 1-1. Illustration of the sequence of ev ents in the induction of autophagy and the formation of autophagosomes in eukaryotic cells.

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24 Figure 1-2. Autophagy proteins (Atg) involved in the elongation of autophagosomes.

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25 CHAPTER 2 MATERIALS AND METHODS Animal Treatment Appropriate pre-and post-injury m anagement guidelines were maintained and these measures were done in compliance with guideline s set forth by the University of Florida (UF) Institutional Animal Care and Use Committee (I ACUC) and the National Institutes of Health (NIH) guidelines detailed in the Guide for th e Care and Use of Laboratory Animals. All experiments were performed using male Sprague -Dawley rats (Harlan, I ndianapolis, IN, USA) that were aged 60 days and weighed between 240 to 270 g. Animals were housed in groups of two per cage and maintained on a 12 h light/dar k cycle (lights on 7 AM to 7 PM). Food and water were available ad libitum. Gel Electrophoresis and Electrotransfer The cell lysa tes were collected at different tim e points after treatment with the appropriate media using lysis buffer containing 1% (v /v) Triton X-100, 5 mM EGTA, 5 mM EDTA, 150 mM NaCl and 20 mM Tris HCl (pH 7.4). The prot ein content was determined using DC Protein Assay (Bio-Rad, Hercules, CA) and the prot ein concentration was standardized to 1 g/ L. Twenty micrograms of protein were subjected to SDS-PAGE gel electrophoresis on 4-20% or 6% Tris-glycine gels (Invitrogen, Carlsbad, CA) and then transferred onto PVDF membrane on a semi-dry electro transferring unit (Bio-Rad). Following the transfer, the membranes were blocked in 5% nonfat dry milk in 1X Tris buffered saline with Tween-20 (TBST) and probed overnight with primary antibody at 4oC. The following day, the membranes were washed with TBST and probed with either secondary peroxidase conjugated anti-rabbit or the biotinylated anti-mouse antibody. Immunoreactivity was detected by either using streptavidin alkaline phosphatase conjugate tertiary antibody or enhanced chemiluminescence (ECL) reaction

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26 (Amersham, Piscataway, NJ). Densitometric qua ntification of the bands was performed using ImageJ software (version 1.29x; NIH, Bethesda, MD). Immunoblot Analysis and Antibodies Immunoblotting m embranes containing tissue prot ein were incubated overnight with the primary antibody at 4oC. On the following day, the membrane s were washed three times with TBST and probed with the secondary antibody for an hour. Immunoreactivity was detected by using streptavidin alkaline phosphatase conjugate tertiary antibody. Monoclonal anti-mouse IIspectrin (Affiniti Research Products, Ltd., UK) and antiactin (Sigma Chemical Co., St. Louis, MO), were used at a dilution of 1:4000 in 5% milk. Antibodies rabbit polyclonal antiIIspectrin and anti-caspase-3 sp ecfic spectrin breakdown produc t of 120 kDa (SBDP120) were made in-house. Anti-NeuN antibody was obtained from Chemicon Laboratories (Temecula, CA) and anti-LC3 antibody from Novus Biologicals (Littleton, CO). Peroxynitrite was purchased from Calbiochem (San Diego, CA). Antibodies anti-GAPDH (EnCor Biotechnology, Alachua, Gainesville), were used at a dilution of 1:1000 in 5% milk. Secondary biotinylated antibodies (Amersham Biosciences, United Kingdom) and streptavidin al kaline phosphatase conjugated tertiary antibody (Amersham Biosciences, United Kingdom) were used at a dilution of 1:3000 in 5% milk. Cell Culture and Amino Acid Starvation Treatment PC-12 cells were incubated in DME M supplem ented with 10% fetal bovine serum (FBS), 10% heat-inactivated ho rse serum (HS), penicillin (60,000U/L ) and streptomycin (60mg/L) and amphotericin B (250 g/ml) at 37oC with 5% CO2. For experimental purposes the cells were plated in complete medium then at 75-80% confluency they were washed twice with PBS and incubated in serum free medium (SFM) w ith or without maitotoxin (MTX, 0.3 nM), staurosporine (STS, 0.5 M), or in amino acid de prived medium Earls balanced salt solution

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27 (EBSS), (a starvation media and autophagy inducer) for th e different time points as discussed for each assay. MTX is a calcium channel opener known to induce oncosis while STS is a broad spectrum inhibitor of protei n kinases inducing apoptosis. Primary Cultures and Treatments Cerebellar cultures were obtained from disso ciated cerebella of 6-8 day old Sprague Dawley rat pups (Harlan Laboratories) and plat ed in Dulbeccos m odified eagles medium (DMEM) supplemented with extra glucose, 25 mM KCl, 10% fetal bovine serum on culture dishes (Nunc plates, Fisher). 1 -arabinofuranosylcytosine (10 M) was added to the culture medium 22 hours after plating to prevent the prol iferation on non-neuronal ce lls for 48 hours. On the 8th day following harvesting, the neurons were exposed to different treatment conditions and subsequent experimental end points. The neurons were treat ed with or without NMDA (200 M) for different time periods and the cells were even tually lysed with triton based lysis buffer for protein immunoblots. The other treatment condition involved a co -treatment of NMDA with 3methyladenine (3-MA, 10 mM). For fluorescent microscopy, the neurons were cultured on glass coverslips coated with poly-l-lysine and treate d in a similar manner as the cultures on plates. MTT Assay Cell death was assayed using the 3-(4,5-dim e thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PC-12 cells were plated in 96-well plates equally and incubated at 370C for 48 hours prior to treatment. At respective time points, the wells we re incubated with 15 L of the dye solution available in the kit (Cell Tite r 96 Non-Radioactive Ce ll Proliferation Assay, Promega, Madison, WI) for 4 hours. After incubation, 100 L solubilizing solution available in the kit was subsequently added to solubilize th e formazan crystals formed in the previous incubation. Following the solubilization of the fo rmazan crystals, absorbance was read at 570nm.

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28 Lactate Dehydrogenase Release Assay (LDH assay) of Cell Death Lactate deh ydrogenase release assay was performed to asse ss cell death by measuring the release of lactate dehydrogenase in the medium from damaged cells due to necrosis and secondary necrosis following apoptosis or autoph agic cell death. The cells at 80% confluency were incubated in SFM (Control), MTX or STS in SFM or EBSS (starvation) media for a period of 5 days in a 24-well plate. Culture medium, 25 L, was collected after 0, 3, 6, 12 h and 1, 2, 3 and 5 days and stored at -20oC in 96-well flat bottom plates. Detection reagent (CytoTox One Reagent, Promega) 25 L was added to each well containing the culture medium and incubated for 10 minutes in the dark at room temper ature. Fluorescence was measured (excitation wavelength: 560 nm and emission wavelength: 5 90 nm) using a fluorescent microplate reader (SpectraMax Gemini, Molecular Devices, Sunnyvale, CA). Six replicates for each time point per experiment were assayed and three such experi ments were performed. Th e arbitrary fluorescent unit values were plotted against time. In a separate set of experiments cells at 80% confluency were pre-incubated for an hour with and wit hout the following inhibi tors: 3-MA (10 mM; autophagy inhibitor), z-VAD-fmk (30 M; caspase inhibitor), Boc-D-fmk (30 M; caspase inhibitor) and SJA-6017 (50 M; calpain inhib itor) before incubation in EBSS supplemented with the inhibitors. The samples were then te sted by the LDH assay after 12 and 24 hours. Six replicates for each time point and for each treatment were assayed. MDC Labeling of Autophagosomes and Nuclear Morphology Using Hoechst 33258 Staining The PC-12 cells were stained with 0.05 m M monodansylcadaverine (MDC) in PBS after 3, 6, 12 hours and 1 day time points at room temp erature (RT) for 10 minutes.26 The cells were washed 2X with phosphate buffered saline (PBS ), mounted using antifade solution (Prolong Antifade, Molecular Probes) and immediately obs erved using the Zeiss fluorescence microscope.

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29 To assess nuclear morphology, the cells were treate d with the appropriate media for either 1 day (STS, starvation, control) or 3 hours (MTX). They were washed twice with 1X PBS and incubated with Hoechst 33258 (0.5 mg/mL) in PBS at room temperature for 10 minutes. Following incubation, the cells were washed tw ice with 1X PBS and observed under the Zeiss fluorescence microscope. Apopto tic cells were characterized by condensed nuclei morphology. Immunocytochemistry PC-12 cells were plated on collag en I coated coverslips and at 80% confluency the cells were treated with EBSS. At va rious experimental time points the cells were fixed in 4% paraformaldehyde for 10 minutes at 4oC, washed with PBS, blocked for 30 minutes in 5% normal goat serum (NGS) in TBST and then incu bated overnight with LC-3 antibody (1:100) in 5% NGS at 4oC. The bound antibody was subsequently detected by incubati on with goat antirabbit Alexa red-conjugated secondary antibody (1: 3000). The cells were ri nsed twice with 1X PBS and subsequently mounted using the moun ting medium for fluorescence containing DAPI (Vectashield, Vector Laboratories, Burlingame CA) and viewed under the Zeiss fluorescent microscope. Cerebellar cells plated on coverslips were fixed using freshly prepared 4% paraformaldehyde solution for 10 mins at 4oC, washed in pure methanol and then permeabilized with 1X tris buffered saline tween (TBST, Sigm a Laboratories, St. Louis, MO). Following TBST washing, the cells were incuba ted in 5% normal goat serum ( NGS) at 37 oC for 30 minutes before incubating with the primary antibody micr otubule associated light chain-3 (LC-3; Atg8; 1:1000) in 5% NGS overnight at 4 oC. On the fo llowing day, the coverslips were washed 3 times with 1X TBST and incubated with the Alexa Fluor (Molecular Probes, Carlsbad, CA) red or green-conjugated secondary antibod ies (1:3000) for 1 hour at 37 oC The coverslips were then

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30 washed with 1X TBST and then mounted with the mounting medium Vectashield (Burlingame, CA) and observed under the microscope. Caspase-3 Activity Assay PC-12 cells were incuba ted in EBSS (starvat ion) and SFM (control) treatment conditions for set periods of time to determine the caspa se-3 activity. Caspase-3 activity was performed using Apo-ONE caspase-3 activity assay kit (Pro mega). The cells were incubated in the ApoONE caspase-3 reagent for 2 hours and fluorescen ce was read at excita tion wavelength of 485 20 nm and an emission wavelength of 530 25 nm. To assay for caspase-3 activity, control, NMDA-treated and NMDA/3-MA co-treated granule neurons from three different wells (12 and 24 h) were scraped in a buffer containing 20 mM Tris-HC1 (pH 7.4 at 4C), 150 mM NaC1, 1 mM dithiothreitol, 5 mM EDTA, 5 mM EGTA, and 1% (vol/vol) Triton X-100 for 1 h. The clea red lysates were mixed with 50% (vol/vol) glycerol. Cell lysates were assayed with 100 M acetyl-Asp-Glu-Val-Asp-7-amido-4methylcoumarin (Ac-DEVD-MCA; Bachem Biosci ence), 100 mM HEPES, 10% glycerol, 1 mM EDTA, 10 mM dithiothreitol. Fluorescence (excitation, 380 15 nm; emission, 460 15 nm) was measured at 60 minutes using a fluorescen t microplate reader (SpectraMax Gemini EM, Molecular Devices) as described pr eviously (McGinnis et al., 1999). Materials Dulbeccos modified eagle m edium (DMEM) Earls Balanced Salt Solution [(EBSS) without calcium or magnesium fetal bovine seru m and horse serum were obtained from Gibco Laboratories (Carlsbad, CA). Staurosporine (S TS), maitotoxin (MTX), monodansylcadaverine (MDC), and 3-methyl adenine (3-MA) were pur chased from Sigma-Aldrich Laboratories (St. Louis, MO). Prolong Antifade was from Mol ecular Probes (Eugene, OR) while the broad spectrum caspase inhibitors zVAD-fmk (Z-Val-Ala-Asp (OMe)-fluoromethyl ketone) and Boc-

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31 D-fmk (BocAsp fluoromethyl ketone) and the cal pain inhibitor SJA-6017 were purchased from Calbiochem (San Diego, CA). Serum free me dium (SFM) was comprised of penicillin (60,000U/L), streptomycin (60mg/L) and amphotericin B (250 g/ml) in DMEM. Chemicals and Antibodies Nmethyl D aspartate (NM DA), 3-methyladenine (MA), monodansylcadaverine (MDC) was purchased from Sigma Laboratories (St. Lo uis, MO). Prolong Antifade was purchased from Molecular Probes (Eugene, OR). Fetal bovine se rum and Dulbeccos modified eagles medium (DMEM) was from Gibco laboratories (Grand Island, NY). Antibodies rabbit polyclonal antiII-spectrin and anti-caspase-3 specfic spectrin breakdown pr oduct of 120 kDa (SBDP120) were made in-house. Anti-actin antibody was purchased from Sigma laboratories (St.Louis, MO), anti-NeuN antibody was obtained from Chemicon Laboratories (Temecula, CA) and anti-LC3 antibody from Novus Biologicals (Littleton, CO). Peroxynitrite wa s purchased from Calbiochem (San Diego, CA). Experimental Paradigm of Traumatic Brain Injury A controlled cortical im pact injury device was used to produce traumatic brain injury (TBI) in adult rats (Dixon et al. 1991; Pike et al. 1998). Rats divided into 2 groups received either TBI injury or sham-injury (n=5). Rats were anesthetized with 4% isoflurane in a carrier gas of 70% N2O/30% O2 followed by maintenance anesthesia of 2% isoflurane in 70% N2O/30% O2. A midline cranial incision was made, the skin and the underlying soft tissues reflected and a unilateral (ipsilateral to the side of impact) craniotomy wa s performed adjacent to the central suture, midway between the bregma and lambda to expose the cortical tissue. Brain trauma was produced by impacting the right co rtex (ipsilateral cortex) with a 6-mm diameter impactor tip at a velocity of 4 m/s with a 1.6 mm compression for 150 ms. Sham animals (n=5) received the craniotomy without the impactor injury and nave control anim als were kept under the same

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32 environmental conditions without receiving a craniotomy or in jury. All procedures and postoperative care for the animals were conducted in accordance with the guidelines set forth for humane handling of the animals by the University of Florida Institute for animal care and use committee and the National Institute of Health. Tissue Collection Post Traumatic Brain Injury Procedures After the appropriate tim e poi nts (2h, 6h, 1d and 2d) following injury, TBI (n=5) and shaminjured (n=5) animals were deeply anaesthetiz ed using sodium pentobarbital (100 mg/kg, i.p). and decapitated at the loss of the toe pinch reflex Cortical brain regions ipsilateral and in close proximity to the cortical impact were rapidly di ssected, snap-frozen in liq uid nitrogen and stored at -80C for further processing. Cerebral cortex tissues from di fferent animals were pulverized and homogenized in a small mortar-pastel to a fine powder set over dry ice. The homogenized cortical tissue powder was then lysed for 90 minutes at 4C with a 1% (v/v) Triton X-100, 5 mM EGTA, 5 mM EDTA, 150 mM NaCl and 20 mM Tr is HCl (pH 7.4), 1 mM dithiothreitol (all chemicals from Sigma-Aldrich, St. Louis, MO) a nd a Complete Mini protease inhibitor cocktail tablet (Roche Biochemicals, Indianapolis, IN). Brain cortex lysates were then centrifuged at 15,000 r.p.m. for 10 minutes at 4C. The supernatant was retained and collected at 4C to prevent proteolysis. The protein content was determined using a DC Protein Assay (BioRad Laboratories, Inc., Hercules, CA USA), after which the protein concentration was standardized to 1 g/ L for immunoblotting analysis. Twenty micr ograms of protein were subjected to SDSPAGE gel electrophoresis on 4-20% or 6% Tris-glycine gels (Invi trogen, Carlsbad, CA) and then transferred onto PVDF membrane on a semi-dry electro transferring un it (Bio-Rad). Following the transfer, the membranes were blocked in 5% nonfat dry milk in 1X Tris buffered saline with Tween-20 (TBST) and probed overnight with primary antibody at 4oC.

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33 Immunoblots were probed with an anti-MA P-LC3 (Novus biological, Littleton, CO) antibody or anti-bcl-2 (BD biosciences, San Jose, CA) or anti-beclin-1 (Santa Cruz biotechnology, Santa Cruz CA) overnight at 4oC. Following the overnight incubation with the primary antibodies, the PVDF membranes were incuba ted with biotinylated anti-rabbit (for bcl-2 and MAP-LC3) and biotinylated anti-sheep (for b eclin-1) for an hour in 5% nonfat dry milk in 1X Tris buffered saline with Tween-20 (TBST). Immunoreactivity was finally detected by using streptavidin alkaline phosphata se conjugate tertiary antibody. Statistical Analysis One-way ANOVA with Tukey post hoc test wa s used to draw co mparisons between groups in the LDH assay. Data was plotted as m eans S.E.M. (standard error of the mean). Students t-test was performed to draw statisti cal comparisons between two treatment groups and a p<0.05 was considered to be statistically signi ficant. Pairwise comparisons were made using the students t-test while one-way ANOVA with Tukey post hoc test was used to draw comparisons between multiple groups. Data was plotted as means S.E.M. (standard error of the mean). A value of p 0.05 was considered to be statistically significant.

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34 CHAPTER 3 AMINO ACID STARVATION INDUCED AUTO PHAGIC CELL DEATH IN PC-12 CELLS: EVIDENCE FOR ACTI VATION OF CASPASE-3 BUT NOT CALPAIN-1 Introduction Cell death, a highly regulated and we ll orches trated process, is classified into three types: apoptotic (type I), autophagic (type II) and necr otic/oncotic (type III) (Baehrecke, 2003; Liu et al., 2004). While mechanisms underlying apopto tic cell death have been well documented (Hengartner, 2000), autophagic cell death is still poorly understood. Autophagy occurs under stress conditions and aids in replenishing ami no acids by degrading intr acellular macromolecules (Meijer and Codogno, 2004). It is characterized by the presence of double membrane vesicles found in the cytoplasm termed autophagosomes Autophagosomes sequester cytoplasmic constituents including organelles and transport them to lysosomes where they are degraded and the amino acids recycled for rebuilding cellular machinery. Deprivation of nutritional support factors for prolonged periods has been reported to culminate in autophagic cell death (Clarke, 1990; Lockshin and Zakeri, 2004). Growth factor or nutrient de privation mediated autophagic cell death has been documented in neuronal cell culture system s (Larsen and Sulzer, 2002; Xue et l., 1999). Autophagic cell death, a conserved integral pathway involve d in mammalian cell development (Levine and Klionsky, 2004) is also activat ed under certain pathologi cal conditions such as the neurodegenerative diseases (Ste fanis et al., 2001). A recent study observed an upregulation of Beclin-1 (Atg6), one of the proteins required for the autophagic process, at the cortical site of injury in mouse brains following closed head in jury (Diskin et al., 2005) Other recent studies suggest there is evidence for th e protective role of autophagy in neurodegenerative disorders that are characterized by aggregate pr oteins such as Huntingtons, Creutzfeldt-Jakob and Alzheimers disease (Ravikumar et al., 2004; Siko rska et al., 2004; Yu et al., 2004).

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35 It has long been believed that the two prime protease families involved in cell death are the calpains [calpain-1 ( -calpain) and calpain-2 (m-calpain)] activated in both apoptosis and oncosis, and the caspases, especial ly caspase-3, believed to be st rictly activated in apoptosis (Chan, 2004; Green, 2005; Wang, 20 00; Liu et al., 2004). Recently there has been increased interest in caspase-independent cell death pathways (Stefani s, 2005). Apoptosis and autophagy appear to share certain common regulatory mechanisms and may in teract in a variety of ways depending upon the cellular environment and tr eatments undertaken. Autophagy may play a role in apoptosis either by preceding and eventually in itiating it or by delaying its onset (Boya et al., 2005; Canu et al., 2005). In other circumstances both pathways may be mutually exclusive (Gozuacik and Kimchi, 2004). Despite the progress made in understanding the precise molecular mechanisms in autophagy, the processes that re gulate autophagic cell death and its relation to apoptosis are still poorly underst ood (Yu et al., 2004). In the pr esent study we evaluated the activation of caspases and calpa ins following amino acid (AA) starvation-induced autophagic cell death in PC-12 cells. Results Autophagy Is Induced under Amino Acid Starvation Conditions PC-12 cells incubated in serum free medium (SFM; control cells) or subjected to amino acid deprivation in Earles Bala nced Salt Solution (EB SS; AA deprived cells) for 3, 6, 12 hours, were subsequently incubated with monodansycad averine (MDC; 0.05 mM), a fluorescent marker for autophagosomes (Biederbick et al., 1995) and analyzed for autophagosomes using fluorescence microscopy (Fig. 31A). The starved cells showed MDC labeled autophagosomes as early as 3 hours and these persisted for at least 12 hours while the SFM treated cells showed little to no MDC incorporation. Th is is consistent with previo usly reported results (Dunn, 1990) demonstrating autophagy induction under AA st arvation conditions. To further confirm

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36 autophagy, immunostaining with LC-3, a mammalian homologue of yeast Atg8 which associates with autophagosome membranes (Kabeya et al., 2000) was performed (Fig. 3-1B). LC-3 expression appeared more intense in AA starved cells when compared to control cells at the various time periods examined. These results confirm induction of autophagy in PC-12 cells under AA starvation conditions. Prolonged Amino Acid Starvation Induces Cell Death and Evidence of II -spectrin Breakdown Having established autophagy induction followi ng AA deprivation, we hypothesized that prolonged AA starvation would eventually lead to autophagic cell death. To assess cell death, lactate dehydrogenase (LDH) re lease assay was performed and the fluorimetric readings at different time points were plotted (Fig. 3-2A). Amino acid deprived cells incubated in EBSS (Starv) were able to su rvive the initial periods of starvation. However, amino acid deprivation, when prolonged for 24 hours or more, resulted in an increase in cell death when compared to controls. The cell death time course was similar to that induced by STS treatment. In contrast cells treated with MTX showed rapid cell death peaking as early as 3 hours. Also observed was an upward trend in cell death in the control cells after appr oximately 80 hours of incubation. These results suggest that AA de prived cells are protected from cell death at earlier time points by the induction of autophagy but prolonged starvati on results in cell death. To confirm the LDH data we also performed the MTT assay for ce ll viability. The cell deat h pattern observed among the amino acid deprived cells (Starv) was similar to that induced by STS treatment and coincided with the LDH data (Fig. 3-2B). To study the possible activation of caspase s and calpains in starvation induced autophagy, cell lysates for the different treatment s were collected at various time points and analyzed by Western blot analysis. We examined the cytoskeletal protein, II-spectrin, a well

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37 established substrate cleaved by both calpains and caspases unde r cell death conditions.29 When compared to control, lysates from the AA star ved cells showed an increase in spectrin breakdown products 120 kDa (SBDP120) and 150 kDa (SBDP150) day 1 through day 5 (Fig. 2C). While the observed SBDP150 band can be repr esentative of either calpain (SBDP150) or caspase-3 (SBDP150i) mediated proteolysis, the SBDP120 proteo lytic fragment is caspases-3 specific.29 The MTX and STS treated cell lysates used as positive controls for calpain and caspase-3 specific spectrin pr oteolysis showed calpain specific SBDP150 and 145 and caspase specific bands SBDP120 and SBDP150i, respectively (Fig. 3-2C). Activation of Caspase-3 and not Calpains in Amino Acid Starvation Mediated A utophagy Based on the II-spectrin proteolysis pattern observed we further examined the roles of calpain-1 and caspase-3 in II-spectrin breakdown and autophagy. The cell lysates obtained from the different treatments were probed with an antibody specific for SBDP150 which in our previous studies have shown to be calpain specific (Nath et al., 1996; Pike et al., 1998). There was no detectable increase in the SBDP150 levels in the starved cells even at the time points when cell death was observed. The MTX (3 hours) and STS (1 day) treated positive controls showed that the antibody is able to detect the calpain-mediated SBDP150 (Fig. 3-3A). Subsequently, the antibody specific for activated calpain-1 was used to directly determine its possible activation in AA starved cells as compared to cells in cubated in SFM. As expected, activated calpain-1 was clearly detected after MTX and STS treatment, as positive controls. In contrast, AA deprived treatment conditions showed the complete absence of calpain-1 activation (Fig. 3-3B). Caspase-3 activation under AA starvation cond itions was also assessed indirectly by measuring the levels of SBDP120 using a caspase-3 specific anti-SBDP1 20 antibody (Nath et al., 1996; Pike et al., 1998). Increase s in SBDP120 levels were first observed to be significant by

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38 densitometric analysis in AA starved cells at 12 hours and this significan ce persisted for the 2 days (p<0.005; Students t-test), the time limit of our study, when compar ed to the untreated controls. A significant in crease in the intensity of SBDP120 was observed in STS treated cells when compared to controls and this increase was comparable to the intensities observed in the AA starved cells (1 day) (Fig. 3-4A). The direct approach to examine caspase-3 activation in starved cells was by immunoblot analysis of the cell lysates for activated caspase -3. Activated caspase-3 bands (19 kDa, 17 kDa) were detectable in starved cells from 12 hour s onward and densitometry analysis showed a significant increase (p<0.005) by da y 2.The expression levels at day 1, while showing a notable upward trend was not statistical significant. The activated caspase-3 levels were comparable between the STS treated and AA star ved cells (1 day) (Fig. 3-4B). To further substantiate the presence of caspase-3, following prolonged starvation, caspase-3 activity was assayed using the Apo-One Caspase-3 assay. Caspase-3 activity in st arved cells was signifi cantly higher than the control cells (Fig. 3-4C ). It peaked at day 1 following starvation which coorelates with the immunoblot data. Activation of Cathepsins Following Starvation Cathepsin B activation had previously been s hown to translocate into the cytosol during apoptosis (Guicciardi et al., 2000). Canu et al. (2005) recently dem onstrated that the cathepsin B translocation into the cytosol was not due to a compromise in the lysosomal membrane integrity or function but was rather cath epsin B independent. Since autopha gy involves lysosomal enzyme activation, the activation of cathepsin B, a lysosomal hydrolas e, during the process of starvation causing cell death was investigate d. We observed activation of activ ation of cathepsin B at the

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39 earlier stages of starvation (3 and 6 hours). This activation appear ed to cease at the later time periods when cell death was observed (Fig. 3-5). Prolonged Amino Acid Starvation Induced Nuclear Morphology and Autophagosome Abnormalit ies To assess nuclear morphology as a measure of cell death followi ng AA starvation, PC-12 cells were maintained under the different conditio ns for 1 day and then stained with the Hoechst 33258 nuclear DNA stain. Both STS treated and AA star ved cells demonstrated the characteristic DNA condensation accompanied by the collapse and shrinkage of the nucleus. Control cells, on the other hand, maintained the well defined oval nuclear morphology of a healthy cell with diffused DNA. In the MTX treated cells a more non-specific disintegration of the DNA was observed (Fig. 3-6A). Furthermore, in the cells deprived of AA, MDC-positive autophagosomes were seen at day 1. This indica tes that autophagy is still taking place in the cells even though cell death is occurring (Fig. 3-6B). The autophagosomes that were previously well defined punctuate stains (Fig. 3-1A) were obser ved as intensely stained aggr egates mostly surrounding the shrunken nucleus at day 1. Cells in cubated in SFM or treated with STS for the same time periods did not show a comparable density of MDC positive cells (Fig. 3-5B). These results are consistent with previous studies that have implicated apoptoti c processes in cell death as a consequence of autophagic inducti on under certain stress stimuli (Canu et al, 2005; Hengartner, 2005). Amino Acid Starvation Mediated Cell Death is Suppressed by Autophagy Inhibitors but not Caspase or Calpain Inhibitors To further evaluate the role of autophagy a nd caspases and calpains in starvation-induced cell death, w e incubated PC12 cells in EBSS with and without 3-methyladenine (3-MA; autophagy inhibitor), zVAD-fmk and Boc-D-fmk (two broad spectrum caspase inhibitors), and SJA-6017 (calpain inhibitor). The LDH assay pe rformed at 12 hours and 24 hours post treatment

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40 showed that starvation-induced cell death significantly decrease d with 3-MA treatment (p<0.05) (Fig. 3-7A, B). Surprisingly, the protective effe cts of caspase inhibi tor Z-VAD was marginal (less than 10%) and not statis tically significant following pr olonged AA starvation. The other broad spectrum caspase inhibito r Boc-D fmk failed to show any protection. Also, there was no protection from calpain inhibitor SJA-6017 confirmi ng our previous results that suggest that calpain is not involved in cell death due to AA starvation. Also, the treatment with 3-MA (10 mM) alone and in combination Z-VAD resulted in a robust and significant protection against cell death following AA starvation. Cell lysates an alyzed for SBDP120 by immunoblotting showed a complete loss of SBDP120 expression in cells treated with broad spectrum caspase inhibitors, MA treatment and a combination of both 3-MA with Z-VAD (Fig. 3-7C). Since cell death was strongly inhibited with the 3-MA treatment alone and considering the morphological data in Fig. 5 it appears that cell death occu rring under AA starvation conditions in PC-12 cells is mediated through autophagy and can be classified as type II autophagic cell death. Discussion Autophagy has been described as a cellular response to stressful stim uli like starvation. One of its primary functions is to recycle amino acids fr om degraded proteins fo r cellular su rvival under nutrient deprived conditions (Cuervo, 2004; Mele ndez et al., 2003; Otto et al., 2003). PC-12 cells have been acknowledged as a neuronal cell line since they respond reversibly to nerve growth factor (NGF) stimulation and acquire sympathetic neuronal phenotype capable of producing cathecholamines as neurotransmitters (Xue et al., 1999). In this study we demonstrated autophagy induction in PC-12 cells under amino aci d starvation conditions (Fig. 3-1). Also, we showed that prolonged starvation resulted in 3MA sensitive autophagic cell death (Fig. 3-7) a process that shares certain co mmon features with the apoptotic pathway (Figs. 3-4, 3-6).

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41 Autophagosomes, double membrane vesicles, have been considered to be one of the hallmark features of autophagy. These autophago somes induced under conditions of stress, are responsible for engulfing cytoso lic organelles and fusing with lysosomes where the engulfed proteins are dismantled into their constituent amino acids. The amino acids are then recycled within the cell (Wang and Klionsky, 2003). Aut ophagosomes, previously reported to accumulate MDC, an autophagolysosome marker (Mizushima, 2004; Munafo et al., 2001), were detected as early as 3 hours in amino acid star ved cells but were not found in the control cells or cells treated with either STS or MTX. While the autophagosomes were observed as punctu ate stains at earlier time periods following starvation, these punctuate markers were later transformed into condensed aggregates around the perinuclear region of the cells. These changes in the morphology of the MDC positive autophagosomes were coincident with the increases observed in cell death and the breakdown of II-spectrin (Fig. 3-2). Autophagosome formation was substantiated by the co-locali zation of MDC with a processed form, LC3-II, of microtubuleassociated protein LC-3, a mamma lian homologue of yeast Atg8 which also associates with outer membrane of the autophagosomes (Mizushima, 2004; Kabeya et al., 2000). The LC-3 labeling of phenotypically normal autophagosomes was likewise observed as early as three hours following starvation (Fig. 3-1B). To determine the involvement of caspas es and calpains, following AA starvation mediated cell death, the pr oteolytic fragments of II-spectrin were studied. The II-spectrin fragmentation pattern is used as a biomarker to specifically identify the role of calpains and caspase-3 in cell death (N ath et al., 1996). Calpain-1 was of intere st due to its role as one of the prime proteases responsible for onc otic cell death and to a lesser extent of apoptotic cell death (Wang, 2000). Calpain-1 has also previously be en found to induce cleavage and release of

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42 apoptosis inducing factor (AIF), in a caspase independent fa shion, triggering the apoptotic pathway (Polster et al., 2005). In examining the role of calpains in AA starvation mediated cell death, there was no increase of calpain-sp ecific spectrin break down product of 150 kDa (SBDP150) (Fig. 3-3A). This was confirmed by th e lack of increase of the activated form of calpain-1 (Fig. 3-3B). Cells treated with MTX, as positive control for onco tic cell death, showed strong calpain activation. These observations pose some interesting questions and the need for further investigation as the cellular concentration of calcium appears to be adequate to activate these proteases. It should be noted that the EB SS starvation media does not provide extracellular Ca2+ (or Mg2+) and this may play a role in the lack of calpain ac tivation under starvation conditions. This may suggest that the release of internal stores of Ca2+, if involved, is insufficient to generate sustained calpain ac tivation in AA starvation induced cell death. To address the involvement of caspases in cell death after prolonged AA starvation, additional tests were performed. The caspase-3 activity assay showed there was considerable caspase-3 activity (Fig. 3-4C) and this was c onfirmed by immunoblots for caspase-3 mediated SBDP120 and activated caspase-3 (Fig. 3-4A,B). These results initially suggested caspase-3 activation may play a role in autophagic ce ll death. In addition, nuclear morphology changes highlighted by Hoechst 33258 staini ng were comparable between th e AA starved cells and those treated with STS (a known apoptosis inducer) at the same time point the aggregated MDC positive autophagosomes were observed in the cells (Fig. 3-6). While cathepsin B activation was observed at the early stages in the cytoplasm following starvation, the l eaking of the lysosomal protease into the cytoplasm was limited to st arvation periods upto 6 hours but not later suggesting that the lysosomal hydrolase is not a participant in the cell death process (Kroemer and Jaattella, 2005). Also the lysosomal membrane integrity appears to remain intact, as the

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43 protease was not observed in the cytosol fo llowing prolonged periods of starvation. Taken together, these data imply that starvation induced aut ophagic cell death shares common features with classic apoptosis. The LDH assay studies of inhi bitor treated cell cultures showed a significant decrease in cell death in the presence of the inhibitor 3-MA. The data also showed a robust decrease in the levels of caspase-3 mediated SBDP120 followi ng 3-MA treatment suggesting that autophagy precedes caspase-3 activation. These findings appear to be in agreement with a previous study which had demonstrated inhibition of apoptotic markers following 3-MA treatment (Canu et al., 2005). The two broad spectrum caspase inhibito rs, zVAD and Boc-D, however, did not show significant protection from cell death under st arvation conditions within the time frame examined. This is consistent with previous findings (Xue et al.1999), which demonstrated that treatment of nerve growth fact or deprived sympathetic neurons treated with pan-caspase inhibitors prevented the morphological changes that are characteristic of apoptosis, but did not prevent cell death nor did they affect autophagic activity. Our data shows that both broad spectrum caspases inhibitors fully suppressed th e formation of SBDP120 band (Fig. 3-7C). The results suggest there is an activation of caspase-3 in autophagic cell death, but the inhibition of caspase-3 is not sufficient by itself to protect against this form of cell death. While we and others have demonstrated that concurrent with caspase-3 activation there is calpain activation in most forms of classical apoptosis including STS treat ment (Fig. 3-4) and NGF deprivation in PC-12 cells (Nath et al., 1996), our results demonstr ated that autophagic cell death following AA starvation does not activate calpain nor does it se em to be required (Fig. 3-3). The data suggests a subtle but important differences between apoptosis (type I) and autophagic cell death (type II).

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44 Conclusion In summ ary, prolonged AA star vation induces autophagic cell death in a rat neuronal-like cell type (PC-12). While benefici al early following stress activ ation, at some point, autophagy unchecked will begin to damage the cellular mito chondria and other vital organelles necessary for its continued survival resul ting in cell death. The neural ce ll autophagic cell death paradigm described in this study will allow fo r further exploration of this fo rm of cell death in neurological disorders such as stroke and traumatic brain injury, and neurodegenerative conditions like Alzheimers disease.

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45 Figure 3-1. Autophagosome staining with MDC and LC3 after amino aci d starvation of PC-12 cells. A) In the PC-12 cells, MDC (50 M) accumulates as a punctate pattern (arrows) predominantly in the cytoplasm under amino acid (AA) starvation conditions (starv; EBSS treated) beginning as early as 3 hours. Inserts highlig ht portions of the cells expressing the MDC positive autophagosom es. Control (Ctrl) cells are incubated in SFM. Photomicrographs are at 400X magni fication and the scale bar is 20 m. B) DAPI (blue) co-localized with LC3 (red) a known autophagosome membrane marker under conditions of AA starvation and cont rol conditions at various time points. Photomicrographs are at 400X magnif ication and the scale bar is 20 m.

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46 0 0.5 1 1.5 2 2.5 3 3.5 03612244872Time (h)MTT AssayOptical Density (570nm) Ctrl Starv MTX STS 100 300 500 700 900 1100 1300 013612244872120 Time (h)LDH Release (arbitrary fluorescent units) Ctrl Starv MTX STSC. A. 3h 1d2d3d 12h 5d 6h SBDP150/150i SBDP120 II-spectrinCtrl Ctrl Ctrl Ctrl Ctrl Ctrl Ctrl Starv Starv Starv Starv Starv Starv Starv MTX Ctrl STS II-spectrin SBDP150/150i SBDP120 SBDP145Ctrl3h1d B. D. 0 0.5 1 1.5 2 2.5 3 3.5 03612244872Time (h)MTT AssayOptical Density (570nm) Ctrl Starv MTX STS 100 300 500 700 900 1100 1300 013612244872120 Time (h)LDH Release (arbitrary fluorescent units) Ctrl Starv MTX STSC. A. 3h 1d2d3d 12h 5d 6h SBDP150/150i SBDP120 II-spectrinCtrl Ctrl Ctrl Ctrl Ctrl Ctrl Ctrl Starv Starv Starv Starv Starv Starv Starv MTX Ctrl STS II-spectrin SBDP150/150i SBDP120 SBDP145Ctrl3h1d B. D. Figure 3-2. Amino Acid starvati on induces cell death. A) i) Qu antification of the LDH (lactate dehydrogenase) assay of cell death. The gra ph depicts cell death under control (SFM; ) and starvation conditions (EBSS; ) from 0 to 120 hours post treatment, with MTX ( ) and STS ( ) acting as the positive controls for calpain and caspase-3 mediated cell death, respectively. The expresse d values are the mean S.E.M. (n=6). B) Cell death was also confirmed using MTT assay. The expressed values are the mean S.E.M. (n=4). C) Repr esentative blots (n=3) of total IIspectrin and its breakdown products (SBDP) observed unde r amino acid starvation and control conditions from 3 hours to 5 days. D) Pos itive controls are for calpain (MTX) and caspase-3 (STS) mediated spectrin breakdow n products. Spectrin breakdown products of 120 kDa, 150 kDa and 145 kDa ar e denoted as SBDP120, SBDP150 and SBDP145, respectively. SBDP150i is the brea kdown product mediated by caspases.

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47 B. A.Ctrl Ctrl Ctrl Ctrl Starv Starv Starv MTXSBDP150 1d 12h 2d3h Ctrl STS1d Activated calpain-1 (76 kDa)Ctrl Starv MTX Ctrl Starv3h1d 3h STS1d B. A.Ctrl Ctrl Ctrl Ctrl Starv Starv Starv MTXSBDP150 1d 12h 2d3h Ctrl STS1d Ctrl Ctrl Ctrl Ctrl Starv Starv Starv MTXSBDP150 1d 12h 2d3h Ctrl STS1d Activated calpain-1 (76 kDa)Ctrl Starv MTX Ctrl Starv3h1d 3h STS1d Activated calpain-1 (76 kDa)Ctrl Starv MTX Ctrl Starv3h1d 3h STS1d Figure 3-3. Evidence for the lack of calpain s under starvation conditions in EBSS. A) Representative immunoblot (n=3) for spectrin breakdown product 150 kDa (SBDP150), a breakdown product that is cal pain specific. MTX and STS incubated cells (3 hours, 1 day respectively) served as positive control for calpain mediated spectrin breakdown. Cell lysates were obtained at 3 hours and 1 day under AA starvation treatment (Starv) and control conditions (Ctrl). There was no SBDP150 for calpain found for any of the time points. B) Representative blots (n=3) of activated calpain-1 (76 kDa) under starvation and MTX treatment conditions. No activated calpain-1 was found under AA starvation trea tment. MTX (3 hours) and STS (1day) incubated cells served as positive controls.

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48 A.Ctrl Starv Ctrl Ctrl Ctrl Starv Starv Starv STSSBDP120 1d 12h 2d 6h 1d 0 10 20 30 40 50Levels of SBDP120 (arbitrary density units)6h12h1d2d1d** *Time* Control Starvation STSA.Ctrl Starv Ctrl Ctrl Ctrl Starv Starv Starv STSSBDP120 1d 12h 2d 6h 1d 0 10 20 30 40 50Levels of SBDP120 (arbitrary density units)6h12h1d2d1d** *Time* Control Starvation STS Control Starvation STS 0 5 10 15 20 25 30 35 40Levels of activated Casp-3 (arbitrary density units)TimeB.Activated casp-3 6h2d1d 12h1d Control Starvation STS*17 kDa 19 kDa Ctrl Starv Ctrl Ctrl Ctrl Starv Starv Starv STS1d 12h 2d 6h -actin (42 kDa) 1d 0 5 10 15 20 25 30 35 40Levels of activated Casp-3 (arbitrary density units)TimeB.Activated casp-3 6h2d1d 12h1d Control Starvation STS Control Starvation STS*17 kDa 19 kDa Ctrl Starv Ctrl Ctrl Ctrl Starv Starv Starv STS1d 12h 2d 6h -actin (42 kDa) 1d

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49 0 1000 2000 3000 4000 5000 6000Caspase-3 activity (arbitrary fluorescent units ) 6h12h16h20h1d2d Starvation Control Time* * *C. 0 1000 2000 3000 4000 5000 6000Caspase-3 activity (arbitrary fluorescent units ) 6h12h16h20h1d2d Starvation Control Time 6h12h16h20h1d2d Starvation Control Time* * * *C. Figure 3-4. Evidence for caspase-3 activation under amino acid starvation conditions. A) Densitometric analysis of immunoblots of SBDP120 indicates caspases play a statistically significant role in spectrin breakdown under am ino acid starvation (Starv) versus control (Ctrl) conditions as evalua ted from 12 hours to 2 days. Staurosporine (STS) treated cells represent positive c ontrol for caspase-3 mediated spectrin breakdown. Students t-test was performed to evaluate statis tical significance, *p<0.005; mean S.E.M; n=3. B) Densitometr ic analysis of activated caspase-3 showed significant increases under amino acid starvation (Starv) vers us control (Ctrl) conditions by 2 days. STS treated cells represent positive control for activated caspase-3 expression (17 and 19 kDa). Students t-test was performed to evaluate statistical significance, *p< 0.005; mean S.E.M; n=3). -Actin acts as a protein loading control indicating even protein lo ading. C) Caspase-3 activation time course in AA starved cells and control (SFM) cells (n=3). Statistical comparisons were made using students t-test and p< 0.05 was considered significant. Ctrl StarvActivated CathepsinB (30 kDa) 3h6h12h1d 3h 1d Ctrl StarvActivated CathepsinB (30 kDa) 3h6h12h1d 3h 1d Figure 3-5. Activation of cathe psin B following starvation. Repr esentative immunoblot shows that the cleaved activated fo rm of Cathepsin B (30 kDa) from whole lysates of cells peaks at 3 and 6 hours following starvation prio r to returning to normal. Cells treated with SFM were employed as controls (n=2).

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50 Starv1d Ctrl 1dSTS 1d MTX 3h Hoechst A. STS 1d MTX 3h Starv1d Ctrl 1d MDCB. Starv1d Ctrl 1dSTS 1d MTX 3h Hoechst A. STS 1d MTX 3h Starv1d Ctrl 1d MDCB. Figure 3-6. Hoechst 33258 nuclear and MDC st aining of cells under different treatment conditions. A) Hoechst 33258 i mmunostaining was performed to observe the nuclear morphology at day 1 for cells incubated unde r amino acid starvation (Starv), control (Ctrl), STS and MTX (3 hours) treatmen t conditions. Apoptotic nuclear morphology is represented by STS (1 day) treated cells while MTX (3 hours) treatment represents positive control for necrosis. Arrows i ndicate condensed DNA. B) MDC stained autophagosomes are still observed in cells under starvation conditions after 1 day (EBSS) when compared to cells incubate d in SFM (Ctrl), STS and MTX (3 hours). Photomicrographs were take n at 400X magnification and sc ale bar represents 20 m. Insert represent (2X) the region of cells showing abnormal autophagosomes.

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51 0 100 200 300 400 500LDH Release (arbitrary fluorescent units)A.** ***Starv+Z +B +SJA +MA +MA+Z 0 200 400 600 800LDH Release ( arbitrar y fluorescent units ) **Starv+Z +B +SJA +MA +MA+ZSBDP120+B +SJA +MA +MA+Z S +Z -actin (42 kDa) B. C.12 h 24 h 0 100 200 300 400 500LDH Release (arbitrary fluorescent units)A.** ***Starv+Z +B +SJA +MA +MA+Z 0 200 400 600 800LDH Release ( arbitrar y fluorescent units ) **Starv+Z +B +SJA +MA +MA+ZSBDP120+B +SJA +MA +MA+Z S +Z -actin (42 kDa) B. C.12 h 24 h Figure 3-7. Effects of caspase, calpain and autophagy inhibitors on AA starvation induced autophagic cell death and SBDP120 formati on. A) and B) Lactate dehydrogenase cell death assay results at 12 (A) and 24 (B) hours after incubation with the caspase inhibitors Z (zVAD, 30 M) and B (Boc-D, 30 M), the calpain inhibitor SJA (SJA6017, 50 M), and the autophagy inhibitor MA (3-MA, 10mM) and the combination of caspase and autophagy inhibito rs MA+Z (3-MA, 10 mM + zVAD, 30 M) in starvation media S (EBSS). The fluorescent m easurements are expressed in arbitrary units. The plates were read at excitati on wavelength 560 nm and emission wavelength 590 nm. The values are mean SEM (n=6) and one-way ANOVA with Tukey posthoc test was performed to evaluate st atistical significan ce (***p<0.006; **p<0.001; *p<0.01). C) Representative imm unoblot demonstrates the e fficacy of the inhibitors on caspase-3 activation by their e ffect on SBDP120 at day 1 (n=3). -Actin acts as a protein loading control indi cating even protein loading.

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52 CHAPTER 4 ACUTE NMDA TOXICITY IN RAT CE REBELLAR GRANULE NEURONS IS ACCOMPANIED BY AUTOPHAGY AND 3-METHYLADENINE-SENSITIVE LATE ONSET AUTOPHAGIC CELL DEATH Introduction Autophagy is an intracellular pathway that is ac tivated in response to cell stress. This is a phenom enon where the cytoplasmic organelles in the cell are engulfed by double membrane vesicles called the autophagosomes and deliver ed to the lysosomes where the organelles are broken down by lysosomal proteases and the amino acids recycled back into the cell machinery to aid cell survival under conditions of stre ss (Dunn, 1990a; Klionsky, 200 4). Some of the key proteins identified to be invol ved in this process are Atg4, Atg8, Atg12 and Atg5 (Klionsky et al., 2003). Autophagy has been reported to be vita l in the development of the central nervous system (Hara et al., 2006; Komatsu et al., 2006). It has also been documented to be constitutively active in the healthy neurons and aid survival (Boland and Nixon, 2006). Programmed cell death among neurons in the central nervous syst em is a regulated process. Depending upon the nature of the insult, the ne urons undergo apoptotic (type I) or autophagic (type II) cell death or oncotic /necrotic (type III) (Baehrecke, 2003; Liu et al., 2004). Acute excitotoxic insults that result fr om using glutamate in primary culture has been shown to induce both oncotic and apoptotic cell death in neurons (Ankarcrona et al., 1995; Nath et al., 1998). Increased excitation of the glutamate receptors by its ligand has been shown to cause an imbalance in the ionic gradient in the neurons, resu lting in an increase in the calcium and sodium levels intracellularly leading to oncosis. At the sa me time, this excessive activation has also been demonstrated to activate the endonucleases causi ng internucleosomal DNA fragmentation, thus resulting in apoptosis in neurons. Though extens ive studies have been conducted on apoptotic

PAGE 53

53 cell death mechanisms, the biochemical mechan isms involving autophagic cell death is poorly understood (Hengartner, 2000; Debnath et al., 2005; Gozuacik and Kimchi, 2007). Autophagic vacuoles have been shown to accumulate in affected neurons of several neurodegenerative diseases such as Alzheimers disease and Parkinsons disease. Wang et al., (2006) recently demonstrated the induction of autophagy was associated with axonal degeneration in Purkinje cells in Lurcher mice. More recent experimental evidence has also shown the upregulation of autophagy protein Beclin-1 (Atg 6) followi ng closed head brain injury in rats (Diskin et al., 2005). Excitotoxicity via N-methyl-D-aspartate (N MDA)-receptor overactivation, is one of the documented hallmark events that occur followi ng acute brain injury. Hence we sought to examine if autophagy is a gene ral response during excitotoxic NMDA challenge by using rat cerebellar granule neuronal cultur es in vitro. In addition, we al so addressed whether autophagic cell death contributes to NMDA excitotoxicity. Results Acute NMDA Exposure Induces Autophagy in Cerebellar Granule Neurons in Culture Rat cerebellar granule neurons were treated with or without N MDA (200 M) in serum free medium (SFM) to achieve excitotoxic and control conditions, respec tively. To assess the possible induction of autophagy following acute NM DA exposure, the neurons were stained with antibody against microtubule associated light chain-3 (LC3) protein, a known autophagosome protein marker (Kabeya et al., 2000). Neurons subjected to NMDA exposure exhibited increased punctate LC3 immunostaining as early as 3 hours th at persisted out to 12 hours when compared to the neurons under control conditions at 8 hours. Co-immunostaining with anti-NeuN antibody, a protein marker of mature neurons was employed to demonstrate that the increase in the LC3 positive autophagosomes was indeed found in neurons following NMDA treatment (Fig. 4-1).

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54 However, in most neurons examined 24 hours post-NMDA exposure, LC3 positive staining was no longer punctate but rather showed a more localized accumulation in LC3 positive bodies suggestive of aggregated autophagos omes in the cell bodies of the neurons. Similarly, the signal from the fluorescent dye monodansylcadaverine (M DC) used to label the autophagosomes also showed a strong increase in autophagosome formation in the NMDA cell cultures 6-12 hours after treatment. The MDC staining displayed the punc tate pattern of autophagosomes in both the cell bodies and the neurites afte r 6-12 hours of NMDA exposure. Th is punctate pattern of MDC staining was replaced by a more intense accumulation of what appeared to be aggregated autophagosomes in the cell bodies by 24 hours (F ig. 4-2). The observati on of LC3 and MDCpositive abnormal autophagosomes at 24 hours coinci ded with the terminal degenerative phase of NMDA-induced neuronal death (see Fig. 4-6 later) This observation might also be suggestive of a breakdown in the recycling machinery involving autophagosomes. Autophagy Protein Marker Beclin-1 is Up -Regulated Follow ing Early Phase NMDA Exposure Having established the induction of autopha gy in neurons exposed to NMDA, we sought to study protein levels of the autophagy prot ein marker beclin-1 (Atg6). We performed immunoblots on cell lysates obtained from culture s following treatment with of without NMDA at different time periods. The beclin-1 levels appear to be increased in the NMDA-treated neurons when compared to controls at time periods ranging from 3h to 24h (Fig. 4-3A). Intriguingly, a decrease in the beclin-1 band intensities was observed at later time periods (12 and 24h) of NMDA exposure with the beclin-1 levels being robustly increased at early time periods of 3h and 6h. However, graphical representation of the band intensities quantified using Image J software, yielded a significant increase in beclin-1 protein levels after NMDA exposure when compared to controls (Fig. 4-3B).

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55 Autophagy Inhibitor 3-Methyladenine (3-M A) Effectively Suppresses NMDA-Induced Autophagy Having observed NMDA-induced autophagy, we exam ined the effects of autophagy inhibitor 3-Methyladenine (3-MA) for its abi lity to suppress the process of autophagy under excitotoxic conditions. Cell lysates from either neurons treated with NMDA or a combination of NMDA and 3-MA were LC3 immu noblot analyzed for the LC3 to LC3-II conversion. Immunoblot data demonstrated a reduction of the LC3-II band in the NMDA+3-MA co-treated neuronal cultures as compared to cultures trea ted with NMDA alone (Fig. 4-4A). As expected, densitometric analysis showed no change in the LC3-I band intensity levels while the LC3-II intensities in the NMDA+3-MA co-treated cultures were significantly lower than that observed in the NMDA-treated at both 12 a nd 24 hours (Fig. 4-4B). Consistent with this observation, there was a robust reduction in the immunostaining of autophagy marker LC3 af ter co-treatment of NMDA and 3-MA in cultures compared to the cu ltures treated with or without NMDA alone (8 and 12 hours) (Fig. 4-5A). This reduced level of expression was furthe r confirmed using MDC staining. The MDC positive autophagosomes were rela tively sparse in cerebellar cultures cotreated with NMDA and 3-MA when compared to cultures treated only with NMDA. We also noted that the aggregated form of aut ophagosomal bodies observed at 16 hours post-NMDA exposure were also absent the neuronal cell cu ltures co-treated with 3-MA (Fig. 4-5B). Cell Death in NMDA-Treated Neur ons w as Alleviated by 3-MA Since sustained and unrelieved autophagy has b een documented to lead to autophagic (type II) cell death, additional studies were carri ed out to determine whether NMDA-induced autophagy contributed to cell d eath in the later stages of NMDA excitotoxicity. Neuronal cultures were treated with or without NMDA (200 M) and a third set was co-treated with NMDA and 3-MA (10 mM). Cell death was assayed by measuring the lactate dehydrogenase

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56 (LDH) enzyme release into the medium. The LDH release plotted over a 24 hours showed an increase in the LDH release in NMDA-treated cultu res compared to controls. This increase in LDH release following NMDA exposure was significan tly alleviated when the cultures were cotreated with 3-MA. The levels of LDH rel ease between NMDA and NMDA+3-MA co-treated cultures were significantly different at 6 hours through 24 hours (p<0.05) (Fig. 4-6A). Representative phase contrast images of the neurons at 16 hours following NMDA-treatment demonstrated unhealthy neurons with retracted neurites and shrunken cell bodies. The NMDAtreated neurons showed apoptotic cell morphology when compared to controls. In contrast, neurons co-treated with NMDA and 3-MA showed a dramatic sparing of both neurite and cell body morphology (Fig. 4-6B). NMDA-Induced Caspase-3 Activation is Suppressed by 3-MA In our previous study (S adasivan et al., 2006) we had demonstrated the activation of caspase-3 under conditions of prolonged ami no acid starvation-induced autophagy in PC-12 cells. Here, we tested our hypothe sis that the neuroprotective effects of 3-MA may have been achieved through caspase-3 suppression. To asse ss caspase-3 activation, we examined the proteolysis of an endogenous caspase -3 substrate (the breakdown of II-spectrin) and by employing a caspase-3 enzymatic assay (Nath et al., 1996; Nath et al., 1998). The II-spectrin breakdown profile using total antiII-spectrin antibody showed an increase of the caspase-3 generated spectrin breakdown pr oduct of 120 kDa (SBDP120) at 24 hours following treatment of cerebellar neurons with NMDA in culture. Incr eases in the calpain-generated SBDP150 and SBDP145 were also observed at 24 hours with NMDA-treated cultures (Fig. 4-7A). Staurosporine (STS) treated cultures were used as positive controls for caspase-3 activation and SBDP120 generation. To further confirm that the 120 kDa band was caspase-3 generated, immunoblots were analyzed using anti-SBDP120 specific antibody developed previously in-

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57 house (Nath et al., 2000). The blot s confirmed the appearance of the SBDP120 at 24 hours in the NMDA-treated cultures and that it was not found in the controls. More importantly, 3-MA cotreatment suppressed the increased SBDP120 levels to near normal (Fig. 4-7A). Consistent with our hypothesis, densitometric analysis of the im munoblots showed a signif icant reduction in the caspase-3 mediated SBDP120 levels in NMDA and 3-MA co-treated cultures as compared to NMDA-treated cells (Fig. 4-7B). To assay the caspase-3 protease activity directly, the caspase-3 preferred substrate Ac-Asp-Glu-Val-Asp-7-a mino-4-methylcoumarin (Ac-DEVD-AMC) was incubated with protease inhibitor-free cell lysa tes under various conditions. Caspase-3 activity was significantly increased in NMDA-treated cultures at 12 and 24 hours when compared to control cultures. On the other hand, this caspa se-3 activity was signifi cantly reduced by 3-MA co-treatment when compared to NMDA-treat ment alone (12 and 24h) (Fig. 4-7C). NMDA Mediated Protein Nitration in Cerebellar Neuron s is also Attenuated by 3-MA NMDA exposure has previously been documen ted to induce oxidative stress in neurons thus contributing to neuronal death (Arundine and Tymianski, 2004; Sanganahalli et al., 2006). We hypothesized that autophagy inhibition (with 3-MA) might also intervene NMDA-toxicity through this pathway. In our experiments oxidative stress due to NDMA was measured by identifying protein nitration at the tyrosine residue. To assess the oxidative modification of proteins by NMDA, we probed prot ein lysates from various conditi ons mentioned above with the anti-nitrotyrosine antibody in immunoblots. Mu ltiple nitrated protei n bands of different molecular weights with varied intensities were observed to be elevated following NMDAtreatment (data not shown). A nitrated protei n of 70 kDa (p70), the most prominent protein observed in the NMDA-treated cells, was thus chosen for densitometric quantification (Fig. 48A). Nitrated protein density values plotted ove r a time scale showed a significant increase at 24

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58 hours following treatment with NMDA when compared to controls (Fig. 4-8A). In cultures cotreated with NMDA and 3-MA, prot ein nitration was attenuated to control levels (Fig. 4-8B). Cerebellar granule neurons were also treated with free radical generating peroxynitrite with or without 3-MA to study th e effects of the autophagy inhibito r, whether it was indeed able to alleviate cellular oxidative stress. An incr ease in cell death measured by LDH release over time was observed in peroxynitrite-treated cultures compared to peroxynitrite and 3-MA cotreatment. In addition, densitometr ic quantification of the peroxynitrite-induced nitrated protein (p70) levels at 24 hours was also significantly suppressed by 3MA co-treatment (see Fig. 4-8 inset). Discussion In this study, we de monstrated that acut e NMDA exposure of cerebellar granule neurons induces autophagy, as established by the autophagy protein marker s LC3 and beclin-1(Figs. 4-1 and 4-3) and the fluorescent dye MDC (Fig. 4-2). We documented that while initial NMDA challenge leads to the induction of autophagy an d classical autophagosome formation (dispersed and punctate vesicles); prolonged exposure of NMDA resulted in abnormal aggregated autophagosomes in the cell bodies of neurons su ggesting the commencement of autophagic cell death. Autophagy inhibitor 3-MA not only effi ciently suppressed the NMDA-induced autophagy but also provided significant neuroprotection ag ainst prolonged exposure to NMDA (Figs. 4 and 5). Apoptosis-linked protease, caspase-3 activ ation and oxidative stress following prolonged NMDA treatment were both observed to be alleviated with 3-MA co-treatment (Figs. 7 and 8). Autophagy induction occurs in the cent ral nervous system under conditions of stress/starvation or protein aggregating neurodegenerative diseases (R avikumar et al., 2002; Webb et al., 2003; Codogno and Meij er, 2005; Komatsu et al., 2006). This study has shown that acute excitotoxicity by NMDA exposure can act as a stressor to induce autophagy in cerebellar

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59 neurons. Glutamate excitotoxicity has previously been documented as on e of the pathways of cell death following experimental traumatic brain injury (Ankarcro na et al., 1995; Ferrer et al., 1995; Portera-Cailliau et al., 1997). (Erlich et al., 2006) demonstrat ed an increase in beclin-1 expression in mice following traumatic brain inju ry suggesting that autophagy is upregulated around the regions of injury to support the cells unde r duress and help disposing off injured components. Recently, there has also been sugges tive evidence for the involvement of autophagy in chronic neurodegenerative diseases such as Parkinsons disease and Huntington disease (Dickson, 2007; Rubinsztein et al., 2007; Sarkar et al., 2007a). LC3 is a well known autophagy marker that exists as a pro-form which is processed at the C-terminal (removal of 5 amino acids) to expose th e glycine residue, which is then conjugated to the phosphatidyl ethanolamine (PE) tail. The PE-conj ugated LC3 then attaches itself to the outer membrane of the autophagosomes where it assists in the formation of mature autophagosomes. An increase in the LC3 immunostaining a nd the monodansylcadaverine (MDC) positive autophagosomes was observed following NMDA treatme nt as compared to control sets (Figs.41, 4-2). The NMDA treatment also increased the leve ls of LC3-I when comp ared to the controls at the earlier time periods (3 and 6 h). This transient enhancemen t in LC3-I protein levels in comparison to control was an indication of an e nhanced capability of the cells to launch an autophagic response (data not shown). Unexpected ly, there was no apparent increase in LC3-II levels following NMDA treatment measured by i mmunoblots at early periods. This suggests that there may be a sub pool of LC3-II that once genera ted was translocated to the outer membrane of the autophagosomes. At this time, the degrada tion and recycling of the autophagosomes inside the neurons seem to function normally, thus maintaining a constant flux of the LC3-PE. This might explain the increase in the LC3 i mmunostaining and MDC-positive autophagosomes

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60 observed, not accompanied with a similar increa se in the LC3-PE levels through immunoblots (Kuma et al., 2007; Mizushima and Yoshimori, 2007). Evidence from studies has the induction of au tophagy and subsequent neuronal death in spinal cord motor neurons and organotypic hippocampal cultures, following glutamate receptormediated injury (Borsello et al., 2003; Tara bal et al., 2005) According to another study, a buildup of autophagosomes could be observed in the axonal terminals of neurons in Lucher mice, a mouse model of excitotoxicity (Wang et al., 2006). The authors speculated that the autophagosomes observed at the terminals or dist al ends of the axon may be due to a breakdown of the retrograde transport. We extend their findings by demonstrating that NMDA in cultured neurons resulted in robust autophagosome formation throughout the cell bodies and neurites. Since prolonged autophagy has been shown to re sult in autophagic cell death (type II), we hypothesized that this form of cell death may be a crucial component to NMDA excitotoxicity (Fig. 4-9). To test this hy pothesis we employed autophagy inhibitor 3-MA and examined whether autophagy inhibition could alleviate NMDA-mediated neuronal death. The results showed effective inhibition of the processing of the LC3-I to the lipidated LC3-II protein, the latter being important in the st abilization of the autophagosomal membrane (Fig. 4-4, 4-5). Also there was a strong decrease in the LC3 imm unostaining and the MDC-positive autophagosome staining with 3-MA co-treatment following NMDA exposure. The treatment of the neurons with 3-MA in this study resulted in significant pr otection against NMDA-induced cell death (Fig. 46). Thus, our data strongly s uggest autophagic cell death to be a component of NMDA-mediated excitotoxic cell death. Apoptotic and autophagy pathways are intricat ely balanced in the cell (Yu et al., 2004; Gonzalez-Polo et al., 2005; Pattingre and Levi ne, 2006). In our experime nts we observed that

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61 NMDA-induced caspase-3 activatio n and breakdown of spectrin was 3-MA sensitive. This observation strongly suggests that NMDA-indu ced 3-MA sensitive autophagy precedes caspase3 activation as illustrated in our schematic (Fig. 4-9). Another well documented component of the pathology following NMDA exposure has be en the generation of reactive oxygen species (ROS) (eg. nitric oxide radicals) which contribu tes to dysfunctional mitochondria and subsequent cell death (Bonfoco et al., 1995; Castilho et al., 1999; Barsoum et al., 2006; Nicholls et al., 2007). NMDA-exposure induces increase in the intr acellular calcium ions which result in ER stresss and mitochondrial stress th at result in the generation of reactive oxygen species (ROS). Interestingly, previous reports (Liu and Lenardo, 2007; Scherz-Shouval and Elazar, 2007) have suggested that oxidative stress is essential for the generation of autophagy, providing a potential positive feedback (Fig. 4-9). The use of 3-MA not only inhibits autophagy but also alleviates the oxidative stress generated due to acute exposure to NDMA. The effects of 3-MA may be due to its ability to inhibit autophagy at the earlier stages which later may be capable of inducing activation of caspase-3 and ROS. In this st udy it was demonstrated that (i) prolonged NMDA exposure (24 hours) enhanced protei n nitration and that (ii) such increased protein nitration was suppressed significantly by autopha gy inhibitor 3-MA (Fig. 48).Thus, these findings place NMDA-induced autophagic pathway ahead of NMDAinduced oxidative stre ss as illustrated in Figure 4-9. Taken together, the data suggest that NMDA excitotoxicity initially induces early autophagy as a self-defense mechanism which when prolonged results in the manifestation of abnormal autophagosome vesicles, leading to autophagic cell death which would involve caspase-3 activation and oxidative stress (Fig. 4-9). Conclusion In summ ary, this study highlights that autopha gy is robustly induced in neurons subjected to excitotoxic NMDA exposure in a simple culture paradigm. In addition, it has also been

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62 demonstrated for the first time that inhibition of autophagy protects against NMDA neurotoxicity in neurons by alleviating the associated oxidati ve stress and caspase-3 activation. Importantly, the data presented in this study, when taken toge ther, strongly suggest th at autophagic cell death might be a significant component of NMDA excitot oxicity. Further studies are now being carried out in our laboratory will examine whether autophagy and autophagic cell death play a significant role in an animal model of excitotoxicity.

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63 NeuNLC3Merged NMDA 3h NMDA 8h NMDA 24h Control 8h (Enlarged) Figure 4-1. NMDA excitotoxicity results in th e induction of LC3-positive autophagosomes in rat cerebellar granule neurons. Representative fluorescent micrographs of cerebellar granule neurons in culture following treatment with NMDA (200 M) show an increase in the MAP-LC3 (red) staining at time periods 3, 8 and 24 hours. NeuN (green) was used to stain mature neurons Arrows (yellow) represent the increased LC3 staining of autophagosomes in the cell bodies of the neurons co-localized with neuronal marker NeuN (green) while arrow heads (yellow) indicate the increase in the LC3-positive autophagosomes along the axon. Re d arrows represent the LC3 staining of aggregated autophagosomes in the neur onal cell bodies at 24 hour s. Neurons in the boxed regions have been magnified. All im ages were taken at 400X magnification. Scale bar represents 20 m.

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64 24 h 12 h ControlNMDA 6 h Figure 4-2. NMDA exposure induces the formation of MDC-positive autophagosomes in cerebellar granule neurons. Representative fluorescence micrographs of granule neurons incubated with monodansylcadaver ine (MDC) show an increase in the labeling of the autophagosomes in both the neurites and the cell bodies. Arrows and arrow heads (yellow) indicate the normal punctate autophagosome staining at 6 and 12hours in the cell bodies and neurites, re spectively. Red arrows indicate the aggregated autophagosomes in the cell bodies following prolonged exposure to NMDA (200 M) (12 to24 hours). All images we re taken at 400X magnification. Scale bar represents 10 m.

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65 0 10 20 30 40 50 60 70Normalized Beclin-1 levels (arb. density units) Figure 4-3. NMDA exposure of neur ons results in an increase in the beclin-1 levels in vitro. A) Lysates were obtained at different time periods 3 and 6 hours of neuronal cultures treated with or without NMDA (200 M). These lysates were analyzed by immunoblots and probed with the anti-beclin -1 antibody (n=3). B) Quantification of the autophagy protein bec lin-1 bands in the immunobl ots was plotted. The band intensities were normalized against the load ing control. Significant increases in the band intensities of the beclin-1 levels were observed after NMDA-treated neuronal cultures as compared to controls. The e xpressed values are means S.E.M. (n=3; *p<0.05). GAPDH was used as a loading control.

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66 0 10 20 30 40 50 60LC3-I density (arb. density units)NMDA 12h NMDA 24h +3MA +3MA 0 10 20 30 40LC3-II density (arb. density units)+3MA +3MA NMDA 12h NMDA 24hA BLC3 (LC3-I) 12h 24h 12h 24hNMDA +3MA LC3 (LC3-II)12h 24h Control Figure 4-4. Autophagy inhibitor 3-MA suppresse s LC3-II formation. A) Lysates were obtained at 12 and 24 hours from neuronal cultures treated with or without NMDA (200 M) and a combination of NMDA+3-MA (200 M+10 mM). These lysates subjected to immunoblotting were probed with antiLC3 antibody. Representative immunoblot demonstrates a reduction in the LC3-II band in the neurons treated with 3methyladenine (3-MA) (n=3). -actin was used as a loading control. B) LC-3 immunoblot band intensities were quantifie d and plotted. A signi ficant reduction in the LC3-II band was observed in NMDA/3-MA co-treated neuronal cultures compared to cultures treated with NMDA alone. The expressed values are means S.E.M. (n=3; *p<0.05 compared to NMDA).

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67 (B) MDC12h 16 h 8h 12h(A) LC3 NMDA+3MA NMDA Control Figure 4-5. NMDA-induced autophagosome formation is inhibited by 3-MA. Representative fluorescence micrographs A) show an incr ease in anti-LC3 immunostaining (red) localizes around DAPI (blue; nucle ar stain) following NMDA (200 M) exposure in cerebellar neurons compared to controls and NMDA+3-MA co-treated cultures. Arrows and arrow heads (yellow) indicate the punctate staining in the cell bodies and axons/neurites, respectively; in neuron s following NMDA exposure for 8 and 12 h. Images are taken at 400X magnifi cation. Scale bar represents 10 m. B) Monodansylcadaverine (MDC; 0.05 mM) labe ling of autophagosomes in neurons increase following acute exposure to NMDA (200 M) compared to control conditions and co-treatment of NMDA and 3-MA. Arrows (red) indicate the accumulation of the autophagosomes in neuronal cell bodies at 12 and 16 hours following NMDA treatment while arrow heads (yellow) and arrows (yellow) indicate the presence of punctate autophagosomes in the existent neurites and cell bodies respectively. The arrows (red) in the image at 16 hours are indicative of the accumulation of the autophagosomes in the cell bodies of neurons following prolonged NMDA exposure. Images are ta ken at 400X magnification. Scale bar represents 20 m.

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68 0 0.1 0.2 0.3 0.4 0.5 0510152025Time (hours)LDH release (arb. denstity units ) Control NMDA NMDA+3MA (A) (B)Control NMDA+3MA NMDA Figure 4-6. Autophagy inhibitor 3-MA protect s neurons against NMDA excitotoxcity. A) Representative phase contrast images i ndicates the changes in the morphology of neurons following treatments with NMDA (200 M), NMDA (200 M) + 3-MA (10 mM) and controls. Arrows (yellow) indi cate surviving cell body morphology from apoptotic cell bodies (red arrows). Arrow heads (yellow) indicate healthy neuritis while red arrow heads indicate degenerati ng neurites. Images are taken at 400X magnification. Scale bar represents 20 m. B) LDH release recorded and plotted after incubating cerebellar neurons in culture in NMDA (200 M) ( ), NMDA+3-MA (10 mM) ( ) and controls ( ). The expressed values are means S.E.M. (n=6; *p<0.05 NMDA vs. controls and #p< 0.05 NMDA vs. NMDA+3MA).

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69 SBDP120 SBDP150 SBDP145 II-spectrin NMDACtrl+3MA24 h 12 h 24 h 3 h 3 h 6 h 24 h 12 h STSSBDP120 AntiII-Spectrin Anti-SBDP120 0 5 10 15 20 25SBDP120 levels (arb. density units)Ctrl NMDA NMDA +3MA STS# 24h 0 20 40 60 80 100 120Ac-DEVD-AMC hydrolysis activity (arb. fluorescent units)Ctrl NMDA NMDA +3MA Ctrl NMDA NMDA +3MA STS*#*# 12h 24h Figure 4-7. NMDA-induced caspase -3 activation is suppressed by 3-MA. A) Representative immunoblot of II-spectrin breakdown profile shows the presence of the caspase-3 specific spectrin breakdown product (SBDP) of 120 kDa in NMDA-treated cultures after 24 hours compared to the controls and NMDA+3-MA (n=3). Representative immunoblot probed with anti-SBDP120 show s a similar profile of the breakdown product in NMDA-treated cultures after 24 hours following treatment but not in controls or NMDA+3-MA co-treatment (n=3 ). B) Densitometric analyses of the immunoblots probed with anti-SBDP120 show a significant increase in the spectrin breakdown product of 120 kDa (SBDP120) after 24 hours following NMDA treatment in cerebellar neuronal cultures but not in controls or NMDA+3-MA cotreated cultures. The expressed values are means S.E.M. (n=3; *p<0.05). C) Caspase-3 enzymatic assay was determined using the caspase substrate Ac-Asp-GluVal-Asp-7-amino-4-methylcoumarin (A c-DEVD-AMC) incubated with proteaseinhibitor free lysates obtained from cultures treated with or without NMDA and NMDA+3-MA co-treatment. An increase in the caspase-3 enzymatic activity was observed in NMDA-treated as opposed to controls or NMDA+3-MA co-treated cultures. The expressed values are mean s S.E.M. (n=3; *p<0.05 compared to control-treated; #p<0.05 compared to NMDA-treated).

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70 (A) 0 5 10 15 20 25 303 6 12 24Time (h)Nitrated protein density (arb. density units)*3h 6h 12h 24 h 24hNMDA Ctrl3h (B)24 h 0 5 10 15 20 25 30Nitrated protein levels (arb.density units)NMDA NMDA +3MA Ctrl*# 0.0 0.4 0.8 1.2 0381224 Time (h)LDH Release (arb. density units) ONOOONOO-+MA 0 10 20 30Nitrated protein (arb. density units)ONOO-+3MA24 hONOOFigure 4-8. Protein nitration in cerebellar granule neurons following NMDA-treatment is alleviated by 3-MA. A) Representative blot probed with anti-nitro-tyrosine, shows the presence of strong nitrated proteins of approximately 70 kDa size under NMDAtreated conditions at 24 hours post-treatment (n=3). Lysates obtained from cultures incubated in SFM at 3 and 24 hours were employed as controls. Densitometric quantifications of the nitrat ed protein (70 kDa) were plotted on a time scale under NMDA ( ) exposure conditions at 3, 6, 12 and 24 hours. Control ( ) values obtained at 3 and 24 hours were plotte d. Values were plotted as the means S.E.M. (n=3, *p<0.05 vs. controls). B) Quantification of th e nitrated protein levels among lysates treated with NMDA showed a significant increase in the values compared to NMDA+3-MA co-treated cultures at 24 hours. The values are expressed as means S.E.M. (n=3, *p<0.05 vs. controls; #p<0.05 vs NMDA-treated). (Inset) Top panel: Autophagy inhibitor 3-MA treatment reduces oxidative damage in peroxynitrite treated cerebellar granule neuronal cultures. Quantification of the intensity values of the nitrated protein at 70 kDa in the Peroxynitrite (0.5 mM, OONO-)+3-MA cotreatment was significantly reduced comp ared to peroxynitrite (OONO-) treatment alone. The values are represented as means S.E.M. (n=3, *p<0.05). Bottom panel: Lactate dehydrogenase (LDH) release wa s assayed following treatment with a combination of peroxynitrite (0.5 mM, OONO-) and 3-MA (10 mM) ( ) and peroxynitrite -treatment ( ) alone. The values are plotted as meansS.E.M. (n=6, *p<0.05).

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71 Figure 4-9. Schematic representation of the i nvolvement of autophagy an d autophagic cell death in neurons following excitotoxic NMDA challenge.

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72 CHAPTER 5 CHANGES IN AUTOPHAGY PROTEINS IN A RAT MODEL OF CONTROLLED CORTICAL IMPACT BRAIN INJURY Introduction Autophagy is an intracellular phenom enon th at has been documented to sustain cell survival under conditions of stress, by lysosomal breakdown of the cytosolic organelles and proteins and recycling the amino acids into the ce ll machinery. It is characterized by the presence of double membrane cytoplasmic vesicles calle d the autophagosomes which sequester the cytosolic components before fusing with th e lysosomes where the lysosomal hydrolases breakdown the sequestered organelles. A number of proteins that intri cately regulate autophagy have been reported (Klionsky et al., 2003)Some of them include beclin-1 (Atg6), Atg4, MAPLC3 (Atg8), Atg7, Atg12 and 5. Recently there has b een tremendous interest in identifying the role of autophagy in the central nervous system. Autophagy has been shown to play an integral role in the neurodevelopment process (Hara et al., 2006; Komatsu et al ., 2006). Other studies have delved into the potential ne uroprotective roles of autophagy in clearing protein aggregates in neurons in animal models expressing polyglutamine phenotype disorder s (Floto et al., 2007; Sarkar et al., 2007a; King et al., 2008). Recent st udies have also studied the alterations in the autophagy protein expression prot eins and involvement of aut ophagy following brain trauma (Zhu et al., 2003; Diskin et al., 2005; He et al., 2007; Koike et al., 2008). In this study, we demonstrate that the autophagy protein MAP-LC3-II is upregulated following controlled cortical impact, a more severe form of brain injury and th ese increases are in the co rtical regions in close proximity to the injury. Also, we develop a co rrelation between the le vels of the autophagy protein beclin-1 and the anti-apopt otic protein bcl-2 at various tim e periods following the cortical injury in rats.

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73 Results Autophagy Induction Increases After Brain Injury in the Cortex Autophagy induction has been studied using the expression levels of the autophagy protein MAP-LC3. To dem onstrate the incr eases in autophagy induc tion after cortical impact injury we probed the cortical lysates obtaine d from the ipsilateral side of the injury with anti-MAP-LC3. We demonstrated increases in the conversion of the LC3-I to LC3-II as opposed to sham-injured or nave animals (Fig. 5-1A). Quantification of the densities of the protein bands revealed a significant increase (p 0.05) in the levels of LC3-II protein as compared to LC3-I in traumatized animals as compared to sham-injured or nave animals (Fig. 5-1B). Beclin-1 Levels are Increa sed Follow ing Brain Injury Beclin-1 (Atg6) is an aut ophagy protein shown to be i nvolved in the regulation of autophagy. To determine if it was affected in acu te brain trauma, cortical lysates from the ipsilateral side of the injury were probed with anti-beclin-1. Also, effect s of the brain trauma on the protein levels of anti-apopto tic protein, bcl-2 was studied. We demonstrat ed that beclin-1 levels are increased in a time-dependent manne r. The levels start to go down 1d and 2d postinjury. The anti-apoptotic protein bc l-2 levels also seem to fluctuat e in the cortex region close to the injury. As observed with the beclin-1 levels the levels of bcl-2 drop towards 1d and 2d postinjury (Fig. 5-2). Immunoblot Detection of Beclin-1 /Bcl-2 Ratio After Brain Injury The ratio of beclin-1 to bcl-2 levels has been demonstrated to be significant in the regulation of autophagy. Densitometr ic quantification of the band intensities of the beclin-1 and bcl-2 do not show any significant differences when plotted individually (Fig. 5-3A,B). The mean ratios of the band intensities of beclin-1/bcl-2 demonstrate a significant in crease at days 1 and 2

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74 (p 0.05) following traumatic brain injury (TBI). Th is increase was significantly higher than the ones observed in sham-injured animals (Fig. 5-3C). Discussion Autophagy has been shown to be neuroprotective in neurodegenerative diseases such as Parkinsons disease, Huntingt ons disease and Alzheim ers disease (Webb et al., 2003; Nixon, 2007; Sarkar et al., 2008). There have also been reports about the relevance of autophagy in traumatic brain injury events such as closed he ad trauma and ischemic stroke (Diskin et al., 2005; Adhami et al., 2006). Here we corroborate th e findings that autophagy is indeed induced after brain trauma, in an acute rat brain trauma model of controlled cortical impact. Our model of brain trauma is a more acute form of the injury and hence an important finding with clinical relevance in severe forms of brain contusions and other penetrating br ain injuries. Through our studies we demonstrated increases in the proc essising of autophagy protein MAP-LC3 and also an increase in the ratio of beclin-1/bcl-2 following the brain trauma. MAP-LC3 has been documented to be one of the most reliable markers to study autophagy induction (Mizushima, 2004; Mizushima et al., 2004) Increase in the processing of the LC3-I protein form to LC3-II has been documented to be one of the hallmarks of autophagy induction. In our results we demonstrated a similar increased processing of the pro-form of MAP-LC3 protein into the LC3-II form which is known to be associated with the autophagosomal membrane in the traumatized animals as compared to sham-injured animals (Fig. 5-1). Other proteins of interest were the au tophagy protein beclin-1 (Atg6) and its inte racting anti-apoptotic protein bcl-2. Beclin-1 is a bcl-2 interacting protein that has been documented to be an important player in the induction of autophagy. Bcl-2, the anti-apopt otic protein has been shown to interact with beclin-1 via the BH-3 domain on b eclin-1. Bcl-2 exerts a controll ing effect on the activity of

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75 beclin-1. Under normal homeostatic conditions, beclin-1 is bound to bcl-2 and hence not available to induce autophagy. Fo llowing a disruption in the ce ll homeostasis due to stress conditions or insults, beclin-1 interaction with bcl-2 is weakened and beclin-1 now becomes available to induce autophagy. This interaction between these protei ns is of utmost significance as it governs the switch between inducing autophagy or apoptosis (Pattingre et al., 2005; Pattingre and Levine, 2006; Feng et al., 2007; Maiu ri et al., 2007a; Maiuri et al., 2007b). Here we demonstrated in our expe riments that though beclin-1 wa s qualitatively increased in traumatized animals compared to sham-injured animals, there was however no statistical significance. A similar observation was reported with the levels of bcl-2 in the traumatized and sham-injured animals (Fig. 5-2). However the ra tio of beclin-1/bcl-2 showed a statistically significant increase in the TBI animals compared to sham animals at days 1 and 2 post-injury. The speculation is that the overexp ression of beclin-1 protein at the site of injury can enhance autophagy induction as a mechanism to discard inju red cells and reduce the extent of damage to cells from the injured components (Erlich et al., 2006). Also, a decline in the levels of beclin-1 and bcl-2 was observed at later time points in the study (Fig. 5-3). This possibly might be because of the loss of neurons in the brain following the injury which coincided with the reduced protein levels. Also, the reduction in the bcl-2 levels noticed might suggest a transition in the cell death pathways from oncotic to apoptotic as well as reduced amounts of protein to inte ract with beclin-1. Thus, the fr ee beclin-1 is now available to induce autophagy. Recently, autophagy-assosciated neuronal death has al so been studied in neuronal injury models. Uchiyama and colleague s demonstrated that hypoxia/ischemia brain injury in the neonatal brain results in energy fa ilure, oxidative stress a nd unbalanced ion fluxes inducing elevated levels of autophagy in the brain neurons (Uchiyama et al., 2008). Due to

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76 commonalities between the pathologies in the two different brain injury etiology, comprehensive studies need to be conducted to elucidate the role of autophagy and autophagic cell death in the acute forms of brain trauma. Conclusion Our studies thus indicate aut ophagy induction in a rat m odel of controlled cortical impact brain injury by demonstrating increases in the le vels of autophagy protein MAP-LC3-II and ratio of beclin-1/bcl-2.

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77 Figure 5-1. Increased levels of MAP-LC3-II are observed following controlled cortical impact. A) Representative immunoblot of cortical lysates probed with anti-MAP-LC3 (n=5). GAPDH has been used as control for even protein loading. B) Densitometric quantification of the LC3-II bands denote a significant increase in MAP-LC3-II levels in the traumatized animals compared to sham animals (p 0.05)

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78 Figure 5-2. Beclin-1 levels increase following co rtical injury. Representa tive immunoblots with cortical lysates probed with anti-beclin-1 and anti-bcl-2 (n=5). GAPDH is used as control for protein loading.

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79 0 20 40 60 80 100 120 140Beclin-1 levels (arb. density units)Naive Sham 2h TBI 2d Sham 6h Sham 1d Sham 2d TBI 1d TBI 6h TBI 2h 0 20 40 60 80 100 120 140Bcl-2 levels (arb. density units)Naive Sham 2h TBI 2d Sham 6h Sham 1d Sham 2d TBI 1d TBI 6h TBI 2h 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Beclin-1/Bcl-2 ratioNaive Sham 2h TBI 2d Sham 6h Sham 1d Sham 2d TBI 1d TBI 6h TBI 2h(A) (B) (C)* Figure 5-3. Increases in the ra tio of beclin-1/bcl-2 indicate autophagy induction. A, B &C) Densitometric quantification of the bands presented in the immunoblots indicates a significant increase in the rati o of beclin-1/bcl-2 represen ting autophagy sustainence n traumatized animals compared to sham-injured animals (n=5; p 0.05).

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80 CHAPTER 6 CONCLUSION Summary and Scientific Applications Our m ain working hypothesis for this project wa s to elucidate the role of autophagy in a rat model of experimental controlled cortical impact (CCI). Results fr om our studies and also studies emerging from other labs have demonstrated the importance of aut ophagy in regulating the neuronal homeostasis in the centr al nervous system. Autophagy ha s been documented to play a critical role in cell survival following conditions of cell stress ranging from nutrient deprivation to cell injury. Though its benefi cial effects in cell maintena nce have been well documented, evidence for it contributing to a novel form of ce ll death autophagic cell death (type II) is emerging. The popular belief is that autophagy is beneficial when it is induced initially following cell stress, but when prolonged left unchecked can result in the sequestrati on and in turn removal of cellular organelles that might be important for cell survival as discussed in our literature review in Chapter 1. In our studies, we used a combination of biochemical protein assays and immunofluroscence techniques to study autophagy and autophagic cell death. Chapter 3 describes our work in establishing the worki ng tools for studying aut ophagy in a neuronal cell line, PC-12 cells. Amino acid starvation induced au tophagy in PC-12 cells and this involved the activation of cysteine proteases caspase-3 but not calpain-1. Calpai ns have been shown to cleave the autophagy protein Atg5 that suppressed au tophagy and activated th e apoptotic cell death pathway. Cell death observed in these cells following prolonged nutrient starvation was accompanied with the presence of monodansylcadaverine (MDC) and LC3 positive autophagosomes. Though apoptotic marker s were observed, autophagy inhibitor 3methyladenine (3-MA) was able to rescue cell deat h. This led us to believ e that though beneficial

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81 initially, autophagy when prolonged can result in a form of cell deat h that shows the presence of autophagosomes as well as is biochemically sim ilar yet morphologically di fferent to apoptosis. We further extended our findings in PC-12 cel ls to a neuronal cell culture paradigm of excitotoxicity in the CNS. We harvested rat ce rebellum and cultured granule neurons that were subsequently exposed to the excitotoxin NMDA as described in Chapter 4. We showed that NDMA exposure induced autophagy as early as 3 hours, detected by i mmunostaining with MAPLC3 and also the presence of MDC-positive autophagosomes and beclin-1 immunoreaction. We demonstrated that cell death resulting from ex citotoxic injury due to NMDA exposure increased at 1day. This cell death was accompanied by mor phological changes in the cell bodies and axons of the neurons and an increase in the presence of MDC-positive autophagosomes. As observed with the PC-12 cells, caspase-3 activity was increased following NMDA exposure. As documented previously, NMDA exposure also indu ces oxidative stress. According to a recent report, oxidative stress positively reinforces th e induction of autophagy (Scherz-Shouval et al., 2006). We observed that 3-methyladenine, a pharmacological inhibitor of autophagy not only inhibited autophagy (evident by the loss of the LC3 -II band), but also was efficient in alleviating the cell damage due to oxidative stress and the resulting possible autophag ic cell death. Thus, we hypothesized that excitotoxic stress induced autophagy and oxidative stress which when prolonged resulted in 3-MA se nsitive autophagic cell death. We further explored the changes in autophagy proteins in our experimental model of controlled cortical impact (CCI) which is an an imal model of severe traumatic brain injury (TBI). We screened for the presence of autophagy proteins MAP-LC3 (Atg8) and beclin-1 (Atg6). Following TBI, autophagy was induced close to the site of the injury in the cortex, evident by the increased levels of autophagy-related proteins. Als o, an increase in the beclin-

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82 1/bcl-2 ratio suggested the induc tion of auophagy after the injury (Patting re et al., 2006). A similar induction of autophagy could not be observed in the underlying hippocampus, suggesting that autophagy might play a more critical role around the site of the injury possibly by clearing the cell debris and arresting th e probable factors that might le ad to a secondary wave of biochemical injury. In a recent study, rapa mycin (autophagy inducer) was reported to be neuroprotective in an animal model of traumatic brain injury when injected at 4hours following injury by the inhibition of the mTOR signaling (Erlich et al., 2007). As we hypothesized, autophagy definitely plays a role in neurotrauma, though further st udies have to be conducted to further elucidate whether its neuroprotectiv e or culminates in autophagic cell death. Future Directions Our work coupled with others has demonstrated a potential role for autophagy after brain injury. Proteom ic studies from our lab has furt her reinforced this thought, as the autophagy protein Atg8 was shown to be up regulated foll owing experimental TBI. The observation that autophagy is oxidative stress furthe r opens the door to investigat e the role of antioxidants as therapy options to either indu ce autophagy or arrest it. A tem poral profile demonstrating the involvement of autophagy in experimental brain injury paradigm would help understand the dual roles of autophagy involving cell su rvival or autophagic cell death.

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92 BIOGRAPHICAL SKETCH Shankar Sadasivan was born and raised in the business capital of India, Mum bai. He completed his high school education in 1995 and em barked on a career in science when he joined the Prin. K.M.Kundnani College of Pharmacy, Wo rli, Mumbai, to earn a B.Pharm. degree in Pharmaceutical Sciences. After completing his studies in pharmaceutical sciences, he became a registered pharmacist in the state of Maharashtra, India. He worked for a year in the Research and Development department as a scientist at Zandu Pharmaceuticals, Mumbai. He came to the U.S.A. in the year 2000 as a M.S. candidate in the field of pharmacology. He acquired his Master of Science diploma in 2003 from the Massachussetts College of Pharmacy and Health Sciences, Boston, MA. His research work focused on the investigating the ther apeutic potential of precursors to the club drug gamma hydroxybutyric acid (GHB), gamma butyrolactone (GBL) and 1,4-butanediol (BD) in a cerebral ischemic m odel of transient and permanent middle cerebral artery occlusion (MCAO) in rats. He came to the University of Florida in th e year 2003, admitted as a doctor of philosophy student to the Interdisciplinary Program (IDP) in the college of Medicine. During his years in the program, he has published several research papers in peer-reviewed journals and was awarded the Bryan W. Robinson Neurological Foundation Grant-in-Aid Achievement Award in 2005. He was also nominated and awarded the Outstanding International Student Award for Research by College of Medicine in 2007. He joined the Depa rtments of Neuroscience and Psychiatry, where he completed his doctoral degree under the mentor ship of Dr. Kevin K Wang, in the fields of autophagy and neurotrauma as part of the Evelyn F. and William L. McKnight Brain Institute.