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Implication of Detergent-Insoluble Aggregates of Superoxide Dismutase 1 in Familial Amyotrophic Lateral Sclerosis

Permanent Link: http://ufdc.ufl.edu/UFE0025156/00001

Material Information

Title: Implication of Detergent-Insoluble Aggregates of Superoxide Dismutase 1 in Familial Amyotrophic Lateral Sclerosis
Physical Description: 1 online resource (252 p.)
Language: english
Creator: Prudencio-Alvarez, Mercedes
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aggregation, amyotrophic, misfolding, motorneuron, neurodegeneration, sclerosis, transgenics
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: IMPLICATION OF DETERGENT-INSOLUBLE AGGREGATES OF SUPEROXIDE DISMUTASE 1 IN FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS Mutations in superoxide dismutase 1 (SOD1) are responsible for 10-20% of the familial cases of amyotrophic lateral sclerosis (ALS). Although it is unknown how these mutations lead to motor neuron degeneration, a yet unknown toxic property of mutant SOD1 is responsible for causing SOD1-associated ALS. One possible toxic property of mutant SOD1 proteins is their ability to misfold and aggregate. Specifically, my research project is focused on further evaluating the mechanism of mutant SOD1 protein aggregation. In particular, we determined the ability of experimental and ALS-associated SOD1 mutations to form detergent-insoluble aggregates, and the effect of these aggregates on disease by using both, cell culture and animal models (Chapters 2 and 3). Additionally, we have established the role of WT SOD1 in modulating aggregation of mutant SOD1 and its repercussions in animal models expressing WT and mutant SOD1 (Chapters 4 and 5). Finally, we have continued to use a cell culture model to further characterize detergent-insoluble aggregates of mutant SOD1 through immunofluorescence and biochemical techniques (Chapter 6). Our studies demonstrate that all mutant SOD1 proteins that form detergent-insoluble aggregates cause ALS-symptoms in animal models. Additionally, higher rates of aggregate formation indicate a higher risk of developing a rapid disease in humans. A modulator of aggregation is WT SOD1; however, its effect on mutant SOD1 aggregation is complicated. While human WT SOD1 slows aggregation of mutant proteins in cell culture, both proteins eventually co-aggregate in cell culture and in endstage ALS mouse models. The effect of WT SOD1 on disease is detrimental in a dose dependent manner, suggesting an important role of WT on disease. Further characterization of detergent-insoluble aggregates of mutant SOD1 indicates that these species may be of very small size, explaining the difficulty of identifying inclusions in cell culture and animal models. Fluorescent-tagged variants can form inclusions in cell culture. However, their large size makes this model not so adequate to establish possible sites of toxic action. In conclusion, we have demonstrated the importance of detergent-insoluble aggregates of mutant SOD1 in ALS. Thus, tools directed to slow down or block aggregate formation could alter the disease course in humans.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mercedes Prudencio-Alvarez.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Borchelt, David Ralph.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025156:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025156/00001

Material Information

Title: Implication of Detergent-Insoluble Aggregates of Superoxide Dismutase 1 in Familial Amyotrophic Lateral Sclerosis
Physical Description: 1 online resource (252 p.)
Language: english
Creator: Prudencio-Alvarez, Mercedes
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aggregation, amyotrophic, misfolding, motorneuron, neurodegeneration, sclerosis, transgenics
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: IMPLICATION OF DETERGENT-INSOLUBLE AGGREGATES OF SUPEROXIDE DISMUTASE 1 IN FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS Mutations in superoxide dismutase 1 (SOD1) are responsible for 10-20% of the familial cases of amyotrophic lateral sclerosis (ALS). Although it is unknown how these mutations lead to motor neuron degeneration, a yet unknown toxic property of mutant SOD1 is responsible for causing SOD1-associated ALS. One possible toxic property of mutant SOD1 proteins is their ability to misfold and aggregate. Specifically, my research project is focused on further evaluating the mechanism of mutant SOD1 protein aggregation. In particular, we determined the ability of experimental and ALS-associated SOD1 mutations to form detergent-insoluble aggregates, and the effect of these aggregates on disease by using both, cell culture and animal models (Chapters 2 and 3). Additionally, we have established the role of WT SOD1 in modulating aggregation of mutant SOD1 and its repercussions in animal models expressing WT and mutant SOD1 (Chapters 4 and 5). Finally, we have continued to use a cell culture model to further characterize detergent-insoluble aggregates of mutant SOD1 through immunofluorescence and biochemical techniques (Chapter 6). Our studies demonstrate that all mutant SOD1 proteins that form detergent-insoluble aggregates cause ALS-symptoms in animal models. Additionally, higher rates of aggregate formation indicate a higher risk of developing a rapid disease in humans. A modulator of aggregation is WT SOD1; however, its effect on mutant SOD1 aggregation is complicated. While human WT SOD1 slows aggregation of mutant proteins in cell culture, both proteins eventually co-aggregate in cell culture and in endstage ALS mouse models. The effect of WT SOD1 on disease is detrimental in a dose dependent manner, suggesting an important role of WT on disease. Further characterization of detergent-insoluble aggregates of mutant SOD1 indicates that these species may be of very small size, explaining the difficulty of identifying inclusions in cell culture and animal models. Fluorescent-tagged variants can form inclusions in cell culture. However, their large size makes this model not so adequate to establish possible sites of toxic action. In conclusion, we have demonstrated the importance of detergent-insoluble aggregates of mutant SOD1 in ALS. Thus, tools directed to slow down or block aggregate formation could alter the disease course in humans.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mercedes Prudencio-Alvarez.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Borchelt, David Ralph.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025156:00001


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1 IMPLICATION OF DETER GENT -INSOLUBLE AGGREGATES OF SUPEROXIDE DISMUTASE 1 IN FAMIL IAL AMYOTROPHIC LATERAL SCLEROSIS By MERCEDES PRUDENCIO LVAREZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Mercedes Prudencio lvarez

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3 To my family for their constant love and support

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4 ACKNOWLEDGMENTS I would like to thank my advisor Dr. David R. Borchelt for the great opportunity that joining his lab has been for my career. I also like to thank Dr. Borchelt for teaching me to work and think independently and to grow as a scientist. I would like to thank my committee members: Dr. Sue Semple-Rowland, Dr. Lucia Notterpek, Dr. Dennis Steindler, and Dr. William Dunn Jr. for their advice and interest on my research and career. In particular, I would like to thank Dr. Semple -Rowland for her constant help and s upport on writing skills, experimental techniques, and guidance on becoming a research scientist. I would like to thank Dr. Notterpek for her scientific and technical support. I also want to thank Dr. Dunn for his constant effort on trying to understand SOD1 associated ALS, and for his research advice. Finally, I want to thank Dr. Steindler for being up to date on my research progress and for finding time to look at my work. Additionally, I want to express my gratitude to all our collaborators: Dr. Julian Whitelegge, Dr. Armando Durazo, Dr. Joan Valentine, Dr. Madhuri Cha ttopadhyay, Dr. Herman Lelie, and Dr. John P. Hart for all their help and their example of wonderful collaborations. Furthermore, I cannot forget to thank past and current members of the Borchelt lab. In particular I want to thank Dr. Celeste M. Karch for all her support as a friend and as researcher, Ms. Hilda Slunt -Brown for her wonderful expertise and help with numerous research techniques, Dr. Guilian Xu and Mr. Andrew Tebbenkamp for t he i r research advice and friendship. I also want to thank all the mem bers of the Borchelt lab for all the good moments that made going to work a pleasure experience. Finally, I want to thank Mr. Ashton B. Manley for all his support and understanding, and to all my family (M ximo, Joaquina and Almudena) that despite the dist ance they are always there for me and I coul d not have done it without their love and support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 9 LIS T OF FIGURES ............................................................................................................ 10 ABSTRACT ........................................................................................................................ 15 CHAPT ER 1 INTRODUCTION ........................................................................................................ 17 Amyotrophic Lateral Sclerosis: A General Overview ................................................ 17 Gen es Associated with ALS ....................................................................................... 18 SOD1 Gene ................................................................................................................ 19 SOD1 Function ........................................................................................................... 20 Structural Properties of SOD1 .................................................................................... 21 SOD1 Mutations ......................................................................................................... 22 Penetrance of SOD1 Mutations ................................................................................. 23 SOD1 Subcellular Location ........................................................................................ 24 Potential Disease Mechanisms of SOD1-Associated ALS ....................................... 25 Loss or Gain of SOD1 Function ........................................................................... 25 Mutant SOD1 Damage through Oxidative Chemistry Mechanisms ................... 26 Mutant SOD1 and Protein Aggregation ............................................................... 28 Implication of WT SOD1 in ALS and in Aggregation ................................................. 30 SOD1 -Associated ALS: A Non Cell Autonomous Disease ....................................... 31 Ast rocytes and Microglia ...................................................................................... 32 Oligodendrocytes and Schwann Cells ................................................................ 33 Muscle Cells ......................................................................................................... 33 Animal Models to Study SOD1-Associated ALS ....................................................... 34 Transgenic Rodents ............................................................................................. 34 Pathology in SOD1-Associated ALS and Rodent Models of the Disease ......... 35 Other Transgenic Animal Models for SOD1 -Associated ALS ............................ 37 SOD1 expressing fruit flies. .......................................................................... 37 Worms models for SOD1 ass ociated ALS ................................................... 38 Other models ................................................................................................. 38 Therapies .................................................................................................................... 39 Riluzole ................................................................................................................. 39 Other Tested Drugs ............................................................................................. 40 Future Clinical Trials ............................................................................................ 42

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6 2 LACK OF TOXICITY IN A NOVEL VARIANT OF HUMAN SOD1 HARBORING ALS -ASSOCIATED AND EXPERIMENTAL MUTATIONS IN METAL BINDING RESIDUES .................................................................................................................. 43 Introduction ................................................................................................................. 43 Materials and Methods ............................................................................................... 44 Results ........................................................................................................................ 45 Discussion ................................................................................................................... 57 SODMD Mice and Expression Levels ................................................................. 57 SODMD Protein Does Not Produce ALS -Like Pathology ................................... 58 Metal Binding, Aggregation and Disease ............................................................ 59 SODMD and WT SOD1 ....................................................................................... 60 Toxicity in ALS ...................................................................................................... 61 Future Directions .................................................................................................. 61 3 VARIATION IN AGGREGATION PROPENSITIES AMONG ALS-ASSOCIATED VARIANTS OF SOD1: CORRELATION TO HUMAN DISEASE .............................. 64 Introduction ................................................................................................................. 64 Materials and Methods ............................................................................................... 65 Results ........................................................................................................................ 65 Discussion ................................................................................................................... 82 Variability in Aggregation Propensity of SOD1 Mutants and Protein Charge .... 83 Variability in Aggregation Propensity of SOD1 Mutants and Protein Stability ... 84 Variability in Aggregation Propensity of SOD1 Mutants and Metal Binding ...... 84 Aggregation vs. Disease ...................................................................................... 85 HEK293FT Cells as a Model to Study SOD1-Associated ALS Aggregation ..... 87 Conclusions .......................................................................................................... 90 4 MODULATION OF MUTANT SUPEROXIDE DISMUTASE 1 AGGREGATION BY CO -EXPRESSION OF WILD -TYPE ENZYME .................................................... 92 Introduction ................................................................................................................. 92 Materials and Methods ............................................................................................... 93 Results ........................................................................................................................ 93 Discussion ................................................................................................................. 104 Human But Not Mouse WT SOD1 Can Co-Aggregate with Mutant SOD1 ...... 104 WT and Mutant Human SOD1 Protein -Protein Interaction: Role of Disulfide Bonding and Cysteine 111. ............................................................................ 105 Role of WT and Mutant SOD1 Interactions in Disease .................................... 106 Con clusions ........................................................................................................ 108 5 A COMPLEX ROLE FOR WILD -TYPE SOD1 IN THE TOXICITY AND AGGREGATION OF ALS -ASSOCIATED MUTANT SOD1 .................................... 110 Introduction ............................................................................................................... 110 Materials and Methods ............................................................................................. 111

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7 Results ...................................................................................................................... 111 Discussion ................................................................................................................. 125 Acceleration of Disease by WT and Mutant SOD1 Overexpression is Independent of Strain Background. ............................................................... 126 Earlier Disease in Mutant/WT Mice is Associated With Aggregated SOD1. ... 127 Co aggregation of WT and Mutant SOD1 is Dependent on SOD1 Protein Levels .............................................................................................................. 128 The Complexity of WT -Mutant Co aggregation ................................................ 130 Critical Levels of Reduced WT SOD1 Protein Can Initiate WT Aggregation. .. 132 Mechanisms of Toxicity ...................................................................................... 133 6 CHARACTERIZATION OF DETERGENT -I NSOLUBLE AGGREGATES OF MUTANT SOD1 IN CELL CULTURE ....................................................................... 137 Introduction ............................................................................................................... 137 Materials and Methods ............................................................................................. 138 Results ...................................................................................................................... 13 8 Discussion ................................................................................................................. 160 Inclusion Formation in Cells Expressing Untagged SOD1 Proteins ................ 161 Subcellular Location of SOD1::YFP Inclusions ................................................. 161 Effect on Detergent Solubility in SOD1::YFP Inclusion Formation ................... 162 WT Modulation of Aggregation and Inclusion Formation ................................. 163 Conclusions ........................................................................................................ 165 7 CONCLUSIONS ........................................................................................................ 166 Detergent Insoluble Mutant SOD1 Aggregates and ALS. ....................................... 167 W T SOD1 as a Modulator of Aggregate Formation ................................................ 167 Effect of WT SOD1 on Aggregation and ALS in Transgenic Mice .......................... 169 Detergent Insoluble SOD1 Aggregates are Difficult to Observe in Cells ............... 170 SOD1 Fluorescent -Tagged Inclusions, Detergent -Insoluble Aggregates and WT -Mutant Interactions. ........................................................................................ 170 Composition of Detergent -Insoluble SOD1 Aggregates .......................................... 171 Summary ................................................................................................................... 175 Future Directions ...................................................................................................... 175 APPEN DIX A LIST OF SOD1 -ASSOCIATED ALS MUTATIONS .................................................. 177 B MATERIALS .............................................................................................................. 179 C METHODS ................................................................................................................ 185 D ANTIBODY LIST ....................................................................................................... 214 E CO -LOCALIZATION STUDIES OF SOD1::YFP INCLUSIONS .............................. 215

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8 LIST OF REFERENCES ................................................................................................. 222 BIOGRAPHICAL SKETCH .............................................................................................. 252

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9 LIST OF TABLES Table page 1 -1 ALS affected areas. ................................................................................................ 18 1 -2 Familial ALS associated genes and loci. ............................................................... 19 1 -3 Penetrance of some SOD1associated mutations. ............................................... 24 1 -4 Transgenic rodent models for several SOD1 mutations. ...................................... 34 1 -5 Past clinical trials conducted in ALS patients. ....................................................... 41 1 -6 Ongoing clinical trials for the treatment of ALS. .................................................... 42 3 -1 Changes in protein charge do not explain aggregation propensity. ..................... 71 3 -2 Biophysical and biochemical charac teristics of SOD1 variants. ........................... 74 3 -3 Clinical data ordered by relative aggregation potential values. ............................ 78 A-1 List of published SOD1associated ALS mutations. ........................................... 177 B-1 Cell culture reagents. ........................................................................................... 179 B-2 Reagents for detergent extraction and centrifugation assay, BCA assay, SDS-PAGE, and Western blotting. ...................................................................... 180 B-3 Histochemistry and cytochemistry reagents. ....................................................... 180 B-4 Reagents for clonning, genotyping, and general DNA work. .............................. 182 B-5 Reagents RNA extraction and northern blotting. ................................................ 184 D -1 List of primary antibodies. .................................................................................... 214 D -2 List of secondary antibodies. ............................................................................... 214

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10 LIST OF FIGURES Figure page 1 -1 Schematic representation of the SOD1 gene location in the human chromosome, the two polyadenylation signals in the SOD1 transcript and the SOD1 protein. ......................................................................................................... 20 1 -2 Sequenti al steps in the dismutase activity of SOD1 ............................................. 21 1 -3 Dimeric structure of human G37R SOD1 .............................................................. 21 1 -4 SOD1 associated ALS mutations represented in red in the SOD1 amino acid sequence ................................................................................................................ 23 1 -5 Peroxidation reaction ............................................................................................. 27 1 -6 Mechanism of covalent nitration of tyrosine re sidues mediated by SOD1. .......... 27 1 -7 An alternative mechanism of covalent nitration of tyrosine residues mediated by SOD1 does not require zinc binding ................................................................. 27 1 -8 Schematic representation of the possible targets of mutant SOD1 aggregates in ALS, interfering with the normal cellular metabolism ........................................ 29 2 -1 Schematic representation of genomic SODMD .................................................... 45 2 -2 Northern blot showing the two lines of SODMD mice with the best expression levels ....................................................................................................................... 46 2 -3 Lower mRNA SOD1 expression levels predict a longer lifespan in mice ............. 48 2 -4 The levels of SOD1 mRNA and lifespan of SOD1 mice statistically correlate ..... 49 2 -5 Protei n levels in the spinal cord of SODMD mice resemble those of L126Z SOD1 mice. ............................................................................................................ 49 2 -6 Spinal cord of old SODMD mice do not contain aggregated SOD1 proteins. ...... 51 2 -7 Myelin abnormalities are observed in symptomatic H46R/H48Q SOD1 mice, while SODMD do not differ fr om non transgenic (NTg) mice ............................... 52 2 -8 SODMD mice, like WT SOD1 mice, lack of any ALS like pathology .................... 55 2 -9 The nonaggregating SODMD cDNA variant present WT -like features in cell culture ..................................................................................................................... 56 2 -10 SODMD co expression with WT SOD1 does not induce a rapid increase in aggregation of either protein after 48 hour transfection interval in cell culture. ... 57

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11 2 -11 Mutations in amino acids 6 and 111 in the context of SODMD mutations can restablish the aggregation propensity of SODMD ................................................. 62 3 -1 Large variability in aggregation among SOD1associated ALS mutants ............. 66 3 -2 Some SOD1 associated ALS mutant proteins aggregate slowly ......................... 70 3 -3 Mutants with low aggregation propensity behave like human WT (hWT) SOD1 in terms of modulating aggregation. ........................................................... 71 3 -4 Changes in the net negative charge of SOD1 do not predict aggregation propensity ............................................................................................................... 72 3 -5 Changes in protein charge do not explain differences in aggregation propensity ............................................................................................................... 73 3 -6 Changes in protein charge do not predict onset or survival ................................. 74 3 -7 Disease onset is not driven by changes in aggregation propensity ..................... 78 3 -8 Mutants possessing a highe r aggregation propensity correlate with shorter disease duration ..................................................................................................... 80 3 -9 Mutants possessing low or moderate aggregation propensities are associated with a large variation in disease duration ........................................... 81 3 -10 Aggregation of mutant SOD1 in mouse N2a cells ................................................ 82 4 -1 Human WT SOD1 modulates the aggregation of mutant SOD1 in cultured cells ......................................................................................................................... 96 4 -2 Comparison of SOD1 molecular mass profiles from S1 and P2 fractions of HEK293FT cells co expressing human WT (hWT) and G93A human SOD1 ...... 97 4 -3 SOD1 mutants with high propensity to aggregate (A4V and G93A SOD1) do not interfere with aggregation of G85R SOD1 ...................................................... 98 4 -4 Alignment of human (h) and mouse (m) SOD1 protein sequences ..................... 99 4 -5 Mouse WT SOD1 also modulates the aggregation of mutant SOD1 in cultured cells, but without evidence of co aggregation ....................................... 102 4 -6 Cysteine 111 is not required for the co aggregation of WT with G85R human SOD1 .................................................................................................................... 103 4 -7 High concentrati ME does not reduce the amount of mutant SOD1 that fractionates to the P2 fraction at 48 hours ................................................... 103

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12 5 -2 WT Gurney lines express higher mRNA and protein levels than WT Wong line ........................................................................................................................ 113 5 -2 All WT SOD1 lines express the same cDNA WT SOD1 sequence .................... 114 5 -3 WT SOD1 protein from Gurneys mice significantly accelerates disease in mice harboring either PrPG37R or L126Z mutations, while the effect of WT SOD1 derived from Wongs mice (L76 WT) is not as strong .............................. 116 5 -4 Symptomatic mice present significant accumulation of detergent -insoluble SOD1 aggregates at endstage ............................................................................ 118 5 -5 WT SOD1 is present in detergent -insoluble fractions of spinal cords of PrPG3 7R/SJL WT mice ....................................................................................... 119 5 -6 Protein levels in the detergent -soluble fraction of heterozygous SJL WT and PrPG37R are not more than two fold different. ................................................... 120 5 -7 Human WT SOD1 slows aggregate formation in cell culture and such effect is stronger for the L126Z SOD1 truncat ion mutant ............................................. 122 5 -8 Low amounts of reduced WT SOD1 protein are present in all WT lines of mice. ..................................................................................................................... 123 5 -9 WT SOD1 from Gurney lines (SJL and Cg), but not from Wong line (L76), forms detergent -insoluble SOD1 aggregates at old ages ................................... 125 5 -10 Hypothetical model on the effect of WT SOD1 on disease and aggregation in mice expressing a mutant SOD1 mutation .......................................................... 129 6 -1 HEK293FT cells expressing SOD1 proteins do not form cellular inclusions ..... 140 6 -2 TK negative cells transfected with SOD1 constructs for 48 hours and stained for human SOD1 as explained in 6 -1 .................................................................. 140 6 -3 HEK293FT transfected cells express higher levels of detergent -soluble SOD1 than detergent -insoluble SOD1 aggregated protein. ............................... 141 6 -4 Saponin eliminates most of the cytosolic SOD1 protein, but does not uncover the presence of SOD1 positive inclusions. .......................................................... 142 6 -5 Digitonin treatment in TK negative cells show a dot -like pattern of SOD1 that is not exclusive of cells expressing mutant SOD1 proteins ................................ 143 6 -6 Similar effects of saponin and digitonin on eliminating soluble SOD1 protein from HEK293FT cells expressing WT or highly aggregating SOD1 mutant proteins. ................................................................................................................ 145 6 -7 Mutant SOD::YFP proteins present variable size and number of inclusions ..... 147

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13 6 -8 Tagged WT::YFP and MD::YFP variants are able to aggregate similarly to slow aggregating D101N::YFP proteins when expressed in cells for 48 hours 148 6 -9 NIH3T3 cells express less SOD1::YFP inclusions than HEK293FT after 48 hour transfections ................................................................................................. 149 6 -10 WT::RFP proteins form inclusions similar to those formed by mutant SOD1::RFP in HEK293FT cells after 24 hour transfections. .............................. 149 6 -11 All HEK293FT cells expressing a SOD1::YFP variant contain detergent insoluble aggregates ............................................................................................ 151 6 -12 Tagged and untagged SOD1 protein co expressions determine different ability of tagged SOD1 to modulate aggregation of A4V SOD1 ......................... 153 6 -13 Tagged and untagged SOD1 protein co expressions determine different ability of tagged protein to modulate aggregation of G85R SOD1 ..................... 154 6 -14 WT SOD1 affects inclusion formation in mutant SOD1::YFP proteins, but not mutant SOD1 on WT::YFP proteins .................................................................... 156 6 -15 Untagged A4V SOD1 does not alter inclusion formation in A4V::YFP expressing cells .................................................................................................... 156 6 -16 WT::RFP can induce inclusion formation of WT::YFP ........................................ 157 6 -17 YFP does not alter inclusion formation ability of A4V::RFP or WT::RFP ........... 158 6 -18 WT and mutant SOD1 prot eins do not easily form hybrid inclusions but both proteins may interact at the soluble level ............................................................ 159 7 -1 SOD1 mutant proteins remain mostly disulfide reduced in cell culture .............. 172 7 -2 Progressive accumulation of disulfide reduced SOD1 proteins in cell culture ... 173 7 -3 The engineered WT SOD1 monomer presents inherent propensity to aggregate, similar to other SOD1 mutant proteins. ............................................. 174 C -1 Scheme of transfer arrangement for northern blotting. ....................................... 188 C -2 Schematic representation of how to make cuts in mouse limbs to extract the sciatic nerve. ......................................................................................................... 192 E-1 SOD1::YFP inclusions do not co localize with mitochondria .............................. 216 E-2 Ubiquitin does not concentrate to SOD1::YFP inclusions .................................. 217 E-3 Mutant SOD1::YFP inclusions do not localize within lysosomes ........................ 218

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14 E-4 Lower expression of dynactin protein p50 in SOD1::YFP containing cells ........ 219 E-5 HEK293FT untransfected cells present similar staining pattern of cellular markers as SOD1::YFP transfected cells ............................................................ 220

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15 Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy IMPLICATION OF DETERGENT-INSOLU BLE AGGREGATES OF SUPEROXIDE DISMUTASE 1 IN FAMILIAL AM YOTROPHIC LATERAL SCLEROSIS By Mercedes Prudencio lvarez December 2009 Chair: David R. Borchelt Major: Medical Sciences Neuroscience Mutations in superoxide dismutase 1 (SOD 1) are responsible for 10-20% of the familial cases of amyotrophic lateral sclerosis (ALS). Although it is unknown how these mutations lead to motor neuron degeneration, a yet unknown toxic property of mutant SOD1 is responsible for causing SOD1-asso ciated ALS. One possibl e toxic property of mutant SOD1 proteins is their ability to mi sfold and aggregate. Specifically, my research project is focused on further evaluating the mechanism of mu tant SOD1 protein aggregation. In particular, we determined the ability of ex perimental and ALSassociated SOD1 mutations to form detergent-insoluble aggregates, and the effect of these aggregates on disease by using both, cell culture and animal models (Chapters 2 and 3). Additionally, we have established the role of WT SOD1 in modulating aggregation of mutant SOD1 and its repercussions in animal models expressing WT and mutant SOD1 (Chapters 4 and 5). Finally, we have continued to use a cell culture model to further characterize detergent-ins oluble aggregates of mutant SOD1 through immunofluorescence and biochemical techniques (Chapter 6).

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16 Our studies demonstrate that all mutant SOD1 proteins that form detergent insoluble aggregates cause ALS -symptoms in animal models. Additionally, higher rates of aggregate formation indicate a higher risk of developing a rapid disease in humans. A modulator of aggregation is WT SOD1; however, its effect on mutant SOD1 aggregation is complicated. While human WT SOD1 slows aggregation of mutant proteins in cel l culture, both proteins eventually co aggregate in cell culture and in endstage ALS mouse models. The effect of WT SOD1 on disease is detrimental in a dose dependent manner, suggesting an important role of WT on disease. Further characterization of deterg ent -insoluble aggregates of mutant SOD1 indicates that these species are of very small size, explaining the difficulty of identifying inclusions in cell culture and animal models. Fluorescent -tagged variants can form inclusions in cell culture. However, th eir large size makes this model not so adequate to establish possible sites of toxic action. In conclusion, we have demonstrated the importance of detergent insoluble aggregates of mutant SOD1 in ALS. Thus, tools directed to slow down or block aggregate fo rmation could alter the disease course in humans.

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17 CHAPTER 1 INTRODUCTION Amyotrophic Lateral Sclerosis: A General Overview Amyotrophic lateral sclerosis (ALS) was first described as a late onset degenerative motor neuron disease by JeanMartin Charcot in 1869 (Charcot JM and AJ, 1869) Nowadays, ALS is one of the most common motor neuron diseases, with an incidence of 1 to 2 every 100,000 people (Cozzolino et al., 2008b) and about 15 new cases are diagnosed per day in North America (Wroe, 2009) ALS is usually diagnose as suspected, possible, probable or definite ALS, according to whether symptoms are identified in one or more areas of the body (bulbar, cervical, thoracic, and lumbar) and whether there is any other supporting information (genetic link, biopsy, pathology, etc). The criteria to diagnose ALS were not described till 1994 and they are commonly known as El Escorial criteria, which was defined at the World Federation of Neurology Research in Spain (Brooks, 1994) These criteria establish that a patient may be diagnosed as an ALS patient when: 1 There is clinical evidence of upper and lower motor neuron degeneration. 2 There are clinical symptoms that progress extending within a certain area or to other areas of the body; and, 3 There are not other symptoms or disease features that can explain the motor neuron degeneration. Pathologically, ALS is characterized by the selective loss of upper and lower motor neurons in the spinal cord, brain stem and cortex. Interestingly the loss of motor neurons that control eye movements and the bladder are rarely affected (Kunst, 2004) Motor neuron degeneration in affected areas generally leads to muscular weakness, atrophy, twitching, and speech disabilities (Bendotti and Carri, 2004) As disease

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18 progresses paralysis occurs, resulting i n death by respiratory failure (Bruijn et al., 2004) A summary of affected areas in ALS patients is represented in Table 1-1 (table adapted from The revised Escorial criteria (Brooks, 1994). Table 1 1. ALS affected areas. Affected areas Lower motor neuron signs Upper motor neuron signs Brain stem Weakness, atrophy, fasciculationsa of : Jaw, face, palate, tongue, larynx. Clonic jaw b gag reflex c exaggerated snout reflexd, pseudobulbar features, forced yawning, p athologic deep tendon reflexese, s pastic tonef. Spinal cord Weakness, atrophy, fasciculations of: Neck, arm, hand, diaphragm, back, abdomen, leg, foot. Clonic deep tendon reflexes, Hoffman reflexg, pathologic deep tendon reflexes, spastic tone, preserved reflex in weak wasted limb, loss of superficial abdominal reflexes aFasciculatio ns = muscle twitching, involunta ry muscle contractions bClonic jaw = rapid repetitive muscular contractions alternated by muscular relaxations of the muscles that control the jaw. cGag reflex = reflex that involves the contraction of the pharyngeal constrictor muscle dSnout reflex = muscle contraction that makes the lips to purse eDeep tendon reflex = contraction of the muscles in response to stretching forces fSpastic tone = permanent muscle contraction that translates into stiffness of the muscles, interfering with movement, and sometimes speech. gHoffman reflex = test to determine corticospinal tract damage by tapping the terminal phalanx of the index, medium or ring fingers. A positive result is indicated by the flexion of the terminal phalanx of the thumb. All ALS cases are clinically indistinguishable. However, in most cases, ALS arises from unknown causes (sporadic ALS), and only about 10% of the cases exhibit autosomal inheritance (familial ALS). Genes Associated with ALS In 1993, Rosen and colleagues found several cases of ALS that were associated with mu tat ions in the gene that encodes the enzyme copper, zinc, superoxide dismutase 1 (SOD1) (Rosen et al., 1993) Since then, additional genes or loci have been associated with familial ALS cases. A list of the genes and loci that have been associated with familial ALS cases is represented on Table 1-2.

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19 Table 1 2. Familial ALS associated genes and loci. Disease Inheritance Gene Chromosomal location References ALS1 a Dominant (exceptions) SOD1, superoxide dismutase 1 21q22.1 (Rosen et al., 1993;Al Chalabi et al., 1998) ALS2 b Recessive ALS2, alsin 2q33 (Hadano et al., 2001;Yang et al., 2001) ALS3 a Dominant Unknown 18q21 (Hand et al., 2002) ALS4 b Dominant SETX, senataxin 9q34 (Chen et al., 2004) ALS5 b Recessive Unknown 15q15.1 q21.1 (Hentati et al., 1998) ALS6 a Dominant FUS/TLS, fused in sarcoma/translated in liposarcoma 16q12 (Vance et al., 2009;Kwiatkowski Jr. et al., 2009) ALS7 a Dominant Unknown 20ptel p13 (Sapp et al., 2003) ALS8 a Dominant VAPB vesicle associated membrane protein/ synaptobrevin associated membrane protein B 20q13.33 (Nishimura et al., 2004) ALS FTD a Dominant Unkown 9q21 22 (Ho sler et al., 2000) ALS D PD a Dominant MAPT, microtubule associated protein tau 17q21 (Lynch et al., 1994;Hutton et al ., 1998) Progressive lower motor neuron disease Dominant DCTN1, dynactin 2p13 (Puls et al., 2003) Sporadic and familial ALS Dominant TDP 43, TAR DNA binding Protein 43 1p36.22 (Gitcho et al., 2008;Sreedharan et al., 2008;Kabashi et al., 2008) aAdult onset, bJuvenile onset FTD: Frontal temporal dementia, D: Dementia, PD: Parkinsons disease. SOD1 Gene The SOD1 gene composes a 11 kb fragment on chromosome 21, with 4 introns and 5 exons (Danciger et al., 1986) T wo different transcripts are formed (0.7 and 0.9 kb) (Danciger et al., 1986) due to the existence of two different polyadenylation sites

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20 (Fig ure 1 1). However, either transcript gives rise to the same SOD1 protein of 153 amino acids, with the same structural and functional features. Figu re 1 1. Sch ematic representation of the SOD1 gene location in the human chromosome, the two polyadenylation signals in the SOD1 transcript and the SOD1 protein. SOD1 Function The human SOD1 protein was first isolated from blood ery throcytes in 1959 while studying the role of copper in erythropoiesis (MARKOWITZ et al., 1959) and it was then known as erythrocuprein. The enzymatic function of human SOD1 as a superoxide scavenger was first described by McCord and Fridovich, ten years after its isolation (McCord and Fridovich, 1969) I n 1973, erythrocuprein was renamed sup eroxide dismutase 1 (SOD1) owing to its dismutase activity (Beckman and Pakarinen, 1973) The function of SOD1 protein is to catalyze the antioxidant reaction that converts superoxide radicals into oxygen and hydrogen peroxide. This reaction takes place

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21 independently of pH (from 5 to 9.5) and occurs in two sequential steps, as indicated in Figure 12. O2 .-+C u2+Z n S O D O2+ C u+Z n S O D O2 .-+ 2 H++ C u+Z n S O D H2O2+ C u2+Z n S O D Figure 12. Sequential steps in the dismutase activity of SOD1. O2-: superoxide radical, Cu2+ZnSOD: oxidized, cupric SOD1, O2: oxygen, Cu+ZnSOD: reduced, cuprous SOD1, H+: proton, H2O2: hydrogen peroxide. Structural P rope rties of SOD1 The 32 kDa SOD1 homodimeric protein is very stable. SOD1 is resistant to heat (90C), detergent (4% sodium dodecyl sulfate, SDS) and chemical (10 M Urea) denaturation (For man and Fridovich, 1973) The stability of this enzyme is due to its structural properties. Each of the two SOD1 monomers that compose the dimer forms a -strands and both SOD1 monomers are connected to each other by non-c ovalent forces (Fig ure 1 3 ). Figure 13. Dimeric structure of human G37R SOD1. The published structure (Resolution 1.90 using PyMOL Software (DeLano Scientific LLC, 2006). Bet a strand connector s not associated wit h any structure Beta strands Copper a tom Zinc atom El ectrostat ic loop Zinc loop

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22 There are two metal binding sites in each monomeric subunit, one for copper and another for zinc. Binding of copper is essential for activity and composes the catalytic site. The electrostatic loop (loop VII, residues 121 144) contains several positive ami no acids that guide the superoxide substrate to the catalytic site. Binding of zinc and the zinc loop (loop IV, residues 49 84) confer structural stability to the folded SOD1 protein (Forman and Fridovich, 1973;Elam et al., 2003;Potter et al., 2007) SOD1 Mutations A total of 14 9 pathogenic mutations in the 153 amino acid SOD1 protein have been identified in ALS patients (Wroe, 2009) however only 113 have been documented in peer -reviewed journals (Fig ure 1 4 ) (See Appendix A for a list of published SOD1associated ALS mutations) The majority of these mutations are single amino acid changes (point mutations); however a few deletion, insertion and frameshift mutations have also been associated to the disease. Point mutations predominate in the beta strand regions while frameshift mutations predominate in the C terminus of SOD1 protein. To date there has not been a SOD1 mutation that produces a null protein. SOD1 associated ALS mutations are spread throughout the 153 amino acid protein sequence with the vast majority of point mutations occurring at highly conserved amino acids (Wang et al., 2006) Almost eight y codons in SOD1 are known to be targets of mutation that give rise to the ALS phenotype; in some cases multiple amino acid substitutions occur at one site (up to 6 for G93) ( Fig ure 1 -4) Some mutations affect the residues that coordinate the binding of copper or zinc, thus affecting the overall SOD1 activity. These mutants are sometimes referred as metal binding mutants. Alternatively, several other mutants do not present reduced activity and have more shared characteristics with the WT SOD1 protein. These

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2 3 mutants are commonly known as WT -like mutants. However, this terminology of metal binding and WT -like mutants is not appropriate as some mutants do not a ccurately fit in one of those two barrel mutants, metal binding region mutants and disulfide loop mutants is more suitable (Seetharaman et al., 2009) (Also see Appendix A). Figure 14. SOD1associated ALS mutations represent ed in red in the SOD1 amino acid sequence. The different structural areas are color coded, as described in F ig ure 1 3. Penetrance of SOD1 Mutations Many SOD1 associated ALS mutations present complete penetrance, that is, all gene carriers of a SOD1 mutation develop ALS. However, a good number of mutations are known to skip generations and disease carriers do not always develop ALS. For a list of SOD1 associated mutations that present either complete or incomplete penetrance can be found in Table 33. A more complete table is represented in Table 1 3 Certain SOD1 mutations are found in recessive cases, for example the D90A mutation causing ALS with recessive inheritance is found in Scandinavian countries,

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24 while in other places one copy of D90A SOD1 mutation is enough to cause disease. Additionally, recessive cases seem to account for very aggressive phenotypes these are the cases of and L126S (Boukaftane et al., 1998;Kato et al., 2001a;Hayward et al., 1998) This data suggest that the levels of SOD1 protein might be important to induce disease development. Table 1 3. Penetrance of some SOD1associated mutations. Complete penetra nce Incomplete penetrance A4V A4T G37R L8Q L38V V14G G41D G16S G41S N19S H43R E21G H46R N65S D76V G72S L84F D76Y L84V N86S G85R A89V N86K D90Aheterozygous G93A G93S G93C A95T E100G E100K D101H D101N I104F S105L G108V I113T C111Y V118L I112M V118ins G114A L126S L126X N139H G127X G141E L144F V148G V148I SOD1 Subcellular Location The SOD1 protein is ubiquitously expressed and it is mainly located in the cytosol (Crapo et al., 1992) In lower quantities, SOD1 can also be found in the nucleus (Crapo

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25 et al., 1992) intermembrane space of mitochondria (Sturtz et al., 2001;Jaarsma et al., 2001) and peroxisomes (Keller et al., 1991) Mutant SOD1 proteins are presumably located in the same compartments, although it is possible that misfolding and aggregation of mutant SOD1 proteins may alter the normal SOD1 location. Some studies suggest that misfolding and aggregation of mutant SO D1 proteins interfere with mitochondrial function/s which would lead then to motor neuron death. Although there are studies that showed mutant SOD1 protein association with the cytoplasmic face of the outer mitochondrial membrane (L iu et al., 2004) it does not appear that mutant SOD1 is internalized into mitochondria. However, it is yet to be elucidated the effect, if any, that protein aggregates may have on mitochondrial metabolism. Additionally, if toxicity derives from mutant SOD1 aggregates targeted to mitochondria, it is unclear how only certain cells are susceptible to this toxicity, or what type of cell death mechanism is triggered. Thus, further studies are required to elucidate the location of mutant SOD1 proteins, includ ing mitochondria and other organelles, in order to further understand the disease mechanism of SOD1associated ALS. Potential D isease M echanism s of SOD1 Associated ALS Loss or Gain of SOD1 Function Several mechanisms have been proposed to explain how muta tions in SOD1 lead to the selective death of motor neurons in familial ALS. Initially, loss or increased protein activity were hypothesized as possible causes of ALS. However, it is now clear that SOD1 activity does not play a role in pathogenesis of famil ial ALS because: 1 Several SOD1associated ALS mutations possess normal SOD1 activity (Ratovitski et al., 1999) ; 2 Some mice require to express the SOD1 mutant protein several folds higher than endogenous mouse WT SOD1 in order to develop familial ALS, thus increasing

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26 overall SOD1 activity (Julien and Kriz, 2006;Jonsson et al., 2006b) ; while other models do not show increase in total SOD1 activity (Jonsson et al., 2004;Jonsson et al., 2006a) ; 3 Mice overexpressing WT SOD1 do not develop ALS (Gurney, 1994;Wong et al., 1995) ; 4 SOD1 knockout mice do not develop ALS (Reaume et al., 1996) ; 5 Reduction of SOD1 activity by depleting the copper chaperone for SOD1 (CCS) in transgenic mice does not change disease onset nor progression (Subramaniam et al., 2002) 6 Eliminating copper binding sites in human SOD1 of experimental transgenic mouse models does not abolish ALS development (Wang et al., 2002b;Wang et al., 2003) Mutant SOD1 Damage through Oxidative Chemistry Mechanisms The accumulation of reactive oxygen species is known to be able to cause cell death, and oxidative stress is known to increase with age and with neurodegenerative processes. In certain cases, free radical damage has been found in postmortem tissue of ALS p atients (Beal et al., 1997;Ferrante et al., 1997;Tohgi et al., 1999) Thus, alteration in the oxidative chemistry has been s uggested to be involved in SOD1associated ALS, as mutations in SOD1 may alter its free radical scavenger activity or provide new functions to SOD1 that leads to increase oxidative damage. The increase in oxidative chemistry may cause damage by peroxidation (Yim et al., 1990;Wiedau-Pazos et al., 1996;Yim et al., 1997) a mechanism by which WT SOD1 is known to react with hydrogen peroxide and produce highly toxic hydr oxyl radicals at low levels (Figure 15). Additionally, SOD1 could cause damage by covalent nitration of tyrosine residues (Beckman et al., 1993;Crow et al., 1997;Estevez et al., 1998) During this process, SOD1 reacts with peroxynitrite, which is produced from the reaction between superoxide and nitric oxide, and tyrosine residues of proteins acquire a nitro group. The

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27 nitration of tyrosines residues may alter protein function or folding patterns. This chemical reaction may occur by one of two different proposed mechanisms that involve SOD1 (Figures 16 and 17). Cu+Z n S O D H2O2 Cu+Z n S O D -O H + O H-O H Figure 15. Peroxidation reaction. Cu+ZnSOD: reduced, cuprous SOD1, H2O2: hydrogen peroxide, OHor OH: hydroxyl radical. O H-+ N O2-T y r-R Cu2+Z n S O D-O O N O H -T y r-R Cu2+Z n S O D O2 .-+ N O Figure 16. Mechanism of covalent nitration of tyrosine residues mediated by SOD1 (Beckman et al., 1993) Cu2+ZnSOD: oxidized, cupric SOD1, O2-: superoxide radical, NO: nitric oxide radical, -OONO: peroxynitrite anion, OH-: hydroxyl radical, NO2: nitrogen dioxide, NO2-Tyr -R: protein with 3nitrotyroxine, H -Tyr R: protein with tyrosine residue. Cu+S O D + O2Cu2+S O D ---O2 .-+ N O Cu2+S O D ---O O N O-N O2-T y r-R( Z n d e pl e t e d) ( Z n d e pl e t e d) ( Z n d e pl e t e d) Figure 17. An alternative mechanism of covalent nitration of tyrosine residues mediated by SOD1 does not require zinc binding (Crow et al., 1997;Estevez et al., 1999) Cu+SOD: reduced, cuprous, zinc depleted SOD1, O2: oxygen, Cu2+SOD: oxidized, cupric, zinc depleted SOD1, O-: superoxide radical, NO: nitric oxide radical, OONO-: peroxynitrite anion, NO2-Tyr -R: protein with 3nitrotyroxine.

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28 The caveat to the se mechanisms of toxicity is that these reactions require mutant SOD1 to bind copper (Yim et al., 1997;Yim et al., 1996;WiedauPazos et al., 1996) the essential cofactor for both dismutase activity and oxidative chemistry. However, a subset of SOD1 mutants (classified as metal binding mutants ) have defects at or near metal binding sites or otherwise lower the enzyme affinity for copper (Valentine and Hart, 2003) Studies in transgenic mice have directly tested this mechanism by producing mutant forms of SOD1 with two (H46R/H48Q) or four (H46R/H48Q/H63G/H120G) of the copper binding residues mutated. Mice expressing either mutant develops ALS -like paralysis (Wang et al., 2003;Wang et al., 2002b) Thus, while these studies have demonstrated that the correct binding of copper is not required for ALS symptoms, the aberrant binding of copper to other residues in the protein to induce motor neuron-specific damage is still a possibil ity (Bush, 2002) Mutant SOD1 and Protein Aggregation SOD1 positive inclusions have been found in SOD1 associated ALS patients (Matsumoto et al., 1996;Shibata et al., 1996b;Sasaki et al., 1998;Kato et al., 1999b;Kato et al., 1999a;Kokubo et al., 1999;Watanabe et al., 2001) and SOD1 transgenic mice (Wong et al., 1995;Dal Canto and Gurney, 1997;Bruijn et al., 1997;Wang et al., 2002a;Wang et al., 2002b;Wang et al., 2003) However, the identification of inclusions has not always been possible and it does not te ll us whether SOD1 -positive inclusions correspond to WT and/or mutant SOD1, or whether it contains misfolded or aggregated protein. Thus, i n our experience, the detection of mutant SOD1 aggregation is best accomplished biochemically, using a detergent extr action and sedimentation technique (Wang et al., 2003) Detergent insoluble species of SOD1 are distinguished by the property of forming structures that are not dissociated by non -ionic detergents and are

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29 of sufficient size to sediment upon centrifugation at high speed; properties associated with protein aggregation (Wang et al., 2003;Wang et al., 2006) For all familial mutant SOD1 proteins studied so far, detergent insoluble and sedimentable forms of mutant SOD1 can also be generated in cell culture models (Wang et al., 2003;Wang et al., 2006) Thus, to date there has been a strong correlation between the aggregation of mutant SOD1 and toxicity. There are different hypothesis that explains aggregate toxicity in ALS through interference with cellular processes ( Figure 1 8). Figure 18. Schematic representation of the possible targets of mutant SOD1 aggregates in ALS, interfering with the normal cellular metabolism. Some of these cellular processes include inhibition of the proteasome (Urushitani et al., 2002;Johnston et al., 2000;Kabashi et al., 2004) accumulation in mitochondria affecting their ov erall activity (Boillee et al., 2006a) interfering with axonal transport (Zhang et al., 1997;Borchelt et al., 1998;Williamson and Cleveland, 1999) or inhibition Axonal transport interference Mitochondrial damage Clogging the proteasome Sequestering proteins

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30 of different cellular proteins by sequestering them into the SOD1 aggregates. Sequestered proteins may include those involved in the chaperone or proteasome system, and in apoptosis (Okado Matsumoto and Fridovich, 2002;Kunst, 2004;Pasinelli et al., 2004) Implication of W T SOD1 in ALS and in Aggregation In SOD1associated ALS, mutant SOD1 proteins are coexpressed with WT SOD1 at 1:1 ratios of synthesis (Borchelt et al., 1994) In some cases, WT SOD1 can heterodimer ize with mutant SOD1protein (Borchelt et al., 1994;Borchelt et al., 1995) Whether toxicity of mutant SOD1 is modulated by interac tions between WT and mutant protein, or by the activity of WT SOD1, has been addressed in several experimental mo dels. Mice over expressing just human WT SOD1 appear largely normal (Gurney et al., 1994;Wong et al., 1995) although there have been reports of mild abnormalities in mice that express very high levels of WT SOD1 (Dal Canto and Gurney, 1994;Tu et al., 1996) In a study of mice that express human G85R SOD1, eliminating the expression of normal endogenous mouse WT SOD1 or over expressing human WT SOD1 [by crossing to a line of m ice produced by Wong and colleagues (Wong et al., 1995) ] had no obvious effects on disease onset, progression, or pathology (Bruijn et al., 1998) However, a later study found that mice co expressing high levels of human WT [by crossing to a line of mice produced by Gurney and colleagues (Gurney et al., 1994) ] and G93A SOD1 showed earlier disease onset than mice expressing the G93A mutant alone (Jaarsma et al., 2000) Recently, Deng and col leagues (Deng et al., 2006) reported that crossing the Gurney human WT SOD1 transgenic mice with mice harb oring three different ALS mutants (A4V, G93A, and L126Z SOD1) caused accelerated disease onset, which was accompanied by the appearance of aggregated

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31 SOD1. In the case of the A4V mutant mouse model, no evidence of mutant protein aggregation was detected in the absence of additional WT SOD1 or disease symptoms. A second interesting outcome was the observation that WT SOD1 protein co -purified with the mutant SOD1 aggregates in mice that co expressed WT and L126Z SOD1 (Deng et al., 2006) Thus, one explanation for the decrease in age to onset could be that the addition of WT SOD1 promoted a more rapid aggregation of m utant protein. Recent studies by Wang and colleagues have shown the results of crossing the same high expressor WT SOD1 mouse line from Gurney and colleagues to G85R SOD1 mice obtaining similar results on accele rated disease onset and progression (Wang et al., 2009c) These results differ from the crosses made by Bruijn and colleag ues with the lower expression WT SOD1 and G85R line (Bruijn et al., 1998) This suggests that the differences may lie in the different WT SOD 1 lines of mice used. However, the G85R SOD1 mouse lines used in both studies are different, thus they may not express the same transgene levels, a feature important in mouse models for SOD1ALS (see Animal Models to Study SOD1-Associated ALS in Chapter 1 and Chapter 2). Additionally, in neither study there is mention on whether aggregation is affected by the expression of WT SOD1 protein. Thus, the effect of WT SOD1 on disease and how they relate to aggregation of mutant SOD1 protein is still unclear, an d further studies should be performed. SOD1 Associated ALS: A N on C ell A utonomous D isease SOD1 associated ALS is known to cause motor neuron degeneration. Several studies demonstrate that motor neurons are primarily targets of mutant SOD1 toxicity: a ) Reducti on of mutant SOD1 from motor neurons slows disease onset (Raoul et al., 2002;Ralph et al., 2005;Miller et al., 2006;Boillee et al., 2006b)

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32 b ) Primary motor neuron cultures, but not of other cell types are susceptible to death pathways when the cells express a SOD1 mutation (Lino et al., 2002) However, these studies do not explain whether the toxicity exerted from mutant SOD1 comes exclusively from motor neurons expressing mutant SOD1. It has been shown that mice with mutant SOD1 expression specifically targeted to motor neurons do not develop a n ALS like phenotype (Pramatarova et al., 2001;Lino et al., 2002) These studies suggest the involvement of other cell types in SOD1 associated ALS. Additionally, decreasing the amount of mutant SOD1 within motor neurons delays dis ease onset in transgenic mice, support ing the notion of motor neurons as the center of toxicity (Boillee et al., 2006b) The Cleveland group has been pioneer in investigating the role of different cell types in SOD1associated ALS pathology. The first indication that SOD1 associated ALS is a non-cell autonomous disease came by the creation of chimeric mice expressing human WT and mutant SOD1 proteins. These mice contained motor neurons with higher survival rates when they were surrounded by nonneuronal cells expressing WT SOD1. And degeneratin g motor neurons were in close proximity to nonneuronal cells expressing mutant SOD1. This study indicates that the motor neuron environment may be critical for their survival, as non-neuronal cells might help to trigger motor neuron death (Gong et al., 2000) Astrocytes and Microglia Gliosis is an early pathologic feature observed in SOD1-ALS mice at early stages of disease development (Feeney et al., 2001) Thus, an early thought was that actrocytes or microglia could be the inducers of motor neuron degeneration in ALS. Transgenic mice expressing mutant SOD1 exclusively in astrocytes do not develop

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33 ALS, demonst rating that ALS might not initiate in this particular cell type. However, gliosis occurring within astroglia that express mutant SOD1 may be involved in aggravating disease once it has initiated (Yamanaka et al., 2008) On the other hand, regarding microglia, depletion of mutant SOD1 from about 25% of microglial cells, but not from astrocytes, translates into longer survival in mutan t SOD1 transgenic mice, with no differences in the number of activated astroglial or microglial cells (Boillee et al., 2006b) This data suggest the involvement of glial cells in ALS, although it is still unclear how they contribute to the disease. Oligodendrocytes and Schwann Cells Oligodend rocytes and Schwann cells form myelin sheets that envelop axons in the central and peripheral nervous system, respectively. Oligodendrocytes do not seem to be implicated in ALS as chimeric mice, which express mutant G37R SOD1 within all motor neurons and oligodendrocytes, do not develop ALS -like disease (Lobsiger et al., 2009) Surprisingly, Sc hwann cells seem to be protective as a 70% reduction of mutant G37R SOD1 transgene from Schwann cells shortens survival by over a month in mice (Miller et al., 2006) Muscle Cells M otor neurons synapse onto muscle cells through the formation of the neuromuscular junction. Retraction of nerve terminals is known to occur in ALS, li kely to a consequence of motor neuron degeneration. However, it is still unclear whether the retraction mechanism starts in the neuron or the muscle cell. In a study of Miller and colleagues, by reducing mutant SOD1 expression from muscle cells alone there is no change in neither onset nor survival of mutant SOD1 transgenic mice (Deng et al.,

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34 2006) These results suggest that mutant SOD1 acting from the muscle cells do not appear to play a role in toxicity. Animal Models to Study SOD1 Associated ALS Transgenic Rodents From all identified genes or loci, mutations in SOD1 represent the largest group in familial ALS, constituting approximately 20% (12% of all ALS cases). Of the more than 100 mutations identified to cause disease in humans, only 11 have been expressed in transgenic animal models in order to unravel the molecular mechanisms of th e disease. The most important models are represented in Table 14. Additionally, several animal models harboring experimental mutations in the SOD1 transgene have also been created: H46R/H48Q (Wang et al., 2003) H46R/H48Q/H63G/H120G or QUAD (Wang et al., 2007) and SODMD: C6G, H43R, H46R, H48Q, H63G, H71R, H80R, C111S, H120G (for information on SODMD mice see Chapter 2). Table 1 4 Transgenic rodent models for several SOD1 mutations. SOD1 mutation Animal model Refere nces A4V* Mouse (Deng et al., 2006) G37R cDNA Mouse (Wang et al., 2005b) G37R Mouse (Wong et al., 1995) H46R Mouse (Chang Hong et al., 2005;Sasak i et al., 2007) H46R Rat (Nagai et al., 2001) L84V Mouse (Tobisawa et al., 2003) G85R Mouse (Bruijn et al., 1997;Wang et al., 2009c) D90A Mouse (Jonsson et al., 2006b) G93A Mouse (Gurney et al., 1994) G93A Rat (Nagai et al., 2001;Howland et al., 2002) G93R Mouse (Friedlander et al., 1997) L126Z or L126stop Mouse (Wang et al., 2005a;Deng et al., 2006) L126delTT(stop 131) Mouse (Watanabe et al., 2005) Gins127TGGG Mouse (Jonsson et al., 2004) *Asymptomatic mice

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35 In all these models, there is evidence suggesting that the levels of expression are critical to induce disease. For example, there are mice that express the G37R or D90A SOD1 that do not develop disease unless bred to homozygosity (Wan g et al., 2005b;Jonsson et al., 2006b) Similarly, disease is absent in mice expressing low levels of A4V SOD1 but evident when the levels of total SOD1 are raised by coexpression with WT SOD1 (Deng et al., 2006) Thus, the levels of expression of SOD1 mutant proteins seem to play an important role in ALS-rodent models. Pathology in SOD 1 Associated ALS and Rodent Models of the Disease A few studies have tried to evaluate the cellular and subcellular changes in familial ALS. The availability of human tissue to evaluate such changes in familial ALS cases is scarce and only provides inform ation of end stage disease since only post mortem tissue is examined. Thus, most studies have been done using asymptomatic and symptomatic tissue of heterozygous transgenic animal models expressing SOD1 mutations associated with familial ALS. Only a few of the SOD1 associated ALS mutations have been studied for such intracellular and pathological changes in animal models, being the G93A SOD1 mice the most extensively studied and followed by G37R, G85R, H46R, H46R/H48Q, Quad and L126Z SOD1 mice. Some of thes e pathological changes include: Vacuolarization in anterior horns of spinal cords has been observed in the high expressor G93A and in G37R SOD1 mice (Dal Cant o and Gurney, 1994;Wong et al., 1995) These vacuoles have been proposed to derive from mitochondria in the case of the G37R SOD1 mice (Wong et al., 1995) or from the Golgi apparatus and the endoplasmic reticulum in the G93A SOD1 mice (Stieber et al., 2000c;Stieber et al., 2000a) However, vacuolar pathology may be a consequence of high SOD1 activity and not a common pathological hallmark of ALS since the vacuolar pathology is also observed in transgenic mouse models expressing human WT SOD1 at high levels (Jaarsma et al., 2000;Sasaki et al., 1998) whereas it is absent in transgenic mice that express SOD1 mutants with none or reduced SOD 1 activity such as G85R SOD1 mice (Bruijn et al., 1997;Watanabe et al., 20 01) H46R/H48Q SOD1 mice

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36 (Wang et al., 2002b) or even in the lower expressor G93A SOD1 mice (Dal Canto and Gurney, 1997) Golgi apparatus fragmentation have also been observed but only in the G93A SOD1 mice (Stieber et al., 1998;Stieber et al., 2000c;Stieber et al., 2000a) Although no other animal model for familial ALS has been used to study the changes in the Golgi apparatus, the same abnormalities have been previously described in some sporadic ALS cases (Wong et al., 1995;Matsumoto et al., 1996;Kato et al., 2000b;Kato et al., 2000a;Kato et al., 1999a;Sasaki et al., 2005;Sasaki et al., 1998;Shibata et al., 1996b) Thus, further studies in other transgenic models for mutant SOD1 should be used to explore the involvement of the Golgi apparatus in ALS. The presence of intracytoplasmic neural inclusions seems to be the common pattern observed in post -mortem human tissue and animal models that develop familial ALS -like disease (Kato et al., 1999b;K ato et al., 1999a;Watanabe et al., 2001;Wang et al., 2002b;Sasaki et al., 2007;Bruijn et al., 1997;Dal Canto and Gurney, 1997;Kokubo et al., 1999;Shibata et al., 1996b) These inclusions are usually observed with haematoxylin and eosin or other unspecif ic stains but some studies have gone further to characterize their composition. The most common inclusions are ubiquitin positive (Seilhean et al., 2004;Matsumoto et al., 1993;Schiffer et al., 1991) or immunoreactive for cystatin C, the latter component of the characteristics Bunina bodies (Shibata et al., 1996a;Okamoto et al., 1993) Familial ALS patients wi th the SOD1 mutations A4V or L126delTT also presented motoneurons filled with SOD1 inclusions (Bruijn et al., 1998;Wong et al., 1995) Likewise, H46R SOD1 ALS patients reveal inclusions that are reactive to SOD1 or ubiquitin (Ohi et al., 2002;Arisato et al., 2003) SOD1 positive inclusions are also observed in motoneurons of G37R (Wong et al., 1995) H46R (Sasaki et al., 2007) G85R (Bruijn et al., 1997;Watanabe et al., 2001) and G93A SOD1 transgenic mice (Watanabe et al., 2001;Stieber et al., 2000b;Sasaki et al., 2005) These SOD1 positive inclusions have been observed surrounding the pathologic vacuoles in G37R and G93A SOD1 mice (Wong et al., 1995;Higgins et al., 2003) and also in astrocytes before the appearance of disease symptoms in G85R SOD1 mice (Bruijn et al., 1997;Watanabe et al., 2001) .The astrocyte SOD1 -positive inclusions have also been reported in an earlier study of a familial ALS frameshift mutation, L126delTT SOD1 (Kato et al., 1996) However, the observation of SOD1 immunopositive inclusions has not always be en possible. For example, poorly SOD1 stain has been reported for G37R, G85R, G93A, H46R/H48Q and Quad SOD1 transgenic mice (Wang et al., 2003) In addition, Watanabe and colleagues als o did not detect any types of inclusions in symptomatic G37R SOD1 mice (Watanabe et al., 2001) Thioflavin S o r T staining has been used to characterize the type of inclusions in familial ALS. Thioflavin is a dye that reacts with stacked beta sheet structures and is a typical amyloid-like inclusions (Kelen yi, 1967;VASSAR and CULLING, 1959) The use of thioflavin stain in sporadic ALS human tissue has not been useful since control samples also show fluorescent material (Wang et al., 2002b;Wang et al., 2003;Wang et al., 2005b) However, thioflavin positive in clusions have been observed in some familial ALS transgenic mice (Wang et al., 2006) but not in the

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37 L126Z SOD1 mouse model (Wang et al., 2005a) These data sugg est that there may be different types of SOD1 aggregates in tissues expressing different SOD1 mutations and the difficulty in some cases to stain these aggregates indicates that further analyses are required. Detergent insoluble aggregates of mutant SOD1: all transgenic animal models expressing SOD1associated ALS mutation consistently form structures of mutant SOD1 proteins that can be isolated with a biochemical assay through detergent extraction and high speed centrifugation. The appearance of these dete rgent insoluble species accumulate significantly at disease endstage of hindlimb paralysis, while they are undetectable when mice are asymptomatic (Wang et al., 2006;Karch et al., 2009) However, there is no evidence that the biochemically isolated aggregates of mutant SOD1 are the SOD1 immunoreactive species (inclusions) seen in mouse or human tissues expressing a SOD1associated ALS mutation. Further studies regarding detergent -insoluble aggregates of mutant SOD1 can be found in the subsequent chapters. Other Transgenic Animal Models for SOD1Associat ed ALS SOD1 -e xpressing fruit flies. The fruit fly, Drosophila melanogaster has been used as a model to study SOD1. Flies null for WT SOD1, present a phenotype characterized by a shortened lifespan, decreased fertility and increased oxidative stress (Parkes et al., 1998) The shortened lifespan is recovered by the expression of WT SOD1, and overexpression of WT SOD1 in motorneurons induces a 40% increase in D. melanogaster lifespan (Mockett et al., 2003) Additonally, flies expressing SOD1associated A LS mutations have also been made (A4V, G37R, G41D, G93C, I113T) (Elia et al., 1999;Mockett et al., 2003) These mutant SOD1 flies can partially restore the shortened lifespan observed in SOD1 null flies (Elia et al., 199 9) However, the mutant SOD1 flies do not present symptoms of paralysis or premature death and do not manifest the toxic gain of function phenotype that is observed in human patients, as in flies the effect on lifespan is a recessive trait (Elia et al., 1999;Mockett et al., 2003) Thus, the use of this model system does not

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38 seem suitable to study ALS, but might be important to better understand how mutations affect the SOD1 protein. Worms m odels for SOD1-a ssociated ALS Recently, transgenic anim al models of Caenorhabditis elegans have been created to express SOD1 proteins (WT, G85R, G93A, G127X) (Wang et al., 2009a;Gidalevitz et al., 20 09) These transgenic worm models express SOD1 proteins with a C terminal yellow fluorescent protein ( YFP )-tag. Worms expressing mutant SOD1::YFP (SOD1 fused to YFP) proteins form visible inclusions, which translates into locomotor dysfunction (Wang et al., 2009a;Gidalevitz et al., 2009) Th us, this model represent s a useful tool to asses s toxicity of SOD1 mutant proteins. Although worms exp ressing WT SOD1::YFP do not present locomotor dysfunction or apparent inclusions, it is uncertain to whether the YFP could modify certain SOD1 properties that would not make this particular worm model useful to replicate all disease related features. Addit ionally, it would be more interesting to study SOD1 in non -tagged scheme in these C. elegans models. Other m odels Other transgenic animal models have been created as an attempt to obtain a better model to study SOD1 associated ALS. An example is the Danish pig, harboring the G93S mutation (Dr. Peter M. Andersen, personal communication). The use such pigs as a model to study ALS may not be, however, cost effective or convenient. In any case, d ata on such models has not yet been published, thus we cannot determine the viability of this possible new ALS model. Recent studies have published the existence of dogs with motor neuron disease that resembles ALS. These dogs express the E40K SOD1 mutation, which might be

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39 equivalent to the E40G ALS mutation in humans. Dogs heterozygous for E40K mutation are asymptomatic but develop motor neuron disease when they are homozygous for the same mutation. If more cases of dogs harboring SOD1 mutations are found and disease features resemble those of ALS, then these animals would represent the first sporadic model to study ALS. Therapies Currently, there are no drugs that can cure ALS. Onl y Riluzole is commonly used to treat ALS. Many different drugs have gone into clinical trials, but no one represents a better alternative treatment. Riluzole Riluzole is the only drug approved by The Food and Drug Administration for treatment of ALS. In th e best of cases, Riluzole extends lifespan by 2 to 3 months (Lacomblez et al., 1996;Lacomblez et al., 2002;Miller et al., 2007b) However, it is still unclear how this drug prolongs survival in ALS patients. Several Riluzole biological effects have been identified: modulation of glutamate release fr om the presynaptic terminal (Albo et al., 2004;Fumagalli et al., 2008) inhibition of G protein dependent processes (Huang et al., 1997a) voltage gated sodium channel blocker (Zona et al., 1998;Urbani and Belluzzi, 2000) voltage gated potassium inhibitor (Zona et al., 1998) and voltage activated calcium channel inhibitor (Huang et al., 1997a;Huang et al., 1997b) More r ecently, Riluzole has been shown to have a protective role against the slowing of neurofilament axonal transport that is induced by glutamate (Stevenson et al., 2009) In ALS patients, Riluzole is thought to act mainly as an anti excitotoxicity agent, inhibiting excessive glutamate release from synaptic terminals. This property of Riluzole

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40 is thought to compensate the abnormal glutamate metabolism, which has been reported in some ALS patients. Additionally, the following studies support this anti excitotoxic role of Riluzole in ALS: The glial glutamate transporter EAA2 is lost in rat models for SOD1associated ALS (Howland et al., 2002) In ALS patients the activity of glutamate transporter GLT -1 is decreased in spinal cord and motor cortex (Rothstein et al., 1992) Protein expression of EAA2 is reduced in the motor cortex of ALS patients (Rothstein et al., 1995) Glutamate transporters are in charge or recycling glutamate released from neurons into glia, where they are transformed into glutamine and transported back to neurons for glutamate neurotransmitter formation. The lost or deficiency of these transporters leads to an increase in extracellular glutamate, which translates into exitotoxicity. This exitotoxicity may include influx of calcium into cells, extensive depolarizations, or other events that may lead to c ell death. However, the exact mechanism by which Riluzole may regulate glutamate release is still unknown Additionally, this particular toxic event does not appear to represent an extremely important factor in ALS as the ben efits of Riluzole are small or n onexistent. Thus, further drugs are still to be discovered to obtain a better treatment to slow or stop ALS development. Other Tested Drugs A wide number of drugs have been tested in human clinical trials. However, none of these drugs h as proven to be beneficial in slowing or curing the disease. A list with tested drugs that have gone into clinical trials is represented in Table 1-5.

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41 Table 1 5. Past clinical trials conducted in ALS patients. Drug Mechanism of action References 3,4 diaminopyridine Slow potassium channel blocker (Aisen et al., 1996;Aisen et al., 1995) Omigapil (TCH346) Anti apoptotic, binds glyceraldehyde 3 phosphate deshydrogenase (Miller et al., 2007a) Brain derived neurotrophic factor Growth factor (Beck et al., 2005;Kalra et al., 2003) Branched chain amino acids Increase muscle protein synthesis (Tandan et al., 1996;Testa et al., 198 9) Celecoxib Cyclooxigenase 2 inhibitor (Cudkowicz et al., 2006) Ciliary neurotrophic factor Motor neuron survival (Al Chalabi et al., 2003) Creatine monohydrate Dietary supplement thought to increase muscle streng th (Groeneveld et al., 2003) Cyclophosphamide Immunosuppressor (Gourie Devi et al., 1997;Brown, Jr. et al., 1986;Smith et al., 1994) Dextromethorphan NMDA glutamate receptor antagonist (Gredal et al., 1997) Gabapentin Glutamate blocker (Miller et al., 2001) Ganglioside Immunomodulator (Harrington et al., 1984) Glutathione Antioxidant (Chio et al., 1998) Insulin like growth factor 1 Growth factor (Sorenson et al., 2008) Interferon beta 1a Immunomodulator (Beghi et al., 2000) Lamotrigine Glutamate release inhibitor (Ryberg et al., 2003) Minocycline Anti apoptotic and anti inflammatory (Gordon et al., 2007) N acet ylcysteine Anti oxidant (Vyth et al., 1996) Nimodipine Calcium channel blocker (Miller et al., 1996a ) Pentoxifylline Anti apoptotic (Levin et al., 2006;Meininger et al., 2006) Physostigmine Interfere with acetylcholine metabolism (Norris et al., 1993) Selegiline Anti o xidant (Lange et al., 1998;Mazzini et al., 1994) Thyrotropin releasing hormone Trophic facto r (Munsat et al., 1992) Topiramate Inhibitor of excitatory neurotransmission (Cudkowicz et al., 2003) Total lymphoid irradiation Immunosuppressor (Drachman et al., 1994) Verapamil Calcium channel blocker (Miller et al., 1996b) Vitamin E Anti oxidant (Graf et al., 2005) Xaliproden Serotonin receptor agonist (Meininger et al., 2004)

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42 Future Clinical Trials Several other drugs are currently being tested in humans or will be in the near future. A list of some of them is indicated in Table 1-6. Note that some trials have been completed, but the studies have not been published yet. Table 1 6. Ongoing clinical t rials for the treatment of ALS. Drug Mechanism of action Status1 Antisense oligonucleaotide SOD1 Reduce SOD1 protein expression levels Unknown Arimoclomol Upregulate heat shock proteins Phase II/III trial (NCT00706147) ongoing Ceftriaxone Anti oxidant Phase III trial (NCT00349622) ongoing Coenzyme Q 10 Cofactor in mitochondrial electron transfer Phase II trial (NCT00243932) completed Diaphragm conditioning Diaphragm conditioning with electrodes Trial (NCT00420719) ongoing MCI 186 Anti oxidant Phase I II trial (NTC00330681) completed Memantine NMDA glutamate receptor antagonist Phase II trial (NCT00353665) completed ONO 2506 Cyclooxigenase2 (COX2) inhibitor Phase II trial (NCT00403204) completed, R(+)pramipexol Anti oxidant Phase II trial (NCT00600873) ongoing Talampanel Regulator of AMPA receptors Phase II trial (NCT00696332) ongoing TRO19622 Mitochondrial transition pore inhibitor Phase II/III trial (NCT00868166) not yet started 1Information on the status of the trials were obtained from the U. S. National Institute of Health web page of clinical trials (Clinical Trials, 2009). T he identifier number was also included.

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43 CHAPTER 2 LACK OF TOXICITY IN A NOVEL VARIANT OF HUMAN SOD1 HARBORING ALSASSOCIATED AND EXPERIMENTAL MUTATIONS IN METAL -BINDING RESIDUES1 Introduction Mutations in SOD1 are found in familial cases o f ALS. The enzymatic function of SOD1 is to catalyze the antioxidant reaction that converts superoxide radicals ( O2 -) into hydrogen peroxide (H2O2) and oxygen (O2) (McCord and Fridovich, 1969) The mature homodimeric SOD1 protein binds copper and zinc ions, which are either required for activity or provides structural stability, respectively (Forman and Fridovich, 1973;Elam et al., 2003;Potter et al., 2007) Histidines coordinate the copper (H46, H48, H63, and H120) and zinc (H63, H71, H80) binding sites. Additionally, an aspartate also forms part of the zinc site (D83). The two monomers in the SOD1 homodimer are maintained together by non -covalent forces, while an intramolecular disulfide bond between cysteines 57 and 146 confers structural stability within each monomer. Two cysteines, C6 and C111, are free in the normally folded protein, and it is known that C111 can bind metals aberrantly (Watanabe et al., 2007) Of t he 148 described SOD1 disease -causing mutations, a small portion presents defects in metal binding capability, thus affecting the overall enzymatic activity of SOD1. However, many other SOD1 mutant proteins do not present significant alterations in metal b inding or activity. In vitro studies suggest that normally folded SOD1 can misfold and aggregate upon the loss of metals and/or reduction of its intramolecular disulfide bond (Chattopadhyay et al., 2008;Oztug Durer et al., 2009) Further studies demonstrate that metal binding prevents the dissociation of the intramolecular disulfide 1 The work presented here is a manuscript in preparation that will be submitted shortly for publication.

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44 bond, suggesting that metal binding is required to prevent aggregation (Tiwari and H ayward, 2005) Transgenic mouse models expressing experimental SOD1 mutant proteins that abolish either two (H46R/H48Q) or four (H46R/H48Q/H63G/H120G or QUAD) of the normal copper binding sites develop ALS, which is accompanied by the characteristic fo rmation of detergent insoluble SOD1 aggregates (Wang et al., 2002b;Wang et al., 2003) These mic e are unable to bind copper in their catalytic site; thus, copper binding do es not appear to be required for aggregation. However, additional studies suggest that copper can interact with free cysteines, in particular cysteine 111 (Watanabe et al., 2007) Thus, the present models may not account for aberrant copper binding that might have a role in aggregation. On the other hand, nothing is known about the role that zinc binding might have in aggregation of mutant SOD1 and/or disease. In order to study the implication of SOD1 metal bindin g and aggregation and its role i n disease, we created mice expressing a SOD1 protein (termed SODMD) in which we abolished all possible sites of metal binding (H43, H46, H48, H63, H71, H80, H120), including the two free cysteine amino acids, C6 and C111. SOD1 in t hese mice is incapable of binding either copper or zinc in its normal binding site, or aberrantly in any of the reported possible sites. We analyzed the effect of the SODMD protein in mice, and we extensively characterized the ability of SODMD and SODMD protein variants to sediment in nonionic detergent. Materials and Methods A list of materials used can be found in Appendix B. Methodology used for the work presented in this chapter is described in Chapter 2 Methods of Appendix C.

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45 Results To study the role of metal binding in aggregation and disease, w e created transgenic mice expressing the metal deficient genomic SOD1 variant, or SODMD. Mutations within this protein comprise all histidine residues known to be involved in metal binding (H43, H46, H48, H63, H71, H80, H120) and the two free cysteines that do not form part of the intramolecular disulfide bond (C6 and C111). A diagram with the SODMD mutations i s represented in Figure. 2 1 Figure 21. Schematic representation of genomic SODMD. The SODMD DNA is mutate d within each of the 5 exons (E) that compose SOD1. Red denotes single amino acid substitutions in the SOD1 protein that have been previously found in different human ALS patients. The rest of the single point mutations in black are experimental mutations. Orange circles indicate copper binding sites, while yellow circles indicate zinc binding sites. The first and last amino acid locations of each exon are also indicated in the figure. A total of seven founder lines were initially produced, from which we selected the two highest expressing lines (I -32 and U -69 lines) to be maintained (Figure 22). The rest of the lines produced very low or non -detectable levels of transgene. To determine whether the expression of mutant SOD1 in either line of these MD mice w as sufficiently high to induce disease, we compared the levels of transgene mRNA in our highest expressor SODMD mice (line U -69) to several of our lines of mutant SOD1 mice (Figure 2 -3) For each line of mice we used 3 mice of similar ages, however even fo r the same E 1 E 2 E 3 E 4 E 5 1 24 79 118 153 23 56 55 80 119

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46 SOD1 mutation, mRNA levels were different among each animal (especially note the error bars for G93A and L126Z line 44 in Figure 23B). Mice expressing the mutations G93A or double histidine (H46R/H48Q) mutants present the highest mRNA levels, f ollowed by WT L76, G37R and two different lines of L126Z SOD1 mice (L44 and L45). A line of L126Z mice (L171) expresses the lowest levels of transgene of all lines analyzed here. Compared to L126Z L171, the SODMD line U 69 mice present equivalent levels of m RNA (Figures 23A and 2-3B). The s e data suggest that mice harboring the SODMD mutations present expression levels that are in the lower range of transgene expression but that are high enough to manifest an ALS phenotype if the mutation is pathogenic. Th e L126Z line 171 mice represent the level of expression that is approximately at the threshold required to induce disease. The SODMD I -32 line expresses mRNA SOD1 at lower levels than L126Z line 171, whereas the SODMD U -69 line expresses at levels equivalent to L126Z line 171 mice (see Figure 22). Thus, for the purpose of these studies we used exclusively animals from the SODMD U -69 line of mice. Figure 22 Northern blot showing the two lines of SODMD mice with the best ex pression levels. The SODMD mRN A levels are compared to those of L126Z L171 which expresses SOD1 at very low levels. This blot was exposed for a long time in order to allow visualization of mRNA levels of the SOD MD line I 32. This figure was kindly provided by Ms. Hilda Slunt -Brown. MD (U 69) MD (I 32) SOD1 mRNA

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47 We have previously observed that the levels of transgene expression relate to disease development in our SOD1 mice. For example, there are cases in which transgenic mice homozygous for a certain mutant SOD1 protein present a shorter lifespan than heterozygous mice for the same SOD1 mutation. However, it is uncertain to whether we can predict when animals develop ALS based on data from different transgenic lines, expressing different mutant SOD1 proteins at different levels. Thus, to determine whether we can use mouse lifespan data available from other SOD1 lines to estimate when SODMD mice should get sick, we collected data on age of disease endstage defined by weakness followed by paralysis of the hindlimbs in different transgenic lines. W hen we compar ed such data with transgene expression levels, w e observed that mice presenting a shorter lifespan, present higher levels of transgene expression ( Figure 2-3 B). For example, G93A mice express the highest levels and develop paralysis at the earliest times, around 5 months old (159.1 4.03 days), while the lowest expression mice (L126Z L171) do not develop symptoms till they are 13 months old (400.3 22.56 days; Fig ure 2 -3 B). Additionally, correlation analys i s demonstrate s that the age of onset of symptoms in transgenic mice is dependent on the level of mutant SOD1 mRNA ( Figure 24 ). Thus, we can expect to observe onset of symptoms in our SODMD mice (line U 69) at similar ages than L126Z L171 mice (around 1 year old), since both lines of mice present simila r levels of SOD1 mRNA In our experience the mRNA levels of SOD1 in m ouse spinal cords predict the levels of SOD1 protein present in s pinal cords Furthermore, we have s hown that mRNA levels are similar for SODMD and L126Z L171 mice. Thus, to corroborate that protein

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48 levels are also similar between these two lines of mice we evaluated total protein levels for mice belonging to each line. Our data demonstrate that protein levels in the low expressor L126Z L171 mice do not differ from that of SODMD mice ( Fi gure 25 ). These results, together with our analysis in mRNA SOD1 levels, assure us that disease development in SODMD would be predicted to occur around the age of onset of L126Z L171 mice if the SODMD protein is a disease causing variant. Figure 23 Lower mRNA SOD1 expression levels predict a longer lifespan in mice. A) Northern blot showing the mRNA levels of different lines of mice expressing WT or mutant SOD1 proteins The 28 S and 18S RNAs and PrP mRNA are also shown, the latter serv ing as a loadi ng control. For these experiments Ms. Hilda Slunt Brown contributed with invaluable help. B) Quantifications of mRNA levels of three different Northern blots are represented by the black bars. White bars indicate the survival times of SOD1 lines calculated from data collected in our lab from animals harvested during more than 4 years. The o symbol over the survival time of SODMD line indicates that the data bar represents mean of lifespan to sacrifice, however no disease symptoms were noted in these mice. In terms of disease development, we did not observe apparent weakness, or paralysis in SODMD mice. At ages closer to 2 years of age (normal lifespan of laboratory mice), animals were monitored daily. In some cases some SODMD mice were found dead, but no s ymptoms of weakness or paralysis were noted. 28S 18S PrP mRNA SOD1 mRNA A B G93A 46/48 L139 G37R L29 L126Z L44 L126Z L45 L126Z L171 MD U69 0 2.5105 5.0105 7.5105 1.0106 1.3106 1.5106 0 200 400 600 800 1000 Levels of SOD1 mRNA (A.U.)Lifespan of SOD1 lines (days)

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49 Figure 24 The levels of SOD1 mRNA and lifespan of SOD1 mice statistically correlate. The levels of SOD1 mRNA were obtained from experiments performed in Figure 23. Data on the lifespan of SOD1 mice were c ollected from a minimum of 10 mice per paralyzed line of mice that were harvested along the course of 4 years. Figure 25 Protein levels in the spinal cord of SODMD mice resemble those of L126Z SOD1 mice. A) Western blot of total protein levels of indicated mouse lines which has been incubated with an antibody that recognizes human SOD1. Note that L126Z truncation mutant and SODMD (MD in figures) run faster than other SOD1 proteins in SDS -PAGE gels. B) Quantification of the total human SOD1 protein levels. Non-significant differences are found between MD and symptomatic L126Z. NTg denotes Non -transgenic animals, N.S. nonsignificant differences, *p #p 0 200 400 0 5.0105 1.0106 1.5106 G93A H46R/H48Q L139 G37R L29 L126Z L44 L126Z L45 L126Z L171 R2 = 0.8518, p 0.01Lifespan of SOD1 lines (days)Levels of SOD1 mRNA (A.U.) NTg H46R/H48Q L126Z MD WT 0 1.0107 2.0107 5.01007 1.01008 1.51008 2.01008Total levels of hSOD1 protein (A.U.) MD A B N.S.#

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50 The levels of transg ene expression are an important feature to cause ALS symptom s in transgenic animal models. For example, mice expressing the G37R mutation under the mouse prion promoter do not develop motor neuron disease unless levels of expression are raised through homozygosity (Wang et al., 2005b) Similar examples occur for a line of mice that express the D90A mutant of SOD1 (Jonsson et al., 2006b) Thus, we self -crossed SODMD mice with the intenti on to increase SOD1 protein levels through homozygosity which should translate into a more rapid disease development. A total of 20 mice were positive for the SODMD transgene, from which approximately 33% should be homozygous. However, we did not observe disease symptoms in any of these mice suggesting that the SODMD variant is not pathogenic. Analysis of the SODMD mice for their ability to form detergent -insoluble aggregates demonstrates that the lack of disease development correlates with the inability o f the mutant protein to aggregate ( Figure 26 ). SODMD proteins appear to turnover rapidly as the SOD1 protein levels in the detergent -soluble fraction (S1) are low. The L126Z truncation SOD1 mutant protein is also rapidly degraded, but when mice become sym ptomatic the aggregated protein is not so easily degraded and accumulates in the cell as detergent -insoluble aggregates (P2), which c an be detected by our biochemical assay ( Figure 2-6 ). However, we did not observe detectable levels of SODMD protein in the insoluble or P2 fraction. Thus, SODMD mice lack of pathology that includes mutant SOD1 aggregates that are detergent -insoluble and centrifuge at high speed.

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51 Figure 26 Spinal cord of old SODMD mice do not contain aggregated SOD1 proteins. Immunoblot of detergent insoluble (P2) and detergent -soluble (S1) protein fractions of mouse spinal cords blotted with a human SOD1 antibody NTg: non-transgenic mice. Note that the L 126Z protein although is quite unstable in spinal cords (undetectable in S1) it is aggregated at detectable levels while SODMD protein does not aggregate. We further analyzed old SODMD mice histologically, in order to look for pathological features that m ay occur at time points previous to significant accumulations of detergent -insoluble aggregates. Axon denervation is an earlier disease feature observed in G93A SOD1 transgenic mice (Fischer et al., 2004) A consequence of this phenomenon is axon demyelinat ion. Thus, we performed myelin staining in sciatic nerve sections from non-transgenic, symptomatic H46R/H48Q, and old SODMD mice ( Figure 2 -7 ). In transverse sciatic nerve sections, only symptomatic H46R/H48Q mice appear to present a lower number of myelinated axons as well as smaller diameter of myelin sheaths, while SODMD sections did not differ from those of nontransgenic mice (Figures 2 7A to 2-7 I ). Similarly, longitudinal sciatic nerve sections showed reduced number of myelinated fibers in sick H46R/H48Q SOD1 mice, while old SODMD mice appear largely normal ( Figures 2 -7J to 2-7R ). Thus, SODMD mice do not present any obvious abnormality in myelinated fibers at old ages. P2 S1 MD

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52 Figure 27 Myelin abnormalities are observed in symptomatic H46R/H48Q SOD1 mice while SODMD do not differ from nontransgenic (NTg) mice A-R) Transverse (A-L) or longitudinal (M -R) sciatic nerve sections stained with myelin basic protein. Nuclear stain was applied with secondary antibody incubations. All micrographs were taken with a 40x objective, bars 50 (A -C, G -I) or 20 (D -F, M -R) m. Myelin Basic Protein (MBP) DAPI MERGE NTg H46R/H48Q MD A D G B E H C F I NTg H46R/H48Q MD J M P K N Q L O R

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53 Gliosis is another of the earliest characterized abnormalities in SOD1 transgenic mice (Feeney et al., 2001) To determine whether SOD MD mice present some early abnormalities at times prior the accumulation of detergent -insoluble SOD1 species or the appearance of hindlimb weak ness/paralysis, we performed studies on histological sections to look for reduced number of motor neurons, reduced axonal fibers and GFAP immunoreactivity. Haemmatoxylin and eosin staining indicates reduced presence of motor neurons in the ventral horn of symptomatic H46R/H48Q SOD1 mice (Figures 28A and 2 -8B). However, the number of motor neurons in WT and SODMD mice do not qualitative differ (Figures 2-8A and 28C). Silver impregnation stains axonal fibers Here, silver staining demonstrates that symptomatic mice expressing mutant SOD1 proteins present reduced number of fibers compared to WT or SODMD mice (Figures 28D to 2 -8F). Additionally, GFAP immunoreactivity is only markedly incre ased in our ssymptomatic H46R/H48Q SOD1 control ( Figures 2 8G to 2-8I ). Thus, our histological analysis demonstrate that mice expressing the SODMD variant are healthy and do not present any of the characteristic mutant SOD1 pathology. One question that might rise from these studies is to whether the 10 single point mutations in the SODMD protein alter too many of the SOD1 protein features. In order to address this issue we created a SODMD cDNA version for its analysis in cell culture. In cell culture, SODMD is more highly expressed such that it is clearly detected in the deter gent -soluble S1 fractions ( Figure 2 -9A, lower panel); however, as in our mouse model, we do not observe significant accumulation of protein that sediment in the detergent -insoluble fraction (P2), not different from WT at 48 h ours following transfection ( Fi gures 2 -9A and 29B ). This data confirms the inability of this mutant to

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54 aggregate. One feature of the WT SOD1 protein in cell culture is its ability to slow the aggregation rates of normally aggregating SOD1 mutants and to ultimately co aggregat e with mut ant proteins at longer, 48 h our transfection intervals (Prudencio et al., 2009a) Taking advantage of the more rapid migrating ability of SODMD protein in SDS-PAGE gels compared to most SOD1 mutant proteins, we co-transfected SODMD with the highly aggregating mutant G93A for 24 h ours At 24 h our transfection intervals, SODMD protein slows aggregation of G93A SOD1 proteins maintaining both proteins in the detergent -soluble S1 frac tion ( Figure 29C ). This significant reduction in aggregation of G93A is not different from the effect of WT on G93A aggregation ( Figure 29D ). Thus, SODMD mutant proteins conserve features of the WT SOD1 protein, suggesting that the amino acid substitutions in SODMD may only alter the metal binding capability and activity of SOD1. Transgenic mice co expressing mutant SOD1 and WT protein at very high level present an accelerated disease phenotype. In particular, WT SOD1 is able to trigger disease and aggreg ation in asymptomatic mice that expresses the A4V SOD1 mutation at very low levels (Deng et al., 2006) In an attempt to induce disease development in SODMD mice, we mated these mice to those expressing WT SOD1 at very high levels. Out of 59 animals that resulted from such mating, 11 presented both, WT and SOD MD transgenes. The youngest doubly transgenic WT/SODMD mice were born in April 2008, and at the time of writing (October 2009 ) such animals do not present visible disease symptoms. Additionally, co expression of WT and SODMD does not induce aggregation in cell culture (Figure 210). These data support the notion of SODMD as being the first

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55 experimental SOD1 mutant protein that is unable to cause an ALS phenotype in transgenic mice. Figure 28 SODMD mice, like WT SOD1 mice, lack of any ALS -like pathology.A -I) Mice expressing WT ( A, D G ), H46R/H48Q ( B, E, H ) and MD ( C, F, I) SOD1 proteins were characterized histologically to determine the appearance of any kind of pathology. A-C ) Heammatoxylin and eosin stain on paraffin embedded sections at low power (10x) demonstrate s significant lower number of motor neurons ( indicated by white arrowheads) in the ventral horn of symptomatic H46R/H48Q ( B) mice than in WT ( A) SOD1 mice. No differences are noted between WT ( A) and SODMD (C ) mice. M: medial portion of the spinal cord, L: lateral porti on of the spinal cord. D -F ) Silver staining of paraffin embedded sections indicat e s the existence of a very low number of axons in symptomatic H46R/H48Q ( E ), while WT ( D ) and MD ( F ) spinal cords appear largely normal. Note that all staining was done simult aneously for all sections thus the low fiber staining in H46R/H48Q mice is due to a lower number of axons present G -I ) GFAP -DAB staining on paraffin embedded sections demonstrate none or low GFAP immunoreactivity in WT ( G ) and MD ( I ) mice, while strong gliosis is apparent in symptomatic H46R/H48Q ( H ) SOD1 mice. Bar represents 50 m in A-C and 20 m in D -I and are located in the ventral portion of the spinal cord. WT L76 H46R/H48Q MD A B C D E F G H I M L M L L M

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56 Figure 2-9. The non-aggregating SODMD cDNA variant present WT-like features in cell culture. A, C) Immunoblot s of detergent-insoluble (P2) and detergent-soluble (S1) protein fractions of HEK293FT cells expressing the indicated SOD1 constructs for 48 (A) or 24 (C) hours. UT denotes untransfected cells. B, D) Quantification of the relative aggregation propensity of the indicated SOD1 mutant proteins expressed in cells fo r 48 hours (B) or of G93A in the 24 hour co-transfection experim ent (D). Paired student t -tests were performed to compare the aggregation propensities of the mutant proteins with the aggregation propensity of the human WT SOD1 protein (hWT or WT), or of indicated pairs. Non-significant differences were found between human WT and SODMD; and SODMD protein signific antly slows aggregate accumulation of G93A SOD1. *p 0.05, #p 0.005. P2 S1 AB hWT A 4 V M D 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 proteins (A.U.)#CD P2 S1 WT A 4 V G 9 3 A G93A + WT G93A + MD 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 mutant proteins (A.U.)* # #

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57 Figure 21 0. SODMD co expression with WT SOD1 does not induce a rapid increase in aggregation of either protein after 48 hour transfection interval in cell culture. A) Immunoblot of detergent -insoluble (P2) and soluble (S1) fractions of indicate d SOD1 proteins expressed in HEK293FT cells for 48 hours. B) Quantification of the aggregation propensity indicates than in this set of transfections, only A4V SOD1 is able to significantly aggregate. Paired student t -tests were performed to evaluate signi ficant differences in terms of aggregation propensity. #p N.S.: non -significant differences. Discussion We present here an experimental SOD1 mutation that eliminates metal binding sites in SOD1 and is unable to cause ALS in animal models. In addition to the lack of disease development, mice overexpressing the SODMD mutant protein are healthy, without any obvious pathology or protein aggregation during their lifespan. Attempts to increase the pathogenicity of this protein by raising the levels of mutant protein or the overall protein levels by overexpressing human WT SOD1, have not been translated into development of an ALS -like disease. Thus, SODMD mice present a human SOD1 protein that is non-toxic. Further understanding of this protein may help us to determine requirements for the SOD1 protein to induce disease. SODMD M ice and E xpression L evels Mice overexpressing experimental SOD1 mutations that abolish copper binding (H46R/H48Q, Quad) have been shown to present the ability to aggregate and produce P2 S1 A B WT A4V MD MD + WT 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 mutants (A.U.) # N.S.

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58 the toxicity observed with disease associated variants (Wang et al., 2002b;Wang et al., 2003) The metal deficient SODMD mice however, do not develop ALS -like symptoms of hindlimb paralysis. We carefully analyzed expression levels on different lines of mice expressing mutant SOD1 and found correlations between mRNA levels and onset of symptoms. The expression levels of the SODMD variant were in the lower end of expression but they were not different from those of L126Z L171 mice, which develop disease at 1 year of age. Additionally the levels of mutant protein in SODMD and L126Z L171 were quite similar. Thus, we are confident that SODMD mice express protein above the normal threshold to induce disease. In addition we crossed transgenic SODMD mice to obtain homozygous SODMD mice. From a total of 20 anim als, 6 should be homozygous mice with higher levels of expression. None of these 20 mice developed disease symptoms or pathology which should have been obvious at earlier time points than heterozygous mice since they have a higher dose of mutant protein. Therefore, the SODMD protein lacks of the toxicity inherent of other SOD1 mutant proteins. SODMD Protein D oes N ot P roduce ALS L ike Pathology The lack of toxicity in SODMD mice is reflected by the absence of detergent insoluble SOD1 aggregates. In recent st udies we have demonstrated that the formation of aggregates of mutant SOD1 in mice are a late event (Karch et al., 2009) and earlier abnormalities are known to occur previous to aggregate formation (Wong et al., 1995;Kennel et al., 1996;Borchelt et al., 1998;Watanabe et al., 2001;Fischer et al., 2004;Hegedus et al., 2007) However, we did not observe any other p athology that might occur at earlier stages in mice generated from SODMD x SODMD cross. In particular, we found no significant changes in myelination, fiber or motor neuron

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59 numbers, or gliosis. Additionally, SODMD does not possess even a mild toxic propert y that could be enhanced by cooverexpressing it with WT SOD1. Thus, we are convinced that SODMD is a non-toxic variant of mutant SOD1. Metal B inding, Aggregation and D isease Relating metal binding to disease is a difficult endeavor since our animals also lack of the aggregate pathology. Eliminating the copper ligands does not abolish aggregation in cell culture or animal models. The binding of zinc is known to confer structural stability t hus abolishing zinc binding could in theory increase the potential of the protein to misfold and aggregate. Rather it seems that the inability of SODMD to aggregate may be related to its lack of pathogenicity. A different explanation would be that metal binding plays a role in disease. From our studies we cannot discard that, however, that metals or aberrant SOD1 activity plays a role in the disease. The inability of SODMD to aggregate could be linked to the presence of an amino acid substitution in cysteine 111 to a serine (C111S). This particular cysteine in position 1 11 has recently shown to play an important role in aggregation and substitution to a serine does not make WT SOD1 to aggregate (Kar ch and Borchelt, 2008;Prudencio et al., 2009a) More importantly, C111 S reduces aggregation rates in combination with other aggregation prone proteins. Thus, it is likely that the lack of aggregating ability of SODMD might come from this particular mutation. Further studies should then be focused on obtaining an SODMD variant that lack of same metal binding sites but is able to aggregate. In this way we would be able to assess whether the lack of toxicity of SODMD comes from the lack of metal binding or the inability of the protein to aggregate.

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60 SODMD and WT SOD1 The 10 point mutations in SODMD make it different from WT in terms of SOD1 activity, since this cannot take place without metal binding. As an experimental mutation, there exists the possibility that this mutant SOD1 protein may be very different from other disease causing mutations. However, when possible, we mutated the metal binding sites to known ALS causing mutations and other models that abolish 2 or 4 of the copper binding sites develop a typical ALS phenotype in mice. Additionally, we were able to determine some similarities of SODMD with the normal WT SOD1 prot ein. SODMD retains the WT SOD1 feature of modulating aggregation, and as WT it lacks of the inherent propensity to aggregate. Additionally, the double or quadruple histidine experimental mutants develop and ALS -like disease in transgenic mice (Wang et al., 2002b;Wang et al., 2003) Thus, we believe that SODMD retains SOD1 properties suitable to study the role of aggregation and metal binding in ALS Previous studies have shown the ability of WT to slow aggregation in cell culture when expressed for short periods of time (Prude ncio et al., 2009a) At longer intervals co aggregation of mutant and WT proteins is a clear event (Deng et al., 2006;Prudencio et al., 2009a) In our hands, SODMD exhibits the aggregation blocking property of WT SOD1 and thus appears to be able to interact with mutant SOD1 in some manner that is si milar to WT SOD1. However SODMD lacks the property of WT that allows it to ultimately co aggregate with mutant SOD1 proteins when incubation periods are extended to 48 hours The mutated amino acids within SODMD may inhibit intramolecular contacts that wou ld make the protein self aggregating. Thus, the SODMD protein could be seen as a way to further stud y likely h otspots within the amino acid sequence that makes SOD1 prone to misfold and aggregate.

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61 Toxicity in ALS As mentioned earlier, aggregation is a late event and does not appear to determine onset in transgenic mice. However, it is possible that more soluble aggregated species, termed oligomers, may be the initiators of disease. D etergent insoluble SOD1 aggregates become detectable as symptoms develop rapidly (Karch et al., 2009) Thus, lack of toxicity from SODM D protein could be due to the inability of this mutant to aggregate. However, we are unable to isolate more soluble protein species of SODMD since this protein is quite unstable. Thus far, this work demonstrates the SODMD protein as another example that supports aggregation as a present feature in symptomatic ALS mice. Future D irections Future studies should be in part focus on obtaining an SODMD variant that can aggregate and still be able to not bind metals. Previous studies have shown the importance of cysteines 6 and 111 in modulating aggregation of mutant SOD1 (Karch and Borchelt, 2008) While SOD 1 with C6G and C111S mutations (also present in SODMD variant) do not produce an aggregating mutant SOD1 protein, modifications in these 2 cysteines can make SOD 1 more prone to aggregate. Thus, as part of future work, we performed modifications in the cDNA sequence of SODMD to produce a protein that is able to aggregate. Res toring either cysteine 6 or 111 allows for this mutant SOD1 protein to aggregate, and when both are restored the overall aggregati o n levels are much higher ( Figure 2 11). M utations in SODMD changing amino acid 111 (in MD a serine) to tyrosine (C111Y is a disease causing mutation) produces a protein which aggregation propensity is similar to that of restoring cysteine 111 in SODMD and when restoring cysteine 6 in S 111Y SODMD variant, the aggregation propensity is even

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62 higher than when restoring both cystein es in SODMD (Figure 211) The C6F SOD1 variant is a highly aggregating mutant, and a phenylalanine in amino acid 6 ( another disease mutation) of SODMD produces a highly aggregating protein. Thus, we created a SODMD variant with amino acid 6 mutated to a phenylalanine, and cysteine 111 changed to a tyrosine ( C 6F H43R, H46R, H48Q, H63G, H71R, H80R, C 111Y H120G) that produces a protein that highly aggregates (see MD -G6F S111Y in Figure 211) In conclusion, it is possible to have a SOD1 protein with all mutations that abolish metal binding but can aggregate. Figure 21 1. Mutations in amino acids 6 and 111 in the context of SODMD mutations can reestablish the aggregation propensity of SODMD. HEK293FT were transfected for 24 ( A, B ) or 48 ( C, D ) hours. A, C ) Immunoblots of detergent extracted HEK293FT expressing the indicated SOD1 proteins. B, D ) Qua n tification of the aggregation propensity at 24 ( B) or 48 ( D ) hours following transfection. All values are normalized to the aggregation propensity of A4V at 24 hours (set to 1). Paired student t -tests were perfor med to compare ag gregation propensities of SOD1 proteins to SODMD. *p #p WT A4V MD MD-G6C MD-S111C MD-S111Y MD-G6C-S111C MD-G6C-S111Y MD-G6F MD-G6F-S111C MD-G6F-S111Y 0.0 1.0 2.0 3.0 4.0Relative aggregation propensity of SOD1 mutants (A.U.) WT A4V G93A MD MD-S111C MD-S111Y MD-G6C MD-G6C-S111C MD-G6C-S111Y 0.0 1.0 2.0 3.0 4.0Relative aggregation propensity of SOD1 mutants (A.U.) P2 S1 P2 S1 A B *#* *#C D

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63 A better understanding of metals on disease would be better addressed through the creation of mice with these two different cysteine modifications. Based on our preliminary cell culture studies, metal binding does not seem to be required for aggregation. We suggest that it might be possible to create mice that express a version of SOD1 that lacks all of the known metal binding sites and which develop typical ALS.

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64 CHAPTER 3 VARIATION IN AGGREGATION PROPENSITIES AMONG ALS-ASSOCIATED VARIANTS OF SOD1: CORRELATION TO HUMAN DISEASE1 Introduction In all mouse models, the manifestation of disease symptoms is accompanied by the accumulation of detergent insoluble aggregated forms of mutant SOD1 (Watanabe et al., 2001;Jonsson et al., 2004;Wang et al., 2005a;Wang et al., 2005b;Deng et al., 2006;Jonsson et al., 2006a) Additionally in Chapter 2 we have seen that an experimental SOD1 mutation unable to form such aggregates cannot cause disease in animal models In human SOD1associated ALS there is similar evidence that mutant SOD1 aggregation is a pathologic feature (Wat anabe et al., 2001) Thus, there seems to be a clear correlation between the presence of detergent -insoluble aggregated forms of mutant SOD1 in spinal cords and disease (Wang et al., 2003) Importantly, aggregated forms of mutant SOD1 that display similar properties of detergent insolubility can be produced in cultured cells (Wang et al., 2003;Wang et al., 2006;Karch and Borchelt, 2008;Prudencio et al., 2009a) representing an efficient system to screen and study aggregation of ALS mutants. It is well established th at specific mutations are associated with disease of short or long clinical course (Cudkowicz et al., 1997) Examples of short disease course include the A4V mutation (< 2 years) (Rosen et al., 1994) whereas mutations such as H46R are associated with a long disease course (> 10 years) (Arisato et al., 2003) A recent study used a variety of biophysical data to calculate aggregation rates for 1The work presented in this chapter has been published in Human Molecular Genetics 18(17):321726 (2009) Mercedes Prudencio and David R. Borchelt designed the experiments, Mercedes Prudencio, Peter M. Andersen and David R. Borchelt interpret ed the data and wrote the manuscript. Mercedes Prudencio carried out all the experiments Peter M. Andersen provided all the clinical data, and P. John Hart provided cDNA SOD1 constructs in a yeast expression vector.

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65 different ALS mutants, suggest ing that aggregation of mutant protein could be a key factor in disease progression (Wang et al., 2008) Here, we have used our cell culture model to analyze a total of 33 SOD1 associated ALS mutations in regards to their ability to form detergent -insoluble aggregates, including different mutation substitutions at the same codon. By this approach, we assess how measured aggregation potentials relate to known biophysical/biochemical characteristics, and examine whether aggregation propensi ties correlate to disease features in human ALS patients. Materials and Methods A list of materials used can be found in Appendix B. Methodology used for the work presented in this chapter is described in Chapter 3 Methods of Appendix C. R esults Our first analysis of 21 mutant SOD1 proteins demonstrated that all are capable of forming detergent insoluble SOD1 aggregates in cells within 24 h ours (Figure 3 1A ). Quantification of the aggregation propensity, which is calculated from the ratio of insoluble to soluble forms of mutant SOD1 in cell lysates of different mutations showed significant differences from WT SOD1. To normalize data from different experiments, we chose to use the aggregation propensity of A4V SOD1 as the reference mutant (assigning 1 to the mean aggregation propensity of A4V, as previously described (Wang et al., 2003;Prudencio et al., 2009a) ). Most mutations analyzed in Figure 3 1A are of similar, or higher, aggregation propensity to A4V. Several mutants possessed aggregation propensities lower than that of A4V (G93D, E21G, E21K and G41D). In cases in which more than one mutation occurred at a particular codon, we often observed significantly different levels of aggregated protein for each individual mutation at one position ( Figure 3 -1B ); examples include A4V vs. T ( p = 0.0168); G93A vs. D ( p =

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66 0.0078), R ( p = 0.0056), or S ( p = 0.0222); G41D vs. S ( p = 0.0128); and E100G vs. K ( p = 0.0160). We also observed examples in wh ich different amino acid changes at the same position did not differentially affect the aggregation propensity: G93A vs C ( p = 0.4934), V ( p = 0.111); V14G vs. M ( p = 0.8766); and E21G vs. K ( p = 0.6213). Further studies on additional SOD1 mut ants involving different amino acid substitutions at the same site demonstrated very high variabili ty in aggregation propensities. Mutation of D101 or V148 to G induced the formation of very high levels of detergent-insoluble proteins in 24 hours (Figure 3-1C). D101G together with E100K SOD1 represent the most aggregation prone protei ns analyzed so far. However, D101N and V148I showed dramatically lower aggregation levels at 24 hours, not differ ent from that of WT SOD1 (Figure 3-1D). Figure 3-1. Large variability in aggregation among SOD1-asso ciated ALS mutants. A, C) Immunoblots of detergent insoluble (P2) and soluble fractions (S1) of HEK293FT cells transfected with WT or mutant SOD1 for 24 hours. UT: untransfected cells. B, D) Quantificati on of the relative aggregation propensity of WT and mutant SOD1 proteins as described in Methods. Bars represent mean SEM of 3 or more independent transfection experiments. p 0.05, #p 0.001. AV 1 4 G V 1 4 M E 2 1 G E 2 1 K G 4 1 D G 4 1 S E 1 0 0 G E 1 0 0 K N 1 3 9 K P2 S1U Th W T A 4 V A 4 T G 9 3 A G 9 3 D G 9 3 R G 9 3 S G 9 3 V L 8 4 V D 9 0 A D 1 2 4 V P2 S1 P2 S1 U Th W T A 4V D 1 0 1 G D 1 0 1 N N L 1 4 4 F L 1 4 4 S V 1 4 8 G V 1 4 8 I U Th W T A 4V D 1 0 1 G D 1 0 1 N N L 1 4 4 F L 1 4 4 S V 1 4 8 G V 1 4 8 I N L 1 4 4 F L 1 4 4 S V 1 4 8 G V 1 4 8 I C BD W T A4V A4 T G93A G9 3 D G93R G93S G93V L 8 4V D90A D1 2 4 V V14G V1 4 M E21G E21 K G41D G41S E100G E1 00K N139K G 93 C H43R 0.0 0.5 1.0 1.5 2.0 2.5 3.0Relative aggregation propensity of SOD1 mutants (A.U.)#*#*# # # #*#*# #*#* *# # WT A4V D 1 0 1 N D101G L 14 4 F L 1 44S V148 G V 1 4 8I 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Relative aggregation propensity of SOD1 mutants (A.U.)*#* *# H 4 3 R

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67 In addition to the slow aggregat ing D101N and V148I mutants, we found other mutations in which aggregation levels resemble those of WT SOD1 protein at 24 h ours (Figure 32A ). Compared to G37R, which to date had been representative of mutants with low potentials to aggregate (Wang et al., 2006) the H80R, D101N, D125H, (Figure 32A upper panels). In all cases, high levels of soluble SOD1 protein w ere detected for each of the mutants indicating robust protein expression ( Figure 32A lower panels). Quantification and statistical analysis indicated that the aggregation propensity of these mutants in 24 h ours was not different from WT SOD1 ( Figure 32 B). Thus, we identified, for the first time, SOD1 mutants that do not readily form detergent insoluble aggregates in 24 h ours showing a similar behavior to WT SOD1. These findings were initially viewed as an indication that aggregation, a priori, may not be necessarily linked to disease development. In order to more rigorously determine whether these mutants remain completely soluble, we extended the interval between transfection and harvest of the cells from 24 to 48 h ours With longer incubation times al l mutants formed detectable levels of detergent -insoluble protein, while WT SOD1 still remained completely soluble ( Figure 3 2C ). At 48 h ours the aggregation propensities of these mutants were significantly different from WT SOD1 ( Figure 3 -2 D ). Although we have shown that all SOD1 mutants ultimately form detergent insoluble aggregates, we were somewhat surprised to find several mutants that were not statistically different from WT SOD1 in regards to aggregate formation in 24 h ours To further explore how closely these mutants resemble WT SOD1, we employed a technique of co -transfection in which the mutants with low aggregation potential were

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68 co transfected with mutants of high aggregation potential. We have previously shown that WT SOD1 is able to slow aggregation of SOD1 mutant proteins in cell culture, while co expression of two highly aggregation prone mutants does not interfere with aggregate formation (Prudencio et al., 2009a) Similar to WT SOD1, co transfection of H80R, D101N, D125H, E133 E, or S134N with G85R SOD1 reduced considerably the amount of detergent insoluble G85R protein present in cells 24 h ours after transfection (Figure 33A) Similar results were obtained with V148I SOD1 (data not shown). The aggregation propensity of G85R was quantified revealing that these WT -like mutants reduced significantly the aggregation of G85R; no longer different from WT SOD1 (Figure 33B) Notably, when we extended the post transfect ion incubation period to 48 h ours then clear evidence of aggregation of both mutants (for all tested here) was observed (Figure 3-3C) At these longer incubation intervals, WT SOD1 can also be captured into mutant SOD1 aggregates (Prudencio et al., 2009a) These results suggest that the H80R, D101N, D125H, E133 E, S134N and V148I SOD1 mutants share two features with WT SOD1: low inherent propensity to aggregate over 24 hours, and the ability to slow the aggregation of mutant SOD1 proteins that exhibit high aggregation rates. It has been suggested that mutations in SOD1 that decrease the net negative charge of the protein (eliminating negatively charged or introducing positively c harged amino acids) causes misfolding and aggregation of mutant SOD1 (Shaw and Valentine, 2007;Sandelin et al., 2007) Many of the ALS associated point mutations in SOD1 reduce the net negative charge of SOD1; however, three ALS mutants would be expected to possess a protein charge more negative than WT SOD1 ( Figure 34, Table

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69 3 -1 ). Thus, we asked whether differences in protein charge among the ALS mutants may explain the variability in the relative rates of mutant SOD1 aggregation. Some of these changes in protein charge can be observed in mutations occurring at amino acid G93. Although some mutations in G93 do not alter the overall protein charge ( Table 31 ), there is a r eduction in the negative charge of SOD1 when mutating G93 to R, increasing the aggregation propensity significantly compared to those mutants that do not produce a change in protein charge (G93R vs. A, p = 0.0056; G93R vs. C, p = 0.0262; G93R vs. S, p = 0. 0012; G93R vs. V, p = 0.0325). Additionally, when G93 is mutated to D, which increases the negative charge of SOD1, then the levels of aggregated protein are significantly reduced compared to neutral change mutants (e.g., G93D vs. A, p = 0.0078). Another example of similar findings is that of mutations at G41, where G41S presented an aggregation propensity higher than that of the more negatively charged G41D ( p = 0.0128). Additionally, when mutations occur in E100 (E100G, E100K) the resulting mutant protei ns present less negative charge than WT SOD1; the E100K mutant shows a more substantial decrease in negative charge and significantly higher aggregation potential as compared to E100G ( p = 0.016). Overall, these findings support the hypothesis that changes in negative charge may play an important role in modulating aggregation of SOD1. However, this apparent correlation does not apply to all mutants that alter the negative charge of SOD1. A good example is the case of mutations in E21, which can be mutated to either G or K, similar to mutations in E100. Although E21K SOD1 has a higher decrease in negative charge, the aggregation propensity levels of E21G and E21K are not statistically different from each other ( p = 0.6213), with both being very low ( see Figure 3-1 ). Another example is that of

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70 changes in D101. Decreasing t he negative charge of SOD1 by substituting amino acid D101 by either G or N produces mutant prot eins in which aggregati on levels are either very high (D101G) or very low (D101N). In th is case the same magnitude of decrease in negative charge produces a very large va riability in aggregation rates (D101G vs. D101N, p = 0.0296). Additional examples, in which a reduction in the negative charge of SOD1 does not produce dramatic increases in aggregation, are the cases of H80R, D125H, and E133 E (Table 3-1). Figure 3-2. Some SOD1-associated ALS mutant proteins aggregate slowly. A, C) Immunoblots of P2 and S1 fractions of HEK293FT cells transfected with human WT (hWT) or mutant SOD1 for 24 (A) or 48 (C) hours. UT: untransfected cells. B, D) Quantificati on of the relative aggregation propensity of SOD1 proteins at 24 (B) and 48 (D ) hours. Bars represent mean SEM of 3 or more independent trans fection experiments. p 0.05, #p 0.001. A B C D U T W TA 4 V G 3 7 RH 8 0 R D 1 0 1 N D 1 2 5 H E 1 3 3 E S 1 3 4 NP2 S1 W T A4V G 3 7R H80R D1 01N D125H E E133 S 134 N V 14 8I G85R 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 mutants (A.U.)#*# U T W TA 4 V G 3 7 RH 8 0 R D 1 0 1 N D 1 2 5 H E 1 3 3 E S 1 3 4 NP2 S1 UTh W T A 4 V V 1 4 8 I G 8 5 R U Th W T A 4 V V 1 4 8 I G 8 5 R W T A 4 V G37R H80R D101N D125H E E 1 3 3 S134N G85R V 1 4 8I 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 mutants (A.U.)#*#*#* *# #

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71 Figure 3-3. Mutants with low aggregation propensity behave like human WT (hWT) SOD1 in terms of modulating aggregati on. A, C) Immunoblots of P2 and S1 fractions of cells singly transfect ed with SOD1 constructs or doubly transfected with G85R and a low aggregatin g SOD1 variant, for 24 (A) or 48 (C) hours. B) Quantificat ion of the aggregation propens ity of G85R when cotransfected for 24 hours. Bars repr esent mean SEM of 3 or more independent transfect ion experiments. #p 0.001. Table 3-1. Changes in protein char ge do not explain ag gregation propensity. WT A4V G85R G85R in WT + G 85R G85R in H80R + G 85R G85R in D 101N + G 85R G 85R in D125H + G85R E + G85R G85R in E133 G 85R in S134N + G 85R 0.0 0.5 1.0 1.5 2.0Relative aggregation propensity of SOD1 mutants (A.U.)# #AB C P2 S1U T h W T G 8 5 R + S 1 3 4 NG 8 5 R + E 1 3 3 E G 8 5 R + D 1 0 1 N G 8 5 R + H 8 0 R G 8 5 R + h W T G 8 5 R G 8 5 R + D 1 2 5 HU T h W TA 4 VP2 S1G 8 5 R G 8 5 R + H 8 0 R G 8 5 R + D 1 0 1 N G 8 5 R + D 1 2 5 H G 8 5 R + E 1 3 3 E G 8 5 R + S 1 3 4 N U Th W T A 4 V G 8 5 R + V 1 4 8 I G 8 5 R Aggregation propensity (24 h) No change in net negative charge Reduce net negative charge Increase net negative charge Low S134N, V148I H80R, D101N, D125H, E133 E Moderate I113T, L144F, L144S, C111Y E21G, E21K, G37R G41D, G93D, N139K High C6G, G93C, V14G, C6F, V14M, A4V, G93A, L84V, G93V, G41S, V148G, G93S H43R, G85R, D90A, E100G, D124V Extreme A4T G93R, E100K, D101G

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72 Figure 34. Changes in the net negative charge of SOD1 do not predict aggregation propensity. Mutations in SOD1 that reduce, increase, or do not modify the negative charge of SOD1 present aggregation propensity values that range from very low to very high; with no pa rticular group representing mutants of high or low aggregation propensities. Unpaired Student t -tests: no change in charge mutants vs. mutants with reduced charge ( p = 0.8711), no change in charge mutants vs. mutants with increased charge ( p = 0.2556), mutants with reduced charge vs. mutants with increased protein charge ( p = 0.5213). To further probe as to whether the location of the mutation within the protein may interact with the change in net charge, we graphed measured aggregation propensity as a function of mutation location and charge ( Figure 3 -5 ). No obvious pattern emerges to link change in net charge to inherent aggregation propensities. Further, there were no obvious correlations between changes in net charge and disease onset or duration ( Figure 3 6 ). Thus, our data suggest that the relative aggregation propensity of mutant SOD1 is not inextricably linked to changes in net protein charge. Additionally we did not find correlations between aggregation propensity and other prot ein features such as metal binding or protein thermostability ( Table 3-2 ). However, we do not discard the possibility that a combination of two or more protein

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73 characteristics ( changes in protein charge, protein stability, metal binding, etc) could explai n the different aggregation propensities of ALS mutants. Figure 35 Changes in protein charge do not explain differences in aggregation propensity. Representation of changes in net negative charge of SOD1 vs. aggregation propensity based on the structural location of the different SOD1associated ALS mutations: in beta strand ( A) vs. nonbeta strand (B ) regions, and by mutations affecting amino acids located on surface of the protein ( C ) vs. amino acids facing the interior of the beta barrel ( D ). Determ ination of the structural location of the mutated amino acids was performed using the pdb online structure of G37R SOD1 mutant protein and visualized with PyMOL software (DeLano Scientific, LLC). Mutations in strand regions Mutations in non strand regionsA B No change Reduced Increased 0.0 0.5 1.0 1.5 2.0 2.5 3.0Net negative chargeRelative aggregation propensity of SOD1 mutants (A.U.) No change Reduced Increased 0.0 0.5 1.0 1.5 2.0 2.5 3.0Net negative chargeRelative aggregation propensity of SOD1 mutants (A.U.) No change Reduced Increased 0.0 0.5 1.0 1.5 2.0 2.5 3.0Net negative chargeRelative aggregation propensity of SOD1 mutants (A.U.) No change Reduced Increased 0.0 0.5 1.0 1.5 2.0 2.5 3.0Net negative chargeRelative aggregation propensity of SOD1 mutants (A.U.) Surface amino acids (loops or facing out from barrel) Non surface amino acids (facing to the inside of the barrel)C D

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74 Figure 36 Changes in protein charge do not predict onset or survival. Mean aggregation vs. mean disease onset ( A, B); or vs. mean disease duration ( C, D ) for SOD1 mutations with a significant number of patients (> 5) ( A, C ), or for all SOD1 mutations with some pati ent data available ( B, D ). Table 3 2. Biophysical and biochemical characteristics of SOD1 variants. SOD1 variant Change in negative charge Aggregation levels at 24 h Copper binding Stability References WT No No High High (Rodriguez et al., 2002;Hayward et al., 2002) V148I No Low ND ND NA C6F No Moderate ND Low a (Lindberg et al., 2002) C6G No Moderate ND ND NA V14G No Moderate ND ND NA V14M No Moderate ND ND NA A B C DMutations with > 5 patients Mutations with > 5 patients All Mutations All Mutations No change Reduced Increased 20 25 30 35 40 45 50 55 60Net negative chargeDisease onset (yrs) No change Reduced Increased 20 25 30 35 40 45 50 55 60Net negative chargeDisease onset (yrs) No change Reduced Increased 0 5 10 15 20Net negative chargeDisease duration (yrs) No change Reduced Increased 0 5 10 15 20Net negative chargeDisease duration (yrs)

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75 Table 3 2. Continued SOD1 variant Change in negative charge Aggregation levels at 24 h Copper binding Stability References G93C No Moderate High c Low a (Lindberg et al., 2002;Borchelt et al., 1994) C111Y No Moderate ND ND NA I113T No Moderate High c Low a (Borchelt et al., 1994;Rodriguez et al., 2005;Valentine et al., 2005) L144F No Moderate High Low (Shaw and Valentine, 2007) L144S No Mode rate High ND (Valentine et al., 2005) A4T No High ND Low b (Nakano et al., 1996) A4V No High Low Low a (Borchelt et al., 1994;Rodriguez et al., 2002;Hayward et al., 2002;Lindberg et al., 2002;Rodriguez et al., 2005) G41S No High High Low a (Rodriguez et al., 2002;Hayward et al., 2002;Shaw and Valentine, 2007) L84V No High Low Low a (Rodriguez et al., 2005;Shaw and Valentine, 200 7) G93A No High High Low a (Rodriguez et al., 2002;Hayward et al., 2002;Lindberg et al., 2002;Shaw and Valentine, 2007) G93S No High ND ND NA G93V No High High ND (Valentine et al., 2005 ) V148G No High High High (Valentine et al. 2005) H80R Reduced Low Low ND (Rodriguez et al., 2002;Valentine et al., 2005) D101N Reduced Low High High a (Rodriguez et al., 2005;Shaw and Valentin e, 2007) D125H Reduced Low Low High a (Rodriguez et al., 2002;Hayward et al., 2002;Rodriguez et al., 2005;Shaw and Valentine, 2007;Valentine et al., 2005) Reduced Low High Low a (Rodriguez et al., 2002;Hayward et al., 2002) S134N Reduced Low Low High a (Rodriguez et al., 2002;Hayward et al., 2002;Rodriguez et al., 2005;Shaw and Valentine, 2007;Valentine et al., 2005)

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76 Table 3 2. Continued SOD1 variant Change in negative charge Aggregation levels at 24 h Copper binding Stability References E21G Reduced Moderate ND ND NA E21K Reduced Moderate ND ND NA G37R Reduced Moderate High c Low a (Borchelt et al., 1994;Rodriguez et al., 2005;Shaw and Valentine, 2007) H46R Reduced Moderate Low High a (Rodriguez et al., 2002;Rodriguez et al., 2005; Antonyuk et al., 2005;Shaw and Valentine, 2007) H48Q Reduced Moderate Low High a (Hayward et al., 2002;Rodriguez et al., 2002;Rodriguez et al., 2005) H43R Reduced High High ND (Valentine et al., 2005) G85R Reduced High Low Low a, b (Borchelt et al., 1994;Borchelt et al., 1995;Rodriguez et al., 2002;Valentine et al., 2005) D 90A Reduced High High Low a (Rodriguez et al., 2002 ;Lindberg et al., 2002) E100G Reduced High High Low a (Rodriguez et al., 2005;Shaw and Valentine, 2007) D124V Reduced High Low High a (Rodriguez et al., 2005;Shaw and Valentine, 2007) G93R Reduced Extreme High Low a (Rodriguez et al., 2005;Valentine et al., 2005;Shaw and Valentine, 2007) E100K Reduced Extreme High High a (Rodriguez et al., 2005;Shaw and Valentine, 2007) D101G Reduced Extreme High ND (Valentine et al., 2005) G41D Increased Moderate High c Low b (Borchelt et al., 1995;Valentine et al., 2005) G93D Increased Moderate ND ND NA N139K Increased Moderate High High a (Rodriguez et al., 2005;Shaw and Valentine, 2007) L126stop Increased Very unstable Low Low NA L126deltt Increased Extreme Low Low b (Ratovitski et al., 1999) aStability calculated of purified apoprotein, or bby its half life when expressed in mammalian cell systems. cCopper binding content high or low based on levels of activity ND: Not determined NA: Not available

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77 A recent study, using mathematical models, suggested that the predicted aggregation propensity of SOD1 mutants correlates with disease duration in humans (Wang et al., 2008) Here we used data from our cell culture model to analyze whether our measured aggregation propensity correlates with patient data on disease onset and/or duration. We imposed a filter on the patient data, focusing on cases in which the number of affected indiv iduals with a particular mutation met or exceeded 5 individuals. We chose 5 individuals as the lower limit because it emerged as a natural breakpoint in our data and because this number of patients allows for a more rigorous estimation of reproducibility o f observed phenotypes. After filtering our patient data set, we were able to identify 21 different mutations for which we possessed data on an adequate number of patients. The data on aggregation propensities of the different mutants were stratified into 4 categories as explained in Figure 3-7 For the 21 mutations in the patient data sets we analyzed, 2 mutations were categorized as exhibiting extreme aggregation propensity, 12 of the mutations fit criteria for high aggregation propensity, 6 fit criteria a s moderate, and one fits criteria for low aggregation propensity ( Table 3 -3 ). No obvious correlation between aggregation propensity and age of onset was noted ( Figure 3-7 ). This outcome was expected because the mean age of onset is 4547 years of age for a ll SOD1 mutants ( Table 3-3 ). When we stratified the mutants by the 4 criteria described above and graphed these groups as a function of disease duration, we noted that mutants exhibiting aggregation propensities equivalent to or greater than the A4V mutat ion largely predicted shorter disease duration in patients ( Figure 3-8A ). Statistical analysis demonstrated significant differences between the groups of high and moderate

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78 aggregation propensities (unpaired t test, p = 0.0008). However, inverse correlations do not exist between aggregation propensity and disease duration ( p = 0.1440, Figure 38B ), possibly because disease duration in patients with mutations of moderate or low aggregation propensity is less predictable. Still, when we focus on the mutants th at show high aggregation propensities, we find that the majority of patients with these mutations exhibit a rapid disease course. Figure 37. Disease onset is not driven by changes in aggregation propensity. SOD1 mutations with a signifi cant number of patients (> 5) (A ), o r all available compiled data (B ) grouped in terms of aggregation propensity: Extreme (produces a level of insoluble mutant protein in 24 h ours that is equal to or greater than twice that of A4V SOD1always set at 1), High (aggregate load is similar to A4V in 24 h ours ; aggregation propensities range between 0.7 and 1.7), Moderate (aggregate load is detectable but < 0.5 in 24 h ours ), Slow (no aggregates detected in 24 h ours only visible at 48 h ours ). No correlation was found between ag gregation and age of onset ( p 0.05). Note that the mean onset for ALS patients with a SOD1 mutation occurs between 4547 years of age. Table 3 3. Clinical data ordered by relative aggregation potential values. Mutation Aggregates 24 h Age of onset Survival time Penetrance N L126deltt Extreme NA NA NA NA E100K Extreme 44.0 6.1 12.0 4.1 Incomplete 16 D101G + Extreme 48.0 9.1 2.5 0.4 NA 3 G93R Extreme 35.8 4.3 5.8 4.5 NA 4 A4T + Extreme 44.0 11 1.2 0.4 Incomplete 14 G93S High 45.9 4.4 8.2 4.0 Incomplete 9 0.0 0.5 1.0 1.5 2.0 2.5 3.0 20 25 30 35 40 45 50 55 60Mean aggregation propensity (A.U.)Mean disease onset (yrs) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 20 25 30 35 40 45 50 55 60Mean aggregation propensity (A.U.)Mean disease onset (yrs) A B p = 0.9219 p = 0.9246Mutations with > 5 patients All Mutations

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79 Table 3 3. Continued. Mutation Aggregates 24 h Age of onset Survival time Penetrance N V148G + High 42.8 10.5 2.0 0.9 Complete 7 G41S + High 46.8 13.5 0.9 0.2 Complete 8 L126X Very unstable 42.0 3.8 Complete 14 G93V High 47.0 4.9 (variable: 4 to >9) NA 3/2 E100G High 46.9 12.0 4.0 2.3 Complete 22 D124V High NA NA NA NA L84V High 53.8 15.3 1.6 0.5 Complete 6 G93A + High 47.9 17.7 2.4 1.4 Complete 11 H43R + High 49.6 15.1 1.4 0.8 Complete 7 D90A mc High 53.0 17.3 < 1 (variable) Incomplete 5/2 A4V + High 47.8 13.3 1.2 0.9 Complete 84 V14M High NA NA NA NA C6F + High 49.5 4.95 0.29 0.06 NA 2 V14G High 39.0 1.9 Apparently sporadic 1 G85R High 55.5 12.6 6.0 4.5 Complete 11 G93C High 45.9 10.6 13.0 4.0 Complete 20 C6G + High 49.5 0.2 NA 2 N139K High NA NA NA 1 G41D Moderate 46.0 7.3 17.0 6.3 Complete 7 C111Y Moderate 49.0 4.4 10.2 3.1 Complete 3 L144S Moderate 42.5 10.6 12.3 3.7 NA 2 G93D Moderate 48.3 16.2 10.5 5.5 NA 3 E21K Moderate NA NA Apparently sporadic 1 L144F Moderate 52.0 6.7 (variable: 3 to 20) Complete 13/11 E21G Moderate 44.9 16.11 Incomplete 18 G37R Moderate 40.0 9.9 18.7 11.4 Complete 8 I113T Moderate 57.8 15.1 4.2 3.2 Incomplete 10 H46R Moderate 45.0 10.2 17.0 7.0 Complete 56 H48Q + Moderate 58.0 1.1 NA 2 D101N Low 41.0 10 2.4 0.9 Incomplete 14 V148I + Low 28.0 3.8 1.8 0.5 Complete 4/3 H80R Low NA NA Apparently sporadic 1 Low NA NA Apparently sporadic 2 S134N + Low NA NA NA 2 D125H + Low 53.0 0.7 NA 2 Moderate aggregate levels when expressed for 48 hours ; High aggregate levels when expressed for 48 hours ; +Very aggressive mutations ; De novo mutation ; mc: indicates D90A heterozygous cases ; NA: not available; N: number of patients analyzed, when more than one number is given in this column, the first number indicates the total number of patients (used to calculate onset) and the second number represents the total number of deceased patients (used for calculation of survival times) ; Mutations in bold font are those in which the number of patients is higher than 5.

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80 Statistical tests indicate that when all av ailable data are includ ed, then the there is no significant correlation between di sease duration and aggregation propensity ( p = 0.2257, Figure 3-9). Whether data on patients with very few cases is reliable is uncertain, but these data are included to allo w the reader to predict what might happen as more data becomes available. The pa ttern that becomes apparent when all data is analyzed is that mutant s with low to moderate inherent aggregation propensities are associated with a wide variation in disease duration. The association between high aggregation propensity and short duration seems to hold; data we interpret as an indication that high aggregation propensity is a risk factor for disease of short duration. Figure 3-8. Mutants possessing a higher aggr egation propensity correlate with shorter disease duration. A) Mutations with a si gnificant number of patients (> 5) grouped in terms or aggregation propensity categories, as explained in Figure 3-7. Mutations associated with shorte r disease durations belong to a group that present high or extreme aggregat ion propensities. B) Non-linear regression of aggregation propensity and disease duration. A statistically significant correlation between aggregat ion and disease duration was not found ( p 0.05). AMutations with > 5 patients Low Moderate High Extreme 0 5 10 15 20Aggregation propensityMean disease duration (yrs)A4T G37R H46R G41D E21G L144F I113T G93S G85R E100G L126X V148G G93A G41S L84V H43R D90A A4V V148G G93A G41S L84V H43R D90A A4V G93C E100K D101N 0.0 0.5 1.0 1.5 2.0 2.5 3. 0 0 5 10 15 20Mean aggregation propensity (A.U.)Mean disease duration (yrs)BMutations with > 5 patients R2= 0.2257 p = 0.1440

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81 Figure 39. Mutants possessing low or moderate aggregation prop ensities are associated with a large variation in disease duration. A) All SOD1 mutations with some patient data available grouped in terms of aggregation propens ity values (see legend in Figure 3 -7 ) and plotted against mean disease duration in years. Significant differences are found between the groups of moderate and high aggregation propensity (unpaired t -test, p = 0.0003). B ) Non -linear regression of aggregation propensity and disease duration. To determine whether the results obtained in human HEK293FT cells are specific to these cells, or are more broadly applicable to other cell types, we have attempted to replicate our findings in a different cell line to determine whether different results are achieved. We chose to use mouse neuroblastoma N2a cells which transfect reasonably well and which have also been used to study mutant SOD1 (Borchelt et al., 1995;Pasinelli et al., 1998;Krishnan et al., 2006) We chose a set of mutants with a range of aggregation propensities (A4V, G37R, G93D, G93R, D101G, D101N, V148G, V148I) to express in N2a cells for 24 h ours (time point in which we can see mutants with low to extreme aggregation propensity ratios in HEK293FT Figure 3 10). Unfortunately, for most of the vectors tested, the levels of human SOD1 expression were much lower than what is obtained in HEK293FT cells and we were unable to ascertain aggregation 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 5 10 15 20Mean aggregation propensity (A.U.)Mean disease duration (yrs) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 5 10 15 20Mean aggregation propensity (A.U.)Mean disease duration (yrs) R2= 0.4541 p =0.0261 R2= 0.4243 p =0.0682A BMutations with > 5 patients All Mutations

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82 propensities. However, we were able to observe good expression of the D101G and D101N mutants, which show extremely high versus extremely low aggregation propensities in HEK293FT cells. In the N2a cells, w e find a similar outcome in that D101G produces a very high level of detergent insoluble SOD1 whereas the D101N mutant remains largely soluble at 24 h ours ( Figure 3-10 ). Figure 310 Aggregation of mutant SOD1 in mouse N2a cells. Mouse neuroblastoma N2a cells were transiently transfected following protocols described in Methods A minimum of 3 repetitions were attempted to assess aggregation propensity in mouse N2a cells at 24 h ours Using the same protocol as HEK293FT cells o nly 2 of the mutants tested reliably produced sufficient levels of expression to allow for assessments of aggregation propensity. A ) Immunoblots of P2 and S1 fractions B) Quantification of aggregation propensity (P2/S1) D iscussion Studies to date, which have examined a relatively small percentage of all ALS mutants, have demonstrated that ALS mutations in SOD1 increase the propensity of the protein to form detergent -insoluble aggregates. Data from our present study raises the total number of SOD1 associated mutants for which we hav e measured aggregation propensities to 30% of all known mutants, providing definitive evidence that increased aggregation propensity is highly likely to be a universal feature of mutant SOD1. However, the inherent propensity of different mutants to produce aggregated protein P2 S1 WT D101G D101N 0.0 0.5 1.0 1.5 2.0 2.5 3.0Relative aggregation propensity of SOD1 proteins (A.U.) A B

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83 varies, even in cases in which multiple mutations target a single amino acid position. We could not identify a specific biochemical or biophysical property of the SOD1 associated ALS mutants that adequately explains the variability in t he propensity of these mutant proteins to aggregate. Although causality for variability is unknown, we find that the inherent aggregation propensity of ALS mutants is related to the duration of disease in ALS patients such that mutants that show high aggregation propensities are associated with a greater ri sk for short disease duration. Variability in Aggregation Propensity of SOD1 Mutants and Protein Charge One aspect of our study sought to determine whether variability in mutant SOD1 aggregation propensi ty could be explained by the nature of the amino acid mutation. Mutations in SOD1 that bring the protein charge to neutrality or decrease the negative charge, have been suggested to make it more prone to aggregate (Chiti et al., 2002;Calamai et al., 2003) More than half of the SOD1 proteins studied here represent mutations affecting a charged residue or introduce a charged amino acid in place of a non-charged ( see Figure 3-4 ). Within this group of mutations that modify the negative charge of SOD1, we can find SOD1 mutants with all defined levels of aggregation propensity (low, moderate, high or extreme), not following a clear correlation between a decrease or increase in prot ein charge and aggregation rates. For example, eliminating aspartate in amino acid 101 produces a low (D101N) or very high (D101G) aggregating protein, with both proteins producing a decrease in negative charge. However, when mutations occur at E100, the m utant with a larger increase in net negative charge has the highest aggregation propensity (E100K vs. G). The E100 and D101 amino acids are in such close proximity that it seems unlikely that the different changes in charged amino acids at these two residu es could have very different structural effects on the

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84 protein. Additionally, we have found no obvious correlation between aggregation propensity and change in charge in amino acids that concentrate to a particular structure (beta strand vs. non beta strand regions, surface vs. interior; see Figure 3-5 ). Finally, we found no obvious correlation between the magnitude of change in charge by mutation and disease onset or duration ( see Figure 3-6 ). Collectively, these data indicate that changes in protein charg e cannot be the only determining factor that drives aggregation of mutant SOD1 and that there is no obvious correlation between charge ch anges and a disease feature. Variability in Aggregation Propensity of SOD1 Mutants and Protein Stability Thermostabilit y is viewed as a measure of the inherent stability of protein conformation. The mutants we identify here as slow to aggregate (H80R, D101N, (see Figure 33 ). Biophysically, thes e mutants show similar H/D exchange kinetics (which assess the exposure of residues in the folded protein to solvent) to WT SOD1 as apo-proteins (Rodriguez et al., 2005) as well as high levels of activity (only when metalla ted) and high thermostability (in both fully metallated or demetallated states) (Rodriguez et al., 2005) (see Table 3 2 ). By contrast, the mutants that show higher aggregation propensities generally show reduced thermostability ( see Table 3 2 ). However, i n our tabulation of data on aggregation levels at 24 h ours in comparison to thermostability, we noted no obvious association between low thermostability of the apo-protein (lacking Cu) and aggregation propensity ( see Table 32 ). Variability in Aggregation Propensity of SOD1 Mutants and Metal Binding Sorting out the role of metal -binding in aggregation propensity is somewhat complicated in that different approaches of assessing metal binding have yielded

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85 different outcomes depending upon whether the protein was produced and isolated from yeast or Sf21 insect cell expression systems. From the available data, we find little evidence to relate poor metal binding capacity to the formation of detergent -insoluble SOD1 aggr egates. Three of the low aggregating SOD1 mutants identified here appear to bind metals weakly ( see Table 3 2 ). Moreover, experimental SOD1 mutants in which copper binding ligands have been abolished do not present a higher propensity to form detergent -ins oluble aggregates than other SOD1associated ALS mutations (Wang et al., 2003) Thus, it does not appear that low metal binding capacity correlates to high aggregation propensity. Aggr egation vs Disease To date, the specific role or impact that aggregates of mutant SOD1 have on disease pathogenesis remains unclear. In some settings, aggregation of mutant protein has been suggested to reduce toxicity by concentrating an otherwise toxic protein to a specific subcellular compartment (Arrasate et al., 2004;Gong et al., 2008) ; however, evidence linking aggregates to toxicity is still largely correlative. All of the SOD1 ALS murine models that have been analyzed accumulate significant amounts of detergent insoluble aggregates in tissues most affected by the disease process (Johnston et al., 2000;Wang et al., 2002b;Wang et al., 2002a;Wang et al., 2003;Jonsson et al., 2004;Wang et al., 2005b;Wang et al., 2006;Karch et al., 2009) In certain cases in which the expression of mutant protein was low, disease development and/or aggregation was not observed (Wang et al., 2005b;Deng et al., 2006) The two interpretations of these findings are that: 1) aggregation of mutant SOD1 is critical to the development of disease, or 2) that some other process initiates disease onset and that cells damaged by the disease process are prone to aggregate mutant SOD1. Our cell model, however,

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86 suggests that a high propensity to aggregate is an inherent characteristic of ALS mutant SOD1. Our studies have shown that SOD1associated ALS mutants display a wide variety of aggregation propensities that are dependent upon the type and location of the mutation. Thus, if aggregates have an important role in disease pathogenesis, then some characteristic of human disease should correlate to the aggregation propensity of the individual mutant. As noted in Results, the mean ag e of disease onset in SOD1associated ALS is between 4547 years of age, and we find no obvious correlation between aggregation propensity and age of onset ( see Figure 3 7). However, in our analysis of clinical data regarding mutations with reliable patien t information ( see Table 3-3 ), we have observed that in general there is an inverse relationship between high aggregation propensity and disease duration (see Fig ure 3 8 ). Mutants with higher aggregation propensities compose a group of mutations in which t he clinical data available is more abundant. In this set of mutants, survival intervals tend to be shorter; all exhibit a survival of less than 12 years, although most of them are characterized by survival times of 4 years or less. By contrast, SOD1 mutant s with moderate aggregation propensities present survival times ranging 1018 years. However, these relationships were not absolute as we found examples of mutants with moderate aggregation propensity in which disease duration is shorter than 10 years: I113T and L144F SOD1 (see Table 3 -3 see Figure 3-8 ). The mutant I113T is known to have incomplete penetrance, thus it might be possible that additional factors regulate disease appearance in certain generations, which would be responsible for the large varia bility in disease duration among patients. Patients harboring the L144F mutation present complete penetrance, but exhibit a wide range of

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87 survival times (see Table 33 ). It is possible that as more data becomes available, the number of exceptions (low aggr egation potential linked to rapid progression) may rise. However, when we include in our analysis of patient data, all available data including cases with only 1 or 2 patients ( see Figure 39) we continue to observe that high aggregation propensity shows a strong association with disease of short duration (< 5 years). Mutants that show low or moderate aggregation potentials are essentially unpredictable. Overall, we regard the high propensity of a particular mutant to aggregate as a risk factor for a more rapidly progressing disease. HEK293FT Cells as a Model to Study SOD1Associated ALS Aggregation All of our data in the present study, as well as data derived from prior studies (Wang et al., 2003;Karch and Borchelt, 2008) have used the HEK293FT cell as the model system. We note that we were able to find one report in the literature in which inducible vectors were used in mouse NSC 34 cell s to express a limited number of human mutant proteins (including A4V, C6F, H46R, G93A, and C146R SOD1) (Cozzolino et al., 2008a) These investigators used a very similar approach of determining the levels of mutant SOD1 in detergent soluble and insoluble fractions. After 48h of induced expression, they reported that A4V and G93A mutants showed ratios of soluble to insoluble SOD1 that were similar to what we report in our current study. We have previously examined the aggregation the C6F and C146R mutants in HEK293FT cells, and found high inherent r ates of aggregation (Karch and Borchelt, 2008) Cozzolino reported a similar high rate of aggregation of these mutants in mouse NSC -34 cells (Cozzolino et al., 2008a) By contrast, the H46R mutant showed much lo wer levels of aggregated mutant SOD1 in NSC 34 cells; which again is similar to what we have previously reported for this mutant in our HEK293FT cell system (Wang et al.,

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88 2003) We al so examined aggregation propensities of human SOD1 by transient transfection of mouse neuroblastoma N2a cells ( see Figure 310 ). One of the more interesting pairs of mutants, D101G and D101N, showed similar aggregation propensities in the N2a cells as was observed in the HEK293FT cells. Thus, we think that our findings in HEK293FT cells reflect inherent aggregation propensities of these mutants that are manifest in other cell types. It is possible that there are factors unique to motor neurons that modulate aggregation; and that such factors could significantly impact mutant SOD1 folding when present at physiologic levels. However, we argue that the HEK293FT cell model, which is dependent upon high levels of expression, overwhelms most of the cellular system s that might otherwise modulate mutant SOD1 aggregation (proteasome degradation, chaperone levels, etc) providing insight in the inherent propensity of these proteins to self associate. Even in the face of other modulating factors, the inherent propensity to aggregate would be the basic force behind aggregation. At this basic level, we find that mutants that exhibit a high aggregation propensity are often associated with disease of short duration. One other factor to consider is that most of the human cases for which we have significant data are examples of disease of relatively short duration. Because the longer duration cases are not equally represented, the apparent association between high aggregation rates and short disease duration could be due to bias in data set. However, statistical analyses of the data indicate a non -random distribution of disease duration among the classes of mutants, with high aggregation propensity more often being found in patients with short duration. Moreover, within the group of patients that show short duration, 9 of the 12 mutants that exhibited high aggregation potential are associated

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89 with disease durations of less than 5 years. However, we clearly identify mutant s that in cell culture show aggregation propensities that do not fit with expectations for disease duration. Whether these exceptions are truly examples of mutants that aggregate slowly and yet are associated with rapidly progressing disease (e.g. D101N mu tant) or mutants that aggregate rapidly and yet are associated with slowly progressing disease (e.g. E100K), is uncertain. These may be examples in which the cell culture system is not accurately predicting the in vivo situation, or these may be examples i n which other modifying factors overshadow the role of aggregation in disease duration. The association between aggregation of mutant SOD1 and symptomatic disease duration, rather than onset, is a particularly intriguing finding. In comparison to other neurodegenerative diseases that have been associated with protein misfolding, SOD1associated ALS appears unique. For example, in neurodegenerative disorders with expansions of glutamine repeats regions there is an association between aggregation and age of d isease onset rather than progression. Individuals with polyglutamine expansions in the huntingtin gene develop Huntingtons disease (HD). In HD, the length of the polyglutamine tract strongly correlates with disease onset, the larger the expansion the earl ier onset (Persichetti et al., 1994;Gusella and MacDonald, 2006) ; and longer repeat lengths correlate with higher aggregation propensities of mutant huntingtin (Scherzinger et al., 1999) In HD, disease duration is not noted to be variable and the length of the polyglutamine repeat does not correlate with disease duration (Gusella and MacDonald, 2006) Another example is Alzheimers disease (AD), which i s defined by amyloid peptides in the brain. Familial forms of AD linked to mutations in amyloid precursor protein or presenilin 1, lead to a net

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90 increase in the amount o amyloid peptide 142 (Duff et al., 1996;Borchelt, 1998) These inherited forms of AD generally show much earlier disease onsets, but disease durations are similar to the sporadic disease (Bertram and Tanzi, 2008;Bird, 2008) Thus, in other examples of neurodegenerative disease associated with protein misfolding and aggregation, the aggregation rates of the causative protein appears to best correlate with disease onset. Conclusions We provide evidence that mutations in SOD1 that are associat ed with a high aggregation propensity generally predict a more rapidly progressing disease. However, several exceptions are noted, and it becomes less predictable for mutants with lower aggregation propensities. Thus, it appears that at least two factors r egulate disease progression; one of which is high inherent aggregation propensity of SOD1 mutant protein. In this view, we would categorize high aggregation propensity as a risk factor for rapidly progressing disease. Studies from our laboratory, and other s, have demonstrated that, in mouse models of SOD1associated ALS, the most significant accumulations of large mutant SOD1 aggregates occurs late in disease (Johnston et al., 2000;Wang et al., 2002b;Wang et al., 2002a;Wang et al., 2003;Jonsson et al., 2004;Wang et al., 2005b;Wang et al., 2005a;Wang et al., 2006;Karch et al., 2009) Importantly, significant accumulation of mutant SOD1 aggregates occurs well after the appearance of multiple pathologic abnormalities in these mouse models (Karch et al., 2009) Thus, we concur with others that have suggested that there must be a toxic form of mutant SOD1 that is distinct from larger protein aggregates with these entities initiating disease. However, aggregation of the mutant protein may be one of the forces capable of modulating the rate of di sease progression. The mechanisms by which

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91 aggregation of mutant SOD1 promotes disease progression are unclear at present. One hypothesis that could apply is the idea that the accumulation of SOD1 aggregates impairs the ability of the cell to maintain prot ein homeostasis (Gidalevitz et al., 2009) The idea is that as chaperones become occupied in unproductive attempts to dissolve protein aggregates, these activities are not available for productive functions in protein folding. There is also evidence that cells accumulating protein aggregates show reduced proteasome function (B ence et al., 2001) Together, the disruption of these critical protein homeostatic processes could cause a feed-forward cascade of impairment in the protein folding and metabolism that could underlie the rapid progression of disease that is seen in many of the mouse models. If these mechanisms apply, then compounds that modulate mutant SOD1 aggregation could be useful therapeutics in slowing the progression of this disease in humans.

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92 CHAPTER 4 MODULATION OF MUTANT SUPEROXIDE DISMUTASE 1 AGGREGATION BY CO EXPRESSION OF WILD -TYPE ENZYME 1 Introduction In SOD1associated ALS, WT and mutant SOD1 proteins are coexpressed at 1:1 ratios of synthesis. Whether toxicity of mutant SOD1 is modulated by interactions between WT and mutant protein, or b y the activity of WT SOD1, has been addressed in several experimental models (for more information about these studies see Implication of WT SOD1 in ALS and Aggregation in Chapter 1). Transgenic mouse studies conducted to determine the effect of human WT SOD1 on the toxicity of mutant human SOD1 have produced conflicted results. In one study mice overexpressing WT and G85R human SOD1 proteins revealed that WT human SOD1 had no effect on disease onset or survival (Bruijn et al., 1998) while in other studies the presence of WT human SOD1 in mice expressing a mutant human SOD1 protein (A4V, G85R, G93A, T116X, or L126Z) was found to accelerate di sease onset (Jaarsma et al., 2000;Deng et al., 2006;Deng et al., 2008;Wang et al., 2009c) Additionally, in one of the latter studies, earlier disease onset was accompanied by the formation of SOD1 protein aggregates that contained both WT human SOD1 proteins (Deng et al., 2006) Thus, one explanation for the decrease in mouse lifespan could be that the addition of human WT SOD1 promoted a more rapid aggregation of mutant protein. Additionally, the lack of correlation between aggregation propensities of slow aggregating SOD1 mutants and 1The work presented in this chapter has been published in The Journal of Neurochemistry 108(4):100918 (2009). Mercedes Prudencio and David R. Borchelt designed the experiments, interpret the data and wrote the manuscript. Mercedes Prudencio carried out all the experiments with the exception of the FMTS analyses which were performed and interpreted by Arma ndo Durazo and Julian Whitelegge at UCLA.

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93 disease duration, studied in Chapter 3, may be due to the fact that WT c ould play an important role in modulating aggregation. In this study we have used our cell culture model of mutant SOD1 aggregation to ask whether WT SOD1 directly promotes the aggregation of mutant SOD1. In order to address this issue, we co expressed cel ls with WT SOD1 (human or mouse) and mutant human SOD1 (A4V, G85R, G93A SOD1) for different periods of time, and asses for aggregation of mutant SOD1 proteins. We observed an outcome not predicted in the mouse studies. Additionally, human and mouse WT SOD1 demonstrated similar, but not identical, ability on modulating mutant SOD1 aggregation. Further analyses are performed to explore such differences between human and mouse WT SOD1. Materials and Methods A list of materials used can be found in Appendix B. Methodology used for the work presented in this chapter is described in Chapter 4 Methods of Appendix C. Results Differential detergent extraction and centrifugation techniques have been demonstrated as an approach to separate mutant SOD1 complexes of high molecular weight (presumed aggregates and defined as such here); which occur in both transgenic mouse tissue and cell culture models (Wang et al., 2003;Wang et al., 2006;Karch and Borchelt, 2008;Karch et al., 2009) } The formation of SOD1 aggregates can be modelled by high level expression of mutant SOD1 in human HEK293FT cells (Wang et al., 2003) Using this model system we sought to examine whether WT SOD1 modulates the aggregation of mutant SOD1. Co expression of WT and mutant human SOD1 proteins in cultured HE K293FT cells reduced the level of detergent -insoluble mutant SOD1 proteins that accumulates in

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94 24 hours ( Figure 4 1 ). C ells transfected with A4V, G85R or G93A SOD1 alone formed detergent -insoluble aggregates that sedimented upon ultracentrifugation, whereas cells expressing both WT and mutant h uman SOD1 produced little or no detergent insoluble SOD1 protein ( Figures 4 1A and 4 -1B upper panel). Instead, both WT and mutant h uman SOD1 proteins were found only in soluble fractions ( Figures 4 -1A and 4 -1B lower panel). To control for non-specific effects of co -transfection, such as reduced mutant protein expression that may have caused a reduction in aggregation, we co expressed the mutant SOD1 constructs (A4V, G85R and G93A SOD1) with a GFP construct and perfor med the detergent extraction and centrifugation assay. In each case, the expression of GFP did not affect the aggregation of mutant SOD1 ( Figures 4 1A and 4 -1B upper panel) ; and a large fraction of the mutant SOD1 remains fully soluble in detergent ( Figur es 4 1A and 4 -1B lower panels). The levels of soluble SOD1 protein provide a good indication of protein expression, indicating that all mutant proteins were expressed at high levels relative to non -transfected control cells. Immunoblot data from at least four experiments for each set of WT and mutant h uman SOD1 co transfections was quantified and analyzed statistically ( Figure 4 -1C ), providing clear evidence that in our cell culture model the co expression of WT SOD1 modulates mutant human SOD1 aggregation To examine the effects of WT h uman SOD1 on mutant SOD1 aggregation over time, we extended the interval between transfection and harvest to 48 hours. Interestingly we found that WT human SOD1 differentially affected the aggregation of the different SOD1 m utants (A4V, G85R and G93A SOD1; Figures 4 1D and 4 1E ). As compared to cells expressing A4V human SOD1 alone, cells co-transfected with vectors

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95 for WT and A4V human SOD1 continued to accumulate less detergent -insoluble mutant protein ( Figure 4 -1D upper p anel ). However, aggregation was not blocked as these cells contained significantly more detergent insoluble SOD1 than cell cells transfected with WT h uman SOD1 ( Figure 4 1E ). The levels of detergent -soluble SOD1 protein in extracts from cells co -transfected with WT and A4V human SOD1 indicate d relatively high levels of expressed protein ( Figure 4 1D lower panel). However, because the WT and A4V human SOD1 proteins c ould not be distinguished by SDS -PAGE and the low amount of detergent insoluble SOD1 in the co transfected cells, we could not determine whether the insoluble human SOD1 is limited to mutant protein. When WT human SOD1 wa s co expressed with G85R h uman SOD1, it wa s possible to differentiate the WT and mutant human SOD1 proteins by SDS -PAGE; the G8 5R variant migrate s anomalously in SDS -PAGE, running slightly faster than the expected size (Hayward et al. 2002; Wang et al. 2003; Wang et al. 2006). In cells coexpressing WT with G85R h uman SOD1, we observed significant accumulation of detergent -insoluble mutant protein at 48 hours. More interestingly, WT human SOD1 wa s clearly detected in the detergent -insoluble fraction ( Figures 4 1D upper panel, and 4 1F). This result i s consistent with the discoveries of detergent insoluble WT human SOD1 in spinal cords of transgenic mice co expressing WT and mutant h uman SOD1 (Deng et al., 2006) Similar to the result with the G85R variant, coexpression of WT and G93A h uman SOD1 for 48 hours showed similar levels of SOD1 protein in the detergent -insoluble fraction as compared to cells transfected with G93A h uman SOD1 expression plasm id alone ( Figures 4 -1D and 4 1E ). However, whether WT human SOD1 co aggregated with G93A SOD1 c ould not be determined by SDS -PAGE alone.

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96 Figure 4-1. Human WT SOD1 modulates the aggregation of mutant SOD1 in cultured cells. A, B, D). Immunoblots of P2 and S1 fractions from HEK293FT cells cotransfected with WT human SOD1 (or GFP) and mutant SOD1 for 24 (A, B) or 48 (D) hours. UT: untransfected cells. hWT: cells transfected with WT human SOD1 construct. C, E, F). Quantification of the rela tive aggregation potentials of the SOD1 mutants, when expressed alone or with hWT (C E); or of hWT when co-expressed with G85R for 48 hour s (F). Bars represent mean SEM. N=3-9. Paired student t -tests vs. hWT, or as indicated. p 0.05, #p 0.005, n.s.: non-significant differences. To determine whether the detergent-insol uble human SOD1 protein fraction of cells co-transfected with WT and G93A hum an SOD1 contain any trace of WT human SOD1 protein, we analyzed such fractions by hybrid linear ion-tr ap Fourier-transform ion cyclotron resonance mass spectrometry (F TMS). FTMS analysis revealed the presence of both WT and G93A human SOD1 in both the detergent-insol uble (P2) and the detergent-soluble (S1) fractions (Figure 4-2). In the detergent-insoluble fractions, there was about 10 fold more G93A than WT human SOD1 detected (Figure 4-2A, P2); while in the soluble fractions, the levels of WT and G93A human SOD1 were similar (Figure 4-2B, S1). These findings indicate that WT human SOD1 co-sediments with the G93A G 9 3 A + G F P 20 kDa 20 kDa 20 kDa 20 kDa U T h W T G 9 3 AG F PG 9 3 A + h W TP2 S1 20 kDa 20 kDa 20 kDa 20 kDab c hWT A 4 V A4V + hWT A 4V + GFP G 8 5R G85R + h W T G85R + G F P G93A G93A + h W T G93A + GFP 0.0 0.5 1.0 1.5 2.0Relative aggregation propensity of SOD1 mutants (A.U.) # # *# # e h W T A4V A4V + h W T G8 5 R G85R + hWT G9 3A G93 A + h W T 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 mutants (A.U.) *n.s. n.s.#* *# # h WT A4 V G85R h W T i n G85 R + h W T 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 proteins (A.U.)#* *f B C F G 8 5 R U T h W TA 4 V G8 5 R + GF P A 4 V + G F PG 85 R + h W TA 4 V + h W TG F PP2 S1 A D 20 kDa 20 kDa U T h W TA 4 VP2 S1G 8 5 R G 8 5 R + h W T G 9 3 A G9 3 A + h W T A 4 V + h W TE

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97 SOD1 mutant protein, but WT protein is not a major component of the detergentinsoluble aggregated protein. Figure 4-2. Comparison of SO D1 molecular mass profiles from S1 and P2 fractions of HEK293FT cells co-expressing human WT (hWT) and G93A human SOD1. A, B) SOD1 was recovered from spinal cord extracts, solubilized and purified (See Methods). An appropriate chromatographic fraction was analyzed by nano-electrospray using FTMS, with resolution 100,000 at m/z=400. Zero charge molecular mass profiles were deconvoluted from raw FTMS spectra of SOD1 recovered from P2 (A) and S1 (B) fractions using Xtract software (Thermo Fisher). Bars represent the individual 13C isotopomers with the most intense approximating the average mass of the protein. Minor sodium (Na) adducts are typical in these experiments. To control for the effects of co-transfection and for the possibility that SOD1 proteins of differing sequences might interf ere with aggregation, we also co-transfected G85R human SOD1 with WT, A4V, and G 93A human SOD1 constructs. In these experiments we took advantage of the anomal ous migration of G85R human SOD1 to examine how the co-expression of two diffe rent SOD1 mutants might affect their aggregation. Cells co-expressi ng G85R SOD1 with either A4 V or G93A SOD1 produced detergent-insoluble forms of each human SOD1 mutant (Figure 4-3A, upper panel). As described above, the presence of mutant human SOD1 protein in the S1 fraction for each transfection indicated that only a porti on of the total protei n adopted the detergentinsoluble conformation (Figure 4-3A, lo wer panel). Quantificat ion of multiple

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98 independent experiments demonstrated that whet her expressed alone or in combination with the A4V or G93A SOD1 variants, the propensity of G85R SOD1 to aggregate was not significantly altered (Figure 4-3B). T hese data suggest that t he apparent reduction in aggregation caused by the co-expression of WT with mutant human SOD1 is not due to some non-specific effect of co-transfection or some non-specific e ffect of interactions between two different SOD1 subunits, but rather appears to be due to a specific property of the WT human SOD1 protein. Figure 4-3. SOD1 mutants with high propensity to aggregate (A4V and G93A SOD1) do not interfere with aggregation of G85R SOD1. A) Immunoblot of P2 (upper panel) and S1 (lower panel) fractions of singly and doubly transfected HEK293FT cells. UT: untransfected cells. No tations are the same as in Figure 4-1. B) Quantificat ion of the relative aggregation potentials of the G85R SOD1 when expressed alone and with other SOD1 constructs. Bars represent mean SEM (N = 4-8). Statisti cal analysis compares t he aggregation of WT human SOD1 to G85R or G85R co-t ransfected with another construct *p 0.05, #p 0.005. Only G85R + WT was signific antly different fr om G85R alone *p 0.05; n.s. indicates non-significant differences. The human and mouse WT SOD1 proteins share approximately 83.6% identity at the level of amino acid sequenc e (Figure 4-4), with 25 amino acid differences in the 153 residue protein. Thus, we next sought to inve stigate whether these differences in protein P2 S1U T h W TG 8 5 R + h W T G 8 5 R G 8 5 R + A 4 VG 8 5 R + G 9 3 A 20 kDa 20 kDa 20 kDa 20 kDaA B W T G8 5 R W T + G85 R A 4V + G 85 R G9 3A + G85 R 0.0 0.5 1.0 1.5Relative aggregation propensity of G85R SOD1 mutant (A.U.)* *#* n.s. n.s.

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99 sequence would affect the ability of WT SOD1 to modulate the aggregation rate of mutant human SOD1. Figure 44. Alignment of human ( h) and mouse (m) SOD1 protein sequences. Below the h uman SOD1 sequence, in bold font, we indicate the 25 differences b etween h uman and mouse SOD1. The ALS mutations used in this study are also indicated above the human SOD1 sequence. HEK293FT cells co transfected with WT mouse SOD1 and mutant human SOD1 proteins (A4V, G85R and G93A SOD1) show ed a significant reduction in the amount of detergent -insoluble SOD1 aggregates produced in 24 hours ( Figures 4 -5A and 4 5B ). Interpretation of the immunoblots o f cells co-transfected with WT m ouse SOD1 and G85R human SOD1 was complicated by the fact that these proteins migrated to very near the same position in SDSPAGE. However these proteins c ould be resolved in gels exposed for short intervals, allowing for t he detection of both WT m ouse SOD1 and G85R human SOD1 in the detergent -soluble protein fraction ( Figure 4 -5A right lower panel). Despite a significant reduction in the amount of insoluble G85R human SOD1 in these co -transfected cells, aggregation wa s not blocked and it wa s possible to demonstrate that the detergent -insoluble fraction contain ed only G85R h uman SOD1 (Figure 4 5A right upper panel). Quantification of the relative aggregation propensity of the h uman SOD1 mutants in cells co -transfected with WT m ouse SOD1 reveal ed a significant reduction in the amount of detergent -insoluble mutant human SOD1 protein ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHF NPLSRKHGGPKD EERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ931 77 V4R85A M T H A17 19 2G24E26 27V L S30 31 32 34Q T36 43Q Q Y49 50 55Q H87 89K A95 90G N96 102R E S109 111 119M Q123WT mSOD1 WT hSOD1 fALS hSOD1 mutations used in this study WT mSOD1 WT hSOD1 fALS hSOD1 mutations used in this study 67 69 75

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100 that accumulated in 24 hours ( Figure 4 -5B ). Thus, WT m ouse SOD1 has the same capacity as WT h uman SOD1 to slow the rate of mutant h uman SOD1 to aggregate. When we extend ed the interval between transfection and harvest to 48 hours, we observed that A4V, G85R and G93A human SOD1, when expressed with WT m ouse SOD1, wer e able to form detectable amounts of detergent i nsoluble SOD1 aggregates (Figure 4 5C ). We observe d that in measures of aggregation propensity, which compensates for any changes in the expression of mutant hSOD1 that may occur when co transfected with WT m ouse SOD1, the presence of WT m ouse SOD1 had no significant effect on aggregation of mutant human SOD1 ( Figure 4 -5D ). Interestingly, WT m ouse SOD1, unlike WT human SOD1, did not seem to co aggregate with any of the mutants even after the longer 48 hour interval ( Figure 4 -5C upper panel). In co transfections of A4V or G93A human SOD1 mutants with WT m ouse SOD1, the amount of m ouse SOD1 detected in the insoluble fraction wa s no t different from that of cells transfected with m ouse SOD1 alone ( Figure 4 5C ; p 0.05, N = 4 ). Moreover, we did not detect m ouse SOD1 in detergent -insoluble fra ctions of cells co expressing WT m ouse SOD1 and G85R h uman SOD1 (p = 0.2028, N = 3) which c ould be observed in gels exposed for short intervals ( Figure 4 5C right panel). Thus, although WT m ouse SOD1 possesses an ability that is similar to WT human SOD1 in modulating the aggregation of mutant proteins, it lacks a feature that allows for co-sedimentation with mutant human SOD1. The differing ability of WT m ouse SOD1 and human SOD1 to co aggregate with mutant human SOD1 is a finding that appears to be consi stent with a recent report suggesting that a specific cysteine residue in h uman SOD1 may mediate the co -

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101 aggregation of WT and mutant h uman SOD1 (Cozzolino et al., 2008a) The cysteine residue at position 111 of human SOD1 has been identified as a potential mediator of disulfide cross -linking between mutant and WT human SOD1 (Cozzolino et al., 2008a) Mouse SOD1 encodes serine at position 111, and thus could not generate such disulfide linkages. To directly test this hypothesis, we used a previously described human cDNA that encodes serine as position 111 (C111S) (Karch and Borchelt, 2008) in co transfection with G85R h uman SOD1 T he mutant C111S human SOD1 is not an ALS mutation; in most species the position equivalent to 111 encodes serine. Previous studies have established that this mutant does not spontaneously aggregate (Cozzolino et al., 2008a;Karch and Borchelt, 2008) In C111S and G85R h uman SOD1 co transfections cells were harvested after 48 hours, which was the interval needed to obser ve WT and G85R human SOD1 coaggregation. Consistent with previous studies, all C111S human SOD1 fractionated to the detergent -soluble fraction ( Figure 4 -6A ). However, in cells co transfected with C111S and G85R h uman SOD1 and harvested 48 hours later, we f ound both proteins in the detergent insoluble fraction Quantification of the aggregation propensity of each SOD1 protein show s significant accumulation of aggregated C111S human SOD1 in the co transfected cells ( Figure 4 -6B ). This finding suggests that the co aggregation of WT human SOD1 with mutant protein is not dependent upon a disulfide linkage between cysteine 111 of WT protein and cysteine residues of mutant h uman SOD1. The implication of disulfide bonding can be further studied by including high ME (30%) in the detergent extraction buffers. In this setting, in cells co transfected with WT and G85R human SOD1, both proteins are detected in the P2

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102 fractions (Figure 4-7, lanes 5 and 7). Not ably, cells expressing WT human SOD1 alone in the presence of -ME present somewhat higher levels of WT human SOD1 in P2 fraction (Figure 4-7, lane 2). This higher backg round of WT in P2 makes it difficult to interpret the meaning behind the presence of WT human SOD1 in insoluble fractions of cells co-expressing WT and G85R human SO D1. Whether some WT human SOD1 is disulfide cross-linked to mutant human SO D1 cannot be excluded by this experiment. However, overall, these data s how that disulfide linkages are unlikely to be important in maintaining aggregate structure. Figure 4-5. Mouse WT SOD1 also modulates the aggregation of mutant SOD1 in cultured cells, but without evidence of co-aggregation. A, D) Immunoblots of P2 and S1 fractions from 24 (A) and 48 (B) hour transfections and cotransfections in of HEK293FT cells. UT: untransfected cells. mWT: cells transfected with WT mouse SOD1 constr uct. B, C) Quantification of the relative aggregation potentials of t he studied SOD1 mutants when expressed alone and with WT mouse SOD1. Bars represent mean SEM. N=3-6. Statistical analysis compares differ ences in aggregation propensity with mWT, or as indicated *p 0.05, #p 0.005, n.s.: non-significant. U T m W TA 4 VP2 S1G 8 5 R G 8 5 R + m W T G 9 3 A G 9 3 A + m W T A 4 V + m W T P2Low exposureG 8 5 R + m W TS1Low exposure G 9 3 AU T m W TA 4 VG 9 3 A + G F PA 4 V + G F PG 9 3 A + m W TA 4 V + m W TG F PP2 S1G 8 5 RG 8 5 R + G F PG 8 5 R + m W T G 8 5 R + m W TP2Low exposureS1Low exposure m WT A 4 V A4V + mWT A 4 V + GFP G8 5 R G8 5 R + mW T G85R + GFP G93 A G 9 3 A + m WT G93 A + G FP 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Relative aggregation propensity of SOD1 mutants (A.U.) # * *a c b mWT A4V A4V + mWT G8 5 R G85R + mW T G9 3 A G 93A + mW T 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 mutants (A.U.) *n.s. n.s.#* *n.s.*d A B C D

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103 Figure 4-6. Cysteine 111 is not required for the co-aggregation of WT with G85R human SOD1. A) Immunoblots of detergent extr acted cells that co-expressed C111S and G85R human SOD1 proteins for 48 hours. Notations are the same as in Figure 4-1. B) Quantification of the relative aggregation propensity of mutant human SOD1 proteins. Bars repr esent mean SEM. N=4. Student t -tests compare the aggregation of WT human SOD1 to each mutant, or as indicated on the figure. *p 0.05, #p 0.005. Figure 4-7. High concentration of -ME does not reduce the am ount of mutant SOD1 that fractionates to the P2 fraction at 48 hours. Detergent extractions followed the same protocols used for previous fi gures except all buffers were adjusted to contain 30% -ME to break all disulfide bonds A) Immunoblots of P2 and S1 fractions. B) Quantificat ion of the relative aggreg ation propensity of WT or mutant human SOD1. Bars represent mean SEM (N = 3). Paired student ttests compare the aggregation of WT hum an SOD1 to each mutant or as noted on the figure, *p 0.05, #p 0.005; n.s.: non-significant differences. U T h W TA4 VP2 S1G 8 5 R G 8 5 R + C 1 1 1 S 20 kDa 20 kDa 20 kDa 20 kDaC 1 1 1 SA B hWT A4 V G8 5 R C111S G8 5 R in G8 5 R + C1 11 S C 111S i n G85R + C1 1 1S 0.0 0.5 1.0 1.5 2.0Relative aggregation propensity of SOD1 mutants (A.U.)#* #* U T h W TA 4 VP2 S1G 8 5 R G 8 5 R + h W T G 8 5 R G 8 5 R + h W T 20 kDa 20 kDa 20 kDa 20 kDa 1234567A B hW T A4 V G 8 5 R G85 R in G 8 5 R + hWT hW T in G8 5 R + hWT 0.0 0.5 1.0 1.5Relative aggregation propensity of SOD1 proteins (A.U.)#* n.s.

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104 Discussion In the present study, we examined the potential for WT SOD1 to influence the aggregation of mutant SOD1 as defined by the formation of structures that are insoluble in non-ionic detergent and sediment upon ultracentrifugation. In a cell culture model of mutant SOD1 aggregation, we found that the presence of WT human SOD1 or mSOD1 significantly lowered the amount of mutant h uman SOD1 (A4V, G85R and G93A) in aggregates after 24 hours. Upon longer incubation (48 hours), we observed significant aggregation of G85R and G93A human SOD1, but continued attenuation of A4V aggregate levels. We also observed that WT human SOD1 can co-sediment with mutant G85R and G93A SOD1 aggregates; however, the predominant species of SOD1 in these aggregates was mutant pr otein. Importantly, these effects of WT human or mouse SOD1 on the aggregation of mutant protein were specific to WT protein. Co expression of G85R h uman SOD1 with either A4V or G93A h uman SOD1 showed no evidence of slowed aggregation rates; detergent -inso luble forms of both mutant proteins were readily detectable in 24 hours. From these findings, we conclude that WT SOD1 possess a capacity to modulate the aggregation of the mutant protein, with the primary effect being to slow aggregation rates. Human B ut N ot Mouse WT SOD1 Can CoA ggregate with M utant SOD1 One mechanism by which WT protein could slow aggregation of mutant protein, but then ultimately become a component of such aggregates, is via direct proteinprotein interactions between the WT and mutant proteins at the level of nucleation, or growth, of the aggregate. In many aggregate structures, the stacking of peptide chains of identical sequence is crucial to the formation of stable oligomeric structures (Petty and Decatur, 2005;Shorter and Lindquist, 2005) Such stacking forces have been

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105 proposed in prion protein conformational changes and it is well established that the presence of two prion proteins with single amino acid substitutions can slow aggregation (Hsiao et al., 1994;Petty et al., 2005) With this notion in mind, we tested whether WT m ouse SOD1 could produce the same effects as WT human SOD1 on the aggregation of mutant h uman SOD1. The WT m ouse SOD1 protein contains 25 amino acid differences from the human protein ( see Figure 4 4 ). Despite these numerous sequence differences, WT m ouse SOD1 retains the ability to slow aggregation of mutant human SOD1, presumably through direct proteinprotein interactions. However, WT m ouse SOD1 does not co -sediment wit h the mutant human SOD1 aggregates. This latter outcome could indicate that the numerous sequence differences between human and mouse SOD1 disrupt the types of close proteinprotein interactions that would be required in the assembly of SOD1 aggregates. WT and Mutant Human SOD1 ProteinProtein Interaction: Role of Disulfide Bonding and C ysteine 111. Our observation that WT m ouse SOD1 does not co aggr egate with mutant h uman SOD1 is consistent with a recently a proposed mechanism of WT and mutant SOD1 co aggregation that suggested a role for inter -subunit disulfide crosslinking between cysteine residues at position 111 (Cozzolino et al., 2008a) In a heterodimer of WT and mutant SOD1 subunits, t hese cysteines would be in close proximity near the dimer interface and thus could mediate an inter -subunit bridge. Mouse SOD1 encodes serine at position 111 and would be incapable of forming such a disulfide bridge. To test the role of disulfide linkages between cysteine 111 residues in the co aggregation of WT and mutant human SOD1, we mutated cysteine 111 of WT human SOD1 to serine and then co transfected this construct with G85R human SOD1; finding that we could still

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106 detect coaggregation of this modif ied WT human SOD1 with mutant protein. We cannot rule out the possibility that cysteine 111 of the mutant h uman SOD1 mediates a disulfide linkage with another cysteine in C111S human SOD1 (cysteines at positions 6, 57, or 146); however, it is clear that th e linkage cannot be between cysteine 111 of the two proteins. Additionally, we have noted that we can supplement the buffers used in mercaptoethanol ( ME a strong reducing agent) without noting sig nificant reductions in the amount of mutant SOD1 that fractionates to detergent insoluble fractions ( see Figure 4 7 ). These data provide compelling evidence that disulfide cross -linking is not a primary mechanism by which the structure of aggregates are maintained [also see (Karch and Borchelt, 2008) ]; and we think it unlikely that disulfide -linkages are responsible for the co -sedimentation of WT h uman SOD1 with mutant h uman SOD1. Rather, we suggest that the co sedimentation of WT human SOD1 with mutant h uman SOD1 is likely to involve more intimate protein-protein interactions. Role of WT and Mutant SOD1 Interactions in D isease In a study by Deng and colleagues (Deng et al., 2006) the co expression of WT and mutant human SOD1 in transgenic animals prod uced by mating two distinct lines of mice, showed earlier onset of disease and earlier age to paralysis; with the symptomatic mice showing high levels of detergent insoluble forms of both WT and mutant protein. If aggregation of mutant SOD1 were one of the driving forces in age to disease onset, then increasing the concentration of total SOD1, through the addition of WT h uman SOD1 protein, could potentially decrease the nucleation phase of protein aggregation; which is well established to be highly concentration-dependent (Jarrett and Lansbury, Jr., 1992) However, in our cell culture model, we find that the presence of

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107 WT h uman SOD1 slows the aggregation of mutant protein. The most informative data in our experiment is a comparison of mutant SOD1 aggregate loads in cells co-transfected with mutant SOD1 constructs and constructs for GFP to cells co -transfected with mutant and WT SOD1 constructs. As compared to GFP, WT SOD1 co expression reduced overall amounts of aggregat ed mutant SOD1 that accumulated in 24 hours. We interpret this finding as evidence that WT human SOD1 does not provide a concentrationdependent enhancement of mutant SOD1 aggregation. Whether the effect of WT SOD1 on mutant SOD1 aggregation occurs at the level of aggregate nucleation is difficult to address in our cell culture system. It is possible that WT SOD1 interferes with the growth phase of aggregation in which small oligomers of protein assemble into larger sedimentable aggregates. If our cell cult ure studies accurately model events that occur in vivo then our data would argue that the basis for accelerated disease onset in the mouse studies of Deng and colleagues (Deng et al., 2006) is not attributable to accelerated rates of mutant SOD1 aggregation. How ever, the foregoing study demonstrated that mice expressing low levels of A4V h uman SOD1 never develop disease and do not develop SOD1 aggregates, whereas mice that co express high levels of WT human SOD1 with low levels of A4V human SOD1 develop disease with spinal cords that contain aggregated SOD1 protein (undetermined whether WT, A4V, or both) (Deng et al., 2006) This latter outcome suggests a direct involvement of SOD1 aggregation in disease pathogenesis. However, other recent studies have demonstrated that aggregation of mutant SOD1 may be dissociable from the toxic events that drive disease onset. Coexpression

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108 of high levels of the copper chaperone for SOD1 (CCS) with G93A SOD1 greatly accelerates the onset of disease while reducing the level of G93A SOD1 aggregation (Son et al., 2007) Moreover, we have recently determined that the accumulation of the larger sedimentable aggregates of mutant SOD1 in ALS mouse models occurs largely after disease onset (Karch et al., 2009) These recent findings indicate that disease onset may not be governed by the rate of mutant protein aggregation. Whether other aspects of disease, such as progression, are related to the rates of mutant protein aggregation is a subject of study Although the cell model we use here has high utility in assessing the aggregation propensity of mutant SOD1, it is not well suited for studies of toxicity. The advantage of the model is that aggregation occurs without an exogenous stimulus, such as in hibition of proteasomes or other toxic insult. However, the levels of expression achieved are admittedly well above physiologic levels and thus we are hesitant to conclude that any toxicity observed in this cell model over a 24 or 48 hour period would equa te to events occurring over a much longer time frame in either mouse models or humans. Deciphering the mechanism by which WT h uman SOD1 overexpression heightens the toxicity of mutant SOD1 will require development of more physiologic ally relevant cell models, or innovative approaches to studying molecular events in animal models. Conclusions In a cell culture model of mutant SOD1 aggregation, we find evidence that WT SOD1 is a direct modulator of mutant h uman SOD1 aggregation, with the predominant effect be ing to slow aggregation rates. More than 100 mutations in SOD1 have been associated with ALS and, given the variability in the biophysical properties of these mutants, we think it is highly likely that the magnitude of the effect of WT protein on the

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109 aggregation r ate of mutant human SOD1 will vary. Indeed in our small sample of mutants in the present study, we find that that the effect of WT SOD1 on the aggregation of A4V h uman SOD1 appears to be distinct from that of the G85R or G93A variants. In human SOD 1 associate d ALS, disease occurs in a setting of equivalent expression of WT and mutant SOD1 subunits. We propose that the modulation of mutant human SOD1 aggregation by WT enzyme may introduce another factor that influences the age to onset, or rate of pr ogression, of disease in humans.

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110 CHAPTER 5 A COMPLEX ROLE FOR W ILD -TYPE SOD1 IN THE TOX ICITY AND AGGREGATIO N OF ALS -ASSOCIATED MUTANT SO D11 Introduction Work presented in the previous chapter (Chapter 4) describes the ability of WT SOD1 to modulate aggregation of mutant proteins in a cell culture model. Results from such studies demonstrate that the coaggregation of WT and mutant human SOD1 proteins is no t a consequence of increased amounts of expressed SOD1 protein, as WT SOD1 slows mutant SOD1 aggregation. However, the role of WT SOD1 on disease remains controversial (see Implications of WT SOD1 in ALS and Aggregation in Chapter 1 and introduction of Chapter 4). The variability in the results of the different studies may be explained by the type of mutant SOD1 protein expressed in conjunction with WT human SOD1, the background of the strain of mice used, and/or the relative expression levels of WT and mutant human SOD1 proteins in the mice. In the present study we have directly compared the WT human SOD1 mouse line used in the Bruijns study to the mouse line used in the other studies (Jaarsma, Deng and Wangs studies) to determine to what degree these mice contributed to the differences observed between these studies. With each of these WT human SOD1 lines of mice, we created double transgenic mice expressing WT human SOD1 and either L126Z or G37R human SOD1 mutant proteins The results of our studies demonstrate that the variability in the rate of disease onset observed in previously published studies can be explained by differences between the WT human SOD1 lines of mice used, and by the specific mutant human SOD1 protein 1The work presented here is a manuscript in preparation that will be submitted shortly for publication.

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111 expressed in these mouse line s. Further, all double WT and mutant human SOD1 transgenic mice created shared the ability to form detergent insoluble SOD1 aggregates of WT and mutant human SOD1 proteins that accumulate at disease endstage. Thus, our data provide: 1) clarification of the effect of WT human SOD1 on disease progression in mutant SOD1 transgenic mice, and 2) insight into the role that WT human SOD1 has modulating aggregation of mutant human SOD1 proteins. Materials and Methods A list of materials used can be found in Appendix B. Methodology used for the work presented in this chapter is described in Chapter 5 Methods of Appendix C. Results A study by Bruijn and colleagues, reported that mice transgenic for both WT and G85R human SOD1 developed disease at the same age as mic e transgenic only for the mutant gene (Bruijn et al., 1998) However, several recent studies have shown that the presence of WT human SOD1 in mice expressing mutant human SOD1 (A4V, G85R, G93A, T116X, or L126Z) accelerates disease onset (Jaarsma et al., 2000;Deng et al., 2006;Deng et al., 2008;Wang et al., 2009c) The study by Bruijn used a different line of WT SOD1 mice than used by the 4 studies that reported WT SOD1 coexpression accelerates disease. To determine if the different lines of WT human SOD1 mice used in these studies produce different outcomes, we crossed three different strains of WT human SOD1 mice to mice expressing either PrPG37R or L126Z human SOD1 proteins The three WT human SOD1 strains of mice used were:1) B6SJLTg(SOD1)2Gur/J hybrid line (SJL WT for abbreviation) (Gurney et al., 1994) [previously shown to accelerate disease when co expressed with A4V, L126Z, G93A (Deng et al., 2006) T116X (Deng et al., 2008) or G85R (Wang et al., 2009c) ], 2) B6/L76 WT SOD1

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112 Wong congenic line (L76 WT for abbreviation) (Wong et al., 1995) [shown not to have an effect o n disease onset or duration when co expressed with G85R) (Bruijn et al., 1998) ], and 3) a variant of the B6SJL-Tg(SOD1)2Gur/J Gurney hybrid l ine, here termed congenic (Cg) WT, that was backcrossed to C57BL/6J (Gurney et al., 1994) to c reate a strain possessing a equivalent background to that of the B6/L76 WT SOD1 Wong congenic line We compared side by side the levels of human SOD1 mRNA and protein in the different WT human SOD1 mouse strains used in this study, using northern and western blot analyses, respectively The mRNA and protein levels of SOD1 in spinal cords isolated from each WT SOD1 strain of mice was normalized using PrP mRNA or -tubulin III protein as loading controls, respectively. The results from these analyses showed t hat both mRNA and protein levels were approximately 30% higher in SJL and Cg WT strains of the Gurney mouse line compared to the L76 WT Wong line ( Figure 5 1 ). We further characterized the three WT mouse strains used in our study by analyzing the coding se quence of SOD1 in these mice. Thus, we performed real time polymerase chain reaction (RT -PCR) analysis from mRNA isolated from spinal cords of each strain. Each of the resulting RT -PCR products (Figure 52A) were then cloned into a vector and sequenced. Th e sequencing results revealed identical SOD1 cDNA sequences ( Figure 5 -2 B), demonstrating that the three strains of the two transgenic WT human SOD1 lines used in this study contain identical WT SOD1 proteins, with the only difference of expressing such transgene at different levels.

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113 Figure 5-2. WT Gurney lines express higher mRNA and protein levels than WT Wong line. A) Northern blot show ing the mRNA levels in t he spinal cords of the WT lines used in this study for which mRNA of the mouse prion protein (PrP) was used as a loading control. B) Quantification of the mRNA levels normalized to the PrP control. C) West ern blot analysis of same animal cords and detected with a human SOD1 antibody. Antibody recognizing -tubulin III was used as a loading protein control. D) Quantification of the human WT protein levels normalized to loading control. Statisti cal differences in B) and D) were assessed thought unpaired student t -tests: p 0.05, #p 0.005. Bars represent mean SEM of three different spinal co rds for each mouse line. N Tg SJL W T Cg WT L76 WT 0 2.0106 4.0106 6.0106 8.0106Band intensity SOD1 protein (A.U.) SOD1 TubulinIII C D#*# PrPmRNA SOD1 mRNA 28S 18S A B NTg SJ L WT C g WT L 76 WT 0 5.0105 1.0106 1.5106 2.0106Band intensity RNA (A.U.)** *#** *

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114 Figure 5 2. All WT SOD1 lines express the same cDNA WT SOD1 sequence. A) Agarose gel showing results from RT -PCR from non-transgenic (NTg) or WT mouse cords. B) Automated sequence analyses of the RT -PCR products indicate identical WT SOD1cDNA sequences (top lines). One line of mutant SOD1 mice used in this study is heterozygous for the G37R SOD1 mutation, which is driven by the mouse prion promoter (PrPG37R SOD1). This promoter drives transgene expression primarily in muscle and neural tissue (Wang et al., 2005b) The expression levels of the PrPG37R SOD1 transgene in heterozygous mice are known to be too low to produce an ALS phenotype i n mice. However, when G37R expression levels are increased by breeding the mice to homozygosity, the resulting mice manifest typical ALS symptoms and pathology (Wang et al., 2005b) Thus, while heterozygous PrPG37R mice do not develop ALS over the course of their 2 year lifespan, homozygous PrPG37R mice develop paralysis at about 8 months of age (Wang et al., 2005b) The other mutant SOD1 mouse line that we also used expresses the L126Z SOD1 transgene, which is controlled by the normal SOD1 human pr omoter and consists of modified genomic SOD1 (Wang et al., 2005a) The survival time for this line of mice is also 8 months (Wang et al., 2005a) SOD1 cDNA 1 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTSAGPHFNPLSR KHGGPKDE SOD1 SJL WT 1 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTSAGPHFNPLSR KHGGPKDE SOD1 Cg WT 1 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTSAGPHFNPLSR KHGGPKDE SOD1 L76 WT 1 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTSAGPHFNPLSR KHGGPKDE SOD1 cDNA 79 ER HVGDLGNVTA DKD G VADVSI EDSVISLSGDH CIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ SOD1 SJL WT 79 ER HVGDLGNVTA DKDGVADVSI EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ SOD1 Cg WT 79 ER HVGDLGNVTA DKDGVADVSI EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ SOD1 L76 WT 79 ER HVGDLGNVTA DKDGVADVSI EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ A B

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115 To study in vivo the effect of WT SOD1 from different mouse lines on disease and aggregation of SOD1 proteins, we performed matings of WT human SOD1 mice to either PrPG37R or L126Z human SOD1 mice. We obtained five different types of double transgenic mice expressing WT and mut ant human SOD1 proteins: PrPG37R/SJL WT, PrPG37R/Cg WT, PrPG37R/L76 WT, L126Z/SJL WT, and L126Z/L76 WT. In PrPG37R/SJL WT mice, we observed survival times of 5 months of age ( 160.1 2.40 days, N = 18), which was even shorter than that of homo zygous PrPG37R SOD1 mice (paraly zed at 8 months, 252.6 4.75 days, N = 20). Mice co expressing G37R protein, and the WT SOD1 from the congenic Gurney strain variant (Cg WT), also became paralyzed very early (190.1 1.01 days, N = 7), but at about a month later than PrPG37R/SJL WT mice (Figure 5-3A). Mice expressing G37R/L76 WT became paralyzed at about 7 months (213.8 4.35 days, N = 13), which represents an earlier age than for paralyzed homozygous PrPG37R mice (Figure 53A). Thus although heterozygous PrPG37R SOD1 mice are asymptomatic, mice expressing G37R/WT human SOD1 proteins developed ALS -like disease and reached endstage at earlier time points than PrPG37R SOD1 homozygous mice. Similar to the outcome described above, in double transgenic mice harboring human WT and the L126Z mutant human SOD1 proteins, were observed earlier onsets of paralysis in mice transgenic for both L126Z and WT human SOD1 that comes from the Gurney WT line of mice. L126Z/SJL WT mice developed disease between 5-6 months of age (170.1 4.74 days, N = 10; Figure 5-3B), which represents about 2 to 3 months earlier than mice expressing just L126Z SOD1 (214.1 3.74 days, N = 53). However, L126Z/L76 WT double transgenic mice did not develop paralysis until 8

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116 months of age (224.8 6.41 days, N = 16; Figure 5-3B), which is the same age of paralysis as for L126Z singly transgenic SOD1 mice. Thus, only the coexpression of WT SOD1 from the SJLWT strain, but not the L76 WT strain, of mice translated into a more rapid development of paralysis in mice harboring the L126Z SOD1 mutation. Figure 53 WT SOD1 protein from Gurney s mice significantly accelerates disease in mice harboring either PrPG37R or L126Z mutations while the effect of WT SOD1 derived from Wongs mice (L76 WT) is not as str ong. A) Survival curves of mice from PrPG37R crosses. B) Survival curves of mice from L126Z crosses. Survival times were estimated as the time comprised between birth and when animals are required to be sacrificed as hindlimb paralysis impeded them reach f ood and water Unpaired t -test demonstrated statistical differences in lifespan between homozygous PrPG37R mice and PrPG37R/SJL WT (p p PrPG37R/L76 WT (p (p 1), but not with L126Z/L76 WT ( p = 0.5177). Previous studies have established that spinal cords of paralyzed mice expressing mutant SOD1 contain detergent -insoluble aggregates of mutant protein (Deng et al., 2006) Additionally, Deng and colleagues observed that the earlier disease phenotype in mice expressing both WT (SJL WT strain) and mutant SOD1 (L126Z) accumulated insoluble forms of WT and mutant SOD1 proteins (Deng et al., 2006) Thus, we sought to determine whether any of our double transgenic mice contain aggregated WT and/or mutant human SOD1 proteins. In order to do that, we use a biochemical assay and western blotting techniques to detect the proportion of SOD1 protein that becomes 0 50 100 150 200 250 0 25 50 75 100 L126Z/L76 WT L126Z L126Z/SJL WT Time (days)Percent survival 0 50 100 150 200 250 0 25 50 75 100 PrPG37R +/+ PrPG37R/SJL WT PrPG37R +/PrPG37R/Cg WT PrPG37R/L76 WT Time (days)Percent survival A B252.6 days 214.1 days 213.8 days 224.8 days 170.1 days 160.1 days 190.1 days

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117 insoluble in non-ionic detergent. With this assay we detected the presence of detergent -insoluble aggregates in the spinal c ords of endstage PrPG37R/WT mice (Figure 54A, P2). The levels of SOD1 in the detergent -insoluble (P2) and soluble (S1) fractions were quantified and graphed as the ratio of P2 to S1; we refer to this value as aggregation propensity. In all PrPG37R/WT mice the accumulation of detergent insoluble SOD1 species is significantly higher than heterozygous PrPG37R mice, and similar to homozygous symptomatic PrPG37R SOD1 mice (Figure 54B). In L126Z/WT mice, we were able to distinguish WT from L126Z protein on immu noblots due to the smaller size of the truncated mutant protein. Thus, in all L126Z/WT mice we detected detergent -insoluble forms of WT and mutant SOD1 proteins (Figure 5 -4C). Note that we were unable to detect detergent -soluble forms of the L126Z protein due to its very rapid turnover (Figure 5 4C). Because we can specifically detect WT SOD1 in detergent soluble and insoluble fraction of these mice, we were able to estimate aggregation propensity (P2/S1). We found that the aggregation propensity of WT in L 126Z/WT mice is much lower than the aggregation propensity of G37R SOD1 in homozygous PrPG37R mice (compare Figures 5 -4B and 54D). Thus, much lower proportions of WT SOD1, compared to mutant SOD1 proteins, are able to become part of detergent insoluble aggregates. In the case of L126Z/WT mice, approximately equivalent levels of WT and mutant SOD1 proteins were present in the detergent -insoluble fraction (Figure 5 -4E). The observation of WT and L126Z human SOD1 proteins co aggregating in double transgenic m ice, is consistent with what has previously been described by Deng and colleagues (Deng e t al., 2006)

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118 Figure 5-4. Symptomatic mice present signi ficant accumulation of detergent-insoluble SOD1 aggregates at endstage. A, C) Western immunoblots of detergentinsoluble (P2) and detergent-soluble (S1) fractions of spinal cord transgenic mice. SOD1 protein was detected with an antibody that recognizes mouse and human SOD1 (A), or an antibody specif ic for human SOD1 (C). Note that the truncation mutant, due to its size, mi grates faster (black arrowheads) than human WT SOD1 (open arrowheads). B, D) Quantificat ion of aggregation propensity of PrPG37R/WT crosses (B ) and human WT in L126Z/WT crosses (D). E) Quantification of the relative protein levels of WT (black bars) and L126Z (white bars) SOD1 present in double transgenic L126Z/WT mice. Statistical differences of aggregation propensity were compared to nontransgenic (NTg) animals by unpaired student t -test: p 0.05, #p 0.005, of at least three different spinal cords per line. N.S.: non-significant differences. As described above, western blot analysis allowed us to determine the presence of WT and mutant human SOD1 in the det ergent-insoluble fractions of L126Z/WT SOD1 mice. However, WT and G37R human SOD1 proteins cannot be distinguished by standard western blot techniques. Thus, to fu rther explore whether WT human SOD1 is also present in the P2 fraction of PrPG37R/WT mice, we analyzed spinal cords of symptomatic PrPG37R/SJL WT mice through hybrid linear ion-trap Fourier-transform P2 S1 A B CD N Tg SJ L W T Cg WT L7 6 W T Pr P G3 7 R + / PrPG37R + / + Pr P G3 7 R/SJ L W T PrPG 3 7R/ C g W T Pr PG 37R / L7 6 W T 0.0 0.2 0.4 0.6 0.8Relative aggregation propensity of SOD1 proteins (A.U.)#*#*# # N T g L126Z/SJL W T L 126Z/ L76 W T 0.00 0.02 0.04 0.06Relative aggregation propensity of human WT SOD1 (A.U.)E T T T T Z 0 2.0106 4.0106 6.0106 8.0106 1.0107Band intensity levels of SOD1 in the P2 fraction (A.U.) WT SOD1 L126Z SOD1 N.S. N.S. N.S. # P2 S1

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119 ion cyclotron resonance mass spectrometry ( FTMS ) analyses. Data from such analyses indicate d the presence of WT and G37R human SOD1 proteins in the detergent insoluble P2 fraction of two different samples from spinal cords expressing PrPG37R and SJL WT transgenes (Figure s 5 -5A and 55B ). Additionally, a portion of WT and G37R SOD1 proteins were also detected in the S1 fractions (Figures 5-5C and 55D). Notably, in both the P2 and S1 fractions, the intensity of the signal for WT SOD1 was much greater than that of the G37R protein. In the S1 fraction, the mass spectrometry data suggested that WT SOD1 was present in much greater quantities than the mutant in the double transgenic animals. However, these data did not appear to be congruent with the data we obtained from immunoblots of PrPG37R/WT mice, in whic h the levels of WT protein should be only about twice more abundant than for the G37R protein (Figure 5 6) Figure 55 WT SOD1 is present in detergent insoluble fractions of spinal cords of PrPG37R/SJL WT mice. A-D) FTMS analyses of P2 (A, B) and S1 ( C, D) fractions from two different sets of spinal cord samples. For each sample, a total of three spinal cords were combined (from different symptomatic PrPG37R/SJL WT mice) and extracted in detergent as explained in methods. Volumes of 1.2 ml for S1 and 100 l for P2 were obtained and sent for FTMS analysis.

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120 Figure 56 Protein levels in the detergent -soluble fraction of heterozygous SJL WT and PrPG37R are not more than two fold different. The amount of S1 protein was calculated from western blot data o f samples presented in Figure 5-4. Although SJL WT protein levels are higher than the levels of G37R protein in heterozygous mice, this difference was not statistically significant. Previous analyses in cell culture have demonstrated that the co expresssi on of WT and mutant SOD1 at 1:1 ratios does not lead to a rapid increase in aggregation of SOD1 proteins. Instead, we have previously demonstrated that WT SOD1 protein slows, but does not block, the aggregation of several human SOD1 variants (A4V, G85R, and G93A) (Prudencio et al., 2009a) However, as early as 48 hours of co expression, both proteins (WT and mutant) co -sediment in the detergent insoluble, aggregated fraction (Prudencio et al., 2009a) Here we evaluated the ability of WT human SOD1 to modulate aggregation of G37R and L126Z human SOD1 mutant proteins in cell culture. Co-transfections of WT and G37R human SOD1 proteins, at 24 hours did not produce detectable levels of aggregated human SO D1 proteins (Figures 5 -7A and 57B, 24 hours), demonstrating the ability of WT human SOD1 in slowing aggregation of G37R SOD1 proteins. However, at 48 hours after transfection we observed significant accumulation of aggregated SOD1 proteins in the WT + G37R SJL WT PrPG37R +/PrPG37R/SJL WT 0.0 2.01007 4.01007 6.01007 8.01007 1.01008Relative levels of SOD1 protein in the S1 protein fraction (A.U.)

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121 human SOD1 co transfection. Additionally, t he total amount of aggregated SOD1 in the P2 fraction was lower when WT and G37R were co expressed as compared to when G37R SOD1 was co expressed with GFP (a control for nonspecific effects of co transfection) (Figures 5 -7A and 57, 48 hours). Thus, it appears that in the cell model, WT human SOD1 slows the aggregation of G37R SOD1. In co transfections of WT SOD1 with the L126Z SOD1 truncation mutant, we evaluated aggregation propensity only at the 48 hour transf ection interval. Cells expressing only L126Z SOD1 presented high levels of aggregated protein and small quantities of soluble SOD1 protein (Figures 57C and 5 -7D). However, in L126Z + WT SOD1 co transfected cells there were virtually undetectable levels of aggregated WT or L126Z human SOD1 proteins (Figures 5-7C and 57D). In the co transfection control (L126Z SOD1 + GFP), we observed levels of aggregated L126Z protein that were significantly higher than the aggregation propensity of L126Z in the WT + L126Z SOD1 co transfection (Figures 57C and 57D). These findings indicate that, at least in our cell model, the presence of WT SOD1 produces a pronounced slowing of L126Z mutant SOD1 aggregation. In previous work, we have demonstrated that a major portion of the mutant SOD1 that accumulates in aggregates lacks the normal intramolecular disulfide bond (Karch et al., 2009) Additionally, high levels of disulfide reduced mutant SOD1 protein have been associated, in specific settings, with a much shorter lifespan of mutant SOD1 mice (Proescher et al., 2008;Son et al., 2009) Thus, we sought to evaluate whether the high level expression of WT SOD1, in the double transgenic mice, might elevate the overall levels of reduced SOD1 in spinal cord. Using previously described western blotting

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122 techniques (Jonsson et al., 2006a;Zetterstrom et al., 2007;Karch et al., 2009), we found that, in the three WT strains of mice, at steady-state levels about 10% of WT SOD1 protein lacked the normal disulfide bond at old ages (Figures 5-8A and 5-8B). Figure 5-7 Human WT SOD1 slows aggregate forma tion in cell culture and such effect is stronger for the L126Z SOD1 truncation mutant. A, C) Aggregate formation determined by western blot of transiently transfe cted cells for 24 (A, upper panels) or 48 (A, lower panels; and C) hours. In co-transfections of WT and mutant SOD1 or mutant SOD1 and GF P (the latter as a control for cotransfection), we used equimolar amount s of each plasmids; with the total amount of transfected protein remaining the same for all reactions (4 g). B, D) Relative aggregation pr opensity of SOD1 proteins were calculated as P2/S1 ratios, and paired student t -test were performed as statistical analyses. Note that relative values of aggregati on propensity are normalized to P2/S1 value of A4V at 24 hour transfection (set to 1). Symbols over the bars indicate differences with human WT SOD1 transfe cted cells, or as indicated in the figure: p 0.05, #p 0.005. Bars repr esent mean SEM of a minimum of 3 independent transfect ion experiments. WT A4V L1 2 6 Z L1 2 6 Z + W T L126Z + GFP 0 1 2 3 4 5Relative aggregation propensity of SOD1 mutants (A.U.)A B P2 S120 kDa 20 kDa 20 kDa 20 kDa24 h 48 h 48 h ##** CD # # # # # # # # #* WT A 4V G37R G37R + WT G37R + GFP 0 1 2 324 h 48 h Relative aggregation propensity of SOD1 mutants (A.U.) P2 S1 P2 S1 20 kDa 20 kDa

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123 Additionally, in younger SJL WT mice, about 10% of the WT protein also migrated on SDS-PAGE as expected for reduced SOD1 (Figures 5-8C and 5-8D). In double transgenic PrPG37R/SJL WT symptomatic mice the levels of reduced SOD1 protein does not differ from aged matched SJL WT SOD1 singly transgenic mice (Figures 5-8C and 5-8D). Thus, it appears t hat the co-expression of WT and mutant SOD1 in double transgenic mice does not lead to an increase in the overall levels of reduced mutant SOD1 protein, and the total levels of reduced SOD1 appear to be the sum of the individual amounts of reduc ed WT and mutant SOD1. Figure 5-8. Low amounts of reduced WT SOD1 protein ar e present in all WT lines of mice. A, C) Immunoblots of reduced an oxidized proteins in spinal cord extracts of different lines of old (> 11 months) WT SOD1 mice (A), or in young (8 months) single and double transgenic mice (C). R: reduced, O: oxidized. B, D) Quantification of th e band intensities of at least three independent experiments from reduced and oxidized WT or mutant proteins in old (B) or young (D) animals. R OA B N T g SJL WT Cg WT L 76 WT 0 2.5106 5.0106 7.5106 1.0107 5.01007 7.51007 1.01008Reduced Oxidized Band intensity of SOD1 protein (A.U.) NTg S JL WT PrPG37R +/ P r PG 3 7R +/+ P rP G 37 R/S JL W T 0 5.0105 1.0106 1.5106 5.0106 1.0107 1.5107 2.0107Reduced Oxidized Band intensity of SOD1 protein (A.U.) R OC D

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124 Recent in vitro studies have demonstrated that small quantities of immature disulfide reduced WT SOD1 protein can be induced to aggregate (Chattopadhyay et al., 2008) Thus, t o investigate whether any of the strains of WT SOD1 mice may accumulate aggregates of SOD1 protein, we performed detergent extraction analyse s and determination of aggregate levels at young (< 8 months) and old (> 11 months) ages. For the Gurney line, in both SJL and Cg WT stra ins, significant accumulations of aggregated WT SOD1 protein w ere found as early as 8 months (SJL WT), and increased to higher levels at 11 (SJL WT) and 17 (Cg WT) months of age (Figures 59 A and 5 -9 C). However, WT SOD1 in the L76 WT SOD1 line did not show significant accumulation of detergent -insoluble protein by 17 months of age (Figures 5-9A and 59C). Importantly, the levels of aggregation propensity for WT human SOD1 protein in any of the lines were not as high as in symptomatic L29 G37R mice (Figures 5 9A and 5 -9C), which accumulates relatively low levels of aggregated SOD1 at disease endstage (Karch et al., 2009) Analys e s of total protein levels at young and old ages showed that the amount of WT SOD1 protein in the different strains of mice increases with age, and the overall WT SOD1 levels at old ages between Gurney and Wong lines remain constant, that is the Gurney line (SJL and Cg WT) express ing about 30% higher protein levels than the Wong L76 WT line (Figures 5 -9 B and 59 D). These data suggest that the higher expression levels of WT human SOD1 in the Gurney mice (SJL and Cg WT) translates into increased amount of misfolded WT SOD1.

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125 Figure 5-9. WT SOD1 from Gurney lines (SJL and Cg), but not from Wong line (L76), forms detergent-insoluble SOD1 aggregates at old ages. A, B) Immunoblots of S1 and P2 fractions (A), or of total SOD1 levels (B) of mice expressing WT SOD1 at young (4 months, or 8 months for SJL WT) or old (17 months, or 11 months for SJL WT) ages. An antibody that recognizes mouse and human SOD1 was used. NTg: non-transgenic mice. -tubulin III was used as a loading control for total protein. C, D) Quantification of the aggregation propensity (C) or of total SOD1 pr otein levels (D). Paired student t -tests were performed to establish significant differ ences with NTg mice, or as indicated: p 0.05, #p 0.005. Bars repr esent mean SEM. Discussion In the present study, we investig ated the apparent discrepancy in studies regarding the potential for WT hum an SOD1 to accelerate the course of disease caused by the expression of mutant SOD1 in trans genic mice. A study by Bruijn and colleagues reported that the course of disease caused by expression of the G85R variant of human SOD1 is not changed by either co-expressi on with human WT SOD1 or elimination of endogenous mouse SOD1 expression (Bruijn et al., 1998). Ho wever, more recently, using a different line of WT human SOD1 mice (B6SJL-Tg(SOD1)2Gur/J, here termed SJL WT), Wang and colleagues r eported that co-expression of WT and mutant SOD1 Total protein TubulinIII NT g yo u ng N T g ol d SJLWT young SJLWT ol d C g W T young Cg W T o l d L 76WT young L76WT old L29 G37R 0 5.0107 1.0108 1.5108Relative protein levels of hSOD1 protein (A.U.)*# # # #* *A B CD P2 S1 NTg young NTg old S JL W T young SJLW T ol d CgWT young C gWT old L 76W T young L7 6W T ol d L2 9 G37R 0.00 0.05 0.10 0.15 0.4 0.5 0.6Relative aggregation propensity of SOD1 proteins (A.U.)*# #* *

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126 accelerated disease caused by transgenic expression of human G85R SOD1 (a different line of G85R mice was used) (Wang et al., 2009c) Additionally, multiple studies using the SJL WT mice have demonstrated that mice transgenic for the transgen e array in this line of WT SOD1 and transgenic for mutant SOD1 constructs, develop disease much earlier than mice transgenic for only the mutant SOD1 genes (Jaarsma et al., 2000;Deng et al., 2006;Deng et al., 2008) Our study clarifies this discrepancy by demonstrating that the effects of the two different mouse lines of WT SOD1 on disease in mice expressing mutant SOD1 is influenced by the nature of the mutation and the level of WT SOD1 expression. We show that in WT and mutant (G37R or L126Z) human SOD1 double transgenic mice, there is acceleration in the development of hindlimb paralysis that is independent of th e strain background in which the WT SOD1 transgenic lines were originally raised. The more rapid disease is associated with the appearance of detergent -insoluble SOD1 aggregates that contain both, WT and mutant human SOD1 proteins at disease endstage. Furt her, an earlier age of hindlimb paralysis correlates with a higher dose of WT human SOD1 protein expressed. These data demonstrate a potential role for WT human SOD1 in SOD1associated ALS. Acceleration of Disease by WT and Mutant SOD1 Overexpression is In dependent of Strain Background. Previous studies of WT and mutant human SOD1 transgenic mice have shown that WT human SOD1 either accelerates or has no effect on disease onset, compared to mice that only overexpress a given human SOD1 mutation (Bruijn et al., 1998;Deng et al., 2006;Deng et al., 2008;Wang et al., 2009c) In our hands, double transgenic mice expressing mutant human SOD1 and either high (from Gurney WT mice) or low (from

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127 Wong L76 WT mice) levels of WT human SOD1 can induce an earlier disease onset of symptoms. However, while all mutant/WT mice with WT human SOD1 protein that derives from the Gurney mouse line (SJL hybrid and Cg congenic strains) present significa nt acceleration in the development of hindlimb paralysis, mutant/WT mice expressing a WT SOD1 protein that derives from L76 WT human SOD1 mice (from the line of Wong and colleagues) suffer a less severe effect (in PrPG37R/L76 WT mice) or no effect at all ( in L126Z/L76 WT mice) in accelerating onset of paralysis. Thus, we observed clear differences on the effect of WT SOD1 derived from two different lines, even when the WT protein comes from mice of equivalent strains (L76 and Cg WT). This finding demonstrat es that the strain background in which the Gurney and Wongs mouse lines were initially raised in does not account for the different outcomes. Rather, the different expression levels of WT human SOD1 from the different mouse lines may explain the stronger effect of the Gurney line vs. the Wong L76 line. Earlier Disease in Mutant/WT Mice is Associated With Aggregated SOD1. Mice expressing different human mutant SOD1 proteins are characterized by a significant accumulation of detergent -insoluble aggregates of mutant SOD1 at disease endstage (Karch et al., 2009) In all our doubly transgenic mutant/WT human SOD1 mice, hindlimb weakness and paralysis is accompanied by the accumulation o f detergent -insoluble species of SOD1 proteins. The overall aggregated protein levels in the different PrPG37R/WT double transgenic mice are more or less equivalent, and similar to the levels of PrPG37R protein in symptomatic mice. For L126Z/WT mice the ov erall amount of detergent -insoluble species were higher than in L126Z singly transgenic mice (see Figure 54), and WT and L126Z human SOD1 are present at similar levels in the P2 fraction. Thus, it appears that in L126Z/WT mice the presence of

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128 WT protein i ncreases the amount of protein that is detergent insoluble, with WT SOD1 protein becoming part of this aggregated fraction. However, in terms of aggregation propensity of L126Z/WT mice, only a small portion of total WT SOD1 protein becomes detergent -insoluble, while the totality of L126Z SOD1 protein that we able to detect by western blotting techniques remains detergent -insoluble. These data indicate that in L126Z/WT mice the ability of the mutant protein to aggregate is much higher than for WT SOD1 protei n, with possibly mutant SOD1 increasing the ability of WT SOD1 to misfold and aggregate. Since double transgenic mice reach endstage at earlier time points than singly transgenic mice, detergent -insoluble aggregated SOD1 species are also detected earlier i n double mutant/WT SOD1 mice. A possible explanation for the shorter lifespan of doubly transgenic mice is by acceleration on the formation of toxic species, which would translate into the appearance of aggregates at earlier times, as they reach the same aggregation propensity levels at endstage. In this case, WT human SOD1 contributes to this more rapid aggregation rates by being included into the aggregated fraction (see Figures 5 -4 and 5-5). Alternatively, rates of aggregation may not change in double tr ansgenic mice and WT SOD1 would only exert a role in disease initiation, thus moving disease development, and aggregate formation events, to earlier times. Still, whether the toxic effect of WT SOD1 in mutant SOD1 transgenic mice has something to do with t he co aggregation phenomenon remains uncertain. Co -aggregation of WT and Mutant SOD1 is Dependent on SOD1 Protein Levels For PrPG37R/WT and L126Z/WT mice, SOD1 aggregates at disease endstage contain WT and mutant human SOD1 proteins. Based on the western b lot data from L126Z/WT mice, it appears that only a small proportion of total levels of WT human SOD1 protein accumulates in the detergent insoluble SOD1 fraction, but constitutes

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129 protein of equal levels of WT and L126Z human SOD1 in the P2 fraction (see F igure 54). However, we cannot distinguish WT and mutant human SOD1 in PrPG37R/WT mice through western blot analysis. To determine whether insoluble aggregates of WT SOD1 are generated in the PrPG37R/WT SOD1 mice, we used FTMS analysis to demonstrate the presence of WT SOD1 in the P2 fractions. Due to uncertainties regarding the relative ability of FTMS analysis to detect the WT and G37R proteins, we are not able to use the FTMS data to establish the relative abundance of WT and G37R SOD1 in the insoluble aggregates of these mice. However, it is possible that aggregates in the PrPG37R/WT SOD1 mice contain more WT than mutant protein. Thus, the larger effect of WT SOD1 on P rPG37R mice might rely on a higher proportion of WT human SOD1 that can be incorporated into detergent -insoluble aggregated protein fraction. Figure 510. Hypothetical model on the effect of WT SOD1 on disease and aggregation in mice expressing a mutant SOD1 mutation. The asterisk (*) indicates a symptomatic m ice with no obvious symptoms of weakness or other abnormalities Paralysis and death Time Mutant SOD1 WT SOD1 WT + Mutant SOD1Asymptomatic* Weakness

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130 The Complexity of WT -Mutant Coaggregation In cell culture mutant/WT SOD1 co expression at 1:1 ratios translates into aggregation levels much lower than mutant alone (compare mutant aggregation propensity in co-transfection with that of mutant + GFP at 24 or 48 hours, see Figure 57). This data demonstrates that when WT human SOD1 is co expressed with mutant SOD1, the rates of mutant SOD1 aggregation are different from those of cells expressing only mutant human SOD1 proteins, indicating that WT human SOD1 is an important modulator of mutant SOD1 aggregation. At longer transfection intervals (48 hours) WT and mutant (G85R or G93A) human SOD1 proteins can be seen to coaggregate in cell culture (Prudencio et al., 2009a) while the aggregation propensity for other mutants is still reduced (see Figure 5-7). Thus, cell culture studies indicate that WT SOD1 protein does not readily co aggregate with mutant SOD1 proteins. A likely explanation on the delay of WT and mutant human SOD1 co aggregation in cell culture may reside on the ability of WT and mutant human SOD1 to interact with each other. G37R and WT SOD1 protein are likely to easy inter act which other, since previous reports demonstrate their ability to form active heretodimers of WT and G37R proteins (Borchelt et al., 1994) Thus, we explain the higher impact of WT human SOD1 protein in asymptomatic PrPG37R mice as a result of a better interaction between WT and G37R proteins. In the case of SOD1 truncation mutants, like L126Z, WT and mutant human SOD1 would be less likely to interact. Additionally, the very rapid turnover of this truncated mutant (hardly detected when soluble in detergent) would make harder for any interactions to occur. However, studies in cell culture and animal models expressing L126Z and WT human SOD1 proteins indicate that some kind of interactions take place between both proteins (Deng et al., 2006;Furukawa et al., 2006)

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131 Thus, higher levels of WT human SOD1 protein (that come from Gurney WT line) are necessary to observe an effect of WT SOD1 on disease in L126Z/WT transgenic mice. A hypothetical model on the effect of WT human SOD1 in disease and aggregation is represented in Figure 5-10. In such model we propose that aggregation occurs in 3 steps: 1) A nucleation phase, in which initial subunits of disulfide immature or misf olded SOD1 come together to form a core from which aggregation can initiate; 2) A growth phase, where detergent insoluble species start accumulating in the cells; and 3) A final phase, which coincides with onset of paralysis in mice and where cells are s aturated with detergent insoluble aggregates and may present other high molecular weight disulfide linked species. According to our model, aggregation of mutant SOD1 proteins (black line) occurs at a rate that varies depending on the SOD1 mutation expressed and that would reach final aggregation stages that coincided with paralysis. However, aggregation of WT SOD1 (blue line) occurs at much lower rates, with significant accumulation of detergent -insoluble species when expressed in cells for long periods of time and at very high levels. This late accumulation of WT SOD1 aggregates would then coincide with the appearance of subtle motor abnormalities. Finally, we propose that when co expression of WT and mutant SOD1 takes place (dotted red line), the nucleation phase of aggregation is increased, compared to mutant protein alone, due to a) longer time required for WT -mutant interactions to occur, b) a more rapid degradation of soluble WT -mutant proteins, c) a less likely chance of mutant aggregating units to com e together due to the existence of WT units, and/or d) nucleation event of WT mutant complexes occurs at slower rates due to the more difficult ability of WT to nucleate and aggregate. Then, once nucleation of WT and mutant SOD1 proteins

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132 occurs, the growth phase would be much faster than for mutant alone. This more rapid growth phase can be explained by either the fact that the presence of WT human SOD1 proteins exerts some kind of toxicity in mutant/WT SOD1 mice that translates into a more rapid accumulation of detergent insoluble aggregates. Or alternatively, the addition of high amounts of SOD1 protein, in this case WT SOD1, allows a more rapid growth phase of aggregation. Critical Levels of Reduced WT SOD1 Protein Can Initiate WT Aggregation. Previously, Jonsson and colleagues showed the ability of WT human SOD1 protein to aggregate in SJL WT mice (Jonsson et al., 2006a) Here, we used higher detergent concentration to determine the portion of SOD1 that is detergent -insoluble. Also, with our assay, we observed that WT SOD1 protein expressed from Gurney line is able to form aggregates at old ages; but not from WT protein expressed from the Wong line (see Figure 5 -9). Previous studies have shown t hat WT SOD1 in the apo, disulfide reduced form can aggregate in vitro (Chattopadhyay et al., 2008) Here, we determined that as much as 10% of the steady state levels of WT human SO D1 protein correspond to WT protein that is in a disulfide reduced state (see Figure 5-7). We have also shown that Gurney WT mouse line expresses about 30% more WT human SOD1 protein that the Wong line (see Figure 5 -1). Thus, the overall levels of disulfid e reduced WT SOD1 in the Gurney line are then higher than in the Wong mouse line. Since, the Wong L76 WT line does not appear to present the same ability to form significant levels of aggregated SOD1 protein at old ages, we may suggest that the overall protein levels of WT SOD1 protein required for aggregation are just below a required threshold. Additionally, it is possible that the required protein threshold could have something to do with the proportion of disulfide reduced SOD1 protein expressed. Overal l, it appears

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133 that in order for WT SOD1 to significantly aggregate, compared to mutant protein, WT SOD1 requires both higher protein expression levels and longer intervals. We also noted that Gurney WT mice (SJL and Cg WT), but not L76 WT Wong mice, presented some motor abnormalities as early as a year of age (data not shown). And in many instances animals died unexpectedly or the veterinary recommended euthanasia before we could age them past 15 months of age. However, none of the WT lines showed ALS -like symptoms in their lifespan, supporting previously published data (Gurney et al., 1994;Wong et al., 1995) Abnormalities in these mice at about 15-20 months of age have been described previously (Tu et al., 1996;Jaarsma et al., 2000;Jaarsma et al., 2008) These include neurofilament inclusions at about 135 days of age (Tu et al., 1996) ; and swollen mitochondria, vacuoles, as well as ubiquitin ated inclusions that appear as early as 280 or 490 days of age respectively (Jaarsma et al., 2000;Jaarsma et al., 2008) However, such abnormalities hav e not been described for the L76 WT line of SOD1 mice (Wong et al., 1995) Thus, the ability of the Gurney WT, but not L76 WT Wong line, to form detergent -insoluble aggregates of SOD1 protein may account for the motor abnormalities observed in this WT SOD1 line. Mechanisms of Toxicity Previous studies by Witan and colleagues have shown that WT/mutant SOD1 heterodimers induce higher toxicity that mutant homodimers in C. elegans whi le the aggregation levels were reduced (Witan et al., 2008) Additionally, they suggest that the role of WT human SOD1 on disease is through stabilization of mutant SOD1 as a soluble protein (Witan et al., 2008;Witan et al., 2009) However, our results on the L126Z truncation mutant indicate that in the presence of WT SOD1, at different levels, soluble L126Z protein remains still undetectable. Thus, the instability of this protein in its

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1 34 soluble state does not support h igher toxicity of mutant through stability of soluble state by WT SOD1. The overexpression of CCS in mutant SOD1 transgenic mice accelerates disease without the formation of SOD1 aggregates (Son et al., 2007;Proescher et al., 2008;Son et al., 2009) In this case it has been suggested that the ability of CCS to transport immature SOD1 into mitochondria translates into a more severe phenotype (Son et al., 2007;Proescher et al., 2008;Son et al., 2009) Additionally, increased mitochondrial toxicity and earlier disease onset due to CCS overexpression has only been observed in G37R and G93A SOD1 transgenic mice (Son et al., 2007;Son et al., 2009) The incorporation of CCS and SOD1 into mitochondria is thought to occur through a disulfide relay system (Ka wamata and Manfredi, 2008) which may explain why in the presence of CCS there is only enhanced toxicity of mutant SOD1 proteins that can form disulfide bonds (Son et al., 2009) An additional study demonstrates that CCS mutant SOD1 interactions can facilitate import of mutant SOD1 proteins into peroxisomes (Islinger et al., 2009) Thus, it is possible that the altered mutant SOD1 localization, due to CCS overexpression explains the enhanced toxicity in CCS/mutant SOD1 mice (Leitch et al., 2009) An interesting point of the CCS/mutant SOD1 studies is that co expression of CCS with ei ther L126Z or G86R mutant human SOD1 proteins in mice, does not change disease course (Son et al., 2009) However, the presence of WT human SOD1 that comes from Gurney WT line of mice induces an earlier onset of hindlimb paralysis in mice expressing either L126Z (Deng et al., 2006) or G85R (Wang et al., 2009c) SOD1 proteins. Additionally, the fact that WT SOD1 plays a role in modulating SOD1 aggregation, while this process is absent in CCS/SOD1 mice,

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135 indicates that the toxic effect exerted by WT human SOD1 protein occurs through different mechanisms than those that might take place in the presence of CCS. Still, it remains unclear whether mutant/WT SOD1 enhanced toxicity is related to any kind of mitochondrial abnormalities. It is known that a very small proportion of WT SOD1 is localized normally in the intermembrane space of mitochondria (Sturtz et al., 2001) Previous studies have found WT and mutant SOD1 accumulates in brain mitochondria, and mutant aggregates are found within this organelle (Vijayvergiya et al., 2005) Additionally, the presence of vacuoles, that in some cases have been shown to derive from mitochondria, have been found in transgenic animal models harboring G37R and G93A mutations (Dal Canto and Gurney, 1994;Wong et al., 1995) as well as in a high expressor WT Gurney line at very old ages (Jaarsma et al., 2000) However, mitochondrial abnormalities are not a common feature of ALS mouse models Additionally, G93A/WT and L126Z/WT transgenic mice created by Deng and colleagues appeared to present some dysfunctional mitochondrial, but no obvious vacuolar pathology was reported in those mice (Deng et al., 2006) Furthermore, previous studies imply that mutant SOD1 does not translocate into mitochondria but it associates with its cytoplasmic membrane (Liu et al., 2004) These data suggest that although some association of mutant SOD1 with mitochondria might normally take place, the toxic effect of WT SOD1 in mice expressing mutant SOD1 proteins does not involve an internalization of mutant SOD1 proteins into these organelles. Rather, alternative toxic mechanisms are more likely to explain mutant/WT SOD1 toxicity. In our studies, PrPG37R/WT mice develop disease at earlier times than PrPG37R homozygous mice (PrPG37R/PrPG37R). Thus, G37R/WT SOD1 complexes appear to

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136 induce a higher toxicity than those composed of G37R/G37R, with G37R/WT aggregates appearing at earlier ages but always concomitant with paralysis. I n the case of L126Z/WT mice, we observed that aggregates of L126Z are more abundant in the presence of WT SOD1 protein. Additionally, in mutant/WT SOD1 mice, WT becomes part of detergent -insoluble aggregates at end stage, suggesting that mutant/WT co expres sion in SOD1 mice increases the levels of misfolded WT SOD1 protein. Thus, we propose that the presence of WT SOD1 protein may stabilize aggregates of SOD1 proteins by some kind of protein -protein interactions. Additionally, co aggregation of WT and mutant proteins is not likely to require normal dimeric interactions, since the L126Z truncation mutant lack amino acids involved in the dimer interface. The presence of detergent -insoluble aggregated species of SOD1 at disease endstage is not likely to explain events that occur earlier that lead to a more rapid onset of paralysis induced by the presence of WT SOD1 protein. However, still remains a possibility that some other type of aggregated species (oligomers, undetectable multimers, etc.) is responsible for disease initiation. Here, we demonstrate that different levels of WT SOD1 protein have different effects on accelerating onset of symptoms in transgenic mice that express a specific SOD1 mutation. Additionally, stronger effects of WT SOD1 in mutant SOD1 to xicity appear to take place with mutants that present more similarities with WT protein. Co aggregation of WT and mutant SOD1 indicates possible protein -protein interactions. Thus, further studies directed to elucidate what kind of mutant WT interactions occur may give insight in the enhanced toxicity provided by the WT SOD1 protein.

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137 CHAPTER 6 CHARACTERIZATION OF DETERGENT -INSOLUBLE AGGREGATES OF MUTANT SOD1 IN CELL CULTURE Introduction Animals models and human ALS patients expressing mutant SOD1 proteins are characterized by the presence of protein inclusions, that in some cases have been identified to contain SOD1 (Shibata et al., 1996b;Kato et al., 2000a;Kato et al., 2001b) However the ability to detect SOD1 -positive inclusions in transgenic mice has not always been easy (see Pathology in SOD1associated ALS and Rodent Models of the Disease in Chapter 1). In cell culture, examples in which protein aggregates can be visualized utilize techniques t hat modify the normal cell homeostasis through the use of proteasome inhibitors (Johnston et al., 2000) or by inducing ER stress (Yamagishi et al., 2007) An alternative t echnique that is increasingly being used to study SOD1 inclusions is by tagg ing SOD1 with fluorescent proteins (Matsumoto et al., 2005;Witan et al., 2008) The drawback of this technique is that either C or N termin al tags localize in the dimer interface of SOD1 thus any kind of tag can possibly affect the normal folding or interactions patterns of SOD1. The presence of detergent -insoluble SOD1 aggregates is part of the pathology of SOD1 associated ALS. T he best way we have to visualize such aggregated species is t hrough a biochemical assay and w estern blotting ( see Chapters 2 to 5). However, i t is unclear whether these species are the same as the inclusions observed in cell culture or animal models Thus, to further stu dy this particular type of mutant SOD1 aggregates we have used fluorescence imaging techniques in cultured cells to a) localize them subcellularly and determine their morphology, and b) determine the implications of the tag in SOD1 aggregate and inclusion formation. These studies provide insight on the

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138 location and morphology of mutant SOD1 aggregates, which could give us clues about their pathogenic role. Materials and Methods A list of materials used can be found in Appendix B. Methodology used for the work presented in this chapter is described in Chapter 6 Methods of Appendix C. Results In order to analyze the morphology and location of mutant SOD1 aggregates, we used our previously described HEK293FT cell culture model to express different SOD1 prot eins. We chose A4V and D101N SOD1 mutant proteins for our studies because their different aggregation propensities A4V high and D101N low (see Chapter 3 for values in aggregation propensity of different mutant SOD1 proteins). The use of these two mutants may help us to understand different stages of inclusion formation. We transiently transfected HEK293FT cells with untagged WT, A4V, or D101N human SOD1 constructs for 24 h ours, and performed immunofluorescence SOD1 staining as described in Methods Cells expressing WT, A4V or D101N SOD1 proteins present uniform SOD1 staining and no particular SOD1 protein inclusion appears to form spontaneously ( Figure 6 -1 ). Thus, in natural conditions (without any additional treatment that would modify protein homeostasis ) we do not observe any obvi ous protein inclusion of SOD1 A possible explanation for our inability to detect mutant SOD1 inclusions resides in the possibility that the amount of endogenous WT SOD1, produced by the human cell line used, may increase the ov erall SOD1 staining and interfere with the visualization of protein inclusions. Thus, to eliminate the background generated by the amount of endogenous WT SOD1 protein expressed by the use of a human cell line (HEK293FT

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139 cells) we performed transient transfections in a mouse fibroblast cell line (TK negative cells also commonly known as L cells ) and used antibodies specific to human SOD1 to detect transfected protein. The t ransfection efficiency of our expression plasmids in this cell type is already quite low and in order to observe detergent insoluble SOD1 aggregates biochemically we need to express such plasmid for at least 48 h ours (data not shown) At 48 h ours mouse TK negative cells expressing WT and A4V SOD1 proteins showed a similar pattern of fluorescence than in HEK293FT cells, indicating that human SOD1 proteins in TK negative cells remain in a protein conformation that does not lead to inclusion formation (Figure 6 2 ). Thus, the elimination of endogenous SOD1 signal in mouse TK negative is not sufficient to allow visualization of inclusions formed by mutant human SOD1. Another possible explanation regarding inclusion visualization may be explained by the amount of detergent -soluble SOD1 protein vs. the amount of detergent insoluble aggregat ed SOD1 protein. Indeed the amount of detergent -soluble SOD1 protein expressed in HEK293FT cells is very high. In transient 24 hour transfections, the levels of detergent -soluble SOD1 protein in HEK293FT cells expressing mutant SOD1 is about 2 (A4V) to 10 (D101N) times higher than the levels of detergent -insoluble aggregated proteins (Figure 63). Thus, it seems reasonable to explain our inability to observe inclusions that might derive from detergent -insoluble SOD1 protein based on the fact that the amount of such aggregated species is too low compared to the overall levels of soluble SOD1 protein that is normally expressed in transfected cell lines.

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140 Figure 61. HEK293FT cells expressing SOD1 proteins do not form cellular inclusions. A-I) Cells were cultured in glass coverslips previously coated with 0.5 mg/ml poly L and transfected with WT ( A-C ), A4V (D -F ), or D101N ( G -H ) SOD1 for 24 h ours Fixed cells were stained with h uman SOD1 antibody overnight A secondary fluorescent (594 nm) antibody was used to visualize SOD1 staining. Co-staining with 4' 6 -diamidino 2 -phenylindole ( DAPI) was performed together with secondary antibody incubation Pictures were taken with either a 100x immersion oil objective, bar 50 m ( A -F); or a 40x obj ective, bar 20 m ( G -H ). Figure 6 2. TK n egative cells transfected with SOD1 constructs for 48 h ours and stained for h uman SOD1 as explained in Fig ure 61. Pictures were taken with a 40x objective. DAPI A4V SOD1 MERGE DAPI WT SOD1 MERGE DAPI D101N SOD1 MERGE A D G B E H C F I WT SOD1 A4V SOD1 UT A B C

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141 Figure 6-3. HEK293FT transfected cells express higher levels of detergent-soluble SOD1 than detergent-insoluble SOD1 aggregated protein. Band intensities were calculated from western blots of P2 (black bars) and S1 (white bars) fractions, and adjusted for the amount of total protein loaded on the SDSPAGE gels. The data represent ed here derives from at least five independent transfection experiments. Significant diffe rences exist between the levels of P2 vs. S1 fractions for each SOD1 pr otein expressed; paired student t -test: #p 0.005. In order to eliminate solubl e SOD1 protein, we chose a technique that consists in treating HEK293FT cells with sa ponin, a mild detergent that open pores on the cellular membranes (Francis et al., 2002). This mild tr eatment does not kill cells right away and it is intended to allow diffusion of soluble prot eins out of the cell. Treatment of cells with such detergent efficiently eliminated part of SOD1 protein that can be visualized through normal immunofluorescence staining techni ques, with most of the SOD1 protein concentrated in, and maybe also around, the nuc lei. However, we did not observe any obvious immunoreactive inclusion (Figure 64). An exception was found in a single cell transfected with A4V SOD1, which presented a few small dot-like inclusions (Figure 64E, arrow heads). We also performed the mild detergent treatment in TK negative cells, observing similar pattern of staining to that found in HEK293 FT cells treated with saponin (Figure 6-5). However, in this case we observed more obvious punctuate structures. This might be due to the slight ly different detergent treatment (digitonin WT A4V D101N 0 1.0108 2.0108 3.0108 4.0108 5.0108P2 S1 Band intensity levels of SOD1 protein (A.U.)# # #

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142 instead of saponin), but t he punctuate structures were not diff erent between cells expressing WT and mutant human SOD1 (Figure 6 5 ). Thus, we believ e that the punctuate staining does not correspond to detergent -insoluble aggregates or inclusions of mutant SOD1 proteins Figure 64. Saponin eliminates most of the cytosolic SOD1 protein, but does not uncover the presence of SOD1 positive inclusions AI) HEK293FT cells were transfected and stained as explained in Figure 6-1. Saponin treatment was performed prior fixation for 30 minutes at a concentration of 0.01% in 1x PBS. Pictures of WT were taken with a 40x objective (A -C), while A4V (D -F) and D10 1N (G -I) pictures were taken with an immersion oil 100x objective. All bars represent 20 m. A D G B E H C F I DAPI A4V SOD1 MERGE DAPI WT SOD1 MERGE DAPI D101N SOD1 MERGE

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143 Figure 6 5 Digitonin treatment in TK negative cells show a dot -like pattern of SOD1 that is not exclusive of cells expressing mutant SOD1 proteins. A F) TK ne gative cells transfected for 48 h ours treated with 0.01% digitonin in 1x PBS, and stained for human SOD1. Transfection, detergent treatment and staining of TK negative cells wa s performed as described for HEK293FT cells in Figure 6 -4 Pictures correspondi ng to WT ( A-C ) and A4V (D F ) were taken using an immersion oil 100x objective. In conclusion, the use of mild detergent treatments on cell s does not allow us to detect aggregates of mutant SOD1. Additionally, t his data provides evidence that demonstrate that detergent insoluble aggregates of mutant SOD1, which we detect through western blot analysis, may be of different nature t han big cellular inclusions. Alternatively, it is possible that such aggregated structures may be washed out by the detergent method applied to the cells. Thus, in order to quantify the efficacy of mild detergents (saponin and digitonin) in eliminating detergent -soluble SOD1 from cultured cells (and leaving aggregates inside), we performed a 30 minute incubation of live cells with either saponin or digitonin prior to harvest of the cells; and then we performed analysis of detergent -solubility as previously desc ribed (Karch and Borchelt, DAPI A4V SOD1 MERGE DAPI WT SOD1 MERGE A D B E C F

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144 2008;Prudencio et al., 2009a; Prudencio et al., 2009b) In both saponin and digitionin treatments, a significant amount of SOD1 protein came out of the cell and into the cell culture media (Figure 6-6). In the case of cells transfected with WT SOD1, it appears that almost the totali ty of the protein (in S1) was released into the media (Figures 6-6A to 6 -6C). However, the overall levels of WT protein expressed are much lower than those of the mutants (Figures 66C to 6-6E), indicating that the apparent more effective treatment on cell s expressing WT SOD1 is due to the overall lower levels of protein expressed. In each case (WT, A4V, or G93A), the levels of SOD1 released are very similar but not high enough to get rid of all soluble SOD1 protein from the cells. Thus, mild detergent treatments were only partially effective in removing the non aggregated forms of mutant SOD1 proteins. However, saponin or digitonin treated cells retained the detergent -insoluble aggregated fraction of mutant SOD1. Thus, the SOD1 immunostaining of saponin or digitonin treated cells suggests that detergent -insoluble SOD1 aggregates of mutant SOD1 may be either smaller than visible cellular inclusions unable to be detected with our current antibodies, or too diffuse to be detected by the described immunocytoche mistry techniques. In view of our observations, we were unable to evaluate the morphology and location of mutant SOD1 aggregates through simple immunofluorescence techniques that do not alter cellular homeostasis or that covalent modify the SOD1 expressed protein. Thus, we decided to study SOD1 inclusions by a protein tagging system We chose SOD1::YFP ( SOD1 fused to YFP) proteins because they are increasingly being u sed to study aggregation and toxicity in C. elegans model (Wang et al., 2009a;Gidalevitz et al., 2009) and in the recently created ALS mouse model expressing

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145 SOD1::YFP (Wang et al., 2009b) The fluorescent tag in these models does not affect SOD1 activity or folding of the YFP tag (Wang et al., 2009a;Gidalevitz et al., 2009) Figure 66. Similar effects of saponin and digitonin on eliminating soluble SOD1 protein from HEK293FT cells expressing WT or highly aggregating SOD1 mutant proteins. A, B) Immunoblots of HEK293FT cells transfec ted with the indicated SOD1 constructs for 48 hours. Prior harvest, cells were incubated 30 minutes in 0.01% detergent, 1x PBS. Then the harvested cell pellets were analyzed by detergent extraction and centrifugation assays. The amount of SOD1 protein rele ased into the cell media was also evaluated. C -E) Quantification of SOD1 protein found in the extracellular space (Media), P2 and S1 fractions of cells untreated or treated with saponin (Sap.) or digitonin (Dig.), and expressing WT (C), A4V (D), or G93A (E). Symbols over bars indicate differences from corresponding non-treated control, or as indicated. Unpaired t -tests: p #p Transient transfections for 24 h ours of WT::YFP (WT SOD1 tagged with YFP) in HEK293FT showed uniform distributions of fluorescence, similar to cells expressing non-tagged WT SOD1 proteins ( Figure 67B ). In this case WT::YFP did not seem to S1 P2 Saponin media S1 P2 Digitonin media G93A G93A + Sap. G93A + Dig. 0.0 5.01007 1.01008 1.51008Media P2 S1 Band intensity levels of SOD1 protein (A.U.) WT WT + Sap. WT + Dig. 0 5.0107 1.0108 1.5108Media P2 S1 Band intensity levels of SOD1 protein (A.U.) A4V A4V + Sap. A4V + Dig. 0.0 5.01007 1.01008 1.51008Media P2 S1 Band intensity levels of SOD1 protein (A.U.) *#* # # *#A B C E D

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146 localize as much protein in the nuclei, being mostly located in the cytosol. We also created a construct to expressed YFP protein by itself. We noted that in this case we can observe a very strong presence of YFP protein in the nuclei (Figure 67A), while all our tagged YFP SOD1 proteins (WT and mutants) are mainly located in the cytosol (Figures 6 7B to 6-7L). This outcome can just sim ply be explained by the bigger size of the fusion protein (about 50 kDa) that impedes SOD1, when tagged, to get easily into the nucleus. In general, cells transfected with high to slow aggregating SOD1::YFP mutants presented from small and many to fewer and larger SOD1 inclusions (Figures 6 -7C to 67L). Closer observation of inclusion formation in high vs. slow aggregating SOD1 mutants suggest possible differences (see Chapters 2 and 3 for more information about selected SOD1 mutants). While YFP or SOD::WT proteins are uniformly spread in the cells, cells expressing the highly aggregating m utants A4V::YFP (Figure 67C), G37R::YFP (Figure 67D), G85R::YFP (Figure 67F), and MDG6FS111Y::YFP (Figure 6 -7L) present multiple small inclusions. However, the slowest aggregating mutants H80R::YFP (Figure 67E), D101N::YFP (Figure 67G), D125H::YFP (Figure 67H), 7I), and S134N::YFP (Figure 6-7J) expressed in cells appeared to be contained in larger discrete areas surrounding nuclei. Interestingly, SODMD protein, which is unable to form detectable levels of detergent -insoluble SOD1 aggregates in cells (see Chapter 2), when tagged with YFP (MD::YFP) produces a fluorescent fusion protein that can be sparsely found concentrating in inclusion structures in different areas of the cell (Figure 67K). Note that the SODMD variant that is modified to increase its aggregation propensity, by altering residues at positions 6

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147 and 111 to encode ALS mutations (MDG6FS111Y, see Chapter 2) produces larger and more obv ious fluorescent inclusions (Figure 6-7L). Figure 67 Mutant SOD::YFP proteins present variable s ize and number of inclusions A-L) HEK293FT c ells were transfected in poly L -lysine coated glass coverslips for 24 hours as described in Figure 6 1. Cells were fixed and observed under a fluorescence microscope. All pictures were taken using a 40x objective, bars10 (G), 20 (A, F, K) or 50 (C -E, H -J, L) m. It is possible that the YFP tag may alter the normal location of mutant SOD1 aggregates. However, the impossibility to determine morphology of inclusions of untagged mutant SOD1 in cell culture by simple immunofluorescence techniques makes it a more difficult task Additionally, the large size of the SOD1::YFP inclusions suggested us that they are unlikely localize in a particular organelle. Thus, we decided to further explore the ability of SOD1::YFP proteins to form inclusions and whether inclusion formation relates to the amount of detergent insoluble aggregates they can accumulate. WT::YFP A4V::YFP G37R::YFP H80R::YFP D101N::YFP D125H::YFP E133 S134N::YFP MDG6FS111Y::YFP MD::YFP B E I C G J D H L K G85R::YFP YFP A F

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148 While analyzing SOD1 ::YFP inclusions we noticed that, in cells expressing WT or the nonaggregating SODMD variant fused to YFP certain cells appear to accumulate SOD1 ::YFP protein similar to slow aggregating mutants (see Figure 6 -7), while they are known not to be able to form detergent -insoluble aggregated species in cell culture. Thus, we decided to express WT::YFP, D101N::YFP (untagged D101N does not form detergent -insol uble aggregates at 24 hours, but do at 48 hours), and MD::YFP constructs in HEK293FT cells for longer transfection intervals (48 hours) to determine whether inclusions may become more prominent. Effectively, we were able to see increased accumulation of SO D1::YFP proteins in all transfected cells ( Figure 6 8 ). This data confirms our suspicion that YFP tagged SOD1 proteins present a higher tendency to misfold and aggregate that is not common of untagged SOD1 protein. Figure 68 Tagged WT ::YFP and MD ::YFP variants are able to form inclusions, similarly to slow aggregating D101N ::YFP proteins when expressed in cells f or 48 hours. A -C) HEK293FT cells were tran s fected for 48 hours with WT::YFP ( A), D101N::YFP (B), or MD::YFP (C ), and visualized as explained i n Figure 6 -7 Bars represent either 10 ( A, C ) or 50 ( B) m, in pictures taken using a 40x objective. We also explored the ability of WT::YFP to form inclusions in other cell types after a 48 h ours transfection. For example, in NIH3T3 cells expressing WT:: YFP no obvious inclusion was found (Figure 6 -9). Additionally, the number of NIH3T3 cells containing mutant SOD1::YFP inclusions was lower (Figure 69), suggesting that the ability of WT:: YFP MD::YFP D101N::YFP A B C

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149 WT::YFP to form inclusions is time and concentration dependent, as the protein expression levels of our selected plasmids are lower in NIH3T3 than in HEK293FT cells. Figure 6 9 NIH3T3 cells express less SOD1::YFP inclusions than HEK293FT after 48 h our transfections. A -C) NIH3T3 cells expressing WT ::YFP (A), A4V::YFP (B), or MDG6FS111Y::YFP (C) for 48 hours. Inclusion visualization was performed as described in Figure 6 -7 Bars represent 50 m, in pictures taken using a 40x objective. Furthermore, we briefly evaluated the ability of other fluorescent tags in inducing mutan t SOD1 inclusion formation. We made WT, A4V and D101N SOD1::RFP variants (RFP fused) and tested them in cell culture. At 24 hours the inclusion patterns of A4V::RFP and D101N::RFP (Figures 6 -10B and 6-10C) are very similar to A4V::YFP and D101N::YFP, respectively. H owever we detected WT::R FP inclusions that resemble those in mutant SOD1::RFP (Figure 6 10A ). Figure 6 10. WT::RFP proteins form inclusions similar to those formed by mutant SOD1::RFP in HEK293FT cells after 24 hour transfections. A C) HEK29 3FT cells expressing WT ::RFP (A), A4V::RFP (B), or MDG6FS111Y::RFP (C) for 24 hours. Inclusion visualization was performed as described in Figure 67 Bars represent 2 0 m, in pictures taken using a 40x objective. WT:: YFP A4V::YFP MDG6FS111Y::YFP A B C A4V::RFP WT::RFP D101N::RFP A B C

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150 This outcome is similar to a recent ly published study that reported the ability of WT SOD1 to for m inclusions when fused to DsRed2 fluorescent protein (Witan et al., 2009) However, it is unclear why this particular tag can induce WT SOD1 to form inclusions. In order to determine the relationship between the inclusion structures formed by mutant SOD1::YFP fusion proteins and the detergent -insoluble aggregates formed by untagged mutant SOD1, we examined the detergent solubility of SOD1:: YFP fusion proteins All SOD1::YFP fusion proteins produce some level of detergent insoluble protein, including the WT SOD1::YFP protein (Figure 6-11), which is normally virtually completely soluble without the YFP tag (see Chapters 2 to 5). However, cells transfected with mutant SOD1::YFP fusion proteins accumulated more detergent insoluble SOD1 protein than WT::YFP (Figure 611). Additionally, the slow aggregating SOD1 mutants D101N and S134N are known to form detectable levels of detergent insoluble SOD1 protein at transfection intervals of at least 48 hour s (Prudencio et al., 2009b) Here, the D101N::YFP and S134N::YFP protein significantly aggregate, now not different from highly aggregating mutants A4V::YFP or G85R::YFP (Figure 6-11). Similarly, SODMD is able to aggregate when fused to YFP protein (Figure 6-11, see inclusions in Figure 68). This data confirms that inclusion formation in HEK293FT cells expressing SOD1::YFP proteins correlates with a higher amount of detergent insoluble SOD1 protein. We have pre viously shown that untagged WT can modulate aggregation of untagged mutant SOD1 proteins (see Chapters 4 and 5). To determine whether the YFP tag in SOD1 interferes with other SOD1 properties, we also examined the effect of WT::YFP and WT SOD1 proteins in modulating aggregation of SOD1 tagged and

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151 untagged A4V and G85R proteins for 24 and 48 hours (Figures 612 and 6 -13). At 48 h ours after transfection, th e differences between WT and WT ::YFP aggregation propensit ies are more obvious, while WT SOD1 (untagged) does not significantly aggregate ( Figure 6-1 2 ). This data suggests that the ability of WT::YFP to form detergent -insoluble SOD1 aggregates may alter its ability to modulate mutant SOD1 aggregation. Figure 61 1 All HEK293FT cells expressing a SOD1::YFP variant contain detergent insoluble aggregates. A) Immunoblot of P2 and S1 protein fractions of cells expressing SOD1::YFP proteins for 24 hours. The SOD1::YFP protein runs at a size corresponding to 50 kDa (filled arrowhead), while endogenous WT SOD1 mon omer runs at 16 kDa (open arrowhead), like untagged SOD1. B) Quantification of the aggregation propensity (P2/S1) of cells expressing SOD1::YFP proteins. Paired student t -tests were performed to compare the aggregation propensity of WT::YFP or mutant SOD1: :YFP with that of untransfected cells: p #p Similarly, the aggregation propensity of tagged A4V::YFP is higher than that of untagged A4V, even at 48 hours (Figure 6-12). While in untagged WT and A4V co UT WT::YFP A4V::YFP G37R::YFP G85R::YFP D101N::YFP S134N::YFP MD::YFP MDG6FS111Y::YFP 0 5 10 15 20 25 30Relative aggregation propensity of SOD1::YFP proteins (A.U.) S1 P2 * *# #A B

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152 transfections A4V aggregation is red uced to WT levels at 24 hours, WT SOD1 when co transfected for 24 hours with A4V::YFP does not seem to slow the ability of A4V::YFP to aggregate (Figures 612A and 6 -12B). A similar outcome is observed at 48 hour co transfection of A4V::YFP with untagged W T, with the only difference of the incorporation of WT SOD1 into the aggregate (Figures 612C and 612D). On the other hand, untagged WT is able to slow aggregation of WT::YFP tagged variant at 24 and 48 hours (Figures 6 12A to 612D). In the case of modul ation of aggregation by tagged variant WT::YFP, the outcome is somewhat different. At 24 hour co -transfections, WT::YFP aggregation is markedly increased in the presence of untagged A4V (Figures 612A and 6 -12B), while the aggregation propensity of WT::YFP at 48 hours does not differ from WT::YFP when co expressed with untagged A4V at 48 hours (Figures 6-12C and 612D). Additionally, the levels of aggregated A4V in the A4V + WT::YFP 48 hour co transfection are the same as A4V SOD1 transfected alone for 48 h ours (Figures 612B and 6 -12D). Similar outcomes are observed when we performed the same experiments using the G85R tagged and untagged variants (Figure 6-13). In this case G85R::YFP and G85R SOD1 aggregation is reduced at 24 hours when they are coexpres sed with either untagged or tagged WT SOD1, respectively (Figures 6-13A and 613C). At the 48 hour transfection interval, the aggregation propensity of G85R::YFP, when co expressed with WT SOD1, appear slightly reduced compared to singly transfected G85R:: YFP (Figures 6 -13B and 6-13D).Moreover, the levels of aggregated WT::YFP and G85R at the 48 hour co transfection represent the sum of the aggregation capability of each individual mutants (Figure 6 13D).

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153 Figure 6-12. Tagged and untagged SOD1 protein co-expressi ons determine different ability of tagged SOD1 to modulat e aggregation of A4V SOD1. A, B) Immunoblots of P2 and S1 fractions of HEK293FT cells co-transfected with the indicated SOD1 constructs for 24 (A) or 48 (B) hours and blotted with an antibody that recognizes mouse and human SOD1. Tagged SOD1::YFP proteins run on SDS-PAGE at a level corresponding to 50 kDa, while untagged SOD1 variants run at about 16 kD a. C, D) Quantif ication of the aggregation propensities (P2/ S1) of tagged and untagged pr oteins from singly or doubly transfected cells. Note that aggregation propensity values have been normalized to the aggregation pr opensity of untagged A4V at 24 hours (set to 1). Additionally, the ba ckground generated by endogenous WT SOD1 has been subtracted from the value of band intensity for each untagged variant. S1 P2 50 kDa, YFP tagged 16 kDa, non tagged SOD1 50 kDa, YFP tagged 16 kDa, non tagged SOD1 24 hours 48 hours W T WT::YFP A 4V A4V:: YFP W T + WT::YFP WT + A4V::YFP WT: : Y F P + A 4V 0 5 10 15 20 25Relative aggregation propensity of SOD1 proteins (A.U.)A C B D W T WT::YFP A 4V A4V::YFP W T + W T: :Y F P WT + A 4V ::YFP WT::YF P + A4V 0 5 10 15 20 25WT::YFP A4V::YFP WT A4V Relative aggregation propensity of SOD1 proteins (A.U.)

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154 Figure 6-13. Tagged and untagged SOD1 protein co-expressi ons determine different ability of tagged protein to modulat e aggregation of G85R SOD1. A, B) Immunoblots of P2 and S1 fractions of HEK293FT cells co-transfected with the indicated SOD1 constructs for 24 (A) or 48 (B) hours and blotted with an antibody that recognized mouse and human SOD1 protein. Tagged SOD1::YFP proteins run on SDS-PAGE at a level corresponding to 50 kDa, while untagged SOD1 variants run at about 16 kDa. C, D) Quantification of the aggregation propensities (P2/S1) of tagged and untagged proteins from singly or doubly transfected cells. Note that aggregation pr opensity values have been normalized to the aggregation pr opensity of untagged A4V at 24 hours (set to 1). Additionally, the background generated by endogenous WT SOD1 has been subtracted from the value of band intensity for each untagged variant S1 P250 kDa, YFP tagged 16 kDa, non tagged SOD1 50 kDa, YFP tagged 16 kDa, non tagged SOD1 24 hours 48 hours W T W T :: Y FP G8 5 R G 85 R :: Y F P WT + G85R::YFP W T : :Y F P + G 8 5 R 0 2 4 6 8 10 12Relative aggregation propensity of SOD1 proteins (A.U.) A C B D WT WT : : Y FP G 85R G85R : :YFP WT + G85R::YFP WT : : Y FP + G 8 5 R 0 2 4 6 8 10 12WT::YFP G85R::YFP WT G85R Relative aggregation propensity of SOD1 proteins (A.U.)

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155 Overall, it appe ars that the delay in aggregate formation in some of the untagged and tagged SOD1 protein co-transfections is just temporary, as such effect in some cases is not present at 48 hour after co transfection. Additionally, the length of time that this interacti on may occur seems to be dependent on the similarity of the proteins coexpressed. For example, WT + WT::YFP SOD1 coexpression delays aggregate formation of WT::YFP even at the 48 hour interval (Figure 612), while little effect is observed with the G85R SOD1 mutant (Figure 613) and none with the A4V SOD1 mutant (Figure 6-12) proteins. These results differ from the outcome observed in untagged SOD1 proteins co transfections, in which the effect of WT SOD1 in slowing A4V SOD1 aggregation was stronger than for the G85R SOD1 mutant (see Chapter 4). Thus, this data indicates that it is possible that the YFP tag may interfere with WT and mutant SOD1 interactions, which could be the explanation for such differences. Next we sought to investigate how the co expre ssion of tagged and untagged SOD1 proteins affects the process of inclusion formation. Analysis of cells co transfected with A4V::YFP or G85R::YFP and untagged WT SOD1 (Figures 6-14B and 6 -14E) were still able to form visible inclusions at 24 hours, like i n singly transfected cells with A4V::YFP or G85R::YFP (Figures 6-14A and 6-14D). However, the number of inclusions per cell appeared to be lower, and such inclusions were of bigger calibers than in cells expressing the SOD1::YFP mutation alone (data not ri gorously quantified). The effect of WT SOD1 on reducing the number of inclusions formed by G85R::YFP protein was more pronounced (Figure 6-14E), which correlates to the effect of WT SOD1 on the formation of detergent insoluble SOD1 aggregates of mutant SOD 1::YFP (see Figure 613). Interestingly, untagged mutant proteins (A4V or G85R) did not affect

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156 the ability of WT::YFP protein to form inclusions (Figures 614C and 6-14F). Additionally, co expression of A4V with A4V::YFP does not alter the ability of A4V:: YFP to form inclusions (Figure 615). Thus, mixtures of untagged and tagged mutant SOD1 proteins can produce fluorescent inclusions, but mixtures of untagged mutant with tagged WT SOD1 do not. Figure 614 WT SOD1 affects inclusion formation in mutant SOD1::YFP proteins, but not mutant SOD1 on WT::YFP proteins. A -F) HEK293FT cells expressing for 24 hours: A4V::YFP (A) or G85R::YFP (D) alone, or with WT SOD1 (B and E); or WT::YFP with either untagged A4V (C) or G85R (F) SOD1. Inclusion visualization was performed as described in Figure 67 Bars represent 2 0 m, in pictures taken using a 40x objective. Figure 615 Untagged A4V SOD1 does not alter inclusion formation in A4V::YFP expressing cells. A, B) HEK293FT cells expressing A4V::YFP alone (A) or w ith untagged A4V SOD1 (B). Inclusion visualization was performed as described in Figure 67 Bars represent 2 0 m, in pictures taken using a 40x objective. A4V::YFP A4V::YFP + WT A4V + WT::YFP G85R::YFP G85R::YFP + WT G85R + WT::YFP A B C D E F A4V + A4V::YFP A4V::YFP A B

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157 To further understand whether tagged SOD1 proteins can template inclusion formation of WT::YFP, we took advantage of our WT::RFP construct which can form visible inclusions. Co transfections of WT::YFP and WT::RFP constructs suggest the ability of WT::RFP inclusion formation capability to interact with WT::YFP and induce inclusion formation containing both of the tagged WT variants (Figures 6-16A to 6-15C). Similarly, cells co -transfected with A4V::YFP and A4V::RFP contain inclusions expressing both proteins (Figures 6 -16D to 6 -16F). This data suggest that the fluorescent tags may not interfere with SOD1 proteinprotein interactions. Figure 616 WT::RFP can induce inclusion formation of WT::YFP. A F) HEK293FT cells co transfected with either WT::YFP + WT::RFP (A -C) or A4V::YFP + A4V::RFP (D -F) for 24 hours. Cells were fixed and visualized under a spinning disc confocal microscope. Pictures were taken using a 60x water immersion objective, bars 10 m. Squared areas in C and F are double amplification of the area in the smaller square to show inclusions in which the core contains SOD1::YFP and SOD1::RFP, surrounded by SOD1::RFP only. White arrowheads indicate inclusions expressing just SOD1::RFP, while open arrowheads indicate inclusions expressing just SO D1::YFP. WT::YFP WT::RFP MERGE A4V::YFP A4V::RFP MERGEA B C D E F 2x 2x

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158 In order to determine whether co -localization of SOD1::YFP and SOD::RFP proteins require the YFP and RFP tags, or whether involve possible SOD1 -SOD1 interactions, we performed co transfections of YFP (no SOD1) and either WT::RFP or A4V::RFP (Figur e 6-17 ). Results from these transfections suggest that YFP does not likely interact with either WT::RFP (Figures 6 17A to 6-17C) or A4V::RFP (Figures 6 17D to 617E) to co localize within inclusions. Figure 617 YFP does not alter inclusion formation ability of A4V::RFP or WT::RFP. A F) HEK293FT cells co -transfected with YFP and either A4V::RFP (A C) or WT::RFP (D -F) for 24 hours. Cells were fixed and visualized under a spinning disc confocal microscope. Pictures were taken using a 60x water immersion o bjective, bars 10 m. Additionally, we performed co transfections of WT::RFP + A4V::YFP and WT::YFP + A4V::RFP to investigate interactions between WT and mutant SOD1 in the ability to form inclusions containing WT and mutant SOD1 (Figure 618). In WT::RFP + A4V::YFP co transfections, we observed inclusions of both WT and mutant SOD1 that did not co localize, but that in some cases A4V::YFP surrounded WT::RFP inclusions (Figures 6YFP A4V::RFP MERGE YFP WT::RFP MERGEA B C D E F

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159 18A to 618C). In the other hand, WT::YFP + A4V::RFP showed a very different o utcome. While in some cells we observed diffuse fluorescence of WT::YFP and A4V::RFP that overlapped completely (Figures 618D to 618F), A4V::RFP inclusions appeared to co localize with more condensed areas of WT::YFP (Figures 6-18G to 618I). However, WT ::YFP did not seem to form a lot of inclusions at the 24 hour co transfection interval. This data suggests that SOD1 inclusion formation is favored when the proteins involved are of the same protein sequence, that is, just WT or just a certain type of SOD1 mutation. Figure 618 WT and mutant SOD1 proteins do not easily form hybrid inclusions but both proteins may interact at the soluble level. A -I) HEK293FT cells co transfected with A4V::YFP + WT::RFP (A -C) or WT::YFP + A4V::RFP (D -I) for 24 hours. Pict ures were taken with a 60x water immersion objective in a spinning disc confocal microscope, bars 10 m. A4V::YFP WT::RFP MERGE WT::YFP A4V::RFP MERGEA B C D E F G H I

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160 Discussion Mutant SOD1 proteins associated with ALS are characterized by their insolubility in non-ionic detergents. Moreover, the formation of such sp ecies is a characteristic pathological feature in mouse models for SOD1associated ALS. However, little is known about the morphology and location of these detergent -insoluble aggregated species within cells. Here, we have used our HEK293FT cell culture model to determine that such aggregated species are cannot be detected through conventional immunofluorescence techniques. However, cytosolic inclusions can be observed by tagging SOD1 mutant proteins with a fluorescent protein. Cells expressing SOD1::YFP pr oteins contain inclusions that are easily visualized, thus may be of larger caliber than the detergent -insoluble aggregates of mutant SOD1. Alternatively, SOD1 antibodies may be incapable of detecting inclusions formed of detergent -insoluble aggregated SOD 1 proteins. We have established that the amount of detergent -insoluble SOD1 mutant protein in cells expressing mutant SOD1::YFP variants is about 6 fold or higher. This data suggest that a higher concentration of the mutant untagged SOD1 protein might be r equired to form detectable inclusions, with the detergent -insoluble aggregates being precursors of inclusions. Alternatively, it is also possible that detergent -insoluble aggregated species are a type of SOD1 aggregates that are different from the inclusions seen by tagging SOD1 with fluorescent tags. In terms of the ability of WT in modulating mutant SOD1 aggregation, tagged proteins do not show as big of an impact when WT and mutant are coexpressed, compared to untagged WT and mutant SOD1 co expression. However, interactions of tagged WT and mutant SOD1 proteins can be observed, with stronger interactions within same WT or mutant proteins and different tags. Additional data suggest that in terms of tagged proteins, it appears that WT and

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161 mutant are more l ikely to interact at the soluble level, defining soluble in this case as the ability to be expressed but without forming cellular inclusions. However, further analyses are under way to support this hypothesis. Inclusion Formation in C ells Expressing Untagg ed SOD1 P roteins Disease associated variants of SOD1 differ from WT SOD1 in their ability to form detergent -insoluble SOD1 aggregates in cell culture an animal models. To asses aggregation propensity of SOD1 proteins we have previously used a biochemical a ssay and western blotting (Karch and Borchelt, 2008;Karch et al., 2009;Prudencio et al., 2009a;Prudencio et al., 2009b) However, we have never before a ssessed their morphology and or location in cells. Using cell culture techniques, we attempted to isolate and visualize such detergent insoluble SOD1 species. However, the amount of soluble SOD1 proteins expressed in the cells may overshadow the low levels of detergent -insoluble SOD1 aggregates. Additionally, techniques that eliminate part of the detergent -soluble SOD1 proteins indicate that aggregates of mutant SOD1 that are insoluble in non-ionic detergents form structures that cannot be observed through the fluorescence microscopy techniques we employed. These data suggest that the difficulty to identify SOD1 inclusions in pathogenic tissue might be due to the usual small size of such aggregates, or to the inability of the antibodies to detect them. Subc ellular Location of SOD1::YFP Inclusions We have observed that tagging SOD1 proteins with fluorescent tags increases the amount of detergent -insoluble SOD1 aggregates that forms in 24 or 48 hour transfection intervals. Additionally, the fluorescent tags in duce inclusion formation in cell culture, even for WT SOD1 (with RFP tag, or with YFP tag and expressed for long period of times). Similar to the detection of detergent insoluble aggregates

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162 biochemically, the number of inclusions depends on the amount of protein expressed. Cells that express our SOD1::YFP vectors with lower efficiency than HEK293FT (NIH3T3 or TK negative cells) contained a lower number of cells with inclusions. Additionally, at long transfection intervals, SOD1 proteins that do not normally form detergent -insoluble species in cell culture of HEK293FT cells (WT, and MD) can be seen within protein inclusions (see Figure 68). Furthermore, the large size of SOD1::YFP inclusions suggested us that they may not likely interact with subcellular organelles. Although, we did not extensively explore this avenue, preliminary data was obtained from a few co localization analyses of SOD1::YFP proteins (see Appendix E). However, the results of these experiments are not conclusive. Effect on Detergent Solu bility in SOD1::YFP Inclusion Formation We have described the higher inherent propensity of SOD1::YFP proteins to become insoluble in non -ionic detergent, with WT::YFP accumulating significant amounts of insoluble aggregates. However, although the levels of detergent -insoluble aggregates of WT:: YFP at 48 hours are not different from those of G85R::YFP at 48 hours (compare G85R::YFP from Figure 6 -1 3 C with WT::YFP from Figure 6-1 3 D), the ability of WT::YFP to form inclusions is very low (see Figure 6-8 A). Thus, it might be that the amount of deterg ent -insoluble WT::YFP protein needed to form inclusions is much higher than for mutant SOD1 proteins, due to a very low inherent propensity of WT to aggregate. Alternatively, it is possible that detergent insoluble SOD1 aggregates and SOD1::YFP inclusions are structures of very different nature, suggesting that other differences between WT and mutant YFP tagged proteins may account for such differences in their ability to form inclusions. Interestingly, we have preliminary data that

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163 suggest a higher ability to aggregate for WT::RFP than for WT::YFP. However we cannot demonstrate whether detergent -insoluble aggregates are precursors of SOD1::YFP inclusions or if they are different species. WT M odulation of Aggregation and Inclusion Formation Interactions of WT and mutant SOD1 proteins are an important feature that modulates aggregation and lifespan in mice expressing a human SOD1 mutation (see Chapters 4 and 5). However, the ability of untagged WT to slow mutant SOD1 aggregation in cell culture differs from t he ability of untagged WT to modulate mutant SOD1::YFP aggregation, or different from the ability of WT::YFP to modulate aggregate formation of untagged mutant SOD1 proteins. These differences may rely on at least two possibilities: 1) the ability of WT::Y FP protein to slow aggregation of mutant proteins may be altered as a consequence of this protein (WT::YFP) to accumulate significant levels of aggregated protein, and 2) the presence of the YFP tag in either WT or mutant SOD1 proteins may introduce subtle modifications in the SOD1 structure, or hide possible exposed amino acid sequences in SOD1, which would translate into weaker ability for WT and mutant SOD1 to interact and modulate aggregate formation. However, we can still observe a smaller effect on m odulating aggregation when one of the SOD1 proteins is tagged with YFP (see Figures 6-12 and 6-13) suggesting that interactions between WT and mutant SOD1 might still occur, but at a much lower level. The amount of detergent insoluble SOD1 aggregates produced by untagged mutant SOD1 proteins (A4V or G85R) does not appear to be sufficient to induce inclusion formation of WT::YFP (see Figures 6-14C and 6-14F) In the other hand, the lower ability of untagged mutant proteins to aggregate (compared to WT:: YFP, see

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164 Chapter 3 and Figure 6-7) may explain that the addition of these detergent -insoluble aggregates of untagged mutant SOD1 to WT::YFP aggregates when co transfected, is not sufficient to make WT::YFP to rapidly create cellular inclusions. Clearly the ability of WT::RFP to form inclusions can induce WT::YFP to be included in such species, while YFP does not in the presence of WT::RFP. Similar results are observed with A4V::YFP and A4V::RFP proteins. Thus, interactions occur between the SOD1 proteins th at do not involve the fluorescent tags. Additionally, the RFP tag induces a higher ability of SOD1 to readily aggregate (data not shown). However, interactions of WT and mutant tagged proteins do appear to easily occur. Interestingly, WT::RFP and A4V::YFP co expression results in the formation of inclusions of each of the expressed proteins, with in some cases A4V::YFP encapsulating the WT::RFP aggregates, but not co-localizing. In the other hand, co expression of WT::YFP and A4V::RFP resulted in a very di fferent outcome. While some cells lacked of any inclusions and presented WT::YFP and A4V::RFP uniformly distributed within cells (note that A4V::YFP has not been observed before as diffused expression, see Figures 6-10B, 616E and 617B), cells presenting A4V::RFP inclusions appear to also recruit WT::YFP. In this latter case, WT::YFP did not easily accumulate within inclusions, as a very high fluorescent intensity was also observed all over the cell. In summary, in terms of WT and mutant interactions with in inclusions we can observe that: 1) Tagged WT and mutant SOD1 proteins do not easily interact with each other to form inclusions, different from WT::YFP + WT::RFP or A4V::YFP + A4V::RFP; but 2) Inclusions expressing both WT and mutant tagged proteins can be observed when the WT SOD1 protein expressed is tagged to YFP, the variant less prone to

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165 inclusion formation. Thus, it is possible that interactions between WT and mutant tagged proteins may take place at state prior to inclusion formation. Then, we would explain the inability of WT::RFP and A4V::YFP to interact within inclusions to the fact that WT::RFP has a very high propensity to form inclusions, not allowing enough time to interact with mutant SOD1. Conclusions In conclusion, the data presented her e is consistent with the idea that the formation of inclusions is accompanied may require an accumulation of a large amount of detergent -insoluble SOD1 protein. WT::YFP seems to show this after 48 hour transfection, where we can observe some inclusions. However, we cannot yet demonstrate whether detergent insoluble SOD1 aggregates could represent a precursor of inclusions, since we cannot normally detect inclusions in cultured cells expressing untagged mutant SOD1 proteins. It is possible that the structure s formed by untagged mutant SOD1 are small and dispersed, but we do not discard the possibility of having a problem of epitope detection with the available antibodies. The presence of a fluorescent tag in SOD1 proteins seems to affect WT and mutant SOD1 interactions, but not WT and WT or mutant and mutant interactions. Thus, inclusions containing tagged WT and mutant SOD1 proteins are less likely to occur, but this process appear to be favored if WT and mutant SOD1 can spend enough time at a state previous to inclusion formation.

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166 CHAPTER 7 CONCLUSIONS SOD1 associated ALS is a devastating neurodegenerative motor neuron disease, with neither effective developed treatments nor cure The main reason is likely explained by the uncertainty of what triggers motor neuron death. Several hypothesis exist that try to explain how mutations in SOD1 provide the protein with a toxic property that kills primarily motor neurons In the work presented here, we have f ocused on one of these hypotheses which suggests tha t some type of aggregated species of mutant SOD1 i s responsible for the toxicity in ALS. In particular, we have performed a exhaustive study to understand the role of aggregates of mutant SOD1 that are insoluble in non-ionic detergents and that can be isol ated through a previously described biochemical assay (Karch and Borchelt, 2008;Prudencio et al., 2009a) The goal s of these stud ies were to determine whether all mutant SOD1 proteins are able to form detergent -insoluble SOD1 aggregates, and whether aggregation is a common feature in SOD1associated ALS. Additionally, we intended to evaluate the role of these structures on disease and determine whether aggregation correlates with any disease feature in humans. ALS is normally dominantly inherited, thus SOD1 associated ALS patients express a SOD1 mutation but also the normal, WT SOD1 protein. Thus, in a second part of this project, w e have investigated whether WT SOD1 plays a role in disease and/or in modulating aggregation. And f inally, we have further characterized in a cell culture model the detergent -insoluble aggregates of mutant SOD1 and how a fluorescent tag SOD1 system may be useful to study WT and mutant SOD1 co aggregation.

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167 Detergent -Insoluble Mutant SOD1 Aggregates and ALS Over 140 mutations in SOD1 are associated with familial ALS. From all these disease causing mutations about 10% had been analyzed in cell culture and animal models previous to the present study. All these analyzed proteins share the common feature of being able to form detergent -insoluble aggregates in cell culture and animal models, while they differ in several other protein features. The work pres ented in Chapter 2 demonstrates that not all mutations in the SOD1 protein induce the formation of such detergent -insoluble SOD1 aggregated structures, and that lack of aggregation correlates with lack of disease development These studies then suggest all SOD1 associated ALS mutations may not have the same inherent propensity to aggregate. Thus, in Chapter 3 we extended the number of studied SOD1associated ALS mutations to over 30%. We demonstrated the different ability of SOD1 disease causing mutations t o aggregate in cell culture. Additionally, the variability in aggregation potential cannot be explained by a single characteristic of the SOD1 protein (stability, metal binding, change in protein charge, etc). However, we determined that higher rates of ag gregation predict shorter disease durations in humans, while aggregation did not seem to direct onset. We were n ot able, however, to find a SOD1 mutation that does not form aggregates but cause ALS in humans or animal models. Thus, it appears that protein aggregation represents at least an important pathological feature of SOD1associated ALS. WT SOD1 as a Modulator of Aggregate Formation In Chapters 2 and 3 we have demonstrated the importance of detergent insoluble SOD1 aggregates in disease. Additionally it appears that high aggregation rates are characteristic of shorter disease durations in humans. However, we did not find

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168 statistically significant correlations between aggregation propensity in cell culture and disease duration in humans. Specially, mu tants with moderate aggregation propensity presented a large variability in disease durations when expressed in humans. Since no intrinsic protein property of mutant SOD1 by itself could explain the variability in aggregation rates, we considered important to determine whether other proteins could be involve in modulating aggregates of mutant SOD1. Thus, we sought to study whether WT SOD1 protein, which is normally present in affected individuals, could modulate aggregation (Chapters 4 and 5). Effectively, WT SOD1 slows aggregation of several SOD1 mutant proteins (A4V, G37R, G85R, G93A and L126Z) expressed in cell culture for 24 hours. This apparent reduction in aggregation is not permanent, and at longer incubation intervals (48 hours), some cells are able to significantly form detergent insoluble aggregates. Additionally in several cases we have determined the presence of both, WT and mutant SOD1 proteins, in such aggregates. This data suggest that interactions of WT and mutant SOD1 proteins may occur to modulate aggregate formation. In Chapter 4, we have also demonstrated that human as well as mouse WT SOD1 proteins can slow aggregation of mutant SOD1 proteins, but only human WT SOD1 is capable of coaggregate with mutant protein. The lack of cysteine 111 i n mouse SOD1 did not explain its inability to co aggregate, as human WT SOD1 without this cysteine can still co aggregate with human mutant SOD1 Additionally, the effect of WT on slowing aggregation of the truncation mutant L126Z in cell culture suggest t hat interactions between WT and mutant human SOD1 protein may take place involving human amino acids present in the first 126 residues of the protein. Further studies

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169 looking at the differences between mouse and human WT SOD1 may help us to understand the co aggregation phenomenon. Effect of WT SOD1 on Aggregation and ALS in Transgenic Mice In Chapter 5 we have shown that in transgenic mice, co aggregation of WT and mutant human SOD1 proteins is a common feature of symptomatic mice overexpressing both human SOD1 proteins. However, in some cases the effect on accelerating disease onset was not as pronounced (compare the effect of WT on PrPG37R and L126Z mice). We have demonstrated that stronger effects on accelerating disease onset are dependent on the dose o f WT SOD1 protein expressed. Additionally, such effect varies from mutant to mutant, which could be explained by the existing differences between WT and mutant SOD1 (in terms of sequence or other properties), and that could account for different ability of WT and mutant SOD1 to interact. Due to a more rapid disease development in mutant/WT SOD1 mice, we can detect WT and mutant co aggregation at earlier time points, coinciding with disease endstage. It is possible that the more rapid disease development cor relates with a more rapid rate of aggregation induced by WT SOD1, rather than an earlier development of aggregates ( see Figure 510). Thus, while WT SOD1 protein initially delays aggregate formation of mutant SOD1, possibly due to WT -mutant SOD1 interactio ns and protein turnover, once a WT/mutant nucleation complex has been formed the rate of aggregation would be increased. At the same time, other abnormalities in the cell, if not caused by some kind of aggregated species, would be accelerated in parallel and leading to a more rapid onset of paralysis symptoms. It is known that several abnormalities occur prior to hindlimb weakness in mutant SOD1 mice (gliosis, muscle denervation, etc), but these cannot be evaluated in alive

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170 mice. Thus, future studies shoul d be focused on elucidating easily markers of disease initiation. This data could help us to understand whether WT SOD1 exert a toxic effect in initial stages of the disease or along disease progression. Overall, our studies demonstrate that the presence o f WT SOD1 in mutant SOD1 transgenic mice is detrimental, with WT SOD1 altering rates of aggregate formation. Detergent -Insoluble SOD1 Aggregates are Difficult to Observe in Cells In order to determine the possible toxic effect of detergent insoluble SOD1 p roteins, we used our cell culture model of HEK293FT to further study these aggregates in vitro However, we were unable to detect aggregates in cell culture using standard fluorescent microscopy techniques. Thus, it is unclear where detergent -insoluble SOD 1 aggregates accumulate and/or exert their toxicity. In the studies presented in Chapter 6 we did not determine whether our SOD1 antibodies are capable of detecting protein that form s part of inclusions. However, due to the fact that mutant SOD1::YFP inclusions can be formed in cells expressing very high levels of detergent -insoluble protein, it is possible that the size of detergent insoluble SOD1 aggregates may be smaller than visible cellular inclusions. SOD1 Fluorescent -Tagged Inclusions Detergent -Ins oluble Aggregates and WT Mutant Interactions. Inclusions of SOD1 proteins can be obtained by using fluorescent tags attached to the C -terminus of SOD1. With such tags, even WT SOD1 can be induced to form inclusions. We have also determined that the addition of the protein tag accelerates the formation of detergent insoluble SOD1 aggregates. In terms of SOD1::YFP inclusions, these are of such large diameters that we think unlikely their presence within any particular organelle. However, we did not present en ough data here to support such

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171 statements (data in Appendix E). Instead, the work presented in Chapter 6 on fluorescent tagged SOD1 demonstrated to be a useful technique to study possibly study proteinprotein interactions between WT and mutant human SOD1 proteins. Our colocalization data suggest that WT and mutant SOD1 can interact but with lower efficiency than WT WT or mutant mutant SOD1 proteins. We were able to observe, that WT and mutant SOD1 co localiz ations when both proteins are tagged, can take place when they are not already forming SOD1 inclusions (WT::YFP + A4V::RFP); but WT::RFP, which forms inclusions very rapidly, does appear to co -localize with A4V::YFP inclusions. From these studies we suggest WT -mutant protein interactions may take place at the soluble level, before the proteins become part of visible cellular inclusions. In order to establish direct protein -protein interactions between WT and mutant SOD1, further experiments should be performed, such as immunoprecipitation or FRET analys es. Composition of Detergent Insoluble SOD1 Aggregates Previous studies on detergent -insoluble SOD1 aggregates have established that they are composed of full length unmodified mutant SOD1 (Shaw et al., 2008) Additionally, other studies have demonstrated that a fraction of different SOD1 proteins is in an immature disulfide reduced state in vivo (Jonsson et al., 2006a;Zetterstrom et al., 2007) Here we have demonstrated that as little as 10% of soluble WT SOD1 protein can be found as a disulfide immature state in mouse spinal cords. Additionally, soluble SOD1 mutant proteins are found mostly in a disulfide reduced state in cell culture, while all detergent -insoluble SOD1 species are in a disulfide reduced state (Figure 71). On the other hand, a larger proportion of WT SOD1 protein is observed as disulfide oxidized protein in cells after 48 hours (Figure 71).

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172 Figure 7-1. SOD1 mutant proteins remain most ly disulfide reduced in cell culture. A-D) Immunoblots of detergent extracted HEK293FT cells that had been transfected for 48 hours with SOD1 constructs and analyzed by western blotting excluding ME (-mercaptoethanol) from the loading buffer. A technique of in-gel reduction has been per formed in C and D that consist in incubating the gels in 2% ME and transfer buffer prior protein transfer. This procedure favors antibody binding to t he oxidized form. A protein control for disulfide reduced (R) and disulfide oxid ized (O) WT SOD1 protein has been included in each blot as controls UT denotes untransfected cells. E) Quantification of the relative levels of SOD1 protein that is disulfide reduced (black bars) and oxidized (white bars) fo rms from immunoblots in C and D. Each experiment was repeated a minimum of 3 times. A very similar figure was included in the published manuscript of Karch et al ., Prot Natl Acad Sci (2009), 106(19):7774-7779. Additionally, we have observed a simila r outcome with several SOD1 mutant proteins in cell culture and animal models, with maybe the latter presenting a higher proportion of mutant oxidized protein in t he detergent-soluble state (Karch et al., 2009). The subtle differences between cells and anim al models could be attributed to the lower availability of factors for the maturation of a very large am ount SOD1 protein expressed (availability of metals or other co-factors). By analyzing the disulfide status of a smaller R ONO GEL REDUCTION 123456789 101112P2 ME S1 MEA R O123456789101112 123456789 101112 123456789101112R O R OP2 ME S1 ME IN GEL REDUCTION C B D 0 2.0107 4.0107 6.0107 8.0107 46/48 S134N E133del D125H D101N H80R G37R A4V WT 46/48 S134N E133del D125H D101N H80R G37R A4V WTReduce d Oxidized P2 S1Band intensity of SOD1 detergent fractions (A.U.)EIN GEL REDUCTION

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173 set of mutant SOD1 proteins at 24 and 48 hours, we observed that in cell culture the am ount of disulfide reduced and oxidized soluble SOD1 proteins increases over time (Figure 72). However, a higher proportion of disulfide reduced protein is more commonly observed in cells expressing mutant SOD1 (Figure 7-2). Interestingly, WT like proteins such as D101N present similar proportions of reduced and oxidized proteins at 48 hours to WT SOD1 (Figure 72). Thus, our studies suggest that over time much of the SOD1 protein may not completely mature and accumulates in the disulfide reduced state. Figure 72 Progressive accumulation of disulfide reduced SOD1 proteins in cell culture. A, B) Immunoblots of detergent -soluble SOD1 proteins expressed for 24 (A) or 48 (B) hours in HEK293FT cells and processed as explained in Figures 72C and 72D. WT -R : WT SOD1 protein control of disulfide reduced protein, WT -O: WT SOD1 protein control of disulfide oxidized protein. C, D) Quantification of the relative levels of SOD1 protein that is disulfide reduced (black bars) and oxidized (white bars) from immunoblo ts in A and D. Each experiment was repeated a minimum of three times. This figure will be published in a manuscript that studies the kinetics of mutant SOD1 aggregation. 48 hours 24 hours WT A4V G85R D101G D101N 0.0 5.01007 1.01008 1.51008 Band intensity of SOD1 soluble fraction (A.U.) WT A4V G85R D101G D101N 0 5.0107 1.0108 1.5108Reduced Oxidized A C B D

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174 Furthermore, we have also observed that for the formation of detergent -insoluble SOD1 aggregates, the protein does not necessarily need to be able to form a dimer. Studies using a WT SOD1 protein that is unable to dimerize, due to a couple amino acid substitutions in the dimer interface (Bertini et al., 1994) is capable of forming detergent insoluble SOD1 aggregates (Figure 73). These studies suggest that monomeric, and possible disulfide reduced, SOD1 protein may be precursors of detergent -insoluble aggregates. However, more ongoing studies in the lab will furt her help to corroborate this hypothesis. Figure 73. The engineered WT SOD1 monomer presents inherent propensity to aggregate, similar to other SOD1 mutant proteins. Immunoblot of detergent insoluble (P2) and detergent -soluble (S1) fractions of HEK293FT transfected with the indicated SOD1 constructs for 24 hours and analyzed by detergent extraction and centrifugation assay, as described in Methods in Appendix C. Another important feature of mutant SOD1 aggregates, is that they do not only contain mutant SOD1 proteins, but human WT SOD1 can also form part of the aggregate. This feature is an intriguing finding since the levels of WT SOD1 protein modulate disease. Thus, the presence of WT SOD1 in mutant SOD1 aggregates should be taken into account to determ ine the specific toxic role of these species. S1 P2

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175 Summary The work presented in this dissertation demonstrates that: 1) All ALS causing SOD1 mutations share the ability to form detergent insoluble structures of SOD1, 2) High aggregation propensity values of mutant SOD1 proteins predict a rapid disease course in humans, 3) The lack of statistical correlation of aggregation propensity in cell culture with disease duration in humans could be explained by the ability of WT SOD1 to modulate aggregation, which has not been considered in those studies, 3) The role of WT SOD1 in modulating mutant SOD1 aggregation is complex, 4) WT and mutant human SOD1 protein can coaggregate in cell culture and animal models, 5) The dose of WT SOD1 protein in mice determines its abi lity to accelerate paralysis, the higher the dose, the earlier the onset of paralysis symptoms in mice; 6) Earlier disease onset in WT/mutant mice correlates with the appearance of detergent -insoluble SOD1 aggregates of both, WT and mutant human SOD1 proteins, 7) Detergent insoluble SOD1 aggregates cannot be easily visualized within cells, 8) Fluorescent tagged SOD1 variants can form visible protein inclusions in cell culture, 9) Fluorescent tagged SOD1 variants form extremely high levels of detergent insol uble SOD1 protein, 10) The presence of the tag alter protein-protein interaction patterns of SOD1 proteins, and 11) Although with low efficiency, WT and mutant tagged SOD1 proteins can interact to form SOD1 inclusions, but such interactions may likely occur prior inclusion formation, as this is not observed when one of the mutants aggregate very rapidly. Future Directions Further studies should be directed to determine the kinetics of aggregate formation and at what time point WT SOD1 intervenes in modulati ng such process. Additionally, it would be important to continue to study WT and mutant SOD1 interactions through

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176 immunoprecipitation assays in which one of the proteins is tagged and/or through FRET analysis. Additionally, further experiments could be con ducted to study what amino acids within the human SOD1 sequence are important for WT -mutant SOD1 interactions. D etermination of the location of detergent insoluble SOD1 structures would provide important information o f the role of these structures i n disea se thus additional techniques should be developed with this goal Finally, screening for drugs that might slow aggregate formation, or disrupt WT and mutant SOD1 interactions may provide a benefit in slowing progression in SOD1 associated ALS patients. Alternatively, the o n going clinical trial of antisense oligonucleotide that targets SOD1 protein to reduce its levels, represents a promising therapy for ALS.

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177 APPENDIX A LIST OF SOD1 -ASSOCIATED ALS MUTATIONS Table A-1. List of published SOD1associated ALS mutations. Mutations Class Principal references Exon 1 1. A4 S, T, V B (Nakanishi et al., 1998;Nakano et al., 1994;Rosen et al., 1994) 2. C6 F G B (Morita et al., 1996;Kohno et al., 1999) 3. V7 E B (Hirano et al., 1994) 4. L8 Q V B (Siddique and Deng, 1996;Andersen et al., 2003) 5. G10 R, V B (Kim et al., 2003) 6. G12 R B (Penco et al., 1999) 7. V14 G M B (Andersen et al., 1997;Deng et al., 1995) 8. G16 A S B (Andersen et al., 2003;Kawamata et al., 1997) 9. N19 S B (Andersen et al., 2003) 10. F20 C B (Andersen et al., 2003) 11. E21 G K B (Siddique and Deng, 1996;Jo nes et al., 1994b) 12. Q22 L B (Andersen et al., 2003) Exon 2 13. V29 B (Shi et al., 2004) 1 4 G37 R B (Rosen et al., 1993) 15 L38 R V B (Boukaftane et al., 1998;Rosen et al., 1993) 16 G41 D S B (Rosen et al., 1993) 17 H43 R B (Rosen et al., 1993) 18 F45 C B (Gellera et al., 2001) 19 H46 R M (Aoki et al., 1993) 20 V47 F B (Andersen et al., 2003) 21 H48 Q R M (Enayat et al., 1995;Andersen et al., 2003) 22 E49 K B (Boukaftane et al., 1998) 23 T54 R D (Andersen et al., 2003) 24 C57 R D (Andersen, 2006) Exon 3 25 S59 I D (Andersen et al., 2003) 26 N65 S M (Garcia Redondo et al., 2002) 27 L67 R M (Boukaftane et al., 1998) 28 G72 C S M (Stewart et al., 2006;Shaw et al., 1998) 29 D76 V Y M (Segovia Silvestre et al., 2002;Andersen et al., 1997) Exon 4 30 H80 R M (Alexander et al., 2002) 31 L84 F V M (Shaw et al., 1998;Aoki et al., 1995) 32 G85 R M (Rosen et al., 1993) 33 N86 D, K, S B (Andersen, 2006;Beck et al., 2007;Hayward et al., 1998) 3 4 V87 A B (Andersen et al., 2003) 35 T88 B (Andersen et al., 2003) 36 A89 T V B (Andersen et al., 2003;Rezania et al., 2003) 37 D90 A V B (Andersen et al., 1995;Chou et al., 2005) 38 G93 A, C, D, R, S, V B (Rosen et al., 1993;Esteban et al., 1994;Elshafey et al., 1994;Siddique and Deng, 1996;Hosler et al., 1996) 39 A95 B (Gellera et al., 2001;Chio et al., 2008) 40 D96 B (Hand et al., 2001)

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178 Table A 1. Continued. Mutations Class Principal references 41 V97 B (Andersen et al., 2003) 42 E100 K B (Rosen et al., 1993;Siddique and Deng, 1996) 43. D101 N Y B (Yulug et al., 1995;Sato et al., 2004;Jones et al., 1994a;Tan et al., 2004) 44 I104 B (Ikeda et al., 1995) 45 S105 B (Andersen et al., 2003) 46 L106 B (Rosen et al., 1993) 47 G108 B (Orrell et al., 1997) 48 D109 B (Naini et al. 2007) 49 C111 B (Shibata et al., 2007) 50 I112 T B (Garcia Redondo et al., 2002;Esteban et al., 1994) 51 I113 T B (Andersen et al., 2003;Rosen et al., 1993) 52 G114 B (Andersen et al., 2003) 53 R115 B (Kostrzewa et al., 1994) 54 T116 B (Andersen, 2006) 55 V118 L ins (stop 122) B (Andersen et al., 2003;S himizu et al., 2000;Jackson et al., 1997) Exon 5 56 D124 V M (Andersen et al., 2003;Hosler et al., 1996) 57 D125 M (Enayat et al., 1995) 58 L126 (stop 131) stop B (Takehisa et al., 2001;Siddique and Deng, 1996;Pramatarova et al., 1994) 59 G127 (stop 133) B (Andersen et al., 1997) 60 E132 (stop 133) B (Orrell et al., 1997) 61 E133 B (Ho sler et al., 1996) 62 S134 M (Watanabe et al., 1997) 63 N139 K B (Nogales Gadea et al., 2004;Pramatarova et al., 1995) 64 A140 B (Naini et al., 2002) 65 G141 stop B (Sato et al., 2004;Andersen et al., 2003) 66 L144 S B (Deng et al., 1993;Sapp et al., 1995) 67 A145 T B (Andersen et al., 2003;Sapp et al., 1995) 68 C146 D (Siddique and Deng, 1996) 69 G147 B (Andersen et al., 2003) 70 V148 I B (Deng et al., 1993;Ikeda et al., 1995) 71 I149 B (Pramatarova et al., 1995) 72 I151 T B (Andersen et al., 2003;Kostrzewa et al., 1996) B: Barrel Mutants; M: Metal binding region mutants; D: Disulfide loop mutants. A similar version of this table has been included in a review paper (Seetharaman et al., 2009)

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179 APPENDIX B MATERIALS Table B -1. Cell c ulture r eagents. Reagent Description Manufacturer Catalog number 10x PBS Phosphate buffered saline solution Invitrogen 14200 166 12 well plates Plates for tissue culture Fisher/Nunc 353043 60 mm normal tissue culture dishes Tissue culture dishes Fisher Scientific 08 772 021 60 mm Poly L lysine Biocoat dishes Poly lysine coated dishes to grow low adherent cell lines Fisher Scientific 356517 Cell lifters Cell scrapers for tissue culture Fisher Scientific 3008 Coverslips 24 x 50 mm Coverslips for microscope slides Fisher Scientific 12 545 88 DMEM Tissue culture media Thermo Scientific SH30022.01 DMSO dimethyl sulfoxide Storage cell reagent Fisher Scientific BP231 1 Fetal bovine serum Supplement for tissue culture media Invitrogen 26140 076 Glass round coverslips Coverslips to grow cells on Fisher Scientific 12 545 84 18CIR 1 D HEK293FT cells Human embryonic kidney cell lines Invitrogen R700 07 Horse serum Supplement for tissue culture media Invitrogen 26050 088 L glutamine Amino acid supplement for tissue culture media Invitrogen 25030 156 Lipofectamine 2000 Transfection reagent Invitrogen 11668 019 TK negative ( L M ) cells Mouse connective tissue cell line ATCC CCL 1.3 MEM Tissue culture media Invitrogen 11140 050 Neuro 2a cells Mouse Neuroblastoma cell line ATCC CCL 131 New born calf serum Supplement for tissue culture media Invitrogen 16010 159 NIH 3T3 cells Mouse Embryonic Fibroblast cell line ATCC CRL 1658 Opti MEM I Tissue culture media for transfections Invitrogen 31985 070 Poly L lysine Coating reagent for tissue culture plates Sigma P2636 Trypsin EDTA 0.25% Trysin Invitrogen 25200 072

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180 Table B -2. Reagents for detergent extraction and centrifugation a ssay BCA assay, SDS-PAGE, and Western blotting. Reagent Description Manufacturer Catalog number 18% Tris Glicine, 12 well SDS PAGE gels Polyacrylamide gels Invitrogen EC6505BOX 96 well plates To measure absorbance Fisher/Nunc 167008 BCA reagents A and B To determine absorbance Thermo Scientific Reagent A: 23224 Reagent B: 23228 Bench mark pre stain Protein ladder marker Invitrogen 10748 010 Bromophenol blue Dye for loading dye Fisher Scientific BP114 25 BSA reagents Bovine Serum Albumin New England Biolabs B9001S Deoxycolate Acid salt Fisher Scientific BP349 100 ECL reagent Membrane protein developer Fisher/Pierce PI32106 EDTA Quelator Fisher Scientific BP120 500 Filter paper, 3MM Filter paper Whatman 3030662 Glycerol Reagent Fisher Scientific BP229 1 Iodoacetamide Thiol modifier agent Sigma I1149 5G Magic XP Marker Protein ladder marker Invitrogen LC5602 Methanol Alcohol Fisher Scientific A412 4 Nitrocellulose membrane, 0,45m OptiTran BA S 85 Reinforced Whatman 10439196 Nonidet P40 (NP 40) Non ionic detergent US Biological N3500 PBS Phosphate buffered saline solution Amresco 0780 2PK Powdered non fat milk Caranation milk for membrane blocking for western blotting Publix Not available Protease inhibitor cocktail (PI) Inhibitor of proteases Sigma P8340 5ML Sodium chloride (NaCl) Salt Fisher Scientific BP358 212 Sodium dodecyl sulfate (SDS) Ionic detergent Fisher Scientific BP166 500 TG SDS Tris Glycine, sodium dodecyl su lfate (for running SDS PAGE ) Mid Scientific 0147 40L Tris Buffer Fisher Scientific BP152 1 Tween 20 Detergent Fisher Scientific BP337 500 Ultracentrifuge tubes Airfuge tubes 5MM PA Beckman 342630 mercaptoethanol Sulfide reduction agent Fisher Scientific BP 176 100 Table B -3. Histochemistry and c ytochemistry reagents. Reagent Description Manufacturer Catalog number 2 2 2 Tribromoethanol, 97% Euthanizing reagent Sigma T48402 25G

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181 Table B 3. Continued. Reagent Description Manufacturer Catalog number 3 methylbutane Alcohol for tissue freezing Fisher Scientific 03551 4 ABC kit Vectastain rabbit IgG for DAB Reagents and antibody for DAB staining Vector Labs PK 6101 Ammonium hydroxide Silver staining reagent Fisher Scientific A 669 Cedarwood oil Paraffin embedding reagent Electron Microscopy Sciences 12420 Citric acid Silver staining reagent Fisher Scientific A940 500 DAB 3,3 Diamino benzidine tetrahydrochlorida Sigma D5905 50TAB DAPI staining solution 4,6 diamidino 2 phenyl indole, dehydrochloride Invitrogen D1306 Digitonin Mild detergent Sigma D141 100MG D Sucrose Sugar Fisher Scientific BP220 1 Eosin Y Tissue staining reagent Fisher Scientific E 511 Ethanol 100% Alcohol 200 proof Fisher Scientific NC9977258 Flash Freeze it Reagent for instant OCT freezing Fisher Scientific 23 022524 Flourescent mounting media Mounting media for fluorescent sections Polysciences, Inc. 18606 F ormaldehyde 37% Reagent Amresco 0493 500ML Glacial acetic acid Acid Fisher Scientific BP2401 212 Glass round coverslips To grow cells for cytochemistry Fisher Scientific 12 545 84 18CIR 1D Histoclear Histological clearing agent N ational Diagnostics HS 200 Hydrogen peroxide (H 2 O 2 ) H 2 O 2 30% Sigma H1009 100ML Methanol Alchohol 100% Fisher Scientific A412 4 Methil Salicylate Paraffin embedding reagent Fisher Scientific O395 500 Modified Meyers Hematoxylin Tissue staining reagent Richard Allan Scientific 72804 Nitric acid (HNO 3 ) Silver staining reagent Fisher Scientific A200 500 Normal goat serum Serum for blocking tissue using secondary antibodies raised in rabbits I nvitrogen 16210 064 OCT Tissue freezing media Electron Microscopy S ciences/Fisher 62550 12 PAP pen Hydrophobic slide marker Research Products International Corp. Not available Paraformaldehyde Fixing cell reagent Electron Microscopy Sciences 19202

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182 Table B 3. Continued. Reagent Description Manufacturer Catalog number Paraplast X tra tissue embedding medium Paraffin Fisher Scientific 23 021 401 PBS Phosphate buffered saline solution Amresco 0780 2PK Permount Mounting media for non fluorescent sections Fisher Scientific SP15 100 Phloxine B Histochemistry dye Fisher Scientific P387 25 Saponin Mild detergent Fluka 47036 Silver nitrate S ilver staining reagent EMD SX0205 5 Sodium thiosulfate pentahydrate (Na 2 S 2 O 3 ) Silver staining reagent Fisher Scientific S78930 1 Superfrost plus slides Microscope slides Fisher Scientific 12 550 15 Tissue Path Cassettes IV white Cassettes for tissue embedding with paraffin Fisher Scientific 22 272416 Tissue Path disposable base molds Molds for paraffin embedding Fisher Scientific 22 038 217 Triton X 100 Detergent to permeabilize cells for staining Fisher Scientific BP151 500 Ultraclean Detergent Spectrum Medical Laboratories 105 544 Table B -4. Reagents for clonning, genotyping, and general DNA w ork Reagent Description Manufacturer Catalog number Agarose Low EEO Agarose for DNA gels Fisher Scientific BP160 500 Alkaline phosphatase Enzyme to eliminate phosphate groups Roche NC9305850 Amonium acetate Reagent Fisher Scientific A637 500 ATP 100mM Adenosine triphosphate 272056 Bacto Agar Agar for bacterial medium plates BD Biosciences 214010 Beta agarase I Agarose digestion enzymes New England Biolabs M0392S Bromophenol blue Dye for loading dyes Fisher BP114 25 Carbenicillin Antibiotic Midwest Scientific/Amresco J358 1G Cesium chloride Plasmid prep reagent Fisher Scientific BP210 500 Chloroform Reagent Fisher Scientific BP1145 1 Circlegrow capsules Bacterial medium reagent BIO101 Systems 3000 131 Deoxiribonucleotides triphosphate (dNTPs) Nucleotides for PCR reaction, 100 mM set Invitrogen 10297 018

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183 Table B 4. Continued. Reagent Description Manufacturer Catalog number efficiency cells Cells for transformation Invitrogen 18265017 DNA columns Ultrafree DA DNA extraction from agarose Millipore 42600 DNA Hyperladder I DNA gel marker Fisher Scientific NC9841467 DNA ladder 1Kb plus DNA gel marker Invitroge n 10787 018 EDTA Quelator Fisher Scientific BP120 500 Ethanol 100% Alcohol 200 proof Fisher Scientific NC9977258 Ethidium bromide Intercalating DNA agent Fisher Scientific BP1302 10 Fast miniprep kit DNA isolation, low scale for sequencing Eppendorff 955150619 Glass beads, 4MM For plating transformed bacteria Fisher Scientific 11 312B Glacial acetic acid Acid Fisher Scientific BP2401 212 Glucose D anhydrous Sugar Amresco 0188 1KG Isopropanol Alcohol Fisher Scientific BP2632 4 LB capsules LB bacterial medium reagent MP Biomedicals, LCC 3002 031 Lysozyme, egg white Enzyme Amresco 12650 88 3 NcoI Restriction enzyme New England Biolabs R0193S Phenol Reagent Amresco 0945 400ML Platinum Pfx DNA polymerase DNA polymerase enzyme Invitrogen 11708 021 Potassium acetate Reagent Fisher Scientific BP364 500 Proteinase K Proteinase enzyme Invitrogen 25530015 PureLink PCR microkit To purify PCR products Invitrogen K310010 RNAse A pancreatic Enzyme Amresco 0675 250MG SalI Restriction enzyme New England Biolabs R0138S SeaPlaque GTG Agarose Low melting point agarose for DNA gels Lonza 50111 Small Kimwipes Task wipers Fisher Scientific 06 666A S.O.C. medium Bacterial medium Invitrogen 15544034 Sodium chloride (NaCl) Salt Fisher Scientific BP358 212 Sodium dodecyl sulfate (SDS) Ionic detergent Fisher Scientific BP166 500 SuperscriptIII RT/Platinum Taq HiFi Enzyme for RT PCR reaction Invitrogen 12574 030 T4 DNA ligase DNA ligase enzyme New England Biolabs M0202S TAE Tris, Tris Acetate, EDTA Amresco 0912 2PK Taq DNA polymerase PCR polymerase New England Biolabs M0273L Tris Buffer Fisher Scientific BP152 1 tRNA yeast Reagent Invitrogen 15401 029 Ultracentrifuge tubes Plasmid prep tubes Beckmann 362185 XhoI Restriction enzyme New England Biolabs R0146S X gal Reagent Amresco 7240 90 6

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184 Table B -5. Reagents RNA extraction and northern blotting. Reagent Description Manufacturer Catalog number Bromophenol blue Dye for loading dyes Fisher BP114 25 BSA for northern Serum albumin Amresco 0332 100G Chloroform Reagent Fisher Scientific BP1145 1 Clear UV 96 well transparent plates To measure RNA concentration Costar / Corning 3635 Diethyl Pyrocarbonate (DEPC) Reagent for Northern Sigma D 5758 DNA Ready to Go label beads dCTP To label DNA probes for Northern GE Healthcare, Amhersan 27 9240 01 Ethidium bromide Intercalating DNA agent Fisher Scientific BP1302 10 Filter paper, 3MM Filter paper Whatman 3030662 Formaldehyde 37% Reagent Amresco 0493 500ML Formamide Reagent Sigma 47670 GeneScreen Plus Nylon membrane Nylon membranes for Northern Perkin Elmer NEF 976 Glycerol Preservative Fisher Scientific BP229 1 Ilustra Probe Quant G 50 MicroColumns Columns to purify radioactive labeled probes GE Healthcare, Amhersan 27 5335 01 Isopropanol Alcohol Fisher Scientific BP2632 4 MOPS buffer, 10x Buffer Amresco E526 500ML Nylon membrane Membrane for Northern GeneScreen Plus NEF976001PK RNAse AWAY Reagent to eliminate RNA Molecular Bioproducts 7002 RNAse free agarose low EEO Free RNA agarose Fisher Scientific BP160 500 Sodium chloride Salt Fisher Scientific BP358 212 Sodium citrate Salt Fisher Scientific BP327 1 Sodium phosphate (NaHPO 4 ) Salt Fisher Scientific TRIzol reagent RNA extraction reagent Invitrogen 15596 026

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185 APPENDIX C METHODS Chapter 2 M ethods Creation of G enomic SODMD C onstruct for Expression in T ransgenic M ice. Mutations in the genomic sequence of human SOD1 were introduced using PCR strategies. The selected mutations are indicated in Figure 2 -1. The resulting 12 kb genomic mutant DNA fragment was then injected into mouse embryos, as previously described for othe r SOD1 transgenic mouse lines (Wong et al., 1995) The elaboration of the genomic SODMD DNA was entirely performed by Ms. Hilda Slunt Brown. Identification of the Presence of the Human SOD1 Transgene/s in M ice: G enotyping of SODMD M ice and M ice from the SODMD x SJL WT Matings. Extraction of DNA from m ouse tails Mouse tails were cut at approximately 0.6 cm in length. The procedures for tail cutting have been reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) at the University of Florida. Collected tails are then digested or stor ed at 20 C. The day before DNA extraction, digest each mouse tail in 600 l of TNES buffer (50 mM Tris base, pH 7.5; 400 mM NaCl; 100 mM EDTA, pH 8.0; 0.5% SDS) and 18 l of Proteinase K at 20 mg/ml (dissolve 20 mg of proteinase K in 1 ml of distilled H2O vortex and store in aliquots at -20 C, once thawed do not reuse). Incubate overnight in a 55 C water bath. Remove tails from the water bath and mix well. Add 167 l of supersaturated 6 M NaCl to each digested tail and mix well for 15 seconds (do not vort ex). Centrifuge at 14,000 rpm for 5 minutes. Remove 650 l of supernatant and place into a new microfuge tube. Try to get below the floating scum on the surface when removing the supernatant. Centrifuge at 14,000 rpm for 5 minutes. Remove 600 l of super natant and place into a new microfuge tube. Add 600 l of cold 100% ethanol to each microfuge tube and mix well for 15 seconds (do not vortex). Centrifuge at 14,000 rpm for 5 minutes.

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186 Discard the supernatant and add 1 ml of cold 70% ethanol to each microf uge tube. Centrifuge at 14,000 rpm for 2 minutes. Discard the supernatant and turn each tube upside-down to air dry for 15 -20 minutes. Resuspend the DNA by adding 200 l of TE buffer (10 mM Tris base, pH 7.5; 1 mM EDTA, pH 8.0) to each microfuge tube. Mix and allow the DNA to dissolve at room temperature for at least 1 hour (can be longer, even overnight). Proceed to PCR analysis or store DNA at -20 C till needed. PCR p rotocol for i dentification of g enomic h uman SOD1 Mix the following reagents for each tail DNA sample: o Distilled H2O 20.14 l o 10x PCR Buffer (with MgCl2 from New England Biolabs) 2.50 l o 5 mM dNTPs 0.50 l o 50 M Hu -S primer 0.38 l o 50 M H/M -AS primer 0.38 l o Taq DNA Polymerase (from New England Biolabs) 0.10 l o Tail DNA 1.00 l o Total reaction volume 25.00 l Hu -S primer: 5 -TCA AGC GAT TCT CCT GCC T -3 H/M -AS primer: 5 -CAC ATT GCC CAR GTC TCC A -3 (R=A/G) Set the above samples into a thermocycler and perform the next PCR program: o Heat blot pre -start o 94oC for 5 minutes o 94oC for 30 seconds o 60oC for 1 minute x 35 cycles o 72oC for 5 minutes o 72oC for 10 minutes o Hold at 10oC The transgenic band has a size of ~1200 bp Note that genotyping of SODMD mice was entirely performed by Ms. Susan E. Fromholt. For SODMD x WT SJL mice, I performed DNA extraction and PCR genotyping as described above In order to distinguish human mutant from human WT SOD1, DNA from mouse tails positive for the human SOD1 transgene were reamplified by the method descri bed above and the resulting DNA product was then purified using the PureLink PCR micro kit from Inivitrogen (Carlsbad, CA), following the manufacturer

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187 protocol. The purified DNA fragments were then sent for automated sequence analysis together with the seq uence primer SOD1-I3 -AS (5CTA TCG AAA GAC CTC AAG TAT AC -3) which allows identification of amino acid sequences in exon 3 RNA Extraction from S pinal C ords of SOD1 T ransgenic M ice The RNA extraction protocol presented here is a more detailed version of the TRIzol Reagent Protocol from Invitrogen (Carlsbad, CA ): Weight the spinal cord tissue and calculate the amount of T RI zol needed per sample (do not add till just before homogenizing each sample) Homogenize tissue samples in 1 ml of T RI zol per 60 -70 mg of tissue using Polytron power homogenizer (minimum speed is 11, start at 11 and increase but do not go over 20). NOTE: use tubes that leave enough space for the homogenizer to go into solution but do not cause overflowing. This step is performed at room temperature. Phase separation: incubate homogenized samples for 5 min utes at room temperature, or till the rest of the samples are homogenized. Then add chloroform: 0.2 ml per ml of T RI zol reagent. Then shake tubes vigorously for 15 seconds and incubate at room temperature for 23 min utes Centrifuge samples at 12000 xg for 8 min utes (at room temperature in bench-centrifuge is fine). RNA precipitation: Transfer aqueous phase to a fresh tube (top), and save the organic phase if isolation of DNA or protein is desired (interfase for both). Precipitate RNA by mixing through vortexing briefly with isopropyl alcohol (use an aliquot obtained in a clean, just opened falcon tube when a new bottle of isopropanol is opened for the first time) at 0.5 ml per ml of T RI zol used. Incubate samples at room temperature for 10 min utes (or over lunch at 4 xg for 10 min utes RNA wash: remove supernatant and wash pellet once with 75% ethanol (1 ml per ml of TRI zol; can do 1ml per sample). Vortex and centrif uge no more than 7500 xg for 5 min utes Redissolving the RNA: air dry pellet for 10-20 min utes It is i mportant not to let pellet dry completely. Resuspend RNA in X l of DEPC treated water (0.1% DEPC in distilled water overnight, autoclave to eliminate DE PC) by incubating for 10 min utes at 50 C (X l = mg tissue/2).

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188 Determination of T otal RNA C oncentration Concentration of resulting RNA can be determined through absorbance in a spectrophotometer microplate reader and using 96well clear UV transparent plates Northern B lotting Run g el, transfer to m embrane an d c rosslink On an RNAse free agarose gel run about 5 or 10 g of RNA with 3x volumes of Northern loading dye (For 730 l: 360 l: formamide, 80 l of 10x MOPS buffer, 130 l 37% formaldehyde, 50 l g lycerol, and 10 l of 1% bromophenol blue). Heat at 100 C, 2 -3 minutes. Cool on ice. Add 0.5 l of 0.5 g/l ethidiu m bromide in DEPC treated water. Load on gel (For 125 ml: weight 1.25 g RNAse free agarose and put into 106.26 ml DEPC treated water, boil to melt agarose and cool, add 12.5 ml 10x MOPS buffer and 6.75 ml of 37% formaldehyde). Run loaded gel at 120 volts, until the bromophenol blue of the loading dye is about two thirds of the way to the bottom (it approximately takes 23 h ours ). Soak gel in distilled H2O for 40 60 minutes with three changes, to remove formaldehyde. Take a picture of the gel with a fluorescent ruler. Transfer RNA from gel to nitrocellulose or nylon membrane by capillary action overnight using 10x SSC (20x SSC stock: 350.6 g of sodium chloride and 174.4 g of sodium citrate in 2 liters of DEPC treated water). We use GeneScreen Plus, Nylon membrane. See scheme of transfer set up on Figure C -1. Figure C -1. Scheme of transfer arrangement for northern blotting. Two sheets of filter paper below the gel, in contact with 10x SSC solution 10x SSC Membrane (with writing up) Two more sheets of filter paper over membrane (gel size) Cover with transparent foil to minimize evaporation (cut square that would cover membrane/filter paper) Agarose gel Stack of napkins and weight on top of everything

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189 Crosslink to membr ane, using the autocrosslink function of the Stratalinker (put membrane onto filter paper. Letters down. Start, about 45 s econds ; then wrap in transparent foil and store at room temperature). Northern b lotting h ybridization p rotocol NOTE: the whole protocol is carried out at 65C Pre -hybridize membrane for 5 minutes to 5 hours (1 hour is good). Use 10-20 ml of hybridization buffer (1% BSA, 1 mM EDTA at pH 8.0, 0.5 M NaHPO4 at pH 7.2, 7% SDS, in distilled water) for the 35 x 150 ml hybridization cylinders/bo ttles (The side of membrane with RNA -face with no writing facing the inside of the cylinder). Discard buffer. Add new hybridization buffer and boiled probe (see next section for labeling the probe). Use 10 ml of hybridization buffer in each hybridization cylinder and 0.5106 to 5106 cpm/ml of boiled probe per cylinder/bottle (add to buffer, not t o the membrane). We used 24-25 l of labeled probe at 1.4106 cpm/ml (total 14106 cpm). Hybridize for 8 to 24 hours. Washes (warm buffers prior washing): pour off hybridization buffer into radioactive waste (it can be saved and re used within two weeks if needed). Rinse blot and bottle with wash buffer 1 (0.1% BSA, 1 mM EDTA at pH 8.0, 40 mM NaHPO4 at pH 7.2, 5% SDS, in distilled water). Pour all buffers into radioactive waste. Perform 3 x 30 minute washes with buffer 1. Wash twice 30 minute each with wash buffer 2 (1 mM EDTA at pH 8.0, 40 mM NaHPO4 at pH 7.2, 1% SDS, in distilled water). Wrap wet blot in transparent foil and expose to film in developing cassett e for 24 h ours at -80C. DNA p robe l abeling with 32P for n orthern b lotting For labeling the DNA probe we use DNA Ready to Go-label beads dCTP from GE Healthcare (Amhersan) to prepare the template and label following the manufacturer protocol. We used DNA insert/fragment as SOD1 probe that has been purified with DNA columns (Ultrafree DA, Millipore). A total of 25 50 ng of DNA probe was used for the labeling reaction. So, for SOD1 at 4 ng/l we used 8 l (32 ng) and denatured it at 95 C for 3 minutes and then put on ice as recommended. For PrP loading control, the probe was a fragment of 4 kb at 1 ng/l purified from a low melting point agarose gel In this

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190 case the PrP DNA was denatured at 95C for 7 minutes, then incubated at 37C for 10 minutes before labeling reaction. Follow labeling reaction as described in manufacturer protocol Once the probe is labeled, the probe is purified using the Illustra ProbeQuant G -50 Micro Columns from GE Healthcare as described by the manufacturer Pre paration of C rude Supernatant (T otal Protein Fraction) from M ouse Spinal C ords Calculate net weight of each spinal cord to analyze and sonicate cords till dissolved (about 2 times for 15 seconds, place on ice in between) in 5 weight volumes of 1x TEN buffe r (10 mM Tris, pH 7.5; 1 mM EDTA, pH 8.0; 100 mM NaCl) with protease inhibitor cocktail at 1:100. Spin down homogenized tissue at 8000 xg for 10 minutes and discard pellet. Keep supernatant as crude supernatant. Detergent Extraction and Centrifugation Assa y Protocol for Mouse Spinal Cords. This assay generates two fractions termed S1 (detergent -soluble cellular protein) and P2 (detergent -insoluble cellular protein). The latter containing detergent -insoluble aggregated forms of mutant SOD1. This assay is performed as follows: A total of 300 l of spinal cord crude supernatant was extracted by adding an equal volume of buffer 1 (to final concentration of 10 mM Tris, pH 7.5; 1 mM EDTA, pH 8.0; 100 mM NaCl; 0.5% NP -40, 1:100 v/v protease inhibitor cocktail), and sonicated 3 times for 10 seconds each. Then the samples are transfer red into airfuge tubes, 200 l per tube, and centrifuge at maximum speed for 5min (> 100000 xg). The s upernatant is saved as the deterg ent soluble (S1) fraction. The p ellet (P1) is fur ther washed with 200 l/tube of the same extraction buffer (sonicate twice, 15 seconds each) and the supernatant is discarded after another 5 minute high speed spin. The remaining pellet represents the detergent insoluble (P2) fraction, which is resuspende d in 3040 l of extraction buffer containing SDS and deoxycolate (10 mM Tris, pH 7.5; 1 mM EDTA, pH

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191 8.0; 100 mM NaCl; 0.5% NP -40, 0.25% SDS, 0.5% deoxycolate, 1:100 v/v protease inhibitor cocktail), and combined into one tube. The total volumes correspond ing to S1 and P2 fractions are of 600 and 100 l, respectively. Protein concentration of S1 and P2 are determined by BCA assay, as described in the corresponding section. For western blot analysis, 5 g of S1 and 20 g of P2 total protein are used to load onto 18% SDS PAGE gels. Western blotting is performed as described in SDS -PAGE Electrophoresis and Western Blotting. BCA Assay for Determination of Protein Concentration Protein concentrations are obtained using the bicinchoninic acid assay (BCA assay) f rom Pierce Biotechnology. We use 96well plates for measur ing protein concentration on a plate reader. For detergent -soluble (S1) and insoluble (P2) fractions, the protein was diluted 1:4 (2.5 l of total protein in 10 l total volume, diluted in water). F or any other lysates a dilution 1:10 is enough. Use 10 l for each BSA standard (range between 0 and 2.4 g/l) To wells containing either a standard or a sample, add 200 l of BCA reagent mixture (50:1, Reagent A: Reagent B). Incubate plate at 37 20 -30 min utes Alternatively, the reaction can take longer at room temperature (1 h our ). Read plate at 562 nm. P rotein concentrations and the standard curves are calculated through the Kinetical KC4 3.4 software (Bio -Tek Instruments, Inc. Winooski, VT). SD SPAGE Electrophoresis and Western Blotting For crude supernatants: Prior loading the gel, prepare the 5 g protein, add 1x TEN up to a 15 l volume. For detergent extracted samples, prepare 5 g of S1 fractions and 20 g of P2 fractions in 1x TEN up to a volume of 15 l. Add 5 l of 4x Laemmli sample buffer to each sample and b oil (or use heat block at 95C) for 5 min utes Load samples on 18% SDS -PAGE gel and run at 125 volts for 110 min utes Protein is then transferred from the gel onto nitrocellulose membranes (Optitran BA S 85 from Whatman Inc ). L abel the bottom of membrane and prewet in distilled water, then wet in transfer buffer. Set into transfer cassette (black electrode, sponge, filter paper, gel, membrane, filter paper, sponge, red electrode). Set transfer at 100

PAGE 192

192 mAmps for overnight transfer in big transfer box. Alternatively you can do 2 hour transfer s at 400 mAmps. To reduce nonspecific antibody binding, membranes were blocked in 5% nonfat milk, 1x PBS for 15 30min before incubating them with the corresponding antibodies. Incubate membrane in milk with the primary antibody overnight (SOD1 can be done for 1 hour ). For antibodies concentrations check the antibody list on Appendix D Wash membrane with 1x PBS-T (0.1% Tween 20 in 1x PBS) for 10 mi n utes 3 times. Incubate membrane in milk with the corresponding secondary antibody (goat anti rabbit HRP in this case) for 1 h our at room temperature. Wash 3 times with 1x PBS-T for 10 min utes each. Secondary chemiluminescence was visualized on a Fujifil m imaging system (FUJIFILM L ife Science ) using ECL reagent mixture (1:1, total of 8 00 l per membrane) Sciatic N erve Extraction for Cryosection from SOD1 Transgenic Mice NOTE: this protocol has been adapted from Dr. Lucia Notterpeks lab protocols. Prepar e a container with liquid nitrogen. Immerse a small beaker with 3methylbutane (it freezes becoming white). Do not let the liquid nitrogen get into the beaker. The liquid nitrogen is just to freeze the 3methylbutane. Extend legs of sacrified mice and hol d with needles. Then, spray animal with water to get hair smoothen down. Cut out skin on the legs and the lower back. Try to avoid getting hair left on the animal. Once the muscle is exposed, cut it with 2 straight cuts, then lift muscle layers to see the sciatic nerve. See scheme in Figure C 2 for instructions on how to extract the sciatic nerve from mice. Figure C -2. Schematic representation of how to make cuts in mouse limbs to extract the sciatic nerve.

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193 Hold the nerve with forceps like in the middle (try to handle it on just one place), and cut underneath the nerve to release it from other tissue s Then cut at the lower end and follow till the upper end and cut. Keep in mind that when it g ets close to the spinal c ord there is a lateral connection that needs to be cut too. Remove extra skin if present and extend the nerve on a piece of filter paper. Get the other nerve and do the same. Get the beaker that contains 3 -methylbutane out of the liquid nitrogen and once i t thaws, immerse the filter paper with the nerve into the 3methylbutane solution. Leave a few seconds and then immerse in liquid nitrogen. Introduce into crystorage tubes and leave into liquid nitrogen till all animals are done. S tore in crystorage, in co ntact with liquid nitrogen. Sciatic N erve C ryosection and Immunostaining NOTE: This protocol has been adapted from Dr. Lucia Notterpeks lab protocol. Cut frozen tissue sections at 5 25C: For longitudinal sections, OCT is applied on the base, then a small piece of sciatic nerve is laid on top of OCT (do not embed). Apply Flash Freezing spray from Fisher (note that if the OCT is very liquid the spray wil l splash OCT away). Carefully put the longitudinally placed sciatic nerve parallel to the blade, so the tissue will not be in an angle with respect to the blade. For transverse sections the tissue needs to be embedded in OCT. A trick to maintain the sciati c nerve perpendicular to the base is to add a little OCT to the base first, then immerse half of a small piece of sciatic nerve in OCT in vertical orientation. Then, fast freeze the sample. This will maintain the sciatic nerve rigid and can be then complet ely covered by OCT. Cut sections are placed on superfrost plus microscope slides at room temperature (touch straightened out tissue with the slide quickly, which allows the tissue to quickly adhere to the slide. Slides can be stored at 80C till staining steps. For staining procedures, let the tissue dry (or come to room temperature if stored at -80C) for about 1 h our at room temperature before beginning staining procedure. Circle entire sample area with a PAP pen (if not available, use rubber cement) F ixation/permeabilization: for MBP in sciatic nerve or mouse tissue the next method works really well o Incubate in 4% paraformaldehyde 10 minutes at room temperature. o Permeabilize 5 -10 minutes with 100% COLD ( -20C) methanol. Rinse 3 times with 1x PBS for 1 0 minutes each.

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194 Blocking: incubate 1 h our at room temperature with 20% goat serum in 1x PBS (can do longer than 2 h ours at 4C). Primary antibody: MBP from Chemicon in 20% goat serum in PBS o/n at 4C (for antibody dilutions check antibody list on Appendix D). Rinse 3 times with 1x PBS for 10 minutes each. Secondary antibody: Alexa fluor goat anti -rabbit 594 nm in 20% goat serum in PBS for 1 h our Also include DAPI (4,6-diamidino -2 phenylindole, dihydrochloride, stock 14.3 mM from Invitrogen) at 1:2000 (if forget to add, it can be done later in 1x PBS for a minimum of 5 minutes, 5 10 minutes is good). Rinse 3 times with 1x PBS for 10 minutes each. Mount cells with Aqua/Poly mount from Polysciences Inc. Mount coverslips over tissue sections. Store slides at 4C in the dark. Paraffin E mbedding Put perfused spinal cord tissue s into small tissue path cassettes from Fisher, and wash in distilled water for 30 minutes. Then follow the next incubation series in the indicated reagents at room temperature with shaking unless otherwise indicated: 70% Ethanol for 15 minutes x 2 80% Ethanol for 15 minutes x 2 95% Ethanol for 15 minutes x 2 100% Ethanol: 4 quick rinses 100% Cedarwood oil for 15 minutes x 3 Pre warmed Cedarwood oil at 37C oven for 2 hours Cedarwood/Methyl Salicylate for 40 minutes x 2 Leave in Cedarwood/Methyl Salicylate overnight Methyl Salicylate for 30 minutes x 3 Paraffin at 60C oven for 30 minutes x 5 Use new paraffin in the last time for 1 hour (tissues cannot be left in hot paraffin for too long, such as overnight .) Embed in 65C paraffin. Blocks can then be stored or sectioned. For sectioning we performed 10 m thick sections and when the tissue s appeared to be dry, we rehydrated them by applying a small piece of paper wet in a solution of 0.01% Tri ton X 100. Deparaffinization and D ehydration of Paraffin S ections. Prior staining of paraffin sections we need to deparaffinize them. After the chosen staining method is performed, sections are dehydrated and permount solution is used to cover the slides.

PAGE 195

195 Deparaffinize slides: incubate slides in a 60C oven till paraffin appears clear. Then pass the slides to a series of solution s in the following order for about 2 minutes in each: histoclear 100% ethanol, 95% ethanol. 70% ethanol, distilled water. Dehydra te by immersing slides for 2 minutes in each of the following solutions in the same order: 70%, 95% and 100% ethanol, and histoclear solution. Mount coverslips with Permount solution. Heammatoxylin and Eosin Staining of Paraffin Sections. Deparaffinize sli des. Immerse slides into filtered Modified Mayers Hematoxylin solution for 5 min utes. Soak in distilled H2O till it turns blue ( or in warm tap water; it takes around 2 min utes ). Check under the miscroscope, if good then go to next step. Immerse in eosin s olution (0.1% eosin, 0.01% phloxine, 74% alcohol, 0,4% glacial acetic acid in water) for 2 minutes, then water for 1 minute. If too dark longer washes can be done in water till the desired color is obtained. Dehydrate and coverslip Silver Staining of Paraffin S ections The day prior staining we need to wash the glassware we will use: slide jars, 200 ml beakers; 250 ml flasks. Wash them in tap water carefully with detergent, rinse carefully. Follow by immersing them in tap water with 20% bleach for at least 2 hours. Then, wash thoroughly in tap water. Place them in U ltraclean overnight (20 m l ultraclean per liter of distilled water). The next day we rinse glassware in running distilled water carefully and perform silver staining as described here. Deparaffinize the slides as previously described. For about 32 sections, prepare 100 ml of ~20% AgNO3 solution (20 g AgNO3 in 100 ml H2O). Place slides in 50 ml of 20% AgNO3 in the dark for 20 minutes for impregnation Titrate the other 50 ml of 20% AgNO3 solution by ammonium hydroxide: add drop by drop until it becomes black and continue till the solution becomes clear again. Wash the slides by distilled water thoroughly, drain the slides carefully Place slides into titrated AgNO3 for 20 minutes in the dark.

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196 Pour the titrated AgNO3 into a beaker and add 2 drops of developer ( 30 ml distilled water, 5 ml of 37% formaldehyde, 1.5 g citric acid, and 1 drop of concentrated HNO3. Mix and return to slide jar. Place the slides into developing solution, normally it takes from 8 to 20 minutes. Be careful to check under a microscope, do not go too dark. Wash by ammonium hydroxide in water (10 drop of ammoni um hydroxide in ~300 ml of distilled water). Wash by distilled water Place slides in 5% Na2S2O3 in water for 30 seconds Wash by distilled water again Dehydrate and coverslip using permount GFAP DAB Staining of Paraffin S ections Deparaffinize sections. The rest of the protocol is performed at room temperature. DAB-pretreatment for 5 min in 0.3% hydrogen peroxide, 10% m ethanol in 1x PBS Wash with 1x PBS for 10 minutes, 3 times. Block in 20% NGS, 0.1% Triton X -100 in 1x PBS for 45 minutes. Primary antibody (rabbit GFAP) incubation in 20% NGS, 0.1% Triton X 100 in 1x PBS overnight. Wash with 1x PBS for 10 minutes x 3 Incubate slides in biotinylated secondary anti -rabbit antibody ( 1:200) in 20% NGS, 0.1% Triton X 100 in 1x PBS for 1 hour. Wash with 1x PBS for 10 minutes once. Make ABC reagent (NOTE MAKE 30minutes BEFORE USE): 2 drops of reagent A into 10 ml of 1x PBS solution, mix and add 2 drops of reagent B, mix well and allow to stand 30 minutes before use Incubation in ABC reagent for 30 minutes Wash with 1x PBS for 10 minutes, x 3 hydrogen peroxide

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197 Put sections in DAB for about 520 minutes (watch for color change, remove i mmediately upon browning) Wash with 1x PBS for 10 minutes, once Counter -stain with haematoxylin (5 minutes) and dehydrate. Use permount to coverslip. Creation of SODMD and SODMD cDNA V ariants The SODMD cDNA was obtained from the genomic version of this mutant protein by RT -PCR. This step was performed by Ms. Hilda Slunt -Brown. Then, by PCR techniques I added XhoI sites at 3 and 5 ends of the cDNA, which allowed for subcloning into a version of pEF BOS vector with a SalI site in the cloning site (XhoI a nd SalI sites are compatible, but ligation is not reversible). For SODMD cDNA variants, the SODMD cDNA was used as a template and new mutations were performed by PCR techniques, in which the primers contain the desire mutation. Additionally, we added to this new versions new restriction sites for cloning into our most updated version of the pEF -BOS vector: NcoI at 5 end and SalI at 3 end. The s e different restriction sites in inserts and the pEF BOS vector allows easier cloning as it allows directional ori entation of inserts. Transfection into HEK293FT C ells for B iochemical A nalysis of Detergent Insoluble A ggregates. HEK293FT cells express the SV40 large T antigen, which allows for episomal replication of the pEF -BOS plasmid as well as strong enhancement of transcription, and were purchased from Invitrogen (Carlsbad, CA, USA). HEK293FT cells were cultured in 60 mm lysine -coated dishes and transiently transfected, at 9095% confluency, with either one SOD1 construct (4 g), or equimolar amounts of two SOD1 constructs (~2 g

PAGE 198

198 each). Transfections were performed using Lipofectamine 2000, following the manufacturers protocol. Prepare the next samples separately: o 4 g DNA + 250 l OptiMEM o 10 l Lipofectamine 2000 + 250 l OptiMEM Then combine both mixes into the same tube and incubate for 20 minutes at room temperature. In the mean time wash cells to transfect with 1x PBS, aspirate and add 2 ml (for untransfected control) or 1.5 ml (for transfected cells) of OptiMEM. Add complex to the cells following the 20 minut e incubation. In some cases, for 24 h our transfections, 3 to 4 hour following addition of DNA complex to the cells, complete DMEM media (with Lglutamine and horse serum) was added (2 ml, total of ~5 ml total media volume). In some other cases, cells were left in OptiMEM media for a total of 24 h ours to enhance transfection efficiency. For 48 h our transfections, cells were left in OptiMEM + DNA complex for 24 h ours at which point it was exchanged by complete cell media (complete DMEM media). Note than when an experiment was performed with a set of conditions the repetitions were carried out following exactly the same conditions. Collect Transfected HEK293FT C ells Cells were harvested 24 or 48 hours after transfection by scraping in 1x PBS. Cell pellets ar e then washed twice in 1.5ml tubes with 1x PBS. When needed, cell pellet s are stored at 20C. Detergent Extraction and Centrifugation Assay of HEK293FT Collected C ell s The method used for HEK293FT cells is the same as decribed for mouse spinal cord ( see Detergent Extraction and Centrifugation Assay for Mouse Spinal Cords). For

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199 cell culture studies, the whole cell pellet is used and lysed by sonication in 200 l of non-ionic detergent (10mM Tris, pH 7.5; 1mM EDTA, pH 8.0; 100mM NaCl; 0.5% NP 40, 1:100 v/v protease inhibitor), 3 times 10 seconds each. This step is equivalent as using 1x TEN and adding equal volumes of detergent extraction buffer 1. The rest of the protocol is followed as described for spinal cord samples, including BCA assay ( see BCA Assay for Determination of Protein Concentration ), SDS -PAGE and western blotting ( see SDS -PAGE Electrophoresis and Western Blotting ). Chapter 3 Methods Creation of SOD1 cDNA E xpression Plasmids All of the WT and mutant human SOD1 cDNAs are expressed from pla smids based on the mammalian pEF -BOS expression vector and cloned through PCR strategies, through either a unique SalI site or a directional 5 NcoI and 3 SalI restriction sites A few of the SOD1 mutants cDNAs used here were previously created into the pEF -BOS system (single SalI site) by Ms. Hilda Slunt -Brown (WT, A4V, G37R, G41D, H46R/H48Q, G85R, G93A, G93C, L126X or L126Z and L126delTT SOD1) (Wang et al., 2005a;Borchelt et al., 1994;Wang et al., 2003) ; however, many other mutants were kindly provided, in the YEp351 yeast ve ctor, by the Hart lab (Dr. Stephen Holloway, University of Texas Health Sciences Center San Antonio, TX) which I subcloned into our pEF -BOS mammalian vector through NcoI and SalI restriction sites (A4T, V14G, V14M, E21G, E21K, G41S, H43R, H80R, G93D, G93R G93S, G93V, L84V, D90A, L144S, V148G and V148I SOD1) The sequences of all mutants in the vectors were verified by automated sequence analysis.

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200 HEK293FT Transfections and Analysis by Detergent Extraction The rest of the methods used in Chapter 3 has been described in Chapter 2 Methods: see HEK293FT T ransfection for D etergent Extraction and C entrifugation Assay Collect Transfected HEK293FT C ells Detergent Extraction and C entrif ugation Assay of HEK293FT Collected C ell P ellets BCA Assay for Determination of P rotein C oncentration, and SDS PAGE Electrophoresis and Western Blotting N2a Cell T ransfection and Analysis of D etergent Insoluble and Soluble Fractions The work of N2a c ells was performed following the same procedures as described for HEK293FT cells. Chapter 4 Methods SOD1 cDNA and GFP Expression Plasmids All SOD1 cDNA constructs used here have been described in Creation of SOD1 cDNA Expression Plasmids from Chapter 3 Methods. Similar techniques were performed to create the mouse WT SOD1 and C111S cDNAs (Karch and Borchelt, 2008) The GFP cDNA was purchased from Clontech (Mountain V iew, CA, USA) and inserted into pcDNA3.1(A) -STOPMyc through a XhoI site prior the vector Myc tag (Invitrogen, Carlsbad, CA, USA) by Ms. Hilda Slunt -Brown. HEK293FT Transfections and Analysis by Detergent Extraction Additional methods used in Chapter 4 ha s been described in Chapter 2 Methods: see HEK293FT T ransfection for D etergent Extraction and C entrifugation Assay (not e that when co -transfections of SOD1 and GFP were performed, equimolar amounts of one SOD1 construct and the GFP construct were used, total 4 g) Collect Transfected

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201 HEK293FT C ells Detergent Extraction and C entrifugation Assay of HEK293FT Collected C ell P ellets (note that for detergent extractions in the presence of high levels BCA Assay for Determination of P rotein C oncentration, and SDS -PAGE Electrophoresis and Western Blotting Analysis of S1 and P2 Fractions by H ybrid Linear I on-trap Fourier -transform Ion C yclotron R esonance M ass Spectrometry (FTMS) Fo r mass spectrometry analysis, six 60 mm culture dishes were co-transfected with WT and G93A h uman SOD1 constructs, then combined and extracted in detergent Ultimately, the S1 and P2 fractions were combined into final volumes of 600 ely. Portions of these fractions were chromatographed by HPLC as previously described (Shaw et al., 2008) SOD1 containing fractions from HPLC chromatography were quickly thawed and 7 l was loaded into nanoelectrospray emitters (Proxeon) for immediate analysis using a nanoelectrospray source equipped mass spectrometer (LTQ -FT Ultra, Thermo, San Jose). Samples were analyzed in positive ion mode with 1.8 kV typically required for stable nanospray performance. Full mass spectra were recorded over a mass range 600-2000 (m/z, Da) with resolution set at 100,000 at m/z = 400. Typically, 50 transients were averaged prior to recording a single MS spectrum. FTMS analyses were repeated twice. The FTMS analyses were performed by Dr. Armando Durazo from the laboratory of Dr. Julian Whitelegge at UCLA.

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202 Chapter 5 Methods SOD1 cDNA Expression Plasmids All SOD1 cDNA constructs used here have been described in Creation of SOD1 cDNA Expression Plasmids from Chapter 3 Methods. HEK293FT Transfections and Analysis by Detergent Extraction The rest of the cell culture methods used in Chapter 5 has been described in Chapter 2 Methods: see HEK293FT T ransfection for D etergent Extraction and C entrifugation Assay (not e that when co -transfections of SOD1 and GFP were performed, equimolar amounts of one SOD1 construct and the GFP construct were used total 4 g) Collect Transfected HEK293FT C ells Detergent Extraction and C entrifugation Assay of HEK293FT Collected C ell P ellets BCA Assay for Determination of P rotein C oncentration, and SDS PAGE Electrophoresis and Western Blotting. Mouse Lines All mouse lines used in this study has been previously described: PrPG37R (Wang et al., 2005b) L126Z (Wang et al., 2005a) SJL WT and Cg WT lines (Gurney et al., 1994) and L76 WT Wong line (Wong et al., 1995) Identification of D ouble Mutant/WT T ransgenic M ice: Genotyping Genotyping and maintenance of individual PrPG37R, L126Z, and L76 WT mouse lines was performed entirely by Ms. Susan E. Fromholt. Extraction of DNA from mouse tails was performed as described in Chapter 2 Methods but d ifferent PCR protocols were used for identification of mutant and WT transgenes in matings resulting from PrPG37R or L126Z crossed to SJL WT, Cg WT, or L76 WT.

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203 PCR p rotocol for i dentification of PrPG37R cDNA Mix the following reagents for each tail DNA sample: o Distilled H2O 20.6 l o 10x PCR Buffer (with MgCl2 from New England Biolabs) 2.5 l o 5 mM dNTPs 0.5 l o 50 M PrP-S 0.2 l o 50 M PrP-AS 0.1 l o Taq DNA Polymerase (from New England Biolabls) 0.1 l o Tail DNA 1.0 l o Total reaction volume 25.0 l PrP-S primer: 5 -GGG ACT ATG TGG ACT GAT GTC GG-3 PrP-AS primer: 5 -CCA AGC CTA GAC CAC GAG AAT GC 3 Set above samples into a thermocycler and perform the next PCR program: o Heat blot pre -start o 94oC for 5 minutes o 94oC for 30 seconds o 60oC for 1 minute x 35 cycles o 72oC for 5 minutes o 72oC for 10 minutes o Hold at 10oC The PrPG37R transgenic band has a size of ~500 bp. For identifying human WT SOD1 from the different PrPG37R x WT matings, I perfor med the PCR Protocol for Identification of Genomic Human SOD1 as described in Chapter 2 Methods. PCR protocol for identification of L126Z and/or WT SOD1 For identifyi ng either L126Z or WT human SOD1 from the different L126Z x WT matings, I perfo r med the PCR Protocol for Identification of Genomic Human SOD1 as described in Chapter 2 Methods, in which the L126Z transgene gives a band of ~500 bp and the WT transgene giv es a band of ~1200 bp. RNA Extraction and Northern Blotting For RNA analysis and northern b lotting of WT lines we followed the same protocols that we described in Chapter 2 Methods: see RNA Extraction from Spinal

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204 Cords of SOD1 Transgenic Mice, Determination of Total RNA Concentration, and Northern Blotting. Protein Analysis and Western Blotting of Mouse Spinal Cords For protein analysis and western blotting of spinal cord samples, we followed protocols that are described in Chapter 2 Methods: see Preparation of Crude Supernantant (Total Protein Fraction) from Mouse Spinal Cords (note that together with TubulinIII, which represents a protein loading control), Det ergent Extraction and Centrifugation Assay Protocol for Mouse Spinal Cords, BCA assay for Determination of Protein Concentration, and SDS -PAGE Electrophoresis and Western Blotting. Analysis of S1 and P2 Fractions by Hybrid Linear Ion-Trap Furier -transform Ion Cyclotron Resonance Mass Specctrometry (FTMS) FTMS analysis of S1 and P2 of spinal cords was performed as described for cell culture samples in Chapter 2 Methods. Here, the spinal cord fractions were prepared by combining three spinal cords into one sample and extracted in detergent to obtain 1.2 ml of S1 and 600 l of P2 fractions. These anal yse s were preformed twice by Dr. Armando Durazo from the laboratory of Dr. Julian Whitelegge at UCLA. Visualization of Reduced and O xidized SOD1 Proteins To observe the redox state of WT and mutant SOD1 proteins, iodoacetamide was included in the extraction buffer (at concentration of 100 mM), which serves as a modifier agent of free SH -groups in order to prevent the random formation of disulfide bonds by air oxidation.

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205 Extracted samples were run onto SDS -PAGE gels as described in SDS -PAGE Electrophoresis and Western Blotting from Chapter 2 Methods, with the following modifications: S amples are boiled in a Laemmli sample buffer lack ing mercaptoethanol. T o allow visualization of the oxidized state, gels were incubated in transfer buffer mercaptoethanol for 10 minutes prior transfer onto membranes (in gel reduction). Alternatively, a in membrane reduction can be done (incubation of memb rane instead of the gel). This step allows for a better binding of the antibody to oxidized forms of the protein (Jonsson et al., 2006a;Zetterstrom et al., 2007) RT -PCR of Mouse WT Lines RNA extracted from WT lines was used in RT -PCR reactions as follows: o 500 ng of RNA o 2x Reaction buffer 25 l o HuSOD -S genomic primer 10 M 1 l o HuSOD -AS genomic primer 10 M 1 l o Enzyme mix (SuperscriptIII RT/Platinum Taq HiFi, from Invitrogen) 1 l o Distilled DEPC treated H2O up to 50 l HuSOD -S genomic primer: 5 -CTA GCG AGT TAT GGC GAC GAA G-3 HuSOD -AS genomic primer: 5 -GAA TGT TTA TTG GGC GAT CCC -3 Set above samples into a thermocycler and perform the next PCR program: o Heat blot pre -start o 55 oC for 30 minutes o 94oC for 2 minutes o 94oC for 15 seconds o 50oC for 30 seconds x 25 cycles o 68oC for 45 seconds o 68oC foor 5 minutes o Hold at 4oC Chapter 6 Methods Creation of SOD1 cDNA Expression Plasmids SOD1 of non-tagged cDNA constructs were described in Creation of SOD1 cDNA Expression Plasmids from Chapter 3 Methods. SOD1 tagged cDNA variants were created from a worm expression SOD1::eYFP pPD30 construct provided from Dr. Rick Morimotos lab. This SOD1::eYFP construct contains a small linker between SOD1 and YFP that was modified by Ms. Hilda Slunt

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206 Brown to introduce a SalI restriction site. This SOD1::YFP DNA was then cloned in to our mammalian pEF -BOS vector. D ifferent SOD1 mutants were introduced in this construct to generate different SOD1::YFP expression plasmids (WT::YFP, A4V::YFP, E::YFP, S134N::YFP, MD::YFP, and MD -G6F -S111Y::YFP). Similarly in some cases, the YFP cDNA was replaced by a Turbo RFP cDNA ( in pTRIPZ empty vector from Open Biosystems, Huntsville, AL) to create WT::RFP, A4V::RFP and D101N::RFP. Transfection into HEK293FT TK -Negative or NIH3T3 C ells for Immunocytochemistry Analysis. Transfection was performed on coverslips previously coated with 0.5 mg/ml poly L -lysine 1x PBS in 12well plates We followed the same protocol as described in Tran s fection into HEK293FT Cel ls for Biochemical Analysis of Detergent -Insoluble Aggregates from Chapter 2 Methods, with the variation that only 2 g of SOD1 cDNA was transfected per sample. Immunocytochemistry of Transfected Cells NOTE: for fluorescent tagged SOD1 transfections that did not need additional staining, only fixation or fixation and DAPI staining was performed. Rinse transfected cells once with 1x PBS. Go directly to step 4 if saponin treatment is not needed. Incubate cells for 30 min utes in 0.01% freshly made saponin (o r alternatively 0.01% freshly made digitonin) in 1x PBS at room temperature. Wash with 1x PBS, 10 min utes once Fix cells in 4% paraformaldehyde in 1x PBS at room temperature for 15 min utes Wash cells 3 times with 1x PBS, 10 min utes each. Permeabilize cell s with cold ( -20C) 100% methanol for 5 minutes and replace by 1x PBS (do not let cells dry).

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207 Block a minimum of 30 minutes in 20% goat serum, 1x PBS (or serum type in which secondary antibody was raised) at room temperature. Incubate cells with primary antibody ( see antibody table in Appendix D for working dilutions) in 10% goat serum, 1x PBS at 4C overnight. Wash cells 3 times with 1x PBS, 10 min utes each. Incubate cells with secondary antibody ( see antibody table in Appendix D for working dilutions) in 10% goat serum, 1x PBS at room temperature for 12 h ours Also add DAPI solution at 1:2000 with secondary antibody incubation (DAPI: 4,6diamidino -2 phenylindole, dihydrochloride, stock 14.3 mM from Invitrogen). NOTE: if adding DAPI is forgotten, it can be added in one of the washes; 5 min utes of DAPI at 1:2000 is enough for good nuclei staining Wash cells 3 times with 1x PBS, 10 min utes each. Mount cells with Aqua/Poly mount from Polysciences Inc. Statistical A nalysis Estimation of A ggregation P ropensit y The aggregation propensity of SOD1 mutants was assessed by comparing the ratio of immunolabeled SOD1 protein in the P2 vs. S1 fractions. Notably, the amount of protein analyzed by immunoblots from these two fractions was not equivalent; in all The intensities of the SOD1 immunoreactive bands in the S1 and P2 fractions establish a ratio value for a particular mutant in a particular immunoblot. To normalize the data from different experiments, each immunoblot that was quantified included a positive control (A4V, with exception of G85R for Figure 43 ), which were used to normalize the data (A4V and G85R show equivalent aggregation propensities and the ratio values for these positive controls was set to 1).

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208 Statistical T ests Statistical analyses included paired and unpaired student t -tests, and correlations studies that were performed as indicated in the corresponding figure or chapter. All these analyses were performed using the GraphPad Prism 5.0 Software (San Diego, CA, USA). In order to perform significant statistical test, each experiment was repeated a minimum of three times. Cloning M ethods A compilation of some of the cloning methods used in this research project is described below. Ligation Ligations are performed using 0.01 pmol of vector and 0.05 pmol of insert and the following reaction components: 1 0mM ATP (no more than 1 month old) 2 l 10x ligase T4 buffer ( from Roche) 2 l Ligase T4 (Roche) 1 l Distil led H2O up to 20 l Incubate ligation reaction 2 h ours at room temperature, or alternatively overnight at 15C. Transformation Thaw DH5 cells on hand and place quickly on ice. Add to 50 l of cells: 4 l of ligation, 2 l of pUC19 control plasmid, or 2 -2.5 g of plasmid DNA. Incubate on ice for 30 min utes Heat shock at 37C for 20-30 seconds. Add 940 l SOC media. Incubate 1 h our at 37C with shaking.

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209 While incubating, plate 100 l of 2% X gal on carbenicillin plates and incubat e at 37C till the 1 hour incubation is over (STEP FOR pBlueScript ONLY). Briefly spin down samples (EXCEPT CONTROL). Eliminate ~850l and resuspend pellet in remaining ~150l media and plate all (PLATE 100l CONTROL). Incubate plate s overnight. Minipreps Pick a (white) colony from transfermed plates into 2.5 ml media with carbenicillin and grow overnight in a 37 C shaking incubator. Transfer ~1.5 ml of overnight growth to a 1.5 ml microfuge tube and spin 30 seconds at full speed. (Use a little less media i f using Circle -Grow, use a little more if using LB.) Discard supernatant. Resuspend cell pellet in 100 l of cold Solution I (50 mM Tris, pH 8; 5 mM EDTA, pH 8; 1% glucose) by vortexing gently. Incubate 2-5 minutes at room temperature. Add 200 l of Soluti on II (0.2 M NaOH, 1 % SDS, make fresh before each use). Mix gently by inverting tube three times. Incubate on ice 25 minutes. Add 150 l Solution III (3M K/5M Ac: for 100 ml mix 29.44 grams of potassium acetate, 11.5 ml of glacial acetic acid in distilled water). Mix thoroughly by inverting tube on vortexer. Incubate on ice 25 minutes. Pellet cell debris for 2 minutes at full speed in microfuge. Transfer supernatant to a new tube and discard the pellet. Optional: Phenol -Chloroform extract. (This is important only when DNA is to be sequenced.). Precipitate DNA by adding 1 ml 100% ethanol. Vortex and incubate at least 2 or more minutes at 20 C (About 30 minutes is good. This is a good place to stop, if necessary. Store samples at -20 C.). Pellet DN A for 5 minutes at full speed in microfuge. Aspirate supernatant. Rinse pellet with 150 l of 70% ethanol. Vortex briefly and spin for 2 minutes at full speed in microfuge. Aspirate supernatant. Allow pellet to dry for 15 20 minutes at room temperature. R esuspend pellet in 50 l distilled H2O.

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210 NOTE: an alternative plasmid DNA isolation protocol can be use following the Fast Plasmid Miniprep kit, especially when this DNA is to be sequenced. Digestion of Plasmid DNA The following reaction works well to diges t about 2 l of miniprep DNA, or 1 g of total DNA (in the latter case use double of restriction enzymes) : 10x NEBuffer 2.5 l RNAase A 1.0 l NEB Restriction enzyme 0.5 l of each ( 1 l for 1 g DNA or 10 l miniprep) Distilled H2O up to 20 .0 l Incubate digestion reaction 1 h our at the corresponding temperature, usually it is at 37C. Plasmid P reps Innoculate 50 ml of autoclaved Circle-Grow with carbenicillin (100 ng/ml) and with miniprep culture. Grow overnight at 37 C in shaking incubator. (For large plasmids, such as PrP, use 100 ml LB). Spin cells down for 5 minutes at 5200 rpm in HS 4 rotor in Superspeed centrifuge at 4 C. Pour off supernatant and resuspend cells in 7.5 ml ice cold Solution I (25 mM Tris, pH 8; 50 mM EDTA, pH 8; 1% glucose; s terile filter) with a pinch of lysozyme added just prior used to ~1 mg/ml. Incubate on ice 510 minutes. Add 15.75 ml Solution II (0.2 M NaOH; 1% SDS, make fresh from stock solutions before each use). Invert three times to mix gently. Incubate on ice 510 minutes. Add 11.6 ml Solution III (3M K/5M Ac: for 100 ml mix 29.44 grams of potassium acetate, 11.5 ml of glacial acetic acid in distilled water). Shake vigorously and incubate on ice for 10 -15 minutes. Spin tubes for 10 minutes at 6000 rpm in HS -4 in Sup erspeed centrifuge at 4 C. Filter supernatant through a Kimwipe funnel (alternatively cheese cloth can be used). Add 2 volumes 100% ethanol. Shake to mix and incubate 5-10 minutes at room temperature. Spin down DNA for 10 minutes at 6000 rpm in HS -4 in Superspeed centrifuge at 4 C.

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211 Pour off supernatant. Allow pellet to dry at room temperature for 1520 minutes. Resuspend pellet in 4 ml distilled H2O, (or TE). Be sure the DNA is completely in solution. Add 4.25 grams of cesium chloride solution (CsCl solution: 42.5 grams CsCl and 40 ml distilled H2O). Mix well to dissolve completely. Pour into ultracentrifuge tube. Top with CsCl solution. Remove 100 l solution from tube and replace with 100 l ethidium bromide (10 mg/ml). Cap and seal tube, balance tubes and place into TV865 rotor immediately. Spin in ultracentrifuge at 20 C, at 45,000 rpm overnight. Tap lower band with a needle and syringe. Add 3 volumes of salt -saturated isopropanol (200 ml isopropanol, 200 ml 5 M NaCl, shake to mix, and allow phases to separate; Add NaCl crystals until it no longer dissolves; Use the top layer only) in the syringe. Shake thoroughly and allow phases to separate. Bend needle and push off the top layer, which is pink. Repeat two more times (till layers are clear). Transfer aqueous phase to a new tube. Add two volumes distilled H2O. Mix. Add two times the resulting volume 100% ethanol. Mix. Incubate at 20 C, for 30 min to overnight. Spin for 20 minutes at 7000 rpm in HS 4 rotor in Superspeed centrifuge at 4 C. Pour off supernatant. Add 2-5 ml of 70% ethanol. Spin for 10 minutes at 7000 rpm in HS 4 rotor in Superspeed centrifuge at 4 C. Pour off supernatant. Allow pellet to dry. Resuspend pellet in 300 l TE or distilled H2O. Check concentration by measuring OD 260 nm of a 1: 100 dilution made in 50 mM Tris, pH 8. Optional: If you dont need Cesium quality DNA, stop before adding CsCl to DNA and add 4 ml of TNES (50 mM Tris, pH 7.5, 100 mM EDTA, 400 mM NaCl, 0.5% SDS).

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212 Add RNase A to 25 g/ml (20 l of a 10 mg/ml stock) and i n cubate 30 minutes at 37 C. Add Proteinase K to 100 g/ml (40 l of a 20 mg/ml stock) and incubate 30 minutes at 37 C. Phenol -Chloroform extraction Precipitate aqueous phase with two volumes 100% ethanol. Resuspend DNA in 300 l TE or distilled H2O. OD at 1:100 dilution. Phenol-C holoform E xtraction Bring the volume of DNA to purify (to eliminate enzymes, etc) to 100 l (with 1x TEN) and transfer your sample to a 1.5 ml eppendorff tube. (60 l of 1x TEN to 40 l of PCR product). Add 100 l of Phenol -Chloroform solution (1:1; mix well 5 ml phenol with 5 ml of chloroform, and centrifuge at 5000 rpm for 5 minutes. Do NOT use upper layer, keep at 4C and discard when the solution gets too pink). Vortex. Centrifuge 3 minutes at maximum speed. Take upper layer to a new eppendorff and add 1 l of tRNA (yeast 10mg/ml). Add 100 l of 5M ammonium acetate (NH4OAc: 19.27grams in 50 ml of distilled water; molecular weight 77.08). Vortex well. Add 2.5 volumes of 100% ethanol (500l). Vortex. Leave on ice (good stop point) at least 10 minutes (usually leave at 20C). Spin down 5 minutes at maximum speed. Discard supernatant. Rinse pellet with 100 l of 70% ethanol and spin down again. Discard supernatant and air dry pellet for 1520 minutes. Resuspend pelleted DNA in 16 l of distilled H2O (variable). Agarase D igestion Weight empty eppendorff Cut out band from low melting point agarose gel and put into eppendorff. Weight band in tube and calculate net weight of band [weight(band+tube) weight(tube)] Assume 1

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213 Add 1 ample: to a 0.12 g ram band ad d 12 Melt band and buffer at 65C, 10 min utes (or till dissolved). Equilibrate band at 42C, for 10 min utes. Add 1 mg band Incubate at 42C, for 1 h our Inactivate enzyme at 95C, for 2 mi nutes. Other U seful P rotocols Freez ing C ells Wash cells from an almost confluent 75 cm2 flask with 5 ml 1x dPBS. Trypsinize with 1 ml of Trypsin. Add media up to 9.5 ml. Take out 9 ml to a 15 ml falcon tube and spin down at 500 rpm for 5 minutes. To the rest 1.5 ml cells + media, add 8.5 ml or 18.5 ml of media to propagate (1:10 or 1:20 dilutions respectively). Resuspend pellet in 5 ml media with 5% DMSO (4.75 ml media + 250 l DMSO). Store at 80C for at least a day and then put cells in liquid nitrogen. Thaw cells after a while to check they grow fine. Thawing C ells Thaw cells in water bath at 37C, then transfer cells to a 15 ml falcon tube and add media up to 10 ml. Spin down cells to get rid of the DMSO (5000 rpm, 5 minutes). Resuspend cell pellet in 5 ml of media and put in 25 cm2 flask. Transfer to a 75 cm2 flask when they get confluent in the 25 cm2 flask.

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214 APPENDIX D ANTIBODY LIST Table D -1. List of primary antibodies. Species Antigen Source Dilution WB Dilution ICC/IHC Rabbit hSOD1 (Borchelt et al., 1994) 1:2500 1:500 Rabbit h/m SOD1 (Pardo et al., 1995) 1:2500 1:500 Rabbit LGP120 Gift from Dr. William Dunn Jr. 1:2000 1:250 Rabbit GFAP Dako, Cat# Z 0334 1:500 Rabbit MBP Gift from Dr. Notterpek ( Chemicon ) 1:500 Mouse Dynactin p50 BD Biosciences, Cat# 611003 1:500 Rabbit hUbiquitin Dako, Cat# Z0458 1:500 Rabbit Tubulin III Covance, Cat# PRB 435P 1:500 Rabbit VDAC Abcam, Cat# ab7783 1:500 WB: Western blot; ICC: Immunocytochemistry; IHC: immunohistochemistry; L GP 120: l ysosome glycoprotein 120; VDAC: Voltage Dependent Anion Channel (commonly known as porin); MBP: Myelin basic protein; GFAP: glial fibrillary acidic protein. Table D -2. List of secondary antibodies. Anti body Source Dilution WB Dilution ICC/IHC Alexa Fluor goat anti rabbit 594 nm Invitrogen, Cat# A11012 1:2000 Goat anti rabbit IgG KPL, Cat# 074 1516 1:5000 WB: Western blot; ICC: Immunocytochemi stry; IHC: immunohistochemistry.

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215 APPENDIX E CO -LOCALIZATION STUDIES OF SOD1::YFP INCLUSIONS Previous studies have determined association of mutant SOD1 with the cytosolic face of mitochondria (Liu et al., 2004). Thus, we sought to determine whether any of these SOD1::YFP fusion proteins appear to accumulate in areas with abundant mitoch ondrial organelles. Expression of SOD1::YFP mutant proteins for 24 hours and staining of such cells with an antibody that recognizes the voltage dependent anion channel (VDAC) of mitochondria, does not appear to indicate strong co localizations of WT or mu tant SOD1 inclusions with mitochondria, nor mitochondrial reorganization derived from mutant SOD1::YFP expression (Figure E -1). Interestingly, apparent accumulations of mitochondria were found in different cells, but in areas devoid of SOD1::YFP inclusions (Figure E 1, open arrowheads). Mitochondrial immunoreactivity appeared to colocalize some with WT::YFP or D101N::YFP proteins (Figures E -1A to 11L). Only in one case we found a cell with an abnormal high number of mitochondrial immunoreactivity, however this cell did not express SOD1::YFP (Figures E -1G and E 1H, filled arrowhead). Thus, our data suggest that SOD1::YFP inclusions do not co localize with mitochondria, however more extensive analysis at higher magnifications may be needed. Ubiquitin-positive inclusions are a common feature in ALS patients. However, ubiquitin and SOD1 co-staining does not appear to take place (Shibata et al. 1996a). Studies in ALS mouse models expressing mutant SOD1 indicate that insoluble SOD1 is partly oligoubiquitinated at disease endstage (Basso et al., 2006) Additionally, other studies in animal models indicate a reduction in proteasome activity in symptomatic mice (Cheroni et al., 2005) In cell culture, studie s have previously shown association of

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216 mutant SOD1, but not WT, with components of the ubiquitin proteasome system (Urushitani et al., 2002) indicating that they might be targeted for proteasomal degradation. In order to study whether our cell model expressing mutant SOD1::YFP also contain ubiquitin positive inclusions, we stained cells expressing fusion SOD1 proteins with an antibody that recognizes ubiquitin. We did not observe ubiquitin inclusions of similar size to SOD1::YFP inclusions (Figure E -2). Additionally, cells expressing a SOD1::YFP protein appeared to be less immunoreactive to ubiquitin (Figures E -2C, E 2D, E -2G, and E -2H), while when bigger ubiquitin positive areas were found, they did not seem to co-localize with SOD1::YFP (Figures E -2K and E -2L). Figure E1. SOD1::YFP inclusions do not co loc alize with mitochondria. AL) HEK293FT transfected for 24 h ours with SOD1::YFP fusion constructs and stained for mitochondrial membrane protein VDAC following a staining procedure as described for Figure 6 1. Pictures were taken using a spinning disk confo cal microscope, with a 6 0x water immersion objective, b ars 10 m. MITOCHONDRIA VDAC DAPI WT::YFP MERGE A D B C DAPI A4V::YFP DAPI D101N::YFP E H F G I L J K 2x 2x 2x 2x 2x 2x VDAC VDAC MERGE MERGE

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217 Figure E2. Ubiquitin does not concentrate to SOD1::YFP inclusions. A-L) HEK293FT cells transfected for 24 h ours with SOD1::YFP fusion constructs and stained like explained on Figure E-1 but stained here with an antibody recognizing ubiquitin. Pictures were taken using a spinning disk confocal microscope, with a 6 0x water immersion objective, b ars 10 m. Thus, it is unclear the role that ubiquitination may play on SOD1::YFP inclusions and further studies should be conducted. In our hands, we did not observe interactions of ubiquitin with either WT or mutant SOD1 when tagged with YFP. An alternative mode of degradation of mutant SOD1::YFP proteins could involve lysosomes. Thus, to determine whether SOD1::YFP inclusions may concentrate into lysosomal compartments, we performed similar immunostaining techniques using an antibody that recognizes LGP120, a lysosomal transmembrane glycoprotein. Similar to mitochondrial staining, WT::YFP proteins were the only ones that we observed to clearly co localize with lysosomes, while mutant SOD1::YFP inclusions did not (Figure E 3). DAPI WT::YFP UBIQUITIN MERGE A D B C DAPI A4V::YFP MERGE E H F G DAPI D101N::YFP UBIQUITIN MERGE I L J K 2x 2x 2x 2x 2x 2x UBIQUITIN

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218 Figure E 3. Mutant SOD1::YFP inclusions do not localize within lysosomes AL) HEK293FT cells transfected for 24 h ours wit h SOD1::YFP constructs and stained as described in Figure E1, using the lysosome marker LGP120. Pictures were taken using a spinning disk confocal microscope, with a 6 0x water immersion objective, b ars 10 m. White arrowhead in K and L indicate strong imm unoreactivity to LGP120, in an area free of SOD1::YFP protein. Additional studies suggest that interactions of SOD1 with components of the axonal transport are required for inclusion formation (Strom et al., 2008) In particular, overexpression of dynactin protein p50 abolishes dynein-SOD1::GFP protein interactions. In our case, we wanted to see whether interactions of p50 dynactin complex with SOD1::YFP inclusions are obvious in our cell culture system. However, we did not observe clear co -localization, and it appeared that a lower immunoreacti vity of p50 was present in cells containing SOD1::YFP inclusions (Figure E -4). Thus, our LYSOSOME LGP120 DAPI WT::YFP MERGE A D B C DAPI A4V::YFP MERGE LYSOSOME LGP120 DAPI D101N::YFP E H F G I L J K 2x 2x 2x 2x 2x 2x LGP120 MERGE

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21 9 data does not provide indication of interactions of SOD1::YFP inclusions with the p50 protein. However, we are aware that the data presented here is too limited to draw conclusions, and additional experiments should be performed to further study this possibility. Figure E 4. Lower expression of dynactin protein p50 in SOD1::YFP containing cells. A L) HEK293FT cells transfected for 24 h ours with SOD1::YFP constructs and stained as described in Figure E 1 using the dynactin marker p50 Pictures were taken using a spinning disk confocal microscope, with a 6 0x water immersion objective, b ars 10 m. In all the described immunofluorescence studies we did not observe co loc alization with SOD1::YFP inclusions. Additionally, we did not see an obvious redistribution of any of the markers that we employed. This can be confirmed when comparing the staining of all these markers in SOD1::YFP expressing cells with untransfected cell s (Figure E 5). DAPI WT::YFP DYNACTIN P50 MERGE A D B C DAPI A4V::YFP DYNACTIN P50 MERGE DAPI D101N::YFP DYNACTIN MERGE E H F G I L J K 2x 2x 2x 2x 2x 2x

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220 Figure E 5. HEK293FT untransfected cells present similar staining pattern of cellular markers as SOD1::YFP transfected cells. A L) HEK293FT cells untransfected and stained as described in Figure E1 after 24 hour of culture using markers for mitochondria (A -C), ubiquitin (D -F), lysosome (G -I), and dynactin (J -L). Pictures were taken using a spinning disk confocal microscope, with a 6 0x water immersion objective, b ars 10 m. Although not extensive studies are presented here, we sus pect the lack of strong co localizations due to the size of the inclusions of SOD1::YFP fusion proteins. Only in certain cases we observed colocalization of WT::YFP with either mitochondria (see Figure E 1) or lysosomes (see Figure E -2). However, we do no t have enough data to DAPI LYSOSOME LGP120 MERGE DAPI DYNACTIN P50 MERGE DAPI MITOCHONDRIA VDAC MERGE DAPI UBIQUITIN MERGE A B C D E F G H I J K L

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221 demonstrate whether such interactions with non-inclusion forming WT::YFP occur on the surface of the organelle, or whether the fusion protein can be internalized. It appears, however, that even WT::YFP has a hard time to get into the nucleus, while YFP is highly expressed there. This suggests that SOD1::YFP proteins are likely not easily being transported inside organelles. Then, interactions may occur at the levels of more soluble states of the protein (not forming inclusions). Thus in the case of SOD1::YFP inclusions, the lack of association with organelles could be explained by their rapid ability to form such structures, which would not allow for interactions to occur at the noninclusion level

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222 LIST OF REFERENCES Aisen, M.L., D.Sevilla, L.Edelstein, and J.Blass. 1996. A double-blind placebo -controlled study of 3,4diaminopyridine in amytrophic lateral sclerosis patients on a rehabilitation unit. J. Neurol. Sci. 138:9396. Aisen, M.L., D.Sevill a, G.Gibson, H.Kutt, A.Blau, L.Edelstein, J.Hatch, and J.Blass. 1995. 3,4diaminopyridine as a treatment for amyotrophic lateral sclerosis. J. Neurol. Sci. 129:21 -24. Al -Chalabi, A., P.M.Andersen, B.Chioza, C.Shaw, P.C.Sham, W.Robberecht, G.Matthijs, W.Cam u, S.L.Marklund, L.Forsgren, G.Rouleau, N.G.Laing, P.V.Hurse, T.Siddique, P.N.Leigh, and J.F.Powell. 1998. Recessive amyotrophic lateral sclerosis families with the D90A SOD1 mutation share a common founder: evidence for a linked protective factor. Hum. Mo l. Genet. 7:20452050. Al -Chalabi, A., M.D.Scheffler, B.N.Smith, M.J.Parton, M.E.Cudkowicz, P.M.Andersen, D.L.Hayden, V.K.Hansen, M.R.Turner, C.E.Shaw, P.N.Leigh, and R.H.Brown, Jr. 2003. Ciliary neurotrophic factor genotype does not influence clinical phenotype in amyotrophic lateral sclerosis. Ann. Neurol. 54:130-134. Albo, F., M.Pieri, and C.Zona. 2004. Modulation of AMPA receptors in spinal motor neurons by the neuroprotective agent riluzole. J. Neurosci. Res. 78:200207. Alexander, M.D., B.J.Traynor, N .Miller, B.Corr, E.Frost, S.McQuaid, F.M.Brett, A.Green, and O.Hardiman. 2002. "True" sporadic ALS associated with a novel SOD -1 mutation. Ann. Neurol. 52:680-683. Andersen, P.M. 2006. Amyotrophic lateral sclerosis associated with mutations in the CuZn sup eroxide dismutase gene. Curr. Neurol. Neurosci. Rep. 6:37 46. Andersen, P.M., P.Nilsson, M.L.Keranen, L.Forsgren, J.Hagglund, M.Karlsborg, L.O.Ronnevi, O.Gredal, and S.L.Marklund. 1997. Phenotypic heterogeneity in motor neuron disease patients with CuZn-su peroxide dismutase mutations in Scandinavia. Brain 120 ( Pt 10):1723-1737. Andersen, P.M., P.Nilsson, V.la-Hurula, M.L.Keranen, I.Tarvainen, T.Haltia, L.Nilsson, M.Binzer, L.Forsgren, and S.L.Marklund. 1995. Amyotrophic lateral sclerosis associated with homozygosity for an Asp90Ala mutation in CuZn-superoxide dismutase. Nat. Genet. 10:61-66. Andersen, P.M., K.B.Sims, W.W.Xin, R.Kiely, G.O'Neill, J.Ravits, E.Pioro, Y.Harati, R.D.Brower, J.S.Levine, H.U.Heinicke, W.Seltzer, M.Boss, and R.H.Brown, Jr. 2003. Si xteen novel mutations in the Cu/Zn superoxide dismutase gene in amyotrophic lateral sclerosis: a decade of discoveries, defects and disputes. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 4:6273.

PAGE 223

223 Antonyuk, S., J.S.Elam, M.A.Hough, R.W.Strange, P.A .Doucette, J.A.Rodriguez, L.J.Hayward, J.S.Valentine, P.J.Hart, and S.S.Hasnain. 2005. Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg. Protein Sci. 14:1201 -1213. Aoki, M., K.Abe, K.Houi, M.Ogasawara, Y.Matsubara, T.Kobayashi, S.Mochio, K.Narisawa, and Y.Itoyama. 1995. Variance of age at onset in a Japanese family with amyotrophic lateral sclerosis associated with a novel Cu/Zn superoxide dismutase mutation. Ann. Neurol. 37:676679. Aoki, M., M.Ogasawara, Y.Matsuba ra, K.Narisawa, S.Nakamura, Y.Itoyama, and K.Abe. 1993. Mild ALS in Japan associated with novel SOD mutation. Nat. Genet. 5:323324. Arisato, T., R.Okubo, H.Arata, K.Abe, K.Fukada, S.Sakoda, A.Shimizu, X.H.Qin, S.Izumo, M.Osame, and M.Nakagawa. 2003. Clini cal and pathological studies of familial amyotrophic lateral sclerosis (FALS) with SOD1 H46R mutation in large Japanese families. Acta Neuropathol. (Berl) 106:561 -568. Arrasate, M., S.Mitra, E.S.Schweitzer, M.R.Segal, and S.Finkbeiner. 2004. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431:805-810. Basso, M., T.Massignan, G.Samengo, C.Cheroni, B.S.De, M.Salmona, C.Bendotti, and V.Bonetto. 2006. Insoluble mutant SOD1 is partly oligoubiquitinated in amyot rophic lateral sclerosis mice. J. Biol. Chem. 281:33325-33335. Beal, M.F., R.J.Ferrante, S.E.Browne, R.T.Matthews, N.W.Kowall, and R.H.Brown, Jr. 1997. Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis. Ann. Neurol. 42:644 -654. Beck, M., P.Flachenecker, T.Magnus, R.Giess, K.Reiners, K.V.Toyka, and M.Naumann. 2005. Autonomic dysfunction in ALS: a preliminary study on the effects of intrathecal BDNF. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 6:100103. Beck, M., M.Sendtner, and K.V.Toyka. 2007. Novel SOD1 N86K mutation is associated with a severe phenotype in familial ALS. Muscle Nerve 36:111114. Beckman, G. and A.Pakarinen. 1973. Superoxide dismutase. A population study. Hum. Hered. 23:346351. Beckman J.S., M.Carson, C.D.Smith, and W.H.Koppenol. 1993. ALS, SOD and peroxynitrite. Nature 364:584. Beghi, E., A.Chio, M.Inghilleri, L.Mazzini, A.Micheli, G.Mora, M.Poloni, R.Riva, L.Serlenga, D.Testa, and P.Tonali. 2000. A randomized controlled trial of

PAGE 224

224 reco mbinant interferon beta-1a in ALS. Italian Amyotrophic Lateral Sclerosis Study Group. Neurology 54:469-474. Bence, N.F., R.M.Sampat, and R.R.Kopito. 2001. Impairment of the ubiquitinproteasome system by protein aggregation. Science 292:15521555. Bendotti C. and M.T.Carri. 2004. Lessons from models of SOD1linked familial ALS. Trends Mol. Med. 10:393 -400. Bertini, I., M.Piccioli, M.S.Viezzoli, C.Y.Chiu, and G.T.Mullenbach. 1994. A spectroscopic characterization of a monomeric analog of copper, zinc superoxide dismutase. Eur. Biophys. J. 23:167176. Bertram, L. and R.E.Tanzi. 2008. Thirty years of Alzheimer's disease genetics: the implications of systematic meta analyses. Nat. Rev. Neurosci. 9:768778. Bird, T.D. 2008. Genetic aspects of Alzheimer disease. Genet. Med. 10:231239. Boillee, S., V.C.Vande, and D.W.Cleveland. 2006a. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52:39 -59. Boillee, S., K.Yamanaka, C.S.Lobsiger, N.G.Copeland, N.A.Jenkins, G.Kassiotis, G.Kollias, and D.W.Cl eveland. 2006b. Onset and progression in inherited ALS determined by motor neurons and microglia. Science 312:13891392. Borchelt, D.R. 1998. Metabolism of presenilin 1: influence of presenilin 1 on amyloid precursor protein processing. Neurobiol. Aging 19 :S15S18. Borchelt, D.R., M.Guarnieri, P.C.Wong, M.K.Lee, H.S.Slunt, Z.S.Xu, S.S.Sisodia, D.L.Price, and D.W.Cleveland. 1995. Superoxide dismutase 1 subunits with mutations linked to familial amyotrophic lateral sclerosis do not affect wild -type subunit function. J. Biol. Chem. 270:3234 -3238. Borchelt, D.R., M.K.Lee, H.S.Slunt, M.Guarnieri, Z.S.Xu, P.C.Wong, R.H.Brown, Jr., D.L.Price, S.S.Sisodia, and D.W.Cleveland. 1994. Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity. Proc. Natl. Acad. Sci. U. S. A 91:8292 -8296. Borchelt, D.R., P.C.Wong, M.W.Becher, C.A.Pardo, M.K.Lee, Z.S.Xu, G.Thinakaran, N.A.Jenkins, N.G.Copeland, S.S.Sisodia, D.W.Cleveland, D.L.Price, and P.N.Hoffman. 1998. Axonal tr ansport of mutant superoxide dismutase 1 and focal axonal abnormalities in the proximal axons of transgenic mice. Neurobiol. Dis. 5:2735. Boukaftane, Y., J.Khoris, B.Moulard, F.Salachas, V.Meininger, A.Malafosse, W.Camu, and G.A.Rouleau. 1998. Identificat ion of six novel SOD1 gene mutations in familial amyotrophic lateral sclerosis. Can. J. Neurol. Sci. 25:192196.

PAGE 225

225 Brooks, B.R. 1994. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinical limits of amyotrophic lateral s clerosis" workshop contributors. J Neurol Sci 124 Suppl:96 -107. Brown, R.H., Jr., S.L.Hauser, H.Harrington, and H.L.Weiner. 1986. Failure of immunosuppression with a tento 14-day course of highdose intravenous cyclophosphamide to alter the progression of amyotrophic lateral sclerosis. Arch. Neurol. 43:383-384. Bruij n, L.I., M.W.Becher, M.K.Lee, K.L.Anderson, N.A.Jenkins, N.G.Copeland, S.S.Sisodia, J.D.Rothstein, D.R.Borchelt, D.L.Price, and D.W.Cleveland. 1997. ALS -linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD 1 -containing inclusions. Neuron 18:327338. Bruijn, L.I., M.K.Houseweart, S.Kato, K.L.Anderson, S.D.Anderson, E.Ohama, A.G.Reaume, R.W.Scott, and D.W.Cleveland. 1998. Aggregation and motor neuron toxicity of an ALS -linked SOD1 mutant independent from wild -type SOD1. Science 281:1851 -1854. Bruijn, L.I., T.M.Miller, and D.W.Cleveland. 2004. Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu. Rev. Neurosci. 27:723749. Bush, A.I. 2002. Is ALS caused by an altered oxidative activity of mutant superoxide dismutase? Nat. Neurosci. 5:919 -920. Calamai, M., N.Taddei, M.Stefani, G.Ramponi, and F.Chiti. 2003. Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins. Biochemistry 42:1507815083. Chang-H ong, R., M.Wada, S.Koyama, H.Kimura, S.Arawaka, T.Kawanami, K.Kurita, T.Kadoya, M.Aoki, Y.Itoyama, and T.Kato. 2005. Neuroprotective effect of oxidized galectin-1 in a transgenic mouse model of amyotrophic lateral sclerosis. Exp. Neurol. 194:203 211. Charcot JM and A J. 1869. Deux cas d'atrophie musculaire progressive avec lsions de la substance grise et des faiseaux antrolatraux de la moelle pinire. Arch Physiol Neurol Path. 744-754. Chattopadhyay, M., A.Durazo, S.H.Sohn, C.D.Strong, E.B.Gralla, J.P.Whitelegge, and J.S.Valentine. 2008. Initiation and elongation in fibrillation of ALS linked superoxide dismutase. Proc. Natl. Acad. Sci. U. S. A 105:18663 18668. Chen, Y.Z., C.L.Bennett, H.M.Huynh, I.P.Blair, I.Puls, J.Irobi, I.Dierick, A.Abel, M.L.Ke nnerson, B.A.Rabin, G.A.Nicholson, M.uer -Grumbach, K.Wagner, J.P.De,

PAGE 226

226 J.W.Griffin, K.H.Fischbeck, V.Timmerman, D.R.Cornblath, and P.F.Chance. 2004. DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am. J. Hum. Genet 74:1128-1135. Cheroni, C., M.Peviani, P.Cascio, S.Debiasi, C.Monti, and C.Bendotti. 2005. Accumulation of human SOD1 and ubiquitinated deposits in the spinal cord of SOD1G93A mice during motor neuron disease progression correlates with a decrease of prot easome. Neurobiol. Dis. 18:509-522. Chio, A., A.Cucatto, A.A.Terreni, and D.Schiffer. 1998. Reduced glutathione in amyotrophic lateral sclerosis: an open, crossover, randomized trial. Ital. J. Neurol. Sci. 19:363 -366. Chio, A., B.J.Traynor, F.Lombardo, M.F imognari, A.Calvo, P.Ghiglione, R.Mutani, and G.Restagno. 2008. Prevalence of SOD1 mutations in the Italian ALS population. Neurology 70:533537. Chiti, F., M.Calamai, N.Taddei, M.Stefani, G.Ramponi, and C.M.Dobson. 2002. Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. Proc. Natl. Acad. Sci. U. S. A 99 Suppl 4:16419 -16426. Chou, C.M., C.J.Huang, C.M.Shih, Y.P.Chen, T.P.Liu, and C.T.Chen. 2005. Identification of three mutations in the Cu,Zn-super oxide dismutase (Cu,Zn -SOD) gene with familial amyotrophic lateral sclerosis: transduction of human Cu,ZnSOD into PC12 cells by HIV 1 TAT protein basic domain. Ann. N. Y. Acad. Sci. 1042:303313. Clinical Trials. 2009. U. S. National Institute of Health C linical Trials, 08/2009. www.clinicaltrials.gov. Cozzolino, M., I.Amori, M.G.Pesaresi, A.Ferri, M.Nencini, and M.T.Carri. 2008a. Cysteine 111 Affects Aggregation and Cytotoxicity of Mutant Cu,Zn-superoxide Dismutase Associated with Familial Amyotrophic Lat eral Sclerosis. J. Biol. Chem. 283:866 -874. Cozzolino, M., A.Ferri, and M.T.Carri. 2008b. Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications. Antioxid. Redox. Signal. 10:405443. Crapo, J.D., T.Oury, C.Rabou ille, J.W.Slot, and L.Y.Chang. 1992. Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc. Natl. Acad. Sci. U. S. A 89:1040510409. Crow, J.P., J.B.Sampson, Y.Zhuang, J.A.Thompson, and J.S.Beckman. 1997. Decreased zinc aff inity of amyotrophic lateral sclerosis associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite. J. Neurochem. 69:1936 -1944.

PAGE 227

227 Cudkowicz, M.E., D.kennaYasek, P.E.Sapp, W.Chin, B.Geller, D.L.Hayden, D.A.Schoe nfeld, B.A.Hosler, H.R.Horvitz, and R.H.Brown. 1997. Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann. Neurol. 41:210221. Cudkowicz, M.E., J.M.Shefner, D.A.Schoenfeld, R.H.Brown, Jr., H.Johnson, M.Qureshi, M.Jacobs, J.D.Rothstein, S.H.Appel, R.M.Pascuzzi, T.D.Heiman Patterson, P.D.Donofrio, W.S.David, J.A.Russell, R.Tandan, E.P.Pioro, K.J.Felice, J.Rosenfeld, R.N.Mandler, G.M.Sachs, W.G.Bradley, E.M.Raynor, G.D.Baquis, J.M.Belsh, S.Novella, J.Goldstein, and J.Hulihan. 2003. A randomized, placebocontrolled trial of topiramate in amyotrophic lateral sclerosis. Neurology 61:456464. Cudkowicz, M.E., J.M.Shefner, D.A.Schoenfeld, H.Zhang, K.I.Andreasson, J.D.Rothstein, and D.B.Drachman. 2006. Trial of celecoxib in amyotrop hic lateral sclerosis. Ann. Neurol. 60:2231. Dal Canto, M.C. and M.E.Gurney. 1994. Development of central nervous system pathology in a murine transgenic model of human amyotrophic lateral sclerosis. Am. J. Pathol. 145:1271 -1279. Dal Canto, M.C. and M.E.G urney. 1997. A low expressor line of transgenic mice carrying a mutant human Cu,Zn superoxide dismutase (SOD1) gene develops pathological changes that most closely resemble those in human amyotrophic lateral sclerosis. Acta Neuropathol. (Berl) 93:537550. Danciger, E., N.Dafni, Y.Bernstein, Z.Laver Rudich, A.Neer, and Y.Groner. 1986. Human Cu/Zn superoxide dismutase gene family: molecular structure and characterization of four Cu/Zn superoxide dismutase-related pseudogenes. Proc. Natl. Acad. Sci. U. S. A 83 :36193623. Deng, H.X., A.Hentati, J.A.Tainer, Z.Iqbal, A.Cayabyab, W.Y.Hung, E.D.Getzoff, P.Hu, B.Herzfeldt, R.P.Roos, and 1993. Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science 261:1047 -1051. Deng, H.X., H.Jia ng, R.Fu, H.Zhai, Y.Shi, E.Liu, M.Hirano, M.C.Dal Canto, and T.Siddique. 2008. Molecular dissection of ALS associated toxicity of SOD1 in transgenic mice using an exon-fusion approach. Hum. Mol. Genet. 17:2310 2319. Deng, H.X., Y.Shi, Y.Furukawa, H.Zhai, R .Fu, E.Liu, G.H.Gorrie, M.S.Khan, W.Y.Hung, E.H.Bigio, T.Lukas, M.C.Dal Canto, T.V.O'Halloran, and T.Siddique. 2006. Conversion to the amyotrophic lateral sclerosis phenotype is associated with intermolecular linked insoluble aggregates of SOD1 in mitochon dria. Proc. Natl. Acad. Sci. U. S. A 103:71427147.

PAGE 228

228 Deng, H.X., J.A.Tainer, H.Mitsumoto, A.Ohnishi, X.He, W.Y.Hung, Y.Zhao, T.Juneja, A.Hentati, and T.Siddique. 1995. Two novel SOD1 mutations in patients with familial amyotrophic lateral sclerosis. Hum. Mol. Genet. 4:11131116. Drachman, D.B., V.Chaudhry, D.Cornblath, R.W.Kuncl, A.Pestronk, L.Clawson, E.D.Mellits, S.Quaskey, T.Quinn, A.Calkins, and 1994. Trial of immunosuppression in amyotrophic lateral sclerosis using total lymphoid irradiation. Ann. Neurol. 35:142150. Duff, K., C.Eckman, C.Zehr, X.Yu, C.M.Prada, J.Perez -tur, M.Hutton, L.Buee, Y.Harigaya, D.Yager, D.Morgan, M.N.Gordon, L.Holcomb, L.Refolo, B.Zenk, J.Hardy, and S.Younkin. 1996. Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1. Nature 383:710 713. Elam, J.S., A.B.Taylor, R.Strange, S.Antonyuk, P.A.Doucette, J.A.Rodriguez, S.S.Hasnain, L.J.Hayward, J.S.Valentine, T.O.Yeates, and P.J.Hart. 2003. Amyloid -like filaments and water -filled nanotubes formed by SOD1 mutant proteins linked to familial ALS. Nat. Struct. Biol. 10:461-467. Elia, A.J., T.L.Parkes, K.Kirby, P.St George -Hyslop, G.L.Boulianne, J.P.Phillips, and A.J.Hilliker. 1999. Expression of human FALS SOD in motorneurons of Drosophila. Free Radic. Biol. Med. 26:13321338. Els hafey, A., W.G.Lanyon, and J.M.Connor. 1994. Identification of a new missense point mutation in exon 4 of the Cu/Zn superoxide dismutase (SOD -1) gene in a family with amyotrophic lateral sclerosis. Hum. Mol. Genet. 3:363-364. Enayat, Z.E., R.W.Orrell, A.Cl aus, A.Ludolph, R.Bachus, J.Brockmuller, K.Ray Chaudhuri, A.Radunovic, C.Shaw, J.Wilkinson, and 1995. Two novel mutations in the gene for copper zinc superoxide dismutase in UK families with amyotrophic lateral sclerosis. Hum. Mol. Genet. 4:1239-1240. Es teban, J., D.R.Rosen, A.C.Bowling, P.Sapp, D.kennaYasek, J.P.O'Regan, M.F.Beal, H.R.Horvitz, and R.H.Brown, Jr. 1994. Identification of two novel mutations and a new polymorphism in the gene for Cu/Zn superoxide dismutase in patients with amyotrophic lateral sclerosis. Hum. Mol. Genet. 3:997-998. Estevez, A.G., J.P.Crow, J.B.Sampson, C.Reiter, Y.Zhuang, G.J.Richardson, M.M.Tarpey, L.Barbeito, and J.S.Beckman. 1999. Induction of nitric oxide dependent apoptosis in motor neurons by zinc -deficient superoxide dismutase. Science 286:2498 -2500. Estevez, A.G., N.Spear, S.M.Manuel, L.Barbeito, R.Radi, and J.S.Beckman. 1998. Role of endogenous nitric oxide and peroxynitrite formation in the survival and death of motor neurons in culture. Prog. Brain Res. 118:269 280. Feeney, S.J., P.A.McKelvie, L.Austin, M.J.Jean -Francois, R.Kapsa, S.M.Tombs, and E.Byrne. 2001. Presymptomatic motor neuron loss and reactive astrocytosis in

PAGE 229

229 the SOD1 mouse model of amyotrophic lateral sclerosis. Muscle Nerve 24:1510 1519. Ferrante, R.J. S.E.Browne, L.A.Shinobu, A.C.Bowling, M.J.Baik, U.MacGarvey, N.W.Kowall, R.H.Brown, Jr., and M.F.Beal. 1997. Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J. Neurochem. 69:20642074. Fischer, L.R., D.G.Culver, P.Tennant, A.A.Davis, M.Wang, A.Castellano -Sanchez, J.Khan, M.A.Polak, and J.D.Glass. 2004. Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp. Neurol. 185:232-240. Forman, H.J. and I.Fridovich. 1973. On the stabi lity of bovine superoxide dismutase. The effects of metals. J. Biol. Chem. 248:2645-2649. Francis, G., Z.Kerem, H.P.S.Makkar, and K.Becker. 2002. The biological action of saponins in animal systems: a review. British Journal of Nutrition 88:587-605. Friedl ander, R.M., R.H.Brown, V.Gagliardini, J.Wang, and J.Yuan. 1997. Inhibition of ICE slows ALS in mice. Nature 388:31. Fumagalli, E., M.Funicello, T.Rauen, M.Gobbi, and T.Mennini. 2008. Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1. Eur. J. Pharmacol. 578:171 176. Furukawa, Y., R.Fu, H.X.Deng, T.Siddique, and T.V.O'Halloran. 2006. Disulfide cross linked protein represents a significant fraction of ALS associated Cu, Zn superoxide dismutase aggregates in spinal cords of model m ice. Proc. Natl. Acad. Sci. U. S. A 103:71487153. Garcia -Redondo, A., F.Bustos, Y.S.Juan, H.P.Del, S.Jimenez, Y.Campos, M.A.Martin, J.C.Rubio, F.Canadillas, J.Arenas, and J.Esteban. 2002. Molecular analysis of the superoxide dismutase 1 gene in Spanish pa tients with sporadic or familial amyotrophic lateral sclerosis. Muscle Nerve 26:274278. Gellera, C., B.Castellotti, M.C.Riggio, V.Silani, L.Morandi, D.Testa, C.Casali, F.Taroni, D.S.Di, M.Zeviani, and C.Mariotti. 2001. Superoxide dismutase gene mutations in Italian patients with familial and sporadic amyotrophic lateral sclerosis: identification of three novel missense mutations. Neuromuscul. Disord. 11:404410. Gidalevitz, T., T.Krupinski, S.Garcia, and R.I.Morimoto. 2009. Destabilizing protein polymorphi sms in the genetic background direct phenotypic expression of mutant SOD1 toxicity. PLoS. Genet. 5:e1000399. Gitcho, M.A., R.H.Baloh, S.Chakraverty, K.Mayo, J.B.Norton, D.Levitch, K.J.Hatanpaa, C.L.White, III, E.H.Bigio, R.Caselli, M.Baker, M.T.Al Lozi, J. C.Morris, A.Pestronk,

PAGE 230

230 R.Rademakers, A.M.Goate, and N.J.Cairns. 2008. TDP -43 A315T mutation in familial motor neuron disease. Ann. Neurol. 63:535538. Gong, B., M.C.Lim, J.Wanderer, A.Wyttenbach, and A.J.Morton. 2008. Time lapse analysis of aggregate format ion in an inducible PC12 cell model of Huntington's disease reveals time-dependent aggregate formation that transiently delays cell death. Brain Res. Bull. 75:146-157. Gong, Y.H., A.S.Parsadanian, A.Andreeva, W.D.Snider, and J.L.Elliott. 2000. Restricted e xpression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause motoneuron degeneration. J. Neurosci. 20:660 665. Gordon, P.H., D.H.Moore, R.G.Miller, J.M.Florence, J.L.Verheijde, C.Doorish, J.F.Hilton, G.M.Spitalny, R .B.Macarthur, H.Mitsumoto, H.E.Neville, K.Boylan, T.Mozaffar, J.M.Belsh, J.Ravits, R.S.Bedlack, M.C.Graves, L.F.McCluskey, R.J.Barohn, and R.Tandan. 2007. Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial. Lancet Neurol. 6:10451053. Gourie-Devi, M., A.Nalini, and D.K.Subbakrishna. 1997. Temporary amelioration of symptoms with intravenous cyclophosphamide in amyotrophic lateral sclerosis. J. Neurol. Sci. 150:167172. Graf, M., D.Ecker, R.Horowski, B.Kramer P.Riederer, M.Gerlach, C.Hager, A.C.Ludolph, G.Becker, J.Osterhage, W.H.Jost, B.Schrank, C.Stein, P.Kostopulos, S.Lubik, K.Wekwerth, R.Dengler, M.Troeger, A.Wuerz, A.Hoge, C.Schrader, N.Schimke, K.Krampfl, S.Petri, S.Zierz, K.Eger, S.Neudecker, K.Traufel ler, M.Sievert, B.Neundorfer, and M.Hecht. 2005. High dose vitamin E therapy in amyotrophic lateral sclerosis as add on therapy to riluzole: results of a placebo-controlled double-blind study. J. Neural Transm. 112:649 660. Gredal, O., L.Werdelin, S.Bak, P .B.Christensen, G.Boysen, M.O.Kristensen, J.H.Jespersen, L.Regeur, H.H.Hinge, and T.S.Jensen. 1997. A clinical trial of dextromethorphan in amyotrophic lateral sclerosis. Acta Neurol. Scand. 96:813. Groeneveld, G.J., J.H.Veldink, d.T.van, I, S.Kalmijn, C.Beijer, V.M.de, J.H.Wokke, H.Franssen, and L.H.van den Berg. 2003. A randomized sequential trial of creatine in amyotrophic lateral sclerosis. Ann. Neurol. 53:437 -445. Gurney, M.E. 1994. Transgenic mo use model of amyotrophic lateral sclerosis. N. Engl. J. Med. 331:1721-1722. Gurney, M.E., H.Pu, A.Y.Chiu, M.C.Dal Canto, C.Y.Polchow, D.D.Alexander, J.Caliendo, A.Hentati, Y.W.Kwon, H.X.Deng, and 1994. Motor neuron degeneration in mice that express a hum an Cu,Zn superoxide dismutase mutation. Science 264:17721775.

PAGE 231

231 Gusella, J.F. and M.E.MacDonald. 2006. Huntington's disease: seeing the pathogenic process through a genetic lens. Trends Biochem. Sci. 31:533540. Hadano, S., C.K.Hand, H.Osuga, Y.Yanagisawa, A.Otomo, R.S.Devon, N.Miyamoto, J.Showguchi Miyata, Y.Okada, R.Singaraja, D.A.Figlewicz, T.Kwiatkowski, B.A.Hosler, T.Sagie, J.Skaug, J.Nasir, R.H.Brown, Jr., S.W.Scherer, G.A.Rouleau, M.R.Hayden, and J.E.Ikeda. 2001. A gene encoding a putative GTPase regu lator is mutated in familial amyotrophic lateral sclerosis 2. Nat. Genet. 29:166173. Hand, C.K., J.Khoris, F.Salachas, F.Gros -Louis, A.A.Lopes, V.Mayeux -Portas, C.G.Brewer, R.H.Brown, Jr., V.Meininger, W.Camu, and G.A.Rouleau. 2002. A novel locus for fami lial amyotrophic lateral sclerosis, on chromosome 18q. Am. J. Hum. Genet. 70:251256. Hand, C.K., V.Mayeux -Portas, J.Khoris, V.Briolotti, P.Clavelou, W.Camu, and G.A.Rouleau. 2001. Compound heterozygous D90A and D96N SOD1 mutations in a recessive amyotroph ic lateral sclerosis family. Ann. Neurol. 49:267271. Harrington, H., M.Hallett, and H.R.Tyler. 1984. Ganglioside therapy for amyotrophic lateral sclerosis: a double blind controlled trial. Neurology 34:1083 -1085. Hayward, C., D.J.Brock, R.A.Minns, and R.J .Swingler. 1998. Homozygosity for Asn86Ser mutation in the CuZn-superoxide dismutase gene produces a severe clinical phenotype in a juvenile onset case of familial amyotrophic lateral sclerosis. J. Med. Genet. 35:174. Hayward, L.J., J.A.Rodriguez, J.W.Kim, A.Tiwari, J.J.Goto, D.E.Cabelli, J.S.Valentine, and R.H.Brown, Jr. 2002. Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis. J. Biol. Chem. 277:1592315931. Hegedus, J., C.T .Putman, and T.Gordon. 2007. Time course of preferential motor unit loss in the SOD1 G93A mouse model of amyotrophic lateral sclerosis. Neurobiol. Dis. 28:154-164. Hentati,A., K.Ouahchi, M.A.Pericak -Vance, D.Nijhawan, A.Ahmad, Y.Yang, J.Rimmler, W.Hung, B. Schlotter, A.Ahmed, M.BenHamida, F.Hentati, and T.Siddique. 1998. Linkage of a commoner form of recessive amyotrophic lateral sclerosis to chromosome 15q15-q22 markers. Neurogenetics 55 -60. Higgins, C.M., C.Jung, and Z.Xu. 2003. ALS associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes. BMC. Neurosci. 4:16. Hirano, M., J.Fujii, Y.Nagai, M.Sono be, K.Okamoto, H.Araki, N.Taniguchi, and S.Ueno. 1994. A new variant Cu/Zn superoxide dismutase (Val7->Glu) deduced from

PAGE 232

232 lymphocyte mRNA sequences from Japanese patients with familial amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 204:572 -5 77. Hosler, B.A., G.A.Nicholson, P.C.Sapp, W.Chin, R.W.Orrell, J.S.de Belleroche, J.Esteban, L.J.Hayward, D.kennaYasek, L.Yeung, A.K.Cherryson, J.E.Dench, S.D.Wilton, N.G.Laing, R.H.Horvitz, and R.H.Brown, Jr. 1996. Three novel mutations and two variants in the gene for Cu/Zn superoxide dismutase in familial amyotrophic lateral sclerosis. Neuromuscul. Disord. 6:361366. Hosler, B.A., T.Siddique, P.C.Sapp, W.Sailor, M.C.Huang, A.Hossain, J.R.Daube, M.Nance, C.Fan, J.Kaplan, W.Y.Hung, D.kennaYasek, J.L.Hain es, M.A.Pericak Vance, H.R.Horvitz, and R.H.Brown, Jr. 2000. Linkage of familial amyotrophic lateral sclerosis with frontotemporal dementia to chromosome 9q21q22. JAMA 284:16641669. Howland, D.S., J.Liu, Y.She, B.Goad, N.J.Maragakis, B.Kim, J.Erickson, J .Kulik, L.DeVito, G.Psaltis, L.J.DeGennaro, D.W.Cleveland, and J.D.Rothstein. 2002. Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant mediated amyotrophic lateral sclerosis (ALS). Proc. Natl. Acad. Sci. U. S. A 99:1604 1609. Hsiao, K.K., D.Groth, M.Scott, S.L.Yang, H.Serban, D.Rapp, D.Foster, M.Torchia, S.J.DeArmond, and S.B.Prusiner. 1994. Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein. Proc. Natl. Acad. Sci. U. S. A 91:91269130. Huang, C.S., J.H.Song, K.Nagata, D.Twombly, J.Z.Yeh, and T.Narahashi. 1997a. G proteins are involved in riluzole inhibition of high voltage activated calcium channels in rat dorsal root ganglion neurons. Brain Res. 762:235 239. Huang, C. S., J.H.Song, K.Nagata, J.Z.Yeh, and T.Narahashi. 1997b. Effects of the neuroprotective agent riluzole on the high voltage activated calcium channels of rat dorsal root ganglion neurons. J. Pharmacol. Exp. Ther. 282:12801290. Hutton, M., C.L.Lendon, P.Riz zu, M.Baker, S.Froelich, H.Houlden, S.Pickering-Brown, S.Chakraverty, A.Isaacs, A.Grover, J.Hackett, J.Adamson, S.Lincoln, D.Dickson, P.Davies, R.C.Petersen, M.Stevens, G.E.de, E.Wauters, B.J.van, M.Hillebrand, M.Joosse, J.M.Kwon, P.Nowotny, L.K.Che, J.Nor ton, J.C.Morris, L.A.Reed, J.Trojanowski, H.Basun, L.Lannfelt, M.Neystat, S.Fahn, F.Dark, T.Tannenberg, P.R.Dodd, N.Hayward, J.B.Kwok, P.R.Schofield, A.Andreadis, J.Snowden, D.Craufurd, D.Neary, F.Owen, B.A.Oostra, J.Hardy, A.Goate, S.J.van, D.Mann, T.Lync h, and P.Heutink. 1998. Association of missense and 5' -splice site mutations in tau with the inherited dementia FTDP 17. Nature 393:702-705. Ikeda, M., K.Abe, M.Aoki, M.Sahara, M.Watanabe, M.Shoji, P.H.St George -Hyslop, S.Hirai, and Y.Itoyama. 1995. Variable clinical symptoms in familial amyotrophic lateral sclerosis with a novel point mutation in the Cu/Zn superoxide dismutase gene. Neurology 45:20382042.

PAGE 233

233 Islinger, M., K.W.Li, J.Seitz, A.Volkl, and G.H.Luers. 2009. Hitchhiking of Cu/Zn Superoxide Dismutas e to Peroxisomes Evidence for a Natural Piggyback Import Mechanism in Mammals. Traffic. Jaarsma, D., E.D.Haasdijk, J.A.Grashorn, R.Hawkins, D.W.van, H.W.Verspaget, J.London, and J.C.Holstege. 2000. Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration, and premature motoneuron death and accelerates motoneuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1. Neurobiol. Dis. 7:623 -643. Jaarsma, D., F.Rognoni, D.W. van, H.W.Verspaget, E.D.Haasdijk, and J.C.Holstege. 2001. CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis -linked SOD1 mutations. Acta Neuropathol. (Berl) 102:293 305. Jaarsma, D., E.Teuling, E.D.Haasdijk, C.I.De Zeeuw, and C.C.Hoogenraad. 2008. Neuron-specific expression of mutant superoxide dismutase is sufficient to induce amyotrophic lateral sclerosis in transgenic mice. J. Neurosci. 28:2075 2088. Jackson, M., A.Al -C halabi, Z.E.Enayat, B.Chioza, P.N.Leigh, and K.E.Morrison. 1997. Copper/zinc superoxide dismutase 1 and sporadic amyotrophic lateral sclerosis: analysis of 155 cases and identification of a novel insertion mutation. Ann. Neurol. 42:803-807. Jarrett, J.T. a nd P.T.Lansbury, Jr. 1992. Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB. Biochemistry 31:12345 -12352. Johnston, J.A., M.J.Dalton, M.E.Gurney, and R.R.Kopito. 2000. Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A 97:1257112576. Jones, C.T., P.J.Shaw, G.Chari, and D.J.Brock. 1994a. Identification of a novel exon 4 SOD1 mutation in a sporadic amyotrophic lateral sclerosis patient. Mol. Cell Probes 8:329 330. Jones, C.T., R.J.Swingler, and D.J.Brock. 1994b. Identification of a novel SOD1 mutation in an apparently sporadic amyotrophic lateral sclerosis patient and the detection of Ile113Thr in three others. Hum. Mol. Genet. 3:649650. Jonsson, P.A., K.Ernhill, P.M.Andersen, D.Bergemalm, T.Brannstrom, O.Gredal, P.Nilsson, and S.L.Marklund. 2004. Minute quantities of misfolded mutant supe roxide dismutase 1 cause amyotrophic lateral sclerosis. Brain 127:73 -88.

PAGE 234

234 Jonsson, P.A., K.S.Graffmo, P.M.Andersen, T.Brannstrom, M.Lindberg, M.Oliveberg, and S.L.Marklund. 2006a. Disulphide-reduced superoxide dismutase1 in CNS of transgenic amyotrophic lateral sclerosis models. Brain 129:451 464. Jonsson, P.A., K.S.Graffmo, T.Brannstrom, P.Nilsson, P.M.Andersen, and S.L.Marklund. 2006b. Motor neuron disease in mice expressing the wild type-like D90A mutant superoxide dismutase1. J. Neuropathol. Exp. Neuro l. 65:1126 1136. Julien, J.P. and J.Kriz. 2006. Transgenic mouse models of amyotrophic lateral sclerosis. Biochim. Biophys. Acta 1762:1013-1024. Kabashi, E., J.N.Agar, D.M.Taylor, S.Minotti, and H.D.Durham. 2004. Focal dysfunction of the proteasome: a path ogenic factor in a mouse model of amyotrophic lateral sclerosis. J. Neurochem. 89:1325-1335. Kabashi, E., P.N.Valdmanis, P.Dion, D.Spiegelman, B.J.McConkey, V.C.Vande, J.P.Bouchard, L.Lacomblez, K.Pochigaeva, F.Salachas, P.F.Pradat, W.Camu, V.Meininger, N. Dupre, and G.A.Rouleau. 2008. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat. Genet. 40:572 -574. Kalra, S., A.Genge, and D.L.Arnold. 2003. A prospective, randomized, placebocontrolled evaluation of corticoneuronal response to intrathecal BDNF therapy in ALS using magnetic resonance spectroscopy: feasibility and results. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 4:2226. Karch, C.M. and D.R.Borchelt. 2008. A Limited Role for Disulfide Cross -linking in the Aggregation of Mutant SOD1 Linked to Familial Amyotrophic Lateral Sclerosis. J. Biol. Chem. 283:1352813537. Karch, C.M., M.Prudencio, D.D.Winkler, P.J.Hart, and D.R.Borchelt. 2009. Role of mutant SOD1 disulfide oxidation and aggregation in the path ogenesis of familial ALS. Proc. Natl. Acad. Sci. U. S. A Kato, M., M.Aoki, M.Ohta, M.Nagai, F.Ishizaki, S.Nakamura, and Y.Itoyama. 2001a. Marked reduction of the Cu/Zn superoxide dismutase polypeptide in a case of familial amyotrophic lateral sclerosis wi th the homozygous mutation. Neurosci. Lett. 312:165-168. Kato, S., S.Horiuchi, J.Liu, D.W.Cleveland, N.Shibata, K.Nakashima, R.Nagai, A.Hirano, M.Takikawa, M.Kato, I.Nakano, and E.Ohama. 2000a. Advanced glycation endproduct modified superoxide dismutase 1 (SOD1) positive inclusions are common to familial amyotrophic lateral sclerosis patients with SOD1 gene mutations and transgenic mice expressing human SOD1 with a G85R mutation. Acta Neuropathol. (Berl) 100:490 505. Kato, S., S.Horiuchi, K.Nakashima, A.Hir ano, N.Shibata, I.Nakano, M.Saito, M.Kato, K.Asayama, and E.Ohama. 1999a. Astrocytic hyaline inclusions contain

PAGE 235

235 advanced glycation endproducts in familial amyotrophic lateral sclerosis with superoxide dismutase 1 gene mutation: immunohistochemical and immu noelectron microscopical analyses. Acta Neuropathol. (Berl) 97:260-266. Kato, S., M.Saito, A.Hirano, and E.Ohama. 1999b. Recent advances in research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 gene mut ations: neuronal Lewy body -like hyaline inclusions and astrocytic hyaline inclusions. Histol. Histopathol. 14:973 -989. Kato, S., M.Shimoda, Y.Watanabe, K.Nakashima, K.Takahashi, and E.Ohama. 1996. Familial amyotrophic lateral sclerosis with a two base pair deletion in superoxide dismutase 1: gene multisystem degeneration with intracytoplasmic hyaline inclusions in astrocytes. J. Neuropathol. Exp. Neurol. 55:1089-1101. Kato, S., H.Sumi Akamaru, H.Fujimura, S.Sakoda, M.Kato, A.Hirano, M.Takikawa, and E.Ohama. 2001b. Copper chaperone for superoxide dismutase coaggregates with superoxide dismutase 1 (SOD1) in neuronal Lewy body -like hyaline inclusions: an immunohistochemical study on familial amyotrophic lateral sclerosis with SOD1 gene mutation. Acta Neuropathol. 102:233-238. Kato, S., M.Takikawa, K.Nakashima, A.Hirano, D.W.Cleveland, H.Kusaka, N.Shibata, M.Kato, I.Nakano, and E.Ohama. 2000b. New consensus research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutations: inclusions containing SOD1 in neurons and astrocytes. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 1:163 -184. Kawamata, H. and G.Manfredi. 2008. Different regulation of wild-type and mutant Cu,Zn superoxide dismutase local ization in mammalian mitochondria. Hum. Mol. Genet. 17:3303 -3317. Kawamata, J., S.Shimohama, S.Takano, K.Harada, K.Ueda, and J.Kimura. 1997. Novel G16S (GGC -AGC) mutation in the SOD 1 gene in a patient with apparently sporadic youngonset amyotrophic later al sclerosis. Hum. Mutat. 9:356 -358. Kelenyi, G. 1967. Thioflavin S fluorescent and Congo red anisotropic stainings in the histologic demonstration of amyloid. Acta Neuropathol. (Berl) 7:336 348. Keller, G.A., T.G.Warner, K.S.Steimer, and R.A.Hallewell. 19 91. Cu,Zn superoxide dismutase is a peroxisomal enzyme in human fibroblasts and hepatoma cells. Proc. Natl. Acad. Sci. U. S. A 88:7381 -7385. Kennel, P.F., F.Finiels, F.Revah, and J.Mallet. 1996. Neuromuscular function impairment is not caused by motor neurone loss in FALS mice: an electromyographic study. Neuroreport 7:1427 -1431. Kim, N.H., H.J.Kim, M.Kim, and K.W.Lee. 2003. A novel SOD1 gene mutation in a Korean family with amyotrophic lateral sclerosis. J. Neurol. Sci. 206:65-69.

PAGE 236

236 Kohno, S., Y.Takahashi, H.Miyajima, M.Serizawa, and K.Mizoguchi. 1999. A novel mutation (Cys6Gly) in the Cu/Zn superoxide dismutase gene associated with rapidly progressive familial amyotrophic lateral sclerosis. Neurosci. Lett. 276:135137. Kokubo, Y., S.Kuzuhara, Y.Narita, K.Kikugawa, R.Nakano, T.Inuzuka, S.Tsuji, M.Watanabe, T.Miyazaki, S.Murayama, and Y.Ihara. 1999. Accumulation of neurofilaments and SOD1 -immunoreactive products in a patient with familial amyotrophic lateral sclerosis with I113T SOD1 mutation. Arch. Neurol. 56:15061508. Kostrzewa, M., U.Burck Lehmann, and U.Muller. 1994. Autosomal dom inant amyotrophic lateral sclerosis: a novel mutation in the Cu/Zn superoxide dismutase1 gene. Hum. Mol. Genet. 3:2261 -2262. Kostrzewa, M., M.S.Damian, and U.Muller. 1996. Superoxide dismutase 1: identification of a novel mutation in a case of familial am yotrophic lateral sclerosis. Hum. Genet. 98:48 -50. Krishnan, J., R.Lemmens, W.Robberecht, and B.L.Van Den. 2006. Role of heat shock response and Hsp27 in mutant SOD1dependent cell death. Exp. Neurol. 200:301 -310. Kunst, C.B. 2004. Complex genetics of amyotrophic lateral sclerosis. Am. J. Hum. Genet. 75:933947. Kwiatkowski, T.J., Jr., D.A.Bosco, A.L.Leclerc, E.Tamrazian, C.R.Vanderburg, C.Russ, A.Davis, J.Gilchrist, E.J.Kasarskis, T.Munsat, P.Valdmanis, G.A.Rouleau, B.A.Hosler, P.Cortelli, P.J.de Jong, Y.Y oshinaga, J.L.Haines, M.A.Pericak Vance, J.Yan, N.Ticozzi, T.Siddique, D.kenna Yasek, P.C.Sapp, H.R.Horvitz, J.E.Landers, and R.H.Brown, Jr. 2009. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323:12051208. Lacomblez, L., G.Bensimon, P.N.Leigh, C.Debove, R.Bejuit, P.Truffinet, and V.Meininger. 2002. Longterm safety of riluzole in amyotrophic lateral sclerosis. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 3:2329. Lacomblez, L., G.Bensimon, P. N.Leigh, P.Guillet, L.Powe, S.Durrleman, J.C.Delumeau, and V.Meininger. 1996. A confirmatory dose -ranging study of riluzole in ALS. ALS/Riluzole Study GroupII. Neurology 47:S242-S250. Lange, D.J., P.L.Murphy, B.Diamond, V.Appel, E.C.Lai, D.S.Younger, and S.H.Appel. 1998. Selegiline is ineffective in a collaborative double-blind, placebo -controlled trial for treatment of amyotrophic lateral sclerosis. Arch. Neurol. 55:93 -96. Leitch, J.M., P.J.Yick, and V.L.Culotta. 2009. The right to choose: Multiple pathways for activating Cu/Zn superoxide dismutase. J. Biol. Chem.

PAGE 237

237 Levin, B.J., G.Thompson, L.H.Mitsumoto, and P.Kaufmann. 2006. Pentoxifylline in ALS: a doubleblind, randomized, multicenter, placebo-controlled trial. Neurology 66:1786 -1787. Lindberg, M.J., L.T ibell, and M.Oliveberg. 2002. Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: decreased stability of the apo state. Proc. Natl. Acad. Sci. U. S. A 99:1660716612. Lino, M.M., C.Schneider, and P.Caroni 2002. Accumulation of SOD1 mutants in postnatal motoneurons does not cause motoneuron pathology or motoneuron disease. J. Neurosci. 22:4825 4832. Liu, J., C.Lillo, P.A.Jonsson, V.C.Vande, C.M.Ward, T.M.Miller, J.R.Subramaniam, J.D.Rothstein, S.Marklund, P.M.Andersen, T.Brannstrom, O.Gredal, P.C.Wong, D.S.Williams, and D.W.Cleveland. 2004. Toxicity of familial ALS -linked SOD1 mutants from selective recruitment to spinal mitochondria. Neuron 43:5 -17. Lobsiger, C.S., S.Boillee, M.onis -Downes, A.M.Khan, M.L.F eltri, K.Yamanaka, and D.W.Cleveland. 2009. Schwann cells expressing dismutase active mutant SOD1 unexpectedly slow disease progression in ALS mice. Proc. Natl. Acad. Sci. U. S. A 106:4465 -4470. Lynch, T., M.Sano, K.S.Marder, K.L.Bell, N.L.Foster, R.F.Defe ndini, A.A.Sima, C.Keohane, T.G.Nygaard, S.Fahn, and 1994. Clinical characteristics of a family with chromosome 17 -linked disinhibition dementia parkinsonism amyotrophy complex. Neurology 44:18781884. MARKOWITZ, H., G.E.CARTWRIGHT, and M.M.WINTROBE. 195 9. Studies on copper metabolism. XXVII. The isolation and properties of an erythrocyte cuproprotein (erythrocuprein). J. Biol. Chem. 234:4045. Matsumoto, G., A.Stojanovic, C.I.Holmberg, S.Kim, and R.I.Morimoto. 2005. Structural properties and neuronal tox icity of amyotrophic lateral sclerosis associated Cu/Zn superoxide dismutase 1 aggregates. J. Cell Biol. 171:75 -85. Matsumoto, S., S.Goto, H.Kusaka, T.Imai, N.Murakami, Y.Hashizume, H.Okazaki, and A.Hirano. 1993. Ubiquitin -positive inclusion in anterior ho rn cells in subgroups of motor neuron diseases: a comparative study of adult onset amyotrophic lateral sclerosis, juvenile amyotrophic lateral sclerosis and Werdnig-Hoffmann disease. J. Neurol. Sci. 115:208 213. Matsumoto, S., H.Kusaka, H.Ito, N.Shibata, T .Asayama, and T.Imai. 1996. Sporadic amyotrophic lateral sclerosis with dementia and Cu/Zn superoxide dismutasepositive Lewy body -like inclusions. Clin. Neuropathol. 15:4146. Mazzini, L., D.Testa, C.Balzarini, and G.Mora. 1994. An open-randomized clinica l trial of selegiline in amyotrophic lateral sclerosis. J. Neurol. 241:223-227.

PAGE 238

238 McCord, J.M. and I.Fridovich. 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:60496055. Meininger, V., B.Asselain, P.Guillet, P.N.Leigh, A.Ludolph, L.Lacomblez, and W.Robberecht. 2006. Pentoxifylline in ALS: a double-blind, randomized, multicenter, placebo -controlled trial. Neurology 66:88 92. Meininger, V., G.Bensimon, W.R.Bradley, B.Brooks, P.Douillet, A.A.Eisen, L.Lacomblez, P.N.Leigh, and W.Robberecht. 2004. Efficacy and safety of xaliproden in amyotrophic lateral sclerosis: results of two phase III trials. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 5:107 -117. Miller, R., W.Bradley, M.Cudkowicz, J.Hubble, V.Mein inger, H.Mitsumoto, D.Moore, H.Pohlmann, D.Sauer, V.Silani, M.Strong, M.Swash, and E.Vernotica. 2007a. Phase II/III randomized trial of TCH346 in patients with ALS. Neurology 69:776784. Miller, R.G., J.D.Mitchell, M.Lyon, and D.H.Moore. 2007b. Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane. Database. Syst. Rev. CD001447. Miller, R.G., D.H.Moore, D.F.Gelinas, V.Dronsky, M.Mendoza, R.J.Barohn, W.Bryan, J.Ravits, E.Yuen, H.Neville, S.Ringel, M.Bromberg, J.Petajan, A.A.Amato, C.Jackson, W.Johnson, R.Mandler, P.Bosch, B.Smith, M.Graves, M.Ross, E.J.Sorenson, P.Kelkar, G.Parry, and R.Olney. 2001. Phase III randomized trial of gabapentin in patients with amyotrophic lateral sclerosis. Neurology 56:843848. Miller, R.G., R.Shepherd, H.Dao, A.Khramstov, M.Mendoza, J.Graves, and S.Smith. 1996a. Controlled trial of nimodipine in amyotrophic lateral sclerosis. Neuromuscul. Disord. 6:101104. Miller, R.G., S.A.Smith J.R.Murphy, J.R.Brinkmann, J.Graves, M.Mendoza, M.L.Sands, and S.P.Ringel. 1996b. A clinical trial of verapamil in amyotrophic lateral sclerosis. Muscle Nerve 19:511515. Miller, T.M., S.H.Kim, K.Yamanaka, M.Hester, P.Umapathi, H.Arnson, L.Rizo, J.R.Mend ell, F.H.Gage, D.W.Cleveland, and B.K.Kaspar. 2006. Gene transfer demonstrates that muscle is not a primary target for non-cell autonomous toxicity in familial amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A 103:1954619551. Mockett, R.J., A. C.Bayne, L.K.Kwong, W.C.Orr, and R.S.Sohal. 2003. Ectopic expression of catalase in Drosophila mitochondria increases stress resistance but not longevity. Free Radic. Biol. Med. 34:207217. Morita, M., M.Aoki, K.Abe, T.Hasegawa, R.Sakuma, Y.Onodera, N.Ichi kawa, M.Nishizawa, and Y.Itoyama. 1996. A novel two-base mutation in the Cu/Zn

PAGE 239

239 superoxide dismutase gene associated with familial amyotrophic lateral sclerosis in Japan. Neurosci. Lett. 205:79 -82. Munsat, T.L., J.Taft, I.M.Jackson, P.L.Andres, D.Hollander, L.Skerry, M.Ordman, D.Kasdon, and L.Finison. 1992. Intrathecal thyrotropin-releasing hormone does not alter the progressive course of ALS: experience with an intrathecal drug delivery system. Neurology 42:1049 -1053. Nagai, M., M.Aoki, I.Miyoshi, M.Kato, P .Pasinelli, N.Kasai, R.H.Brown, Jr., and Y.Itoyama. 2001. Rats expressing human cytosolic copper -zinc superoxide dismutase transgenes with amyotrophic lateral sclerosis: associated mutations develop motor neuron disease. J. Neurosci. 21:9246-9254. Naini, A ., M.Mehrazin, J.Lu, P.Gordon, and H.Mitsumoto. 2007. Identification of a novel D109Y mutation in Cu/Zn superoxide dismutase (sod1) gene associated with amyotrophic lateral sclerosis. J. Neurol. Sci. 254:17 -21. Naini, A., O.Musumeci, L.Hayes, F.Pallotti, B .M.Del, and H.Mitsumoto. 2002. Identification of a novel mutation in Cu/Zn superoxide dismutase gene associated with familial amyotrophic lateral sclerosis. J. Neurol. Sci. 198:1719. Nakanishi, T., M.Kishikawa, A.Miyazaki, A.Shimizu, Y.Ogawa, S.Sakoda, T. Ohi, and H.Shoji. 1998. Simple and defined method to detect the SOD -1 mutants from patients with familial amyotrophic lateral sclerosis by mass spectrometry. J. Neurosci. Methods 81:41-44. Nakano, R., T.Inuzuka, K.Kikugawa, H.Takahashi, K.Sakimura, J.Fujii N.Taniguchi, and S.Tsuji. 1996. Instability of mutant Cu/Zn superoxide dismutase (Ala4Thr) associated with familial amyotrophic lateral sclerosis. Neurosci. Lett. 211:129 131. Nakano, R., S.Sato, T.Inuzuka, K.Sakimura, M.Mishina, H.Takahashi, F.Ikuta, Y. Honma, J.Fujii, N.Taniguchi, and 1994. A novel mutation in Cu/Zn superoxide dismutase gene in Japanese familial amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 200:695703. Nishimura, A.L., M.Mitne-Neto, H.C.Silva, A.Richieri -Costa, S.Middl eton, D.Cascio, F.Kok, J.R.Oliveira, T.Gillingwater, J.Webb, P.Skehel, and M.Zatz. 2004. A mutation in the vesicle-trafficking protein VAPB causes lateonset spinal muscular atrophy and amyotrophic lateral sclerosis. Am. J. Hum. Genet. 75:822831. Nogales -Gadea, G., E.Garcia Arumi, A.L.Andreu, C.Cervera, and J.Gamez. 2004. A novel exon 5 mutation (N139H) in the SOD1 gene in a Spanish family associated with incomplete penetrance. J. Neurol. Sci. 219:1 6. Norris, F.H., Y.Tan, R.J.Fallat, and L.Elias. 1993. Trial of oral physostigmine in amyotrophic lateral sclerosis. Clin. Pharmacol. Ther. 54:680-682.

PAGE 240

240 Ohi, T., K.Saita, S.Takechi, K.Nabesima, H.Tashiro, K.Shiomi, S.Sugimoto, T.Akematsu, T.Nakayama, T.Iwaki and S.Matsukura. 2002. Clinical features and neuropathological findings of familial amyotrophic lateral sclerosis with a His46Arg mutation in Cu/Zn superoxide dismutase. J. Neurol. Sci. 197:7378. Okado Matsumoto, A. and I.Fridovich. 2002. Amyotrophic lateral sclerosis: a proposed mechanism. Proc. Natl. Acad. Sci. U. S. A 99:90109014. Okamoto, K., S.Hirai, M.Amari, M.Watanabe, and A.Sakurai. 1993. Bunina bodies in amyotrophic lateral sclerosis immunostained with rabbit anti -cystatin C serum. Neurosci. Lett. 162:125-128. Orrell, R.W., J.J.Habgood, I.Gardiner, A.W.King, F.A.Bowe, R.A.Hallewell, S.L.Marklund, J.Greenwood, R.J.Lane, and J.deBelleroche. 1997. Clinical and functional investigation of 10 missense mutations and a novel frameshift insertion mutati on of the gene for copper zinc superoxide dismutase in UK families with amyotrophic lateral sclerosis. Neurology 48:746751. Oztug Durer, Z.A., J.A.Cohlberg, P.Dinh, S.Padua, K.Ehrenclou, S.Downes, J.K.Tan, Y.Nakano, C.J.Bowman, J.L.Hoskins, C.Kwon, A.Z.Ma son, J.A.Rodriguez, P.A.Doucette, B.F.Shaw, and V.J.Selverstone. 2009. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase. PLoS. ONE. 4:e5004. Pardo, C.A., Z. Xu, D.R.Borchelt, D.L.Price, S.S.Sisodia, and D.W.Cleveland. 1995. Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons. Proc. Natl. Acad. Sci. U. S. A 92:954-958. Parkes, T.L. A.J.Elia, D.Dickinson, A.J.Hilliker, J.P.Phillips, and G.L.Boulianne. 1998. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat. Genet. 19:171-174. Pasinelli, P., M.E.Belford, N.Lennon, B.J.Bacskai, B.T.Hyman, D.Trotti, and R.H.Brown, Jr. 2004. Amyotrophic lateral sclerosis associated SOD1 mutant proteins bind and aggregate with Bcl 2 in spinal cord mitochondria. Neuron 43:19 30. Pasinelli, P., D.R.Borchelt, M.K.Houseweart, D.W.Cleveland, and R.H.Brown, Jr. 1998. Caspase -1 is activated in neural cells and tissue with amyotrophic lateral sclerosisassociated mutations in copper zinc superoxide dismutase. Proc. Natl. Acad. Sci. U. S. A 95:1576315768. Penco, S., A.Schenone, D.Bordo, M.Bolognesi, M.Abbruzzese, O.Bugiani, F.Ajmar, and C.Garre. 1999. A SOD1 gene mutation in a patient with slowly progressing familial ALS. Neurology 53:404-406. Persichetti, F., J.Srinidhi, L.Kanaley, P.Ge, R.H.Myers, K.D'Arrigo, G.T.Barnes, M.E.MacDonald, J.P.Vonsattel, J.F.Gusella, and 1994. Huntington's disease

PAGE 241

241 CAG trinucleotide repeats in pathologically confirmed post mortem brains. Neurobiol. Dis. 1:159 166. Petty, S.A., T.Adalsteinsson, and S.M.Decat ur. 2005. Correlations among morphology, beta -sheet stability, and molecular structure in prion peptide aggregates. Biochemistry 44:4720 -4726. Petty, S.A. and S.M.Decatur. 2005. Intersheet rearrangement of polypeptides during nucleation of {beta} -sheet aggregates. Proc. Natl. Acad. Sci. U. S. A 102:14272 14277. Potter, S.Z., H.Zhu, B.F.Shaw, J.A.Rodriguez, P.A.Doucette, S.H.Sohn, A.Durazo, K.F.Faull, E.B.Gralla, A.M.Nersissian, and J.S.Valentine. 2007. Binding of a single zinc ion to one subunit of copper z inc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein. J. Am. Chem. Soc. 129:4575 -4583. Pramatarova, A., D.A.Figlewicz, A.Krizus, F.Y.Han, I.Ceballos Picot, A.Nicole, M.Dib, V.Meininger, R.H.Brown, and G.A.Rouleau. 1995. Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis. Am. J. Hum. Genet. 56:592596. Pramatarova, A., J.Goto, E.Nanba, K.Nakashima, K.Takahashi, A.T akagi, I.Kanazawa, D.A.Figlewicz, and G.A.Rouleau. 1994. A two basepair deletion in the SOD 1 gene causes familial amyotrophic lateral sclerosis. Hum. Mol. Genet. 3:20612062. Pramatarova, A., J.Laganiere, J.Roussel, K.Brisebois, and G.A.Rouleau. 2001. Neuron specific expression of mutant superoxide dismutase 1 in transgenic mice does not lead to motor impairment. J. Neurosci. 21:3369 -3374. Proescher, J.B., M.Son, J.L.Elliott, and V.C.Culotta. 2008. Biological effects of CCS in the absence of SOD1 enzyme ac tivation: implications for disease in a mouse model for ALS. Hum. Mol. Genet. 17:1728-1737. Prudencio, M., A.Durazo, J.P.Whitelegge, and D.R.Borchelt. 2009a. Modulation of mutant superoxide dismutase 1 aggregation by co expression of wild -type enzyme. J. N eurochem. 108:10091018. Prudencio, M., P.J.Hart, D.R.Borchelt, and P.M.Andersen. 2009b. Variation in aggregation propensities among ALS associated variants of SOD1: Correlation to human disease. Human Molecular Genetics ddp260. Puls, I., C.Jonnakuty, B.H.L aMonte, E.L.Holzbaur, M.Tokito, E.Mann, M.K.Floeter, K.Bidus, D.Drayna, S.J.Oh, R.H.Brown, Jr., C.L.Ludlow, and K.H.Fischbeck. 2003. Mutant dynactin in motor neuron disease. Nat. Genet. 33:455456.

PAGE 242

242 Ralph, G.S., P.A.Radcliffe, D.M.Day, J.M.Carthy, M.A.Lerou x, D.C.Lee, L.F.Wong, L.G.Bilsland, L.Greensmith, S.M.Kingsman, K.A.Mitrophanous, N.D.Mazarakis, and M.Azzouz. 2005. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat. Med. 11:429433. Raoul, C., A.G.Estevez, H.Nishimune, D.W.Cleveland, O.deLapeyriere, C.E.Henderson, G.Haase, and B.Pettmann. 2002. Motoneuron death triggered by a specific pathway downstream of Fas. potentiation by ALS linked SOD1 mutations. Neuron 35:1067 1083. Ratovitski, T., L.B.C orson, J.Strain, P.Wong, D.W.Cleveland, V.C.Culotta, and D.R.Borchelt. 1999. Variation in the biochemical/biophysical properties of mutant superoxide dismutase 1 enzymes and the rate of disease progression in familial amyotrophic lateral sclerosis kindreds Hum. Mol. Genet. 8:1451-1460. Reaume, A.G., J.L.Elliott, E.K.Hoffman, N.W.Kowall, R.J.Ferrante, D.F.Siwek, H.M.Wilcox, D.G.Flood, M.F.Beal, R.H.Brown, Jr., R.W.Scott, and W.D.Snider. 1996. Motor neurons in Cu/Zn superoxide dismutase deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat. Genet. 13:43 47. Rezania, K., J.Yan, L.Dellefave, H.X.Deng, N.Siddique, R.T.Pascuzzi, T.Siddique, and R.P.Roos. 2003. A rare Cu/Zn superoxide dismutase mutation causing familial amyotroph ic lateral sclerosis with variable age of onset, incomplete penetrance and a sensory neuropathy. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 4:162 166. Rodriguez, J.A., B.F.Shaw, A.Durazo, S.H.Sohn, P.A.Doucette, A.M.Nersissian, K.F.Faull, D.K.Eg gers, A.Tiwari, L.J.Hayward, and J.S.Valentine. 2005. Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis. Proc. Natl. Acad. Sci. U. S. A 102:1051610521. Rodriguez, J.A., J.S.Valentine, D.K.Eggers, J .A.Roe, A.Tiwari, R.H.Brown, Jr., and L.J.Hayward. 2002. Familial amyotrophic lateral sclerosis associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase. J. Biol. Chem. 277:15932-15937. Rosen, D.R., A.C.Bowling, D.Patterson, T.B.Usdin, P.Sapp, E.Mezey, D.kenna Yasek, J.O'Regan, Z.Rahmani, R.J.Ferrante, and 1994. A frequent ala 4 to val superoxide dismutase 1 mutation is associated with a rapidly progressive familial amyotrophic lateral sclerosis. Hum. Mol. Genet. 3:981-987. Rosen, D.R., T.Siddique, D.Patterson, D.A.Figlewicz, P.Sapp, A.Hentati, D.Donaldson, J.Goto, J.P.O'Regan, H.X.Deng, and 1993. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lat eral sclerosis. Nature 362:5962.

PAGE 243

243 Rothstein, J.D., L.J.Martin, and R.W.Kuncl. 1992. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N. Engl. J. Med. 326:14641468. Rothstein, J.D., K.M.Van, A.I.Levey, L.J.Martin and R.W.Kuncl. 1995. Selective loss of glial glutamate transporter GLT -1 in amyotrophic lateral sclerosis. Ann. Neurol. 38:73 -84. Ryberg, H., H.Askmark, and L.I.Persson. 2003. A double-blind randomized clinical trial in amyotrophic lateral sclerosis usin g lamotrigine: effects on CSF glutamate, aspartate, branched -chain amino acid levels and clinical parameters. Acta Neurol. Scand. 108:1 8. Sandelin, E., A.Nordlund, P.M.Andersen, S.S.Marklund, and M.Oliveberg. 2007. Amyotrophic lateral sclerosis associated copper/zinc superoxide dismutase mutations preferentially reduce the repulsive charge of the proteins. J. Biol. Chem. 282:2123021236. Sapp, P.C., B.A.Hosler, D.kenna Yasek, W.Chin, A.Gann, H.Genise, J.Gorenstein, M.Huang, W.Sailer, M.Scheffler, M.Valesky J.L.Haines, M.Pericak -Vance, T.Siddique, H.R.Horvitz, and R.H.Brown, Jr. 2003. Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis. Am. J. Hum. Genet. 73:397403. Sapp, P.C., D.R.Rosen, B.A.Hosler, J.Esteban, D.kenna Yasek, J.P.O'Regan, H.R.Horvitz, and R.H.Brown, Jr. 1995. Identification of three novel mutations in the gene for Cu/Zn superoxide dismutase in patients with familial amyotrophic lateral sclerosis. Neur omuscul. Disord. 5:353 357. Sasaki, S., M.Nagai, M.Aoki, T.Komori, Y.Itoyama, and M.Iwata. 2007. Motor neuron disease in transgenic mice with an H46R mutant SOD1 gene. J. Neuropathol. Exp. Neurol. 66:517-524. Sasaki, S., Y.Ohsawa, K.Yamane, H.Sakuma, N.Shi bata, R.Nakano, K.Kikugawa, T.Mizutani, S.Tsuji, and M.Iwata. 1998. Familial amyotrophic lateral sclerosis with widespread vacuolation and hyaline inclusions. Neurology 51:871873. Sasaki, S., H.Warita, T.Murakami, N.Shibata, T.Komori, K.Abe, M.Kobayashi, and M.Iwata. 2005. Ultrastructural study of aggregates in the spinal cord of transgenic mice with a G93A mutant SOD1 gene. Acta Neuropathol. (Berl) 109:247-255. Sato, T., Y.Yamamoto, T.Nakanishi, K.Fukada, F.Sugai, Z.Zhou, T.Okuno, S.Nagano, S.Hirata, A.Sh imizu, and S.Sakoda. 2004. Identification of two novel mutations in the Cu/Zn superoxide dismutase gene with familial amyotrophic lateral sclerosis: mass spectrometric and genomic analyses. J. Neurol. Sci. 218:7983. Scherzinger, E., A.Sittler, K.Schweiger, V.Heiser, R.Lurz, R.Hasenbank, G.P.Bates, H.Lehrach, and E.E.Wanker. 1999. Self assembly of polyglutamine -containing

PAGE 244

244 huntingtin fragments into amyloidlike fibrils: implications for Huntington's disease pathology. Proc. Natl. Acad. Sci. U. S. A 96:4604 -4 609. Schiffer, D., L.utilio -Gambetti, A.Chio, P.Gambetti, M.T.Giordana, F.Gullotta, A.Migheli, and M.C.Vigliani. 1991. Ubiquitin in motor neuron disease: study at the light and electron microscope. J. Neuropathol. Exp. Neurol. 50:463-473. Seetharaman, S.V. M.Prudencio, C.Karch, S.P.Holloway, D.R.Borchelt, and P.J.Hart. 2009. Immature Copper zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis. Exp. Biol. Med. (Maywood. ) Segovia -Silvestre, T., A.L.Andreu, C.Vives -Bauza, E.Garcia-Arumi, C.Cervera, and J.Gamez. 2002. A novel exon 3 mutation (D76V) in the SOD1 gene associated with slowly progressive ALS. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. 3:69 -74. Seilhean, D., J.Takahashi, K.H.El Hachimi, H.Fujigasaki, A.S.Lebre, V.Biancalana, A.Durr, F.Salachas, J.Hogenhuis, T.H.de, J.J.Hauw, V.Meininger, A.Brice, and C.Duyckaerts. 2004. Amyotrophic lateral sclerosis with neuronal intranuclear protein inclusions. Acta Neur opathol. (Berl) 108:81 -87. Shaw, B.F., H.L.Lelie, A.Durazo, A.M.Nersissian, G.Xu, P.K.Chan, E.B.Gralla, A.Tiwari, L.J.Hayward, D.R.Borchelt, J.S.Valentine, and J.P.Whitelegge. 2008. Detergent insoluble aggregates associated with amyotrophic lateral scleros is in transgenic mice contain primarily full length, unmodified superoxide dismutase 1. J. Biol. Chem. 283:8340 -8350. Shaw, B.F. and J.S.Valentine. 2007. How do ALS associated mutations in superoxide dismutase 1 promote aggregation of the protein? Trends B iochem. Sci. 32:78 85. Shaw, C.E., Z.E.Enayat, B.A.Chioza, A.Al -Chalabi, A.Radunovic, J.F.Powell, and P.N.Leigh. 1998. Mutations in all five exons of SOD -1 may cause ALS. Ann. Neurol. 43:390-394. Shi, S.G., L.S.Li, K.N.Chen, and X.Liu. 2004. [Identification of the mutation of SOD1 gene in a familial amyotrophic lateral sclerosis]. Zhonghua Yi. Xue. Yi. Chuan Xue. Za Zhi. 21:149-152. Shibata, N., K.Asayama, A.Hirano, and M.Kobayashi. 1996a. Immunohistochemical study on superoxide dismutases in spinal cords f rom autopsied patients with amyotrophic lateral sclerosis. Dev. Neurosci. 18:492-498. Shibata, N., A.Hirano, M.Kobayashi, T.Siddique, H.X.Deng, W.Y.Hung, T.Kato, and K.Asayama. 1996b. Intense superoxide dismutase1 immunoreactivity in intracytoplasmic hyal ine inclusions of familial amyotrophic lateral sclerosis with posterior column involvement. J. Neuropathol. Exp. Neurol. 55:481-490.

PAGE 245

245 Shibata, N., M.Kawaguchi, K.Uchida, A.Kakita, H.Takahashi, R.Nakano, H.Fujimura, S.Sakoda, Y.Ihara, K.Nobukuni, Y.Takehisa, S.Kuroda, Y.Kokubo, S.Kuzuhara, T.Honma, Y.Mochizuki, T.Mizutani, S.Yamada, S.Toi, S.Sasaki, M.Iwata, A.Hirano, T.Yamamoto, Y.Kato, T.Sawada, and M.Kobayashi. 2007. Proteinbound crotonaldehyde accumulates in the spinal cord of superoxide dismutase1 muta tionassociated familial amyotrophic lateral sclerosis and its transgenic mouse model. Neuropathology. 27:49 -61. Shimizu, T., A.Kawata, S.Kato, M.Hayashi, K.Takamoto, H.Hayashi, S.Hirai, S.Yamaguchi, T.Komori, and M.Oda. 2000. Autonomic failure in ALS with a novel SOD1 gene mutation. Neurology 54:1534 -1537. Shorter, J. and S.Lindquist. 2005. Prions as adaptive conduits of memory and inheritance. Nat. Rev. Genet. 6:435 -450. Siddique, T. and H.X.Deng. 1996. Genetics of amyotrophic lateral sclerosis. Hum. Mol. Genet. 5 Spec No:14651470. Smith, S.A., R.G.Miller, J.R.Murphy, and S.P.Ringel. 1994. Treatment of ALS with high dose pulse cyclophosphamide. J. Neurol. Sci. 124 Suppl:84 87. Son, M., Q.Fu, K.Puttaparthi, C.M.Matthews, and J.L.Elliott. 2009. Redox suscep tibility of SOD1 mutants is associated with the differential response to CCS over expression in vivo. Neurobiol. Dis. 34:155162. Son, M., K.Puttaparthi, H.Kawamata, B.Rajendran, P.J.Boyer, G.Manfredi, and J.L.Elliott. 2007. Overexpression of CCS in G93A -SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology. Proc. Natl. Acad. Sci. U. S. A 104:60726077. Sorenson, E.J., A.J.Windbank, J.N.Mandrekar, W.R.Bamlet, S.H.Appel, C.Armon, P.E.Barkhaus, P.Bosch, K.Boylan, W.S.David, E.Feldman, J.Glass, L.Gutmann, J.Katz, W.King, C.A.Luciano, L.F.McCluskey, S.Nash, D.S.Newman, R.M.Pascuzzi, E.Pioro, L.J.Sams, S.Scelsa, E.P.Simpson, S.H.Subramony, E.Tiryaki, and C.A.Thornton. 2008. Subcutaneous IGF 1 is not beneficial in 2 year ALS tria l. Neurology 71:17701775. Sreedharan, J., I.P.Blair, V.B.Tripathi, X.Hu, C.Vance, B.Rogelj, S.Ackerley, J.C.Durnall, K.L.Williams, E.Buratti, F.Baralle, B.J.de, J.D.Mitchell, P.N.Leigh, A.Al -Chalabi, C.C.Miller, G.Nicholson, and C.E.Shaw. 2008. TDP -43 mut ations in familial and sporadic amyotrophic lateral sclerosis. Science 319:1668-1672. Stevenson, A., D.M.Yates, C.Manser, K.J.De Vos, A.Vagnoni, P.N.Leigh, D.M.McLoughlin, and C.C.Miller. 2009. Riluzole protects against glutamateinduced slowing of neurofi lament axonal transport. Neurosci. Lett. 454:161 -164.

PAGE 246

246 Stewart, H.G., I.R.Mackenzie, A.Eisen, T.Brannstrom, S.L.Marklund, and P.M.Andersen. 2006. Clinicopathological phenotype of ALS with a novel G72C SOD1 gene mutation mimicking a myopathy. Muscle Nerve 33 :701-706. Stieber, A., Y.Chen, S.Wei, Z.Mourelatos, J.Gonatas, K.Okamoto, and N.K.Gonatas. 1998. The fragmented neuronal Golgi apparatus in amyotrophic lateral sclerosis includes the trans -Golgi network: functional implications. Acta Neuropathol. (Berl) 95:245253. Stieber, A., J.O.Gonatas, J.Collard, J.Meier, J.Julien, P.Schweitzer, and N.K.Gonatas. 2000a. The neuronal Golgi apparatus is fragmented in transgenic mice expressing a mutant human SOD1, but not in mice expressing the human NF -H gene. J. Neur ol. Sci. 173:6372. Stieber, A., J.O.Gonatas, and N.K.Gonatas. 2000b. Aggregates of mutant protein appear progressively in dendrites, in periaxonal processes of oligodendrocytes, and in neuronal and astrocytic perikarya of mice expressing the SOD1(G93A) mu tation of familial amyotrophic lateral sclerosis. J. Neurol. Sci. 177:114 -123. Stieber, A., J.O.Gonatas, and N.K.Gonatas. 2000c. Aggregation of ubiquitin and a mutant ALS linked SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus. J. Neurol. Sci. 173:5362. Strom, A.L., P.Shi, F.Zhang, J.Gal, R.Kilty, L.J.Hayward, and H.Zhu. 2008. Interaction of amyotrophic lateral sclerosis (ALS) -related mutant copper zinc superoxide dismutase with the dynein -dynactin complex contribute s to inclusion formation. J. Biol. Chem. 283:2279522805. Sturtz, L.A., K.Diekert, L.T.Jensen, R.Lill, and V.C.Culotta. 2001. A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage. J. Biol. Chem. 276:38084 38089. Subramaniam, J.R., W.E.Lyons, J.Liu, T.B.Bartnikas, J.Rothstein, D.L.Price, D.W.Cleveland, J.D.Gitlin, and P.C.Wong. 2002. Mutant SOD1 causes motor neuron disease independent of copper chaperonemediated copper loading. Nat. Neurosci. 5:301 -307. Takehisa, Y., H.Ujike, H.Ishizu, S.Terada, T.Haraguchi, Y.Tanaka, T.Nishinaka, K.Nobukuni, Y.Ihara, R.Namba, T.Yasuda, M.Nishibori, T.Hayabara, and S.K uroda. 2001. Familial amyotrophic lateral sclerosis with a novel Leu126Ser mutation in the copper/zinc superoxide dismutase gene showing mild clinical features and lewy body -like hyaline inclusions. Arch. Neurol. 58:736 740. Tan, C.F., Y.S.Piao, S.Hayashi, H.Obata, Y.Umeda, M.Sato, T.Fukushima, R.Nakano, S.Tsuji, and H.Takahashi. 2004. Familial amyotrophic lateral sclerosis with bulbar onset and a novel Asp101Tyr Cu/Zn superoxide dismutase gene mutation. Acta Neuropathol. 108:332-336.

PAGE 247

247 Tandan, R., M.B.Bromberg, D.Forshew, T.J.Fries, G.J.Badger, J.Carpenter, P.B.Krusinski, E.F.Betts, K.Arciero, and K.Nau. 1996. A controlled trial of amino acid therapy in amyotrophic lateral sclerosis: I. Clinical, functional, and maximum isometric torque data. Neurology 47:1220 1226. Testa, D., T.Caraceni, and V.Fetoni. 1989. Branched-chain amino acids in the treatment of amyotrophic lateral sclerosis. J. Neurol. 236:445 447. Tiwari, A. and L.J.Hayward. 2005. Mutant SOD1 instability: implications for toxicity in amyotrophic lat eral sclerosis. Neurodegener. Dis. 2:115-127. Tobisawa, S., Y.Hozumi, S.Arawaka, S.Koyama, M.Wada, M.Nagai, M.Aoki, Y.Itoyama, K.Goto, and T.Kato. 2003. Mutant SOD1 linked to familial amyotrophic lateral sclerosis, but not wild-type SOD1, induces ER stres s in COS7 cells and transgenic mice. Biochem. Biophys. Res. Commun. 303:496 503. Tohgi, H., T.Abe, K.Yamazaki, T.Murata, E.Ishizaki, and C.Isobe. 1999. Remarkable increase in cerebrospinal fluid 3-nitrotyrosine in patients with sporadic amyotrophic lateral sclerosis. Ann. Neurol. 46:129 -131. Tu, P.H., P.Raju, K.A.Robinson, M.E.Gurney, J.Q.Trojanowski, and V.M.Lee. 1996. Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions. Proc. Natl. Acad. Sci. U. S. A 93:3155-3160. Urbani, A. and O.Belluzzi. 2000. Riluzole inhibits the persistent sodium current in mammalian CNS neurons. Eur. J. Neurosci. 12:35673574. Urushitani, M., J.Kurisu, K.Tsukita, and R.T akahashi. 2002. Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis. J. Neurochem. 83:1030-1042. Valentine,J.S., P.A.Doucette, and S.Z.Potter. Copper Zinc Superoxi de Dismutase and Amyotrophic Lateral Sclerosis 2005. Ref Type: Generic Valentine, J.S. and P.J.Hart. 2003. Misfolded CuZnSOD and amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A 100:36173622. Vance, C., B.Rogelj, T.Hortobagyi, K.J.De Vos, A.L.Nishimura, J.Sreedharan, X.Hu, B.Smith, D.Ruddy, P.Wright, J.Ganesalingam, K.L.Williams, V.Tripathi, S.Al Saraj, A.Al -Chalabi, P.N.Leigh, I.P.Blair, G.Nicholson, B.J.de, J.M.Gallo, C.C.Miller, and C.E.Shaw. 2009. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323:1208 -1211. VASSAR, P.S. and C.F.CULLING. 1959. Fluorescent stains, with special reference to amyloid and connective tissues. Arch. Pathol. 68:487-498.

PAGE 248

248 Vijayvergiya, C., M.F.Beal, J.Buck and G.Manfredi. 2005. Mutant Superoxide Dismutase 1 Forms Aggregates in the Brain Mitochondrial Matrix of Amyotrophic Lateral Sclerosis Mice. Journal of Neuroscience 25:2463 -2470. Vyth, A., J.G.Timmer, P.M.Bossuyt, E.S.Louwerse, and J.M.de Jong. 1996. Su rvival in patients with amyotrophic lateral sclerosis, treated with an array of antioxidants. J. Neurol. Sci. 139 Suppl:99 103. Wang, J., A.Caruano Yzermans, A.Rodriguez, J.P.Scheurmann, H.H.Slunt, X.Cao, J.Gitlin, P.J.Hart, and D.R.Borchelt. 2007. Disease associated mutations at copper ligand histidine residues of superoxide dismutase 1 diminish the binding of copper and compromis e dimer stability. J. Biol. Chem. 282:345 352. Wang, J., G.W.Farr, D.H.Hall, F.Li, K.Furtak, L.Dreier, and A.L.Horwich. 2009a. An ALS -linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neur ons of Caenorhabditis elegans. PLoS. Genet. 5:e1000350. Wang, J., G.W.Farr, C.J.Zeiss, D.J.Rodriguez -Gil, J.H.Wilson, K.Furtak, D.T.Rutkowski, R.J.Kaufman, C.I.Ruse, J.R.Yates, III, S.Perrin, M.B.Feany, and A.L.Horwich. 2009b. Progressive aggregation despite chaperone associations of a mutant SOD1 YFP in transg enic mice that develop ALS. Proc. Natl. Acad. Sci. U. S. A 106:13921397. Wang, J., H.Slunt, V.Gonzales, D.Fromholt, M.Coonfield, N.G.Copeland, N.A.Jenkins, and D.R.Borchelt. 2003. Copper binding -site -null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature. Hum. Mol. Genet. 12:2753 2764. Wang, J., G.Xu, and D.R.Borchelt. 2002a. High molecular weight complexes of mutant superoxide dismutase 1: age dependent and tissue -specific accumulation. Neurobiol. Dis. 9:139 148. Wang, J., G.Xu, and D.R.Borchelt. 2006. Mapping superoxide dismutase 1 domains of non-native interaction: roles of intraand intermolecular disulfide bonding in aggregation. J. Neurochem. 96:1277 1288. Wang, J., G.Xu, V.Gonzales, M.Coonfield, D.Fromholt, N.G.Copeland, N.A.Jenkins, and D.R.Borchelt. 2002b. Fibrillar inclusions and motor neuron degeneration in transgenic mice expressing superoxide dismutase 1 with a disrupted copper binding site. Neurobiol. Dis. 10:128138. Wang, J., G.Xu, H.Li, V.Gonzales, D.Fromholt, C.Karch, N.G.Copeland, N.A.Jenkins, and D.R.Borchelt. 2005a. Somatodendritic accumulation of misfolded SOD1L126Z in motor neurons mediates degeneration: alphaB -crystallin modulates aggregation. Hum. Mol. Genet. 14:2335 -2347.

PAGE 249

249 Wang, J., G.Xu, H .H.Slunt, V.Gonzales, M.Coonfield, D.Fromholt, N.G.Copeland, N.A.Jenkins, and D.R.Borchelt. 2005b. Coincident thresholds of mutant protein for paralytic disease and protein aggregation caused by restrictively expressed superoxide dismutase cDNA. Neurobiol. Dis. 20:943-952. Wang, L., H.X.Deng, G.Grisotti, H.Zhai, T.Siddique, and R.P.Roos. 2009c. Wild -type SOD1 overexpression accelerates disease onset of a G85R SOD1 mouse. Hum. Mol. Genet. 18:1642 1651. Wang, Q., J.L.Johnson, N.Y.Agar, and J.N.Agar. 2008. Protein aggregation and protein instability govern familial amyotrophic lateral sclerosis patient survival. PLoS. Biol. 6:e170. Watanabe, M., M.Aoki, K.Abe, M.Shoji, T.Iizuka, Y.Ikeda, S.Hirai, K.Kurokawa, T.Kato, H.Sasaki, and Y.Itoyama. 1997. A novel missense point mutation (S134N) of the Cu/Zn superoxide dismutase gene in a patient with familial motor neuron disease. Hum. Mutat. 9:6971. Watanabe, M., M.Dykes -Hoberg, V.C.Culotta, D.L.Price, P.C.Wong, and J.D.Rothstein. 2001. Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues. Neurobiol. Dis. 8:933941. Watanabe, S., S.Nagano, J.Duce, M.Kiaei, Q.X.Li, S.M.Tucker, A.Tiwari, R.H.Brown, Jr., M.F.Beal, L.J.Hayward, V.C.Culotta, S.Yoshiha ra, S.Sakoda, and A.I.Bush. 2007. Increased affinity for copper mediated by cysteine 111 in forms of mutant superoxide dismutase 1 linked to amyotrophic lateral sclerosis. Free Radic. Biol. Med. 42:1534 -1542. Watanabe, Y., K.Yasui, T.Nakano, K.Doi, Y.Fukad a, M.Kitayama, M.Ishimoto, S.Kurihara, M.Kawashima, H.Fukuda, Y.Adachi, T.Inoue, and K.Nakashima. 2005. Mouse motor neuron disease caused by truncated SOD1 with or without C -terminal modification. Brain Res. Mol. Brain Res. 135:1220. Wiedau -Pazos, M., J.J .Goto, S.Rabizadeh, E.B.Gralla, J.A.Roe, M.K.Lee, J.S.Valentine, and D.E.Bredesen. 1996. Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 271:515 518. Williamson, T.L. and D.W.Cleveland. 1999. Slowing of axonal transport is a very early event in the toxicity of ALS -linked SOD1 mutants to motor neurons. Nat. Neurosci. 2:5056. Witan, H., P.Gorlovoy, A.M.Kaya, I.Koziollek -Drechsler, H.Neumann, C.Behl, and A.M.Clement. 2009. Wild -type Cu/Zn superoxide dismutase (SOD 1) does not facilitate, but impedes the formation of protein aggregates of amyotrophic lateral sclerosis causing mutant SOD1. Neurobiol. Dis.

PAGE 250

250 Witan, H., A.Kern, I.Koziollek -Drechsler, R.Wade, C.Behl, and A.M.Clement. 2008. Heterodimer formation of wild typ e and amyotrophic lateral sclerosis causing mutant Cu/Zn-superoxide dismutase induces toxicity independent of protein aggregation. Hum. Mol. Genet. 17:1373 -1385. Wong, P.C., C.A.Pardo, D.R.Borchelt, M.K.Lee, N.G.Copeland, N.A.Jenkins, S.S.Sisodia, D.W.Clev eland, and D.L.Price. 1995. An adverse property of a familial ALS -linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14:1105 1116. Wroe, R. 2009. The ALS association online database (alsod), 8/20 09, http://als od.iop.kcl.ac.uk/Als/index.aspx. Yamagishi, S., Y.Koyama, T.Katayama, M.Taniguchi, J.Hitomi, M.Kato, M.Aoki, Y.Itoyama, S.Kato, and M.Tohyama. 2007. An in vitro model for Lewy body -like hyaline inclusion/astrocytic hyaline inclusion: inductio n by ER stress with an ALS linked SOD1 mutation. PLoS. ONE. 2:e1030. Yamanaka, K., S.Boillee, E.A.Roberts, M.L.Garcia, M.onis -Downes, O.R.Mikse, D.W.Cleveland, and L.S.Goldstein. 2008. Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice. Proc. Natl. Acad. Sci. U. S. A 105:7594 7599. Yang, Y., A.Hentati, H.X.Deng, O.Dabbagh, T.Sasaki, M.Hirano, W.Y.Hung, K.Ouahchi, J.Yan, A.C.Azim, N.Cole, G.Gascon, A.Yagmour, M.Ben-Hamida, M.Pericak Vance, F.Hentati, and T.Siddique. 2001. The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat. Genet. 29:160-165. Yim, H.S., J.H.Kang, P.B.Chock, E.R.Stadtman, and M.B.Yim. 1997. A familial amyotrophic lateral sclerosis associated A4V Cu, Zn -superoxide dismutase mutant has a lower Km for hydrogen peroxide. Correlation between clinical severity and the Km value. J. Bio l. Chem. 272:8861 -8863. Yim, M.B., P.B.Chock, and E.R.Stadtman. 1990. Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide. Proc. Natl. Acad. Sci. U. S. A 87:50065010. Yim, M.B., J.H.Kang, H.S.Yim, H.S.Kwak, P.B.Chock, and E.R.Stadtman. 1996. A gainof -function of an amyotrophic lateral sclerosis associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide. Proc. Natl. Acad. Sci. U. S. A 93:5709 5714. Yulug, I.G., N.Katsanis, B.J.de, J.Collinge, and E.M.Fisher. 1995. An improved protocol for the analysis of SOD1 gene mutations, and a new mutation in exon 4. Hum. Mol. Genet. 4:1101 -1104.

PAGE 251

251 Zetterstrom, P., H.G.Stewart, D.Bergemalm, P.A.Jonsson, K. S.Graffmo, P.M.Andersen, T.Brannstrom, M.Oliveberg, and S.L.Marklund. 2007. Soluble misfolded subfractions of mutant superoxide dismutase 1s are enriched in spinal cords throughout life in murine ALS models. Proc. Natl. Acad. Sci. U. S. A 104:1415714162. Zhang, B., P.h.Tu, F.Abtahian, J.Q.Trojanowski, and V.M.Y.Lee. 1997. Neurofilaments and Orthograde Transport Are Reduced in Ventral Root Axons of Transgenic Mice that Express Human SOD1 with a G93A Mutation. The Journal of Cell Biology 139:1307-1315. Zona, C., A.Siniscalchi, N.B.Mercuri, and G.Bernardi. 1998. Riluzole interacts with voltageactivated sodium and potassium currents in cultured rat cortical neurons. Neuroscience 85:931938.

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252 BIOGRAPHICAL SKETCH Mercedes Prudencio lvarez was born in Badajoz (Spain) in 1982 to Mximo and Joaquina. She graduated with Honors from I.E.S. Maestro Domingo Cceres in 2000, and obtained an English degree at the Escuela Oficial de Idiomas that same year. In 2000, she attended Universidad de Extremadura for her undergraduate studies, where she graduated with a 5year Bachelor of Science in biology in 2005. Mercedes began her graduate studies in biomedical sciences (neuroscience) at the University of Florida in 2005, where s he joined the laboratory of Dr. David R. Borchelt in 2006.