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Characterization and treatment of mouse models of Huntington disease Van Raamsdonk, Jeremy Michael 2005

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Characterization and Treatment of Mouse Models of Huntington Disease by Jeremy Michael V a n Raamsdonk B . S c , University o f British Columbia, 1997 M . S c , McMaster University, 1999  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FORTHE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES  (Medical Genetics)  THE UNIVERSITY OF BRITISH C O L U M B I A September 2005  © Jeremy Michael Van Raamsdonk, 2005  ABSTRACT Huntington disease ( H D ) is an adult onset neurodegenerative disorder that is characterized by motor dysfunction, cognitive impairment and neuropsychiatric disturbances. H D patients exhibit progressive and selective neurodegeneration primarily in the striatum and cortex. There is currently no treatment that can prevent the development o f H D or alter its progression. The major objectives o f this thesis were to determine which symptoms o f H D are recapitulated in Y A C transgenic mouse models o f the disease, to develop a standardized protocol for therapeutic trials in these mice and to investigate potential treatments for H D .  Two transgenic mouse models o f H D were examined that express huntingtin (htt) with either 72 ( Y A C 7 2 mice) or 128 ( Y A C 128 mice) glutamines from a yeast artificial chromosome transgene. While Y A C 7 2 mice exhibit a mild phenotype, Y A C 1 2 8 mice show quantifiable abnormalities that recapitulate the motor and cognitive deficits in H D . Importantly, Y A C 128 mice also exhibit selective and progressive degeneration in the brain, including neuronal loss.  T o determine the feasibility o f genetic modulation o f the disease phenotype, we investigated the ability o f over-expression o f w i l d type htt to prevent striatal neuropathology in Y A C 128 mice based on a putative pro-survival function o f wild type htt. We demonstrate for the first time that wild type htt is neuroprotective in the brain. In Y A C 128 mice, over-expression o f wild type htt prevented atrophy o f striatal neurons(but did not significantly improve striatal volume or striatal neuronal numbers.  To determine the feasibility o f pharmacologic therapeutic trials in Y A C 1 2 8 mice we treated mice with cystamine, a transglutaminase inhibitor with other beneficial characteristics. While cystamine treatment did not improve motor symptoms, this treatment ameliorated striatal volume loss, striatal neuronal loss and striatal neuronal atrophy. This trial validates the use o f Y A C 128 mice in therapeutic trials for H D as we reproduced all o f the differences between Y A C 128 and W T mice in this therapeutic trial. Overall, this thesis demonstrates that the Y A C 128 mouse model o f H D recapitulates the progressive motor dysfunction, cognitive deficits and selective neurodegeneration o f H D . A s such, these mice can be used for studies of H D pathogenesis and in preclinical therapeutic trials for H D .  ii  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF T A B L E S  vii  LIST OF FIGURES  viii  LIST OF ABBREVIATIONS  x  PREFACE  xii  ACKNOWLEDGEMENTS  xvii  1.0 I N T R O D U C T I O N  1  1.1 Clinical description o f Huntington disease  1  1.2. Neuropathology o f Huntington disease  2  1.3 Genetics o f Huntington disease 1.3.1 Huntington disease is caused by a C A G expansion in Huntington disease gene 1.3.2 Expression o f the Huntington disease gene  4 4 5  1.4 Mouse models o f Huntington disease 5 1.4.1 Neurotoxin models o f Huntington disease 6 1.4.2 Huntington disease mouse models expressing N-terminal fragments o f mutant htt 6 1.4.3 Knock-in mouse models o f Huntington disease 9 1.4.4 F u l l length transgenic mouse models o f Huntington disease 10 1.5 Preclinical therapeutic trials for Huntington disease  12  1.6 Huntingtin function 1.6.1 Transcription 1.6.2 Transport 1.6.3 Neuroprotection  17 19 20 21  1.7 Loss o f huntingtin function in Huntington disease  22  1.8 Objectives 2.0 M A T E R I A L S A N D M E T H O D S 2.1 M i c e  ....23 26 .26  in  2.2 Behavioural assessments 2.2.1 Rotarod test o f motor coordination 2.2.2 Assessment o f motor learning on the rotarod 2.2.3 Open field activity test 2.2.4 Open field habituation test o f learning and memory 2.2.5 Simple swimming test 2.2.6 Pre-pulse inhibition and habituation to acoustic startle 2.2.7 Beam crossing test 2.2.8 Footprint analysis 2.2.9 Swimming T-maze test with reversal  26 26 27 27 27 28 28 29 .....29 30  2.3 Physiologic measurements 2.3.1 B o d y weight 2.3.2 Organ weights 2.3.3 Survival analysis 2.3.4 Histological examination o f the testis  31 31 31 31 31  2.4 Neuropathological assessments 2.4.1 Volume o f brain structures 2.4.2 Neuronal numbers 2.4.3 Striatal neuronal cross-sectional area 2.4.4 Striatal D A R P P - 3 2 expression 2.4.5 Nuclear localization o f mutant huntingtin  32 32 33 33 34 34  2.5 Neurotoxicity 2.5.1 Delivery o f kainic acid 2.5.2 Delivery o f quinolinic acid 2.5.3 Assessment o f hippocampal damage 2.5.4 Assessment o f striatal damage  34 34 35 35 36  2.6 Treatment 2.6.1 Treatment with over-expression o f wild type huntingtin 2.6.2 Treatment with cystamine  36 36 36  2.7 Molecular biology 2.7.1 Western blotting 2.7.2 Measurement o f transglutaminase activity  37 37 37  2.8 Statistical Analysis  38  3.0 T O W A R D S T H E R A P E U T I C T R I A L S : M O U S E M O D E L S A N D O U T C O M E MEASURES 3.1 Characterization o f the Y A C 7 2 mouse model o f Huntington disease 3.1.1 Motor dysfunction in Y A C 7 2 mice 3.1.2 Neuropathology in Y A C 7 2 mice  40 40 40 43  iv  3.2 Characterization o f the Y A C 1 2 8 mouse model o f Huntington disease 3.2.1 Motor dysfunction in Y A C 1 2 8 mice  43 43  3.2.2 Cognitive deficits in Y A C 1 2 8 mice 47 3 . 2 . 2 . 1 Y A C 1 2 8 mice show impaired motor learning on the rotarod 47 3.2.2.2 Y A C 1 2 8 mice show impaired memory in a test o f open field habituation 50 3.2.2.3 Y A C 1 2 8 mice show cognitive deficits in a simple swimming test 52 3.2.2.4 Y A C 1 2 8 mice show multiple cognitive deficits in swimming T-maze test....54 3.2.2.5 Deficits o f Y A C 1 2 8 mice in swimming tests are primarily cognitive 57 3.2.2.6 Presymptomatic Y A C 1 2 8 mice show difficulty in shifting strategy 59 3.2.2.7 Y A C 1 2 8 mice show decreased sensorimotor gating in pre-pulse inhibition test 61 3.2.3 Neuropathology in Y A C 1 2 8 mice 64 3.2.3.1 Selective atrophy and neuronal loss in Y A C 1 2 8 brain 64 3.2.3.2 Progression o f neuropathology in Y A C 1 2 8 mice 66 3.2.3.3 Striatal neuronal atrophy and D A R P P - 3 2 down-regulation in Y A C 1 2 8 mice69 3.2.3.4 Regional differences in the expression and nuclear localization o f mutant huntingtin in Y A C 1 2 8 mice 71 3.2.3.5 Transglutaminase activity is selectively increased in the forebrain o f Y A C 1 2 8 mice 76 3.2.4 Decreased survival in Y A C 1 2 8 mice  76  3.2.5 Y A C 1 2 8 mice exhibit atrophy and degeneration in the testis  78  3.2.6 Outcome measures for therapeutic trials  ,  4.0 W I L D T Y P E H U N T I N G T I N F U N C T I O N  80 84  4.1 W i l d type huntingtin is neuroprotective in the hippocampus  84  4.2 W i l d type huntingtin is neuroprotective in the striatum  86  4.3 Huntingtin expression influences weight  88  5.0 P R E - C L I N I C A L T H E R A P E U T I C T R I A L S I N T H E Y A C 1 2 8 M O U S E M O D E L OF HUNTINGTON DISEASE 94 5.1 Genetic treatment o f Y A C 1 2 8 mice by over-expression o f wild type huntingtin 5.1.1 Rationale for treatment with wild type huntingtin 5.1.2 Generation o f Y A C 1 2 8 mice that over-express wild type huntingtin 5.1.3 Effect o f w i l d type huntingtin on neuropathology in Y A C 1 2 8 mice  94 94 94 96  5.2 Pharmacologic treatment o f Y A C 1 2 8 mice with cystamine  98  v  5.2.1 5.2.2 5.2.3 5.2.4  Rationale for treatment with cystamine Delivery o f therapeutic agent: cystamine Effect o f cystamine on neuropathology Effect o f cystamine on motor impairment.......  6.0 D I S C U S S I O N  6.1 The Y A C 7 2 mouse model o f Huntington disease  98 98 99 102 105  106  6.2 The Y A C 1 2 8 mouse model o f Huntington disease .106 6.2.1 Motor dysfunction in the Y A C 1 2 8 mouse model o f Huntington disease 107 6.2.2 Cognitive deficits in the Y A C 1 2 8 mouse model o f Huntington disease 108 6.2.3 Decreased survival in Y A C 1 2 8 mouse model o f Huntington disease 112 6.2.4 Selective degeneration in Y A C 1 2 8 mouse model o f Huntington disease 114 6.2.5 Mechanism o f selective degeneration in the Y A C 1 2 8 mouse model o f Huntington disease 117 6.2.6 Testicular degeneration in the Y A C 1 2 8 mouse model o f Huntington disease 120 6.2.7 Comparison o f Y A C 1 2 8 mouse model with human Huntington disease 121 6.3 W i l d type huntingtin function 122 6.3.1 W i l d type huntingtin is neuroprotective in the brain 126 6.3.2 Novel function o f huntingtin influences weight 128 6.3.3 Possible contribution o f w i l d type huntingtin to weight loss in Huntington disease 129 6.4 Treatment o f Y A C 1 2 8 mice with the over-expression o f wild type huntingtin  131  6.5 Treatment o f Y A C 1 2 8 mice with cystamine 133 6.5.1 Transglutaminase activity in the Y A C 1 2 8 mouse model o f Huntington disease.133 6.5.2 Cystamine treatment in the Y A C 1 2 8 mouse model o f Huntington disease 134 6.5.3 Mechanism responsible for beneficial effects o f cystamine in Huntington disease 136 6.5.4 Implications for treatment o f Huntington disease 137 6.6 Contribution o f loss o f huntingtin function to Huntington disease  137  6.7 Conclusions  140  6.8 Future directions  141  7.0 R E F E R E N C E S  148  vi  LIST O F T A B L E S  Table 1.1 Mouse models o f Huntington disease  7  Table 1.2 Summary o f Treatment Trials in Genetic Mouse Models o f Huntington Disease .15 Table 1.3 Decreasing w i l d type huntingtin levels results in phenotypic abnormalities  18  Table 3.1 Summary o f phenotypic difference between Y A C 128 and W T mice that can be used as outcome measures for therapeutic trials 83 Table 6.1 Neuropathological phenotypes among mouse models o f Huntington disease  116  Table 6.2 Comparison o f Y A C 128 mouse model with human Huntington disease  124  Table 6.3 Criteria based comparison o f Y A C 128 mice to other H D mouse models  125  vn  LIST OF FIGURES Figure 1.1 Model for the contribution o f loss o f huntingtin function to Huntington disease. 24 F i g u r e 3.1 Motor function in Y A C 7 2 mice  42  F i g u r e 3.2 Neuropathology in Y A C 7 2 mice  44  F i g u r e 3.3 Motor deficits in Y A C 128 mice  46  F i g u r e 3.4 Y A C 128 mice show deficits in motor learning on the rotarod  48  Figure 3.5 Y A C 128 mice show decreased open field habituation  51  F i g u r e 3.6 Y A C 1 2 8 mice show a cognitive deficit in a simple swimming test  53  Figure 3.7 Y A C 128 mice show cognitive and motor deficits in swimming T-maze  55  F i g u r e 3.8 Y A C 128 mice show cognitive deficits in the reversal phase o f the swimming Tmaze test 58 F i g u r e 3.9 Cognitive deficits are primarily responsible for increased latencies to reach the platform in swimming tests 60 F i g u r e 3.10 Presymptomatic Y A C 128 mice show cognitive deficit in strategy shifting  62  Figure 3.11 Y A C 128 mice show decreased pre-pulse inhibition and decreased habituation to acoustic startle 63 Figure 3.12 Selective degeneration in the brains o f Y A C 128 mice  65  F i g u r e 3.13 Progressive neuropathology in Y A C 128 mice  68  Figure 3.14 Striatal'neuronal atrophy in Y A C 1 2 8 mice  70  F i g u r e 3.15 Down-regulation o f striatal D A R P P - 3 2 expression in Y A C 128 mice  70  F i g u r e 3.16 Selective neuropathology in Y A C 128 mice is not correlated with mutant huntingtin expression  72  F i g u r e 3.17 Selective nuclear localization o f mutant huntingtin in the brain o f Y A C 128 mice 75 F i g u r e 3.18 Y A C 128 mice show a forebrain specific increase in transglutaminase activity. 77 Figure 3.19 Y A C 1 2 8 mice show a male specific deficit in survival  79  viii  \  F i g u r e 3.20 Expression o f mutant huntingtin results in testicular degeneration in Y A C 128 mice 81 Figure 4.1 Over-expression o f wild type huntingtin protects neurons from kainic acid toxicity in the hippocampus  85  F i g u r e 4.2 Over-expression o f w i l d type huntingtin protects neurons from quinolinic acid toxicity in the striatum 87 F i g u r e 4.3 Dose dependent wild type huntingtin mediated neuroprotection in the striatum..89 Figure 4.4 W i l d type huntingtin influences body weight  91  F i g u r e 4.5 W i l d type huntingtin influences organ weight  93  F i g u r e 5.1 Generation of Y A C 128 mice that over-express wild type huntingtin  95  Figure 5.2 Over-expression o f wild type huntingtin in Y A C 128 mice results in mild improvements in striatal neuropathology  97  F i g u r e 5.3 Cystamine treatment ameliorates striatal neuropathology in Y A C 128 mice  100  F i g u r e 5.4 Cystamine treatment does not impact motor dysfunction in Y A C 128 mice  103  F i g u r e 6.1 Natural history o f abnormalities in Y A C 128 mice  123  ix  LIST O F ABBREVIATIONS  Analysis o f Variance ( A N O V A ) Brain-derived neurotrophic factor ( B D N F ) cAMP-response element binding protein ( C R E B ) Complementary D N A ( c D N A ) C R E B binding protein ( C B P ) Cytomegalovirus ( C M V ) Decibel (dB) Diaminobenzidine ( D A B ) Dopamine- and cAMP-regulated phosphoprotein, 32 k D a ( D A R P P Eicosapentaenoic acid ( E P A ) Ethylenediamine tetra-acetic acid ( E D T A ) Figure (Fig.) Gamma-aminobutyric acid ( G A B A ) Huntingtin (htt) Huntingtin associated protein ( H A P ) Huntingtin interacting protein (HIP) Huntingtin interacting protein protein interactor (HIPPI) Huntington disease (HD) Hypoxanthine phosphoribosyltransferase gene ( H P R T ) Kainic acid ( K A ) Kilo-basepair (kb) Kilodalton (KDa) Magnetic resonance imaging ( M R I ) Magnetic resonance spectroscopy ( M R S ) Messenger R N A ( m R N A ) N-acetyl aspartate ( N A A ) Neuron restrictive silencer element ( N R S E ) N-methyl-D-aspartate ( N M D A ) Nuclear Corepressor protein ( N C o R ) Nuclear export signal ( N E S )  Nuclear localization signal ( N L S ) Phenyl methyl sulfonyl fluoride ( P M S F ) Phosphate buffered saline (PBS) Polymerase chain reaction ( P C R ) Postsynaptic density-95 (PSD-95) Pre-pulse inhibition (PPI) Quinolinic acid ( Q A ) Repressor element-1 transcription factor/neuron restrictive silencer factor ( R E S T / N R S F ) Revolutions per minute ( R P M ) R N A interference ( R N A i ) Specificity protein 1 ( S p l ) Standard error o f the mean ( S E M ) Suberoylanilide hydroxamic acid ( S A H A ) T A T A binding protein ( T B P ) Tdt-mediated dUTP-biotin nick end labeling ( T U N E L ) Tissue transglutaminase (tTG) Transglutaminase (TG) Trichloroacetic acid ( T C A ) Wild-type ( W T ) Yeast artificial chromosome ( Y A C )  xi  PREFACE  Work from this thesis has resulted in several manuscripts and abstracts as indicated on the following pages. A traditional format was chosen for this thesis so that a coherent story could be presented. I designed all o f the experiments contained in this thesis, analyzed all o f the results and composed all o f the text for this thesis. Aside from the exceptions noted, I performed all o f the experiments in this thesis. In some cases, work from this thesis was included in manuscripts written by other researchers.  Technical assistance was obtained as follows. Jacqui Pearson assisted with behavioural testing for assessing cognitive function in Y A C 1 2 8 mice and in the therapeutic trials. Danny Rogers measured striatal D A R P P - 3 2 expression in Y A C 1 2 8 mice. Zoe Murphy measured the volume o f the hippocampus and cerebellum and performed western blots measuring the levels o f huntingtin in mice. Ge L u perfused all o f the animals for which neuropathology was assessed and injected quinolinic acid into the striatum o f mice. Photographs o f hippocampal damage following delivery o f kainic acid were taken by Dr. Blair Leavitt. Transglutaminase activity was assayed by our collaborator Dr. Craig Bailey. Testis samples were processed by Dr. A . Wayne V o g l .  xn  PUBLICATIONS F R O M W O R K IN THIS THESIS 1. V a n R a a m s d o n k J M , Pearson J, Rogers D , Bissada N , V o g l A W , Hayden M R Leavitt B R (2005). Loss o f w i l d type huntingtin influences motor dysfunction and survival in the Y A C 1 2 8 mouse model o f Huntington disease. Hum Mol Gen 10(14): 1379-1392. . 2. V a n R a a m s d o n k J M , Pearson J, Slow E J , Hossain S M , Leavitt B R , Hayden M R (2005). Cognitive Dysfunction Precedes Neuropathology and Motor Abnormalities in the Y A C 1 2 8 Mouse Model o f Huntington's Disease. J. Neurosci 25(16): 4169-80. 3.  Slow, E J , V a n R a a m s d o n k J M , Rogers D , Coleman S H , Graham R K , Deng Y , O h R, Yang Y - Z , Bissada N , L i X - J , Simpson E M , Gutekunst C - A , Leavitt B R , Hayden MR(2003). Selective striatal neuronal loss in a Y A C 128 mouse model o f Huntington disease. Hum Mol Genet 12(13): 1555-1567.  4. Pinto JT, V a n R a a m s d o n k J , Leavitt, B R , Hayden M R , Krasnikov B F , Cooper A J L (2005). Treatment o f Y A C 128 M i c e and Their Wild-Type Littermates with. Cystamine Does not Lead to Its Accumulation in Plasma or Brain: Implications for the Treatment o f Huntington Disease. J. Neurochem. 94(4): 1087-101. 5. V a n R a a m s d o n k J M , Pearson J, Bailey C D C , Rogers D , Johnson G V W , Hayden M R , Leavitt B R . Cystamine Treatment Ameliorates Striatal Neuropathology in Y A C 1 2 8 Mouse Model o f Huntington's Disease. J. Neurochem. (in press). 6. V a n R a a m s d o n k J M , Pearson J, Rogers D A , L u G , Hayden M R , Leavitt B R . EthylE P A Treatment Improves Motor Dysfunction, but not Neurodegeneration in H D M i c e . Submitted to Experimental Neurology (in press).  xiii  MANUSCRIPTS F R O M W O R K IN THIS THESIS  1. Leavitt B R * , V a n R a a m s d o n k J * , Shehadeh J, Fernandes H , Graham R K , Wellington C L , Raymond L A , Hayden M R . Wild-Type Huntingtin Protects Neurons from Excitotoxicity. Submitted to Journal ofNeurochemistry. *equal contribution 2. V a n R a a m s d o n k J M , Murphy Z , Slow E J , Leavitt B R , Hayden M R Selective Degeneration and Nuclear Localization o f Mutant Huntingtin in the Y A C 1 2 8 Mouse Model o f Huntington Disease. Submitted to Human Molecular Genetics. 3.  V a n R a a m s d o n k J M , Pearson J, Rogers D A , Bissada N , Hayden M R , Leavitt B R . Over-Expression o f W i l d Type Huntingtin Does N o t Prevent Striatal Atrophy in the Y A C 1 2 8 Mouse Model o f Huntington Disease. Submitted to European Journal of Neuroscience.  4. V a n R a a m s d o n k J M , Murphy Z , Gibson B , Pearson J, L u G , Leavitt B R , Hayden M R . Effect o f W i l d Type Huntingtin on Weight Gain is Not Disrupted by Polyglutamine Expansion. In preparation.  xiv  A B S T R A C T S F R O M W O R K IN THIS T H E S I S 1. Van Raamsdonk J M , Murphy Z , Slow E J , Leavitt B R , Hayden M R Selective Degeneration and Nuclear Localization o f Mutant Huntingtin in the Y A C 1 2 8 Mouse Model o f Huntington Disease. World Congress for Huntington Disease. Manchester England (September, 2005) 2. Cooper A J , Krasnikov B F , Pinto JT, Van Raamsdonk J M , Hayden M R , Leavitt B R , Jeitner T M . Transglutaminases in Huntington disease ( H D ) . International Society o f Neurochemistry meeting. Innsbruck, Austria (August, 2005) 3. Van Raamsdonk J M , Pearson J , Rogers D , Bissada N , V o g l A W , Leavitt B R , Hayden M R . Levels o f wild type huntingtin expression modulate motor dysfunction and survival in the Y A C 1 2 8 mouse model o f Huntington disease. Gordon Conference on C A G Triplet Repeat Disorders. Mount Holyoke, U S A (July, 2005) 4. Van Raamsdonk J M , Murphy Z , Hayden M R . Region specific toxicity o f mutant huntingtin in human Huntington's disease brain is directly replicated in Y A C 1 2 8 mouse model. American Society o f Human Genetics, Toronto, O N , Canada (October, 2004). 5. Van Raamsdonk J M , Pearson J, Slow E , Leavitt B R , Hayden M R , V a n Raamsdonk J M . Impaired lifespan and cognitive dysfunction in the Y A C 1 2 8 mouse model o f Huntington's disease. Hereditary Disease Foundation Huntington's Disease 2004: Change Advances and Good News ( C A G ) Huntington's Disease Research Conference, Cambridge, M A , U S A (August, 2004). n  6. Van Raamsdonk J M , Rogers D , Pearson J, L u G , Slow E J , Hayden M R , Leavitt B R . Pre-clinical trials o f experimental therapeutics in the Y A C 1 2 8 transgenic mouse model o f Huntington's disease. Hereditary Disease Foundation Huntington's Disease 2004: Change Advances and Good News ( C A G ) Huntington's Disease Research Conference, Cambridge, M A , U S A (August, 2004). n  7. Leavitt B R , Van Raamsdonk J M , Shehadeh J, Fernandes H , Graham R K , Wellington C L , Raymond L A , Hayden M R . Wild-Type Huntingtin Protects Neurons from N M D A - M e d i a t e d Excitotoxicity. Hereditary Disease Foundation Huntington's Disease 2004: Change Advances and Good News ( C A G ) Huntington's Disease Research Conference, Cambridge, M A , U S A (August, 2004). n  8. Van Raamsdonk J M , Leavitt B R , Slow E , Hayden M R . Therapeutic Trials in the Y A C 1 2 8 Mouse M o d e l o f Huntington's Disease. Gordon Conference on C A G Triplet Repeat Disorders. Barga, Italy (May, 2003) 9.  Leavitt B R , Van Raamsdonk J M , Wellington C L , Fernandes H , R o y S, Raymond L A , Nicholson D W , Hayden M R . Pro-Survival Effects o f W i l d Type Huntingtin. Gordon Conference on C A G Triplet Repeat Disorders. Barga, Italy (May, 2003)  xv  10. V a n R a a m s d o n k J M , Leavitt B R , Slow E , Hayden M R . Therapeutic Trials in the Y A C 1 2 8 Mouse M o d e l o f Huntington's Disease. Molecular Mechanisms of Neurodegeneration Meeting. M i l a n , Italy (May, 2003) 11. Leavitt B R , V a n R a a m s d o n k J , Slow E, Hayden M R . Experimental Therapeutics in Transgenic Mouse Models o f Human Neurodegenerative Diseases. C I H R new investigators meeting. 12. Slow E , V a n R a a m s d o n k J , Rogers D , Graham R , Bissada N , Oh R, Simpson E , Leavitt B , Hayden M R . Decreased Brain Weight in the Y A C 1 2 8 Huntington Disease Mouse M o d e l . Hereditary Disease Foundation Huntington's Disease 2002: Change Advances and Good News (CAG) Huntington's Disease Research Conference(Hereditary Disease Foundation General Meeting), Cambridge, M A , U S A (August, 2002). n  13. Leavitt B R , V a n R a a m s d o n k J , Slow E, Devon R S , Simpson E M , Hayden M R . Cautionary "Tails": Genetic Factors Affecting the Behavioral Phenotype o f the Y A C Transgenic Mouse M o d e l of Huntington Disease. Huntington's Disease 2002: Change Advances and Good News (CAG) Huntington's Disease Research Conference(Hereditary Disease Foundation General Meeting), Cambridge, M A , U S A (August, 2002). n  14. Slow E , V a n R a a m s d o n k J , Rogers D , Graham R, Bissada N , O h R, Simpson E , Leavitt B , Hayden M R . Decreased Brain Weight, Striatal Volume and Striatal Neuron Count in the Y A C 128 Huntington Disease Mouse M o d e l . Huntington Disease Society o f America-Coalition for the Cure, Chicago, I L , U S A ( A p r i l , 2002) 15. Slow E , V a n R a a m s d o n k J , Rogers D , Bissada N , Hossain S, Simpson E , Leavitt B R , Hayden M R . Decreased Brain Weight, Striatal Volume and Striatal Neuron Count Subsequent to the Onset o f a Motor Co-ordination Deficit in the Y A C 128 Huntington Disease Mouse M o d e l . Canadian Genetic Diseases Network Meeting, Montreal, Quebec, Canada (April, 2002).  xvi  ACKNOWLEDGEMENTS  First o f all, I would like to thank my supervisor, Michael Hayden, for providing an excellent research environment, for giving me the freedom to design and carry out experiments independently and for his critical reading o f my manuscripts. I would also like to thank the remainder o f my thesis committee - Jane Roskams, Wendy Robinson and Blair Leavitt - for their guidance along the way. I would especially like to thank my co-supervisor Blair Leavitt for sharing an interest in therapeutic trials, for helping to make them a reality in our mouse model and for making time to discuss even the negative results. Second o f all, I would like to thank the people who have helped me along the way. I would like to thank Jacqui Pearson, a true super-technician, who has made my life easier with the consistent quality o f her assistance. I would like to thank Zoe Murphy, another supertechnician, who has helped me considerably in the later stages o f my degree by taking care o f all o f the odds and ends o f experiments that have allowed me to devote more time to writing manuscripts. I would like to thank Ge L u for his hard work and doing the thankless job o f perfusing mice. I would like to thank Danny Rogers for his help with D A R P P - 3 2 intensity measurements and help with the microscopes. I would like to thank Rona Graham and Simon Warby for sharing their knowledge o f Western blotting. I would like to thank Elizabeth Slow for our early collaborative efforts studying the Y A C 128 mouse model. I would like to thank Nagat Bissada for her management o f mouse operations. Finally, in addition to the names mentioned above, I would like to thank Tami Yamashita, Jen Witmer, Scott Neal, Soo Sen, Jenny Thiele, C J Opina, Tara Davidson, Y u Deng, Jeff Carroll, Jen Schmidt, Jamie Lepard, Claudia Schwab and E d Chan for making my long hours in the lab more pleasant.  Finally, I would like to thank my family and friends for providing pleasant and needed distractions from my thesis work. I would like to thank my mom, Angela, and dad, Ray, for their love, support and encouragement for the past 29 years. I would like to thank my brother Mark and his family, Cathy and Paul, for providing a local dose o f family whenever necessary and also my little sisters, Raeann, Rachelle and Remei, for providing a not so local dose o f family. Last, but not least, I would like to thank M a i y a Geddes for her love and for making me happy throughout the ups and downs o f my thesis work.  xvii  DEDICATION  To Maiya for her love, support and the happiness she brings me.  xviii  1 . 0 INTRODUCTION Huntington disease  ( H D ) is an adult onset, neurodegenerative  disorder caused by a  trinucleotide repeat expansion in the gene encoding the huntingtin (htt) protein. Symptoms normally arise between the ages o f 35 and 50 with progression to death approximately 15 years later (Harper, 1996). The disease is an autosomal dominant disorder and affects approximately one in 10,000 individuals (Conneally, 1984).  1.1 Clinical description of Huntington disease Symptoms o f H D include motor dysfunction, cognitive impairment and neuropsychiatric manifestations. There are two components to the movement disorder: an increase in involuntary  movements  and  a progressive  decline in voluntary motion. The  most  characteristic feature o f H D is chorea. This excessive, involuntary movement is seen in early phases o f the disease. A s the disease progresses, patients show an increased inability to perform voluntary movements. Bradykinesia, a difficulty in initiating voluntary movements or slowness in performing motor tasks, eventually progresses into dystonia, rigidity and a complete inability to initiate voluntary movements.  During this progression,  patients  experience difficulties in both speaking (dysarthria) and swallowing (dysphagia). Abnormal eye movements and gait disturbances are also present in H D patients.  While the clinical diagnosis o f H D is dependent on the presence o f specific motor signs on the neurologic exam, persons carrying the disease mutation can demonstrate cognitive dysfunction prior to the onset o f motor symptoms. Specifically, presymptomatic H D carriers have been shown to have deficits in shifting strategy, psychomotor speed, recognition memory, planning and verbal fluency (Paulsen et al, 2001; Berrios et al, 2002; Hahn-Barma et al,  1998; Lawrence et al,  1998A; Lawrence et al,  1998B; Snowden et al,  2002).  However, presymptmatic cognitive impairments are subtle as many studies have been unable to detect any difference in cognitive function prior to motor onset (Blackmore et al, 1995; De Boo et al, 1997; De B o o et al, 1999; Strauss and Brandt, 1990).  1  As the disease progresses through motor onset, cognitive deficits worsen and become more widespread. Symptomatic HD patients show deficits in procedural learning (Schmidtke et al, 2002, Heindel et al, 1988, Heindel et al, 1989, Gabrieli et al, 1997, Knopman and Nissen, 1991) and have been shown to have deficits in both long term (Wilson et al, 1987; Rohrer et al, 1999) as well as working memory (Lawrence et al, 2000; Brown et al, 2001). One of the most common findings in HD patients is a difficulty in tasks requiring a shift in strategy (Lawrence et al, 1999; Ho et al, 2003). This type of response may stem from an inability to inhibit the previously-learned response, a process thought to be mediated by the striatum (Mink, 1996). The loss of inhibition in HD patients is also demonstrated by impairments in pre-pulse inhibition (Swerdlow et al, 1995). HD patients also exhibit neuropsychiatric disturbances which can be quite varied. Studies examining the prevalence of psychiatric disorders in HD patients have found that anywhere from 36-81% of patients are affected (summarized in Harper, 1996). Irritability and aggressive outbursts have commonly been reported in HD patients. Depression, anxiety and apathy are also frequently observed. In the early phases of the disease, patients will often exhibit personality changes such as an exacerbation of already present personality traits. Patients with HD have also been reported to exhibit psychosis, paranoia, schizophrenic symptoms and obsessive compulsive disorder. In addition, HD patients lose weight as the disease progresses (Djousse et al, 2000). Even when placed on high calorie diets HD patients either fail to gain weight or continue to lose weight, perhaps suggesting either a defect in metabolism throughout the body or specific damage to a region of the brain controlling metabolism. 1.2. Neuropathology of Huntington disease  The neuropathology of HD is. characterized by selective and progressive atrophy and neuronal loss occurring primarily in the striatum (caudate and putamen) and cortex. Overall, HD patients can exhibit decreases in total brain mass of up to 30% (Sharp and Ross, 1996) that result from degeneration in select regions of the brain. Striatal volume loss in early-mid  2  stages o f H D has been measured as 53% compared to controls using magnetic resonance imaging ( M R I ) or post-mortem volume displacement (Rosas et al, 2003; Halliday et al, 1998). Although the magnitude o f atrophy is less than in the striatum, significant volume losses o f 4 1 % and 23% were reported in the globus pallidus and cortex o f H D brains respectively (Rosas et al, 2003; Halliday et al, 1998). In contrast, the hippocampus and the cerebellum are relatively spared. H D patients show a 9% decrease in hippocampal volume and no change at all in cerebellar volume compared to unaffected control subjects (Rosas et al, 2003).  Neuronal loss is also selective in H D as cell loss is prominent in the striatum and cortex but not in other regions o f the brain such as the hippocampus and cerebellum. Neuronal loss in H D patients approaches 90% in the striatum and 40% in the cortex (MacDonald and Halliday, 2002). Neuronal loss has also been reported in the C A 1 region o f the hippocampus (Spargo et al, 1993). l n the striatum, G A B A e r g i c medium spiny projection neurons are most affected (Vonsattel et al, 1985; A l b i n et al, 1992) while large neurons in layers III, V and V I are lost in the cortex (Cudkowicz and K o w a l l , 1990; Hedreen et al, 1991). In addition to cell loss, the striatum shows several markers o f inflammation including astrocytosis, microgliosis and complement activation (Singhrao et al, 1999).  Microscopically, H D is characterized by the formation o f neuronal protein aggregates. A role for aggregate formation in H D pathology was originally proposed based on the appearance o f aggregates in a mouse model o f H D which preceded the development o f neurologic abnormalities (Davies et al, 1997). Re-examination o f brains from H D patients revealed that aggregates primarily occur in the striatum and cortex, the two regions most affected by the disease, and that the frequency o f aggregates is much higher in the most severe form o f H D juvenile H D (DiFiglia et al, 1997). While it is plausible that a protein mass in the cell could alter critical functions in neurons (eg. axonal transport (Sapp et al, 1999; L i et al, 2003)), the association o f aggregates with pathology does not indicate causality. Evidence is accumulating that large aggregates are not harmful to cells and may be beneficial (Arrasate et al, 2004; Saudou et al, 1998; Kuemmerle et al, 1999; H o d g s o n ^ al, 1999). However, it has not been possible to rule out aggregation intermediates as the toxic entity in H D brain.  3  1.3 Genetics of Huntington disease  The HD gene, originally known as IT 15 (interesting transcript 15), was first identified by the HD Collaborative Research Group in 1993 (MacDonald et al, 1993) and is located on the short arm of chromosome 4 at position 4pl6.3. The 210 kb HD gene contains 67 exons and encodes two mRNA products of 10.3 kb and 13.6 kb, with the size difference accounted for by differential polyadenylation (Lin et al, 1993). The larger mRNA predominates in the brain while the smaller transcript predominates elsewhere. The HD gene encodes a 350 KDa protein called huntingtin which is expressed ubiquitously with highest levels occurring in the brain and testis (Sharp et al, 1995). The mutant htt protein is also expressed indicating that the pathogenesis of HD does not result from a simple absence of htt protein expression (Stine etal, 1995; Sharp etal, 1995). 1.3.1 Huntington disease is caused by a C A G expansion in Huntington disease gene  The dominant mutation that causes HD is a CAG expansion in the polyglutamine-encoding CAG tract in exon 1 of the HD gene (HD Collaborative Research Group, 1993). While the length of the CAG tract is polymorphic in normal individuals, there is a strikingly sharp CAG threshold for the development of HD (Kremer et al, 1994). Unaffected individuals have between 9 and 35 CAG repeats, while individuals with HD have repeat sizes greater than 35 (Brinkman et al, 1997). Among HD patients, a longer CAG repeat size is associated with an earlier onset and an increased severity of symptoms (Trottier et al, 1994; Brinkman et al, 1997). Interestingly, the size of the CAG tract shows somatic and intergenerational instability with both contractions and expansions being observed (Duyao et al, 1993). Between generations, longer CAG repeat sizes, especially those in the disease range, show both greater instability and a greater tendency towards expansion (Leeflang et al, 1995). As a result, two unaffected parents can give rise to a child who will develop HD. Furthermore, due to the expansion bias of disease alleles and the CAG size dependence of disease severity, there is a progressive increase in disease severity between generations of affected individuals. This is the molecular mechanism that explains the clinical phenomenon of anticipation (Mahadevan et al, 1992; Trottier et al, 1994).  4  1.3.2 Expression of the Huntington disease gene Examination of htt expression in the brain has revealed no correlation between htt expression and disease pathology. Both in situ hybridization and western blotting has revealed that htt expression is highest in the cerebellum which is relatively unaffected in HD (Sharp et al, 1995; Strong et al, 1993; Trottier et al, 1995; Li et al, 1993; Landwehrmeyer et al, 1995). Similarly, htt expression is high in the hippocampus where neuropathology is present but limited (Sharp et al, 1995; Strong et al, 1993; Landwehrmeyer et al, 1995). In contrast, htt expression in the cortex is similar to hippocampal htt expression but the cortex shows more widespread and earlier atrophy and neuronal loss (Sharp et al, 1995; Landwehrmeyer et al, 1995). Finally, htt expression in the striatum is less than in cerebellum, hippocampus or cortex, while this is the region of greatest degeneration (Sharp et al, 1995; Li et al, 1993; Landwehrmeyer et al, 1995). Thus, the regional differences in htt expression observed within the brain do not account for the selective degeneration in HD.  1.4 Mouse models of Huntington disease An ideal animal model of HD can be used both for studying disease pathogenesis and for preclinical therapeutic trials. Since there is no naturally occurring animal model of HD, it has been necessary to engineer appropriate models of the disease. In most cases, mice have been chosen. These animals are advantageous as a genetic model because protocols are available for incorporating a transgene into the genome and for making targeted modifications to the genome. Following genetic manipulation, the availability of inbred strains allows for the assessment of only changes induced by the manipulation, independent of variation elsewhere in the genome. Furthermore, mice reproduce rapidly (21 day gestation), produce large litters (average litter size of 8 with FVB/N mice), have reasonably short lifespans (approximately 2 years) and take up relatively little space. While some aspects of HD can be studied in simpler genetic models (eg. cell models, Drosophila), mouse models permit the study of pathogenesis in a complex brain. Four basic types of HD mouse models have been generated: neurotoxin models, models expressing an N-terminal fragment of mutant htt, knock-in mouse models of HD and full  5  length transgenic mouse models of HD (summarized in Table 1.1). The ideal mouse model for HD should reproduce the progressive motor deficits, cognitive impairment and psychiatric disturbances that characterize the human disease.  The mice should also  demonstrate selective and progressive atrophy and neuronal loss in the same regions of the brain that are affected in HD. In order to study and develop treatments against the early stages of disease pathogenesis, it is important to reproduce the genetic defect in H D (ie. the tissue and developmentally specific expression of full length mutant htt). Finally, in order for a mouse model of HD to be useful for therapeutic trials it must exhibit quantifiable, biologically relevant differences from WT mice that can be used as outcome measures in assessing the benefits of potential treatments for HD.  1.4.1 Neurotoxin models of Huntington disease Prior to the discovery of the H D gene, multiple neurotoxin models of HD were developed. Excitotoxic neurotoxins such as kainic acid (Coyle and Schwarcz, 1976; McGeer and McGeer, 1976) and quinolinic acid (Beal et al, 1986; Beal et al, 1991) have been shown to generate striatal lesions similar to those present in HD. Similarly, inhibitors of mitochondrial function such as 3-nitroproprionic acid and malonate have also been used to model HD (Beal et al, 1993). While acute delivery of these neurotoxins results in neuronal loss and in some cases behavioural changes that are similar to HD, these models cannot be used to study the process of neuronal damage in H D and may be of limited use in developing treatments for the disease. Nonetheless, these models may be useful in high throughput screening of treatments prior to preclinical therapeutic trials in more accurate mouse models of HD (eg. Andreassen et al, 2000; Ona et al, 1999; Chen et al, 2000).  1.4.2 Huntington disease mouse models expressing N-terminal fragments of mutant htt Transgenic models of H D have been created that express an N-terminal fragment of htt with an expanded C A G tract. These mice show obvious behavioural abnormalities, intranuclear inclusions, hypoactivity, weight reductions and unselective degeneration in the brain (Mangiarini et al, 1996; Schilling et al, 1999; Yamamoto et al, 2000; Laforet et al, 2001).  6  Table 1.1 Mouse models of Huntington disease. Four basic types of HD mouse model have been produced. Neurotoxin models involve injecting mice with excitotoxic or metabolic neurotoxins such as kainic acid, quinolinic acid, 3NP or malonate. N-terminalfragmentmodels are transgenic models of HD that express an N-terminal fragment of htt with an expanded polyglutamine tract. Knock-in mouse models are generated by the targeted insertion of CAG repeats into the mouse endogenous HD gene. Full length mouse models are transgenic mouse models that express full length htt with an expanded polyglutamine tract. Model  Neurotoxin  N-terminal fragment  Knock-in  Full Length  Generation  Advantages  Disadvantages  Examples  Striatal lesioning with neurotoxins  Exhibits neuronal death in striatum Can rapidly screen neuroprotective treatments  Acute damage does not model the genetic disorder Cannot study disease pathogenesis  Kainic acid (Coyle and Schwarcz, 1976; McGeer and McGeer, 1976) Quinolinic acid (Beal et al, 1986) 3-nitroproprionic acid (Beal et al, 1993)  Transgenic expression of N-terminal fragment of mutant htt  Clear behavioural abnormalities with early onset Can complete therapeutic trials relatively rapidly Can use survival as an endpoint  Do not express full length mutant htt Neuropathology is not selective No neuronal loss  R6/2, R6/1 (Mangiarini etal, 1996) N-171-82Q (Schilling et al, 1999) HD100 (Laforet et al, 2001)  Targeted insertion of CAG repeats into mouse HD gene  Precise genetic model of HD Express full length mutant htt Can examine early events in disease pathogenesis  Mild behavioural phenotype Mild, late neuropathology No neuronal loss Cannot use for therapeutic trials  HdhQ (White et al, 1997) HdhQ , HdhQ' (Wheeler et ai, 2000) CAG71, CAG94 (Levine et al, 1999) CHL2 - Hdh< > (Lin et al, 2001) CAG140 (Menalled et al, 2003)  Transgenic expression of full length mutant htt  Express full length mutant htt Exhibit both behavioural abnormalities and neuropathology Show neuronal loss Can use for therapeutic trials Can study disease pathogenesis  Appropriate expression is not present in cDNA mice Onset is late in YAC mice Express 2 copies of wild type HD gene  HD46, HD89 (Reddy et al, 1998) YAC46, YAC72 (Hodgson et al, 1999) YAC 128 (Slow et al, 2003)  50  92  11  CAG  150  The phenotypic similarity o f these mice (Mangiarini et al,  1996; Schilling et al,  1999) to  mice that express an expanded polyglutamine tract in the H P R T protein (Ordway et  al,  1997) suggest that the phenotype may result from polyglutamine toxicity and may not model the specific pathogenesis o f H D .  The R6/1 and R6/2 mouse models were generated through the microinjection o f a 1.9 kb genomic fragment o f human D N A containing exon 1 from the H D gene with approximately 130 C A G repeats and about 1 kb o f the 5' untranslated region (Mangiarini et al,  1996).  Initial characterization o f R6/2 mice revealed a constant tremor, age onset weight loss, a decreased survival o f 10-13 weeks and a non-specific 19% decrease in brain volume in absence o f neuronal loss (Mangiarini et al, 1996). Subsequently, dark cells (defined by their enhanced affinity for staining with toluidine blue or osmium), which were proposed to be s  degenerating neurons, have been demonstrated in aged R6/2 mice, but not quantifiable neuronal loss (Turmaine et al,  1999). R6/2 mice also show a non-selective decrease in  striatal volume and striatal neuronal cross-sectional area. Behavioural characterization o f R6/2 mice shows the development o f learning impairments beginning at 3-4 weeks o f age which precede the onset o f motor deficits at about 5 weeks o f age (Carter et al, 1999; Lione et al, 1999). Neuronal intranuclear inclusions are present throughout the brain o f R6/2 mice (Davies et al, 1997; L i et al, 2000). The N171-82Q mouse model expresses a c D N A encoding the first 171 amino acids o f htt with 82 C A G repeats under the control o f the prion promoter (Schilling et al, 1999). N 1 7 1 82Q mice show age onset weight loss, decreased survival, motor impairment on the rotarod, widespread aggregates but do not show selective neuronal loss (Schilling et al,  1999).  Interestingly, the addition o f a nuclear localization signal ( N L S ) to the N171-82Q construct did not alter the phenotype o f the mice, suggesting that the neuronal dysfunction in these animals is caused by the action o f the mutant htt fragment in the nucleus and that the fragment is small enough that it can easily enter the nucleus without the N L S (Schilling et al, 2004).  8  A conditional N-terminal fragment model o f H D was developed to determine  whether  cessation o f mutant htt expression could reverse the symptoms o f the disease. These mice were created to express exon 1 o f htt with 94 C A G repeats under a minimal C M V promoter that requires induction by a tetO operator (Yamamoto et al, 2000). M i c e expressing mutant htt with 94 C A G repeats showed aggregation in the brain, a general decrease in brain size, decreased striatal volume, reactive astrocytosis, clasping and hypoactivity. Turning off the mutant H D gene prevented the progression o f the disease and encouragingly resulted in reversion towards wild-type (Yamamoto et al, 2000).  More recently a mouse model has been developed that expresses a c D N A containing 3 kb o f the H D gene transcript with 100 C A G repeats under the rat neuron-specific enolase promoter (Laforet et al, 2001). These mice showed clasping, abnormal gait, decreased activity and decreased performance on the rotarod. In the brain, dystrophic neurites and increased nuclear htt staining were detected primarily in the striatum and cortex and much less in other areas o f the brain. Striatal neuronal atrophy was detected in these animals but not cell loss. Striatal neurons in these mice exhibited altered electrophysiology which may underlie the motor abnormalities (Laforet et al., 2001).  Overall, N-terminal fragment mouse models develop early motor abnormalities that are similar to symptoms o f human H D . However, changes in the brain are generally not selective, most likely as a result o f not expressing the full length protein and missing all or part o f the endogenous H D gene promoter. In addition, none o f these models exhibited the selective striatal neuronal loss that is present in human H D . Thus, these mice can be used in therapeutic trials to identify treatments that can limit the effects o f polyglutamine toxicity but are not ideal for studying H D pathogenesis because the protein context o f the C A G expansion in H D is not replicated in these models.  1.4.3 Knock-in mouse models of Huntington disease Knock-in models o f H D have been generated through the targeted insertion o f additional C A G repeats into the mouse H D gene (Hdh). A s in the human condition, knock-in mice were  9  engineered to express one copy o f wild-type htt and one copy o f mutant htt. The first H D knock-in mouse was generated with 50 C A G repeats - a repeat size at the high end o f the range for adult onset H D . However, in these mice no phenotypic differences from W T mice were detected even when the mice were bred to homozygosity (White et al,  1997).  Subsequent H D knock-in mice were generated with C A G repeats ranging from 72 to 1 1 1 repeat sizes that would result in juvenile H D in humans. Combined these mice demonstrated only mild abnormalities including decreases in brain size (Shelbourne et al., 1999), increased aggressive behaviour (Shelbourne et al., 1999), increased neuronal sensitivity to N M D A (Levine et al., 1999), increased rearing early followed by hypoactivity (Menalled et al., 2002), increased nuclear localization o f mutant htt (Wheeler et al., 2000) and late onset aggregation (Menalled et al., 2002). None o f the models showed evidence o f neuronal loss. Most recently, H D knock-in mice with C A G repeat lengths that are larger than is seen in human H D patients have been generated (140-150 C A G repeats). Again, even in the homozygous state these mice displayed only mild abnormalities including a rotarod deficit, abnormal activity, decreased weight, nuclear localization o f mutant htt, aggregation and astrocytosis (Lin et al., 2001; Menalled et al., 2003). Overall, knock-in models o f H D reproduce some o f the changes present in H D but the mildness o f the phenotypes observed and the absence o f neuronal loss in these mice limit their utility in therapeutic trials for H D . Nonetheless, the accurate replication o f the genetic defect in H D makes these models suitable for studying the early steps in H D pathogenesis.  1.4.4 Full length transgenic mouse models of Huntington disease Two full length transgenic mouse models o f H D have been developed. Due to the difficultly of generating transgenic mice with a gene that is 210 kb in size, one group generated mice using only the c D N A  from the H D gene while our laboratory used yeast, artificial  chromosomes in order to maintain the proper genetic context o f the full length gene. The full length c D N A transgenic mouse model o f H D was created to express mutant htt with 89 glutamines under the control o f a C M V promoter (Reddy etal, 1998). In addition to nuclear inclusions, limited behavioral changes, hyperactivity and reduced weight, these mice showed  10  neurodegeneration and astrocytosis. However, these lines of mice have been lost and are no longer available for study.  Our laboratory has generated Y A C (yeast artificial chromosome) transgenic mouse models of H D that contain a mutant HD gene with 24 kb of sequence 5' of the H D gene transcript (Hodgson et al, 1999). This approach was chosen to facilitate the inclusion of the entire H D gene, including all of the introns and adjacent regulatory regions, and ensure appropriate expression of the mutant htt transgene. The fact that htt expressed from the Y A C transgene can rescue mice homozygous for the targeted inactivation of the H D gene from embryonic lethality (Hodgson et al, 1996; Hodgson et al, 1999; Leavitt et al, 2001) suggests that the Y A C transgene does contain all of the endogenous regulatory elements of the H D gene required for tissue specific and developmentally appropriate htt expression. Originally, Y A C mice with 46 and 72 C A G repeats (YAC46 mice, YAC72 mice) were generated to model adult onset and juvenile onset H D respectively. YAC46 mice failed to show any differences from WT mice aside from abnormal hippocampal electrophysiology at 10 months of age. In contrast, YAC72 mice show abnormal electrophysiology in the hippocampus at 6 months of age, hyperactivity starting at 7 months of age and nuclear translocation of mutant htt and dark cell neurodegeneration at 12 months of age (Hodgson et al, 1999). Nonetheless, YAC72 mice are not ideal for therapeutic trials since the HD-like symptoms in these mice are mild and primarily qualitative.  Accordingly, our laboratory has developed the Y A C 128 mouse model of H D which expresses mutant htt with approximately 128 glutamines (Slow et al, 2003). Three lines of Y A C 128 mice were generated as previously described (Hodgson et al, 1996; Hodgson et al, 1999). Y A C 128, line 53 mice were shown to have more copies of the Y A C transgene present than the other two Y A C 128 lines and accordingly express higher levels of mutant htt (Slow et al, 2003). As such, the characterization of the Y A C 128, line 53 mice (hereafter called Y A C 128 mice) is described here. Elsewhere characterization of the Y A C 128, line 55 mice has replicated many of the phenotypes observed in the line 53 mice at later time points indicating that the phenotype reported here does not result from a site of integration effect  11  and that the toxicity of mutant htt in Y A C mouse models is dose dependent (Graham et al., 2005).  Overall, an examination of mouse models of H D indicates that it is necessary to increase the toxicity of the mutant htt transgene by increasing the C A G size, increasing the levels of protein expression or decreasing the size of the htt protein in order to observe a clear phenotype in mice. O f all of the models generated, only the full length transgenic mouse models of HD show both behavioural abnormalities and selective neurodegeneration and of these only the Y A C model has appropriate temporal and spatial expression of htt and is currently available for study. Nonetheless, the severity of symptoms in Y A C 7 2 mice is very mild compared to the human condition and the differences from WT mice are not quantifiable and significant enough to use as robust outcome measures for therapeutic trials.  1.5 Preclinical therapeutic trials for Huntington disease While various symptomatic treatments are available for patients with HD, there is currently no treatment for H D that can prevent the onset of the disease or alter disease progression. The two basic forms of treatment are conventional pharmacologic therapy and gene therapy. Pharmacologic therapy involves the delivery of a therapeutic product that is produced outside of the patient's body. While this approach allows the product to be characterized before delivery and the cessation of treatment at any point in time, multiple treatments are generally needed and the products may have difficulty in reaching and or entering target cells. Gene therapy offers an alternative approach to treatment where a therapeutic gene is delivered to the patient rather than the product it produces. This form of therapy is effective for both therapeutic proteins and R N A . The major advantage of gene therapy compared to pharmacologic therapy is that the therapeutic product is continuously produced within the patient after a single treatment and tissue specific expression can be obtained. In mice the efficacy of gene therapy using a particular gene can be predicted based on crosses with transgenic mice engineered to over-express the particular gene.  12  In addition to,how the therapeutic product w i l l be delivered, considerations that are critical to the design o f therapeutic trials include: when to deliver the treatment; where to deliver treatment and how much treatment to deliver. While the time course o f events that lead to neuronal dysfunction and death in H D is currently unknown, it is promising that a recent conditional model o f H D suggests that the progression o f the disease can be stopped and the deficits reversed by eliminating expression o f mutant htt (Yamamoto et al, 2000). The most obvious site for the delivery o f treatment would be the striatum since this area shows the most neuronal death but the possibility that neuronal damage begins in the cortex suggests that it may also be necessary to treat cortical neurons. Finally, the optimum dose o f treatment must balance the potential toxicity o f excessive treatment with the reduced effect o f low levels o f treatment.  The exact mechanism o f pathogenesis in H D is currently unknown. While many o f the steps have been elucidated, the order o f these steps remains uncertain. The pathogenesis may follow a linear progression, in which case, prevention o f the earliest step in the disease mechanism would be sufficient to prevent the disease, or multiple mechanisms in parallel could be involved, possibly necessitating combination therapies for complete amelioration o f disease phenotypes. The earliest step in H D pathogenesis is the expression o f mutant htt which is sufficient to cause H D . Subsequently, the following events are thought to be involved: misfolding o f mutant htt, cleavage o f mutant and wild-type htt by caspases, calpains and aspartyl proteases (Goldberg et al. 1996; Wellington et al, 2000; K i m et al, 2001; Gafni et al, 2002), aggregation o f mutant and wild-type htt (Davies et al, D i F i g l i a et al,  1997), nuclear localization o f mutant htt (Peters et al,  1997;  1999), alterations in  transcription (Cha et al, 2000), alterations in transport within the cell (Gauthier et al, 2004; Trushina et al, 2004), caspase activation (Sanchez et al, 1999; Ona et al, 1999), proteasome inhibition, decreased mitochondrial function (Panov et al, increased sensitivity to excitotoxicity (Young et al,  2002; Gines et al,  1988; Zeron et al,  2003),  2000), disturbed  calcium signaling (Bezprozvanny and Hayden, 2004; Tang et al, 2005), oxidative damage (Browne et al,  1999), altered interaction with htt interacting proteins ( L i and L i , 2004),  decreased levels o f neurotrophic factors (Ferrer et al, 2000; Zuccato et al, 2001; Zuccato et al, 2003) and loss o f wild-type htt function (Cattaneo et al, 2001). While some o f these  13  steps can be directly connected (eg. cleavage of htt leads to its nuclear localization which results in alterations in transcription), connecting all of the steps has proven difficult. Nonetheless, these insights into the pathogenesis of HD, have provided numerous targets for potential treatments against HD. Prior to testing these possible treatments in humans, the efficacy and safety of potential treatments should be tested in an appropriate animal model. As outlined below, most of the treatments assessed in animal models have been mechanism based (Table 1.2). In addition, all of the therapeutic trials in genetic mouse models of HD thus far have been completed in N-terminal fragment mouse models. These models provide the opportunity to rapidly assess the effectiveness of a treatment on a well characterized behavioural and neuropathological phenotype and to compare the effectiveness of different treatments based on standardized outcome measures such as survival, neuronal size and rotarod performance. A combination therapy of coenzyme Q10 and remacemide has shown the greatest impact on survival increasing the lifespan of treated R6/2 mice by 32% (Ferrante et al, 2002). The treatments with the greatest impact on neuronal size (increased 131%) and rotarod performance (increased 77%) were mithramycin and creatine respectively (Ferrante et al, 2004; Ferrante et al, 2000). As summarized in Table 1.2, treatments aimed to prevent aggregation (congo red, trehalose), to restore normal transcription (SAHA, phenylbuyrate, sodium butyrate, mithramycin), to limit apoptosis (dominant negative caspase-1, zVAD-fmk, minocycline, YVAD-cmk with DEVD-fmk, Bcl-2), to limit excitotoxicity (riluzole), to increase antioxidant activity (lipoic acid, BN82451) and treatments with multiple beneficial effects (tauroursodeoxycholic acid, creatine, lithium chloride, coenzyme Q10 with remacemide, cystamine, caloric restriction) have all shown benefits in the N-terminal fragment models of HD thereby supporting a role for these mechanisms in HD pathogenesis. The fact that none of the treatments completely ameliorated the symptoms in the mice suggests that the earliest step in HD pathogenesis wasriotprevented or that multiple early steps occur simultaneously.  A promising alternative to these treatments would be to design a treatment that eliminates the expression of mutant htt. Since continued expression of mutant htt appears to be required for disease progression (Yamamoto et al, 2000), even preventing mutant htt expression in  14  Table 1.2. Summary of Treatment Trials in Genetic Mouse Models of Huntington Disease. Treatment Ascorbate  Mechanism(s) Restore ascrobate levels  Model  Benefits  Reference  R6/2  M i l d I motor s y m p t o m s  R e b e c etal., 2003  Antidepressant; Paroxetine  inhibits uptake o f  t survival 10% N171-82Q  serotonin  t b o d y w e i g h t , rotarod  Duane/o/.,2004  J. v e n t r i c u l a r enlargement t m o t o r function  Environmental  R6/1  Enrichment  I clasping  V a n D e l l e n e r a/., 2 0 0 0  | forebrain v o l u m e 1 3 %  Environmental  R6/1  Enrichment Environmental Enrichment  N171-82Q  T rotarod, B D N F , D A R P P - 3 2 T rotarod 7 3 % | b o d y weight  Spires etal., 2 0 0 4 S c h i l l i n g et al., 2 0 0 4  t survival 16% C o n g o red  Prevent aggregation  R6/2  t rotarod, b o d y w e i g h t  S a n c h e z et al., 2003  I aggregation, c l a s p i n g T s u r v i v a l 11% Trehalose  Prevent aggregation  R6/2  Suberoylanilide h y d r o x a m i c acid (SAHA)  Restore transcription  R6/2  Phenylbutyrate  Restore transcription  N171-82Q  S o d i u m butyrate  Restore transcription  t b o d y w e i g h t , brain weight, rotarod J, v e n t r i c u l a r enlargement, aggregates  t rotarod  Tanakae/a/.,2004  H o c k l y etal., 2003  t survival 2 3 %  Mithramycin  Restore transcription ( A n t i t u m o r antibiotic)  R6/2  zVAD-fmk  L i m i t apoptosis  R6/2  Minocycline  L i m i t apoptosis  R6/2  L i m i t apoptosis  R6/2  L i m i t apoptosis  R6/2  L i m i t apoptosis Tauroursodeoxycholic acid  t brain w e i g h t 2 3 %  Ferrante et al, 2004  t survival 2 0 % R6/2  Bcl-2  Fen-ante et al., 2003  | neuronal size 1 3 1 %  L i m i t apoptosis  fmk  t survival 2 1 % T rotarod 3 5 % t brain w e i g h t 16% t neuronal size 9 8 %  G a r d i a n etal., 2004  t survival 2 9 % R6/2  Dominant Negative Caspase-1  YVAD-cmk + DEVD-  t cerebral v o l u m e 2 2 % | neuronal size 1 1 5 %  T survival 2 5 % t rotarod t s u r v i v a l 14% t rotarod t survival 12% t rotarod 17% f survival 10% j rotarod  Ona etal., 1999  O n a etal., 1999 C h e n et al., 2 0 0 0 C h e n etal., 2 0 0 0 Z h a n g etal,  2003  I T U N E L p o s i t i v e striatal neurons  Antioxidant Improve  t rotarod, b o d y w e i g h t I aggregation  R6/2  mitochondrial  t striatal v o l u m e 1 2 % | aggregates  K e e n e d a/., 2 0 0 2  t rotarod, a c t i v i t y  function  t survival 17% t rotarod 7 7 %  Restoring cellular Creatine  energy levels  R6/2  Inhibits M P T  t brain weight 1 7 % t neuronal size - 5 7 %  Ferrante e r a / . , 2 0 0 0  t body weight I aggregation t s u r v i v a l 14% t rotarod 2 3 %  Restoring cellular Creatine  energy levels  R6/2  Inhibits M P T  t neuronal size 6 0 % T body weight 19%  D e d e o g l u et al, 2003  t brain weight 1 7 % i aggregation t survival 19%  Restoring cellular Creatine  energy levels Inhibits M P T  N171-82Q  | rotarod, b o d y w e i g h t t brain w e i g h t 1 3 %  A n d r e a s s e n et al, 2001  I aggregation  15  Table 1.2. Summary of Treatment Trials in Genetic Mouse Models of Huntington Disease(continued) Treatment  Mechanism(s)  Model  Riluzole  Anti-excitotoxic  R6/2  Coenzyme QI 0 and remacemide  Improve mitochondrial function Anti-excitotoxic  N17I-82Q  Coenzyme Q10 and remacemide  Improve mitochondrial function Anti-excitotoxic  R6/2  a-Lipoic Acid  Improve mitochondrial function Anti-excitotoxic Anti-oxidant  a-Lipoic Acid  Anti-oxidant  NI71-82Q  BN82451  Anti-oxidant  R6/2  Essential Fatty Acids  Membrane integrity  R6/1  Cystamine  Inhibits T G activity Anti-apoptosis Anti-oxidant  Coenzyme Q10 and remacemide  Cystamine  Cystamine  Inhibits T G activity Anti-apoptosis Anti-oxidant Inhibits T G activity Anti-apoptosis Anti-oxidant  NI71-82Q R6/2  R6/2  R6/2  R6/2  Tissue T G knockout  Eliminate tissue T G activity  R6/1  Tissue T G knockout  Eliminate tissue T G activity  R6/2  Dietary Restriction  Decrease ROS Increase B D N F  Striatal transplantation  N171-82Q  Benefits t survival 10% t body weight J. hyperactivity I size of aggregate t rotarod 28%  t survival 32% j rotarod 62% j body weight 20% t brain weight 18% t neuronal size 87% J. aggregation f survival 17% f body weight 8% t survival 7% t survival 8% t body weight t survival 15% j striatal volume 22% 1 neuronal size 65% J. aggregation | survival 19% M i l d I motor symptoms j rotarod 27% t body weight 15% t brain weight 20% f neuronal size 61 % | aggregation T survival 12% M i l d J, motor symptoms f body weight ] dopamine D2 receptor function I size of aggregates t neuronal morphology t survival 12% T aggregation i degenerating neurons 60-70% t rotarod t survival 20% t aggregation 30-35% t rotarod | | t | J.  survival body weight rotarod ventricular enlargement aggregation  Reference Schiefer era/., 2002  Schilling  Hsp70  R6/2  R N A Interference  Decrease mutant htt levels  N171-82Q  GDNF  Neuroprotection  R6/2  CGS21680  A A adenosine receptor antagonist  R6/2  Lithium Chloride  Limit apoptosis Anti-excitotoxic  R6/2  2  M i l d I motor symptoms t body weight  I aggregation t rotarod •T gait no benefit t rotarod I ventricular enlargement I size of aggregates t rotarod  2001  Ferrante et al., 2002  Ferrante et al., 2002  Andreassen et al., 2001 Andreassen et al., 2001 Klivenyi  Clifford  etal.,  etal.,  2003  2002  Dedeoglu et al, 2002  Karpuj et al., 2002 Wang et al., 2005 Mastroberardino et ai, 2002  Bailey and Johnson, 2004  Duan  etal.,  2003  etal.,  R6/2 Promote proper folding and elimination of mutant htt  eta!.,  Dunnett 1998 Hansson et ai, 2003  Harper  etal., etal.,  Popovic Chou era/., 2005  2005  2005  Wood and Morton, 2003  16  symptomatic patients should provide benefit. Early attempts to decrease the expression of htt by antisense gene therapy resulted in only small reductions in the levels of htt (Haque and Isacson, 1997). Recently, the approach of RNA interference (RNAi) in combination with delivery using an adeno-associated virus gene therapy vector was successful in decreasing mutant protein expression, improving motor performance and improving brain morphology in a mouse model of another polyglutamine expansion disorder (Xia et al, 2004). If a similar, RNAi strategy could prevent mutant htt expression then this may provide an ideal treatment for the disease. As a proof of principle, it has recently been demonstrated that decreasing the expression of the mutant htt fragment in N171-82Q mice by 50% using RNAi resulted in mild motor improvements and decreased aggregation (Harper et al, 2005). 1.6 Huntingtin function Wild-type htt function is essential for embryonic development and normal function in adulthood (summarized in Table 1.3). The complete loss of htt expression results in embryonic lethality (Nasir et al, 1995; Duyao et al, 1995; Zeitlin et al, 1995). Reducing wild-type htt levels by half leads to behavioural abnormalities, cognitive dysfunction and neuronal loss (Nasir et al, 1995; O'Kusky et al, 1999). Further reduction of wild-type htt levels results in gross brain abnormalities and perinatal lethality (White et al, 1997; Auerbach et al, 2001). The continued importance of wild-type htt function in adulthood is indicated by the progressive neurological phenotype observed in mice with a conditional adult inactivation of wild-type htt in the forebrain (Dragatsis et al, 2000). Wild-type htt is a multi-functional protein that acts in both the nucleus and cytoplasm. As wild-type htt expression is decreased in HD patients, it is important to define the normal functions of wild-type htt as these functions may be disrupted in HD. Since mutant htt is able to rescue mice homozygous for the targeted inactivation of the mouse HD gene from embryonic lethality (Hodgson et al, 1999; Leavitt et al, 2001; Van Raamsdonk et al, 2005), it is clear that mutant htt maintains some of the essential functions of wild-type htt. The currently defined functions of wild-type htt fall under three main categories: transcription, transport and neuroprotection.  17  Table 1.3. Decreasing wild-type huntingtin levels results in phenotypic abnormalities. Levels of wild-type htt that are 50% or less of normal levels show clear abnormalities in mouse models. Mouse  Expression of Wild-type htt  Heterozygous for targeted inactivation of HD gene  50%  Adult Inactivation of htt in testis and forebrain  10-16% in adult brain  H d h  neo50Q  H d h  neo50Q  h  h  o  e  m  o  z  m  i  z  y  y  g  g  o  o  t  t  e  e  s  s  Homozygous for the targeted inactivation of the HD gene  -33% -16% None  Phenotype  Reference  Hyperactivity, cognitive defects, neuronal loss Reduced size, clasping, hypoactivity, neuronal degeneration Gross brain abnormalities, do not survive more than 2 days Gross brain abnormalities, embryonic lethal  Nasire/a/., 1995 O'Kusky et al, 1999  Embryonic Lethal  Dragatsis et al, 2000  White etal, 1997 White et al, 1997 Nasire/a/., 1995 Zeitline/a/., 1995 Duyao etal, 1995  18  1.6.1 Transcription A role for wild-type htt in transcription is suggested by the fact that several proteins that interact with wild-type and mutant htt are involved in transcription. These proteins include: CBP (Steffan et al. 2000; Nucifora et ai, 2001), Spl (Li et al, 2002; Dunah et al, 2002), NCoR (Boutell et al, 1999), REST-NRSF (Zuccato et al, 2003), p53 (Steffan et al, 2000), TBP (Huang et al, 1998), C-terminal binding protein (Kegel et al, 2002), TAFII130 (Dunah et al, 2002), NF-kB (Takano and Gusella, 2002) and CA150 (Holbert et al, 2001). The interaction of htt with CBP and Spl has been particularly well studied since these transcription factors regulate genes that are critical for neuronal function and thus alteration of this function may significantly contribute to the pathogenesis of HD. While the effect of CAG expansion on CBP-mediated and Spl-mediated transcription has been well characterized, the precise role of wild-type htt in their normal functioning has not been elucidated. In contrast, the role of wild-type htt in promoting BDNF transcription has been clearly determined. Initial studies demonstrated that over-expression of wild-type htt increased transcription of BDNF both in vitro and in transgenic mice engineered to over-express wildtype htt (Zuccato et al, 2001). Further investigation revealed that the increase in expression resulted from htt's interaction with REST/NRSF (repressor element-1 transcription factor/neuron restrictive silencer factor). REST/NRSF binds to NRSE (neuron restrictive silencer elements) to inhibit transcription of neuronal genes including BDNF. Thus, increased levels of wild-type htt result in increased sequestration of REST/NRSF, decreased silencing of genes regulated by NRSE and consequently increased expression of neuronal genes such as BDNF (Zuccato et al, 2003). An examination of gene expression changes that occur in HD also suggests a role for wildtype htt in transcription as does the presence of wild-type htt in the nucleus. Gene expression studies in mouse models of HD have revealed numerous changes in N-terminal fragment models of the disease (Luthi-Carter et al, 2002) with a much smaller number of changes in mice expressing full length mutant htt (Chan et al, 2002). This follows from the fact that  19  smaller fragments of mutant htt can' enter the nucleus with greater ease and provides a possible explanation for why N-terminal fragments may be more toxic than full length mutant htt. While the ability of mutant htt to alter gene expression does not necessarily indicate that wild-type htt is involved in normal gene expression, wild-type htt is clearly involved in the regulation of genes under NRSE. 1.6.2 Transport  >  A role for wild-type htt in intracellular transport is suggested by its interaction with several proteins that are involved in trafficking and endocytosis including HAP1 (Li et al, 1995), HIP1 (Kalchman et al, 1997; Wanker et al, 1997), HIP 14 (Singaraja et al, 2002), PACSIN1 (Modregger et al, 2002) and PSD-95 (Sun et al, 2001). The role of wild-type htt in axonal transport has been most clearly described. The involvement of wild-type htt in axonal transport was first demonstrated in Drosophila. Decreasing wild-type htt levels using RNAi resulted in defects that were characteristic of deficits in axonal transport and these defects were exacerbated by reductions in kinesin heavy or light chains (Gunawardena et al, 2003). A specific role for wild-type htt in the transport of vesicles carrying BDNF was demonstrated by modulating wild-type htt levels in cell culture (Gauthier et al, 2004). Increasing levels of wild-type htt resulted in increased movement of BDNF containing vesicles while decreasing htt levels by RNAi resulted in a decreased velocity of transport (Gauthier et al, 2004). The interaction of htt with HAP1 and pl50Glued was shown to be important for htt's role in facilitating axonal transport (Gauthier et al, 2004). Similarly, a role for wild-type htt in mitochondrial transport along axons has been demonstrated since decreased htt levels resulted in decreased movement of mitochondria along axons (Trushina et al, 2004). A role for htt in fast axonal transport is supported by the fact that htt along with HAP1 are themselves moved by fast axonal transport in the cell (Block-Galarza etal, 1997).  ••  •  20  1.6.3 Neuroprotection A protective role for wild-type htt against cell death was originally proposed based on findings o f extensive apoptosis leading to embryonic lethality in mice homozygous for the targeted inactivation o f the mouse H D gene (Zeitlin et al, 1995). Subsequently, several in vitro studies have confirmed a pro-survival effect o f wild-type htt. The over-expression o f wild-type htt in immortalized striatal neurons protected these cells from death induced by either serum deprivation at 39°C or exposure to the mitochondrial toxin 3-nitroproprionic acid (Rigamonti et al, 2000). Investigation into the mechanism o f protection by wild-type htt suggested an anti-apoptotic function since there was a reduction in D N A laddering in the cells over-expressing wild-type htt and these cells were also protected from multiple inducers of apoptotic death (Rigamonti et al, 2000). Further, the effect o f wild-type htt was thought to be downstream o f cytochrome c release from the mitochondria (Rigamonti et al, 2001). These findings o f in vitro protection by wild-type htt were extended to show that wild-type htt could protect against the toxicity o f polyglutamine expansion. Cells transfected with wildtype htt were shown to be more resistant to cell death caused by the expression o f an N terminal fragment of mutant htt containing 72 glutamines than cells transfected with a control protein (Ho et al, 2001). Most recently, our laboratory has demonstrated that in addition to a general protective effect, wild-type htt can specifically protect against excitotoxic cell death in vitro (Leavitt et al, submitted).  Our laboratory has also extended in vitro findings to demonstrate that wild-type htt can protect cells from death in vivo. The elimination o f wild-type htt expression in Y A C 4 6 or Y A C 7 2 mice that express mutant htt with either 46 or 72 glutamines respectively resulted in massive degeneration in the testis which increased with C A G size (Leavitt et al, 2001). Addition o f one copy o f wild-type htt eliminated the testicular phenotype in Y A C 4 6 mice and improved the phenotype in Y A C 7 2 mice. Similarly, addition o f two copies of wild-type htt completely restored testicular morphology in the Y A C 7 2 mice indicating that the expression o f wild-type htt protects against cell death in vivo: •  21  The mechanism responsible for the protective effect of wild-type htt is currently unknown but may be mediated through BDNF. BDNF is a neurotrophic factor that promotes the survival of striatal neurons and both its transcription and transport in the cell are increased with increased levels of wild-type htt (Zuccato et al, 2001; Gauthier et al, 2004). Another possible mechanism for wild-type htt's pro-survival effects would be the sequestration of HIP1 (htt interacting protein 1). Free HIP1 protein has been shown to be pro-apoptotic through interaction with HIPPI and activation of caspase 8 (Gervais et al, 2002; Hackam et al, 2000). Since binding of HIP 1 to htt prevents its interaction with HIPPI, increased levels of wild-type htt can lead to decreased levels of free HIP1 and decreased caspase activation. A third possibility, is that htt's protective effect results from htt's ability to inhibit either the expression or activity of caspase 3, an enzyme involved in programmed cell death (Zhang et al, unpublished). 1.7 Loss of huntingtin function in Huntington disease Despite extensive study into the role of mutant htt in HD, the possible contribution of loss of wild-type htt function to the natural history and pathogenesis of HD has not been fully elucidated. Clearly, wild-type htt has multiple important functions within the cell. Since HD patients express both reduced levels of wild-type htt and mutant htt, it is important to determine which of htt's functions are disrupted by polyglutamine expansion and thereby predict the contribution of loss of htt function to HD. It appears that most, if not all, of the currently defined functions of wild-type htt are decreased or lost in mutant htt. Moreover, in many cases mutant htt has a detrimental effect which goes beyond a simple loss of function. Wild-type htt's role in transcription, transport and neuroprotection are all disrupted by polyglutamine expansion. Rather than increasing BDNF expression, mutant htt expression decreases BDNF expression (Zuccato et al, 2001). Similarly, while wild-type htt facilitates axonal transport, mutant htt decreases axonal transport velocities Of vesicles, mitochondria and organelles leading to the accumulation of organelles in axons and slowness to replenish pre-synaptic BDNF (Szebenyi et al, 2003; Gunawardena et al, 2003; Gauthier et al, 2004; Trushina et al, 2004). With respect to htt's pro-survival function, polyglutamine expansion  22  in mutant htt not only eliminates the protection provided by wild-type htt but also makes cells more susceptible to death (Rigamonti et al, 2000; Leavitt et al, submitted). In addition, polyglutamine expansion alters the interaction of htt with at least 17 proteins within the cell which likely has detrimental functional effects (Li and Li, 2004). While reducing levels of wild-type htt alone is not sufficient to cause HD (Ambrose et al, 1994, Nasir et al, 1995, Duyao et al, 1995, Zeitlin et al, 1995), the fact that mutant htt is unable to perform many of the functions of wild-type htt suggests that loss of wild-type htt may significantly contribute to the pathogenesis of HD. Patients who are heterozygous for the HD mutation start out expressing wild-type htt at 50% of the level in unaffected individuals. However, these levels can be further reduced by the disease process either through sequestration into aggregates, through increased cleavage by caspases and calpains or through mislocalization to the nucleus. In support of this, transgenic mouse models of HD expressing an N-terminal fragments of mutant htt have been shown to have decreased levels of wild-type htt as the disease progresses (Ona et al, 1999; Zhang et al, 2003). Given the clear demonstrations of htt's ability to protect cells from death (Rigamonti et al, 2000; Ho et al, 2001; Leavitt et al, 2001), a simple model of HD pathogenesis is suggested where the loss of wild-type htt makes cells more susceptible to the toxic effects of mutant htt (Fig. 1.1). 1.8 Objectives The primary objectives of this thesis were (1) to characterize genetic mouse models of HD, (2) to develop a protocol for therapeutic trials in an appropriate mouse model and (3) to demonstrate the feasibility of performing therapeutic trials in these mice. In addition to these goals, the protective effect of wild-type htt was examined in vivo to determine whether the loss of this function might contribute to HD. This thesis focuses on YAC mouse models of HD. Initial characterization of these mice sought to determine the extent to which YAC mice recapitulate the symptoms observed in human HD. Accordingly, motor function and cognitive function of the mice was assessed using behavioural analysis. Subsequently, the brains of these mice were examined to  23  Neuronal dysfunct Neuronal loss Decreased transport Altered transcription  Figure 1.1 Model for the contribution of loss of huntingtin function to Huntington disease. H D patients express decreased levels of wild-type htt and increased levels of mutant htt compared to unaffected individuals. Since mutant htt has been shown to be toxic and wild-type htt has been shown to be neuroprotective both changes may contribute to the pathogenesis of H D . In addition to possible effects of transport and transcription, the loss of wild-type htt would result in decreased neuroprotection. This may make neurons more susceptible to the toxic effects of mutant htt accelerating the development of neuronal dysfunction and neuronal loss. In addition, mutant htt expression may lead to further decreases in wild-type htt levels through increased caspase or calpain cleavage of wild-type htt, sequesteration of wild-type htt into aggregates and altered localization of wild-type htt.  24  determine i f the selective degeneration that characterizes H D brains was present in these mice. Once the phenotypic differences between Y A C mice and W T mice had been identified, quantifiable, biologically relevant outcome measures from this characterization were selected to develop a standardized protocol for therapeutic trials.  Having established a protocol for therapeutic trials, we sought to determine whether the initial findings from the phenotypic characterization o f the Y A C 128 mice could be reproduced in a therapeutic trial. For this purpose, two therapeutic trials were carried out: genetic treatment by over-expression o f wild-type htt and pharmacologic treatment with cystamine. A genetic treatment and a pharmacologic treatment were selected to demonstrate that both types o f trial are feasible in the Y A C 128 mouse model.  25  2.0 MATERIALS AND METHODS  2.1 Mice Experiments were carried out on yeast artificial chromosome ( Y A C ) transgenic mice that express the human H D gene with 18 C A G repeats ( Y A C 18 mice; Hodgson et al, 1996), 72 C A G repeats ( Y A C 7 2 mice; Hodgson et al,  1999) or approximately 128 C A G repeats  ( Y A C 1 2 8 mice; Slow et al, 2003). T w o separate lines o f Y A C 1 8 mice were used: the low expressing B60 line and a higher expressing line 212. A l l mice were maintained on the F V B / N background strain (Charles River, Wilmington, M A ) . M i c e were group housed in a clean facility and given free access to food and water. A l l experiments were carried out with the approval o f the University o f British Columbia's animal care committee.  2.2 Behavioural assessments M i c e were tested during the light phase o f a normal light-dark cycle where lights were turned off at 8:00 P M and on at 6:00 A M . Experimenters were blind to the genotype o f the mice.  2.2.1 Rotarod test of motor coordination M i c e were trained at 2 months o f age with three trials per day over three days at 24 R P M on the rotarod ( U G O Basile). O n the first training day, i f a mouse fell off the rotarod before 2 minutes, the mouse was placed back on the rotarod and allowed to continue running. M i c e were tested bimonthly from 2 to 12 months o f age at either a fixed speed (12 R P M , 24 R P M , 36 R P M , 40 R P M ) or accelerating (rotarod accelerated from 5 to 40 R P M over 4 minutes). M i c e were removed from the rotarod after falling or reaching a maximum time o f 60 seconds on the fixed speed test or 300 seconds on the accelerating test. The amount o f time that the mouse could stay on the rotarod for three trials spaced 2 hours apart was' recorded and averaged for the mouse's score at each time point.  26  2.2.2 Assessment of motor learning on the rotarod Motor learning was assessed using the rotarod apparatus. In this test, mice must learn to run when placed on a rotating rod to prevent them from falling. Separate cohorts o f mice were trained at 2 months, 7 months or 12 months o f age with 3 trials per day spaced 2 hours apart for three days. 2 month old mice were trained at a fixed speed o f 24 R P M . 7 month and 12 month mice were trained with the rotarod accelerating from 5 to 40 R P M to facilitate learning in older animals. Learning was assessed by comparing the performance o f mice during training. M i c e trained at 2 months and 7 months were tested bimonthly and monthly respectively until 12 months o f age. A t 12 months o f age, the performance o f mice trained initially at 2 months, 7 months and 12 months was compared in the accelerating rotarod task.  2.2.3 Open field activity test Open field activity was measured using an automated open field system (San Diego Instruments). Activity was measured as the total number o f beams crossed during a given time period either 10 minutes, 1 hour or 8 hours. Activity was measured in an empty mouse cage in the dark during the mouse's light cycle. For each trial, mice were placed in a new cage and activity was measured immediately with no training sessions.  2.2.4 Open field habituation test of learning and memory The same apparatus was used to measure open field habituation. Since mice demonstrate less exploratory activity in a familiar environment, this simple test assesses the ability o f the mouse to learn and remember the open field chamber. Intrasession habituation is measured by the relative decrease in activity over time in a single open field trial and is calculated as the difference in activity from the first interval divided by the activity o f the first interval. Intersession habituation is the relative decrease in activity between repeated open field trials and is calculated as the decrease in activity from the first trial divided by the level o f activity in the first trial. The activity o f mice in the open field was assessed in the dark during the light cycle and was measured automatically as the number o f photobeam breaks during each trial. M i c e that were naive to the open field chamber were given 5 trials o f 30 minutes  27  duration. The first three trials were given at 9:00 A M on three consecutive days with the remaining trials given at 11:00 A M and 1:00 P M on the third day. O n the first trial, activity was recorded for the duration o f the 30 minutes period in order to assess intrasession habituation in 5 minute intervals. The activity during the first 10 minutes o f each o f the 5 open field trials was used to assess intersession habituation. For each trial, mice were allowed to remain in the testing chamber with the lights out for a total o f 30 minutes to facilitate habituation.  2.2.5 Simple swimming test A simple swimming test was designed to test procedural learning. In this test mice are placed in the middle o f a linear swimming chamber (76 cm X 13 c m ; water depth: 9 cm; platform: 6 cm X 13 cm) facing away from an escape platform. M i c e are trained to reach the platform in the shortest amount o f time in order to escape from the water. Completion o f this task involves learning and remembering the location o f the platform or the route followed to reach the platform. O n subsequent trials, mice must plan to turn around immediately for the shortest route to the platform. The amount o f time required for the mouse to reach the platform and the initial swimming direction were recorded for each trial. Swimming towards the platform was arbitrarily given a score o f 0 while swimming away from the platform was initially given a score o f 1. M i c e were trained at 2 months o f age with 3 pairs o f 2 consecutive trials spaced 2 hours apart for 2 days. M i c e were tested bimonthly with 3 tests per day spaced 2 hours apart until 12 months o f age.  2.2.6 Pie-pulse inhibition and habituation to acoustic startle Acoustic startle pre-pulse inhibition (PPI) and habituation to acoustic startle were assessed using two SR-Lab Systems (San Diego Instruments). PPI is a test o f sensorimotor gating and does not involve learning. PPI is measured as the percentage decrease in startle intensity from a pulse alone startle when a pre-pulse is given prior to the pulse. Habituation to acoustic startle is a test o f learning o f memory. If mice remember that there is no biologic consequence associated with a repeated loud sound they w i l l show decreased startle in response. Prior to testing, the sensitivity o f the two startle chambers was calibrated using a  28  vibrating standardization unit at 700 volts (San Diego Instruments). M i c e were then placed into each startle chamber and given a 5 minute acclimatization period with background noise alone (65 dB). Each mouse was presented with 6 trials (block 1) o f a 40 msec 120 dB noise burst (pulse alone). Subsequently, the mice experienced 8 blocks o f 6 trials (48 trials total), each block consisting o f the following trial types: 1) no stimulus (background noise only), 2) a 40 msec 120 dB noise burst alone or 3-6) a 40 msec 120 dB noise burst preceded 100 msec by a 20 msec pre-pulse (2, 4, 8 and 16 d B above background noise). The order o f trials within each block o f 6 trials was pseudo-randomized and 4 o f the 8 blocks contained an extra pulse alone trial. The mice then received another 6 trials (block 10) o f 40 msec 120 dB noise burst (pulse alone). The intertrial interval was randomized throughout the entire session and ranged between 8 and 23 seconds.  Each animal enclosure was wiped clean with ethanol  between test subjects. Habituation analysis was measured using the average o f 4 blocks o f pulse alone scores (block 1, pulse alone startles from blocks 2-5, pulse alone startles from block 6-9, block 10) and is calculated as the difference in startle between the first block and subsequent blocks divided by the startle in the first block. PPI was calculated from the average o f 6 trials per pre-pulse as follows: PPI = [(pulse alone startle)-(pre-pulse + pulse startle)]/pulse alone startle.  2.2.7 Beam crossing test M i c e were trained at 2 months o f age with three sets o f 2 consecutive trials. For each trial mice were placed on the beam facing a darkened chamber on the other end o f the beam. The lights in the room were turned off 10 minutes prior to testing. A bright light was pointed at the starting end o f the pole. The amount o f time it took the mouse to reach the darkened chamber was recorded as well as the number o f falls, i f any. M i c e were tested monthly from 2 to 10 months o f age with three trials per day spaced 2 hours apart. Scores for the three trials were averaged.  2.2.8 Footprint analysis For the footprint analysis, mice were given a dry training run where they were allowed to walk from the starting end to the finish end o f the linear walking chamber.^Paper was placed  29  in the chamber prior to training to simulate the testing conditions. Immediately after the training run, the forepaws and hindpaws o f the mice were painted orange and purple respectively and the mouse was allowed to run down the papered walking chamber again. Distances between the resulting footprints were measured and averaged for 2 trials spaced 2 hours apart. Stride length was measured as the distance between consecutive footprints by the same foot for a minimum o f 6 strides.  s 2.2.9 Swimming T-maze test with reversal A swimming T-maze test was developed to assess procedural learning. In this test, mice were placed in the base o f a water-filled T-maze with an escape platform located in the right arm o f the maze (T-maze dimensions - arms: 38 cm X 14 cm, water depth: 7 cm, platform: 10 cm X 14 cm). M i c e must learn to turn right upon reaching the top o f the T in order to reach the platform directly. The time to reach the platform and the path taken to reach the platform were recorded. Swimming right was arbitrarily given a score o f 0 while swimming left was given a score o f 1. M i c e received 4 trials per day spaced 45 minutes apart for 3 days. To successfully complete this task, mice must remember either the location o f or the path to the escape platform. Since F V B / N mice have severe retinal degeneration at the age tested (Taketo et al,  1991; Huerta et al,  1999), learning to swim to the correct arm o f the maze  likely relies on internal rather than external cues.  Following one day o f rest, a reversal phase to the swimming T-maze test was included to assess the ability o f the mice to replace a previously learned strategy. For this test, the platform was switched to the left arm o f the T-maze and again the amount of time and the path to reach the platform were recorded. Swimming towards the platform was arbitrarily given a score o f 0 while initially swimming away from the platform was given a score o f 1. In addition, the total number o f arm entries was noted for each mouse. M i c e received 4 trials per day spaced 45 minutes apart for 3 days. After 3 days o f reversal testing, the swimming speed o f the mice was measured by blocking the stem of'the T-maze and measuring the amount o f time the mice take to swim the length o f the top o f the T to reach the platform.  30  M i c e were given 5 trials spaced 45 minutes apart. The last four trials were used to calculate swimming speed.  2.3 Physiologic measurements 2.3.1 Body weight Body weights were taken biweekly at 9:00 A M on the same day each week.  2.3.2 Organ weights M i c e were injected with heparin, terminally anesthetized by intraperitoneal injection o f 2.5% avertin and perfused with 3% paraformaldehyde in phosphate buffered saline (PBS). Organs were excised from the perfused mice and post-fixed in 3% paraformaldehyde for 24 hours. Organs were then washed briefly and equilibrated with P B S for 24 hours prior to weighing. Brains were left in the skull for the post-fixation and then removed after transfer to P B S . The olfactory bulbs and the brain stem were removed from the brain before weighing. A l l the organs were patted dry with Kim-wipes before weighing.  2.3.3 Survival analysis Cohorts o f mice were followed up until 12 months o f age. The cause o f death was noted for all mice that did not survive to 12 months. In cases where the death was caused by fighting or human intervention, the mouse was not used in the survival analysis. In some cases where mice were sick and the animal care facility deemed that the mice would not survive, mice were euthanized to prevent their suffering and recorded as a death in the survival analysis. Kaplan-Meier analysis was used to assess survival significance ( L o g Rank test).  2.3.4 Histological examination of the testis Testes from mice perfused with 3% paraformaldehyde were punctured with a small needle and then post-fixed in 1.5% paraformaldehyde,  1.5% glutaraldehyde, 0.1 M sodium  cacodylate, p H 7.3. Testis were subsequently cut into small pieces and fixed further in 1%.  31  osmium tetroxide on ice for 1 hour, washed in water and stained in 1% aqueous uranyl acetate. Finally, sample were washed with water, dehydrated and embedded in J E M B E D 812 (J.B. E M Services). Section o f approximately 1 um were cut and stained with toluidine blue for examination o f testicular morphology.  2.4 Neuropathological assessments Brains from mice perfused with 3% paraformaldehyde were infiltrated with sucrose (25% in P B S ) and frozen on dry ice before mounting with Tissue-TEK O . C . T . compound (Sakura). Twenty-five um coronal sections were cut on a cryostat (Microm H M 500M) and collected in P B S in a 24 well plate.  2.4.1 Volume of brain structures A series o f 25 um coronal sections spaced 200 um apart were stained with N e u N for determination o f brain structure volumes. Endogenous peroxidase activity was blocked by incubating sections with phenylhydrazine ( l u l / m l P B S ) for 45 minutes at 36°C. Sections were then washed twice for 10 minutes with P B S before blocking in P B S with 10% N G S and 0.1% T-X-100 for 1 hour at room temperature. Incubation in N e u N primary antibody (1:100 dilution in 5% N G S , 0.1% T-X-100, P B S , M A B 3 7 7 ; Chemicon) was carried out overnight at room temperature or overnight at 4 ° C followed by 2 hours at room temperature. Unbound primary antibody was washed off with three 10 minute washes with P B S before putting the sections into a biotinylated anti-mouse secondary antibody (1:200 dilution in 1% N G S , 0.1% T-X-100, P B S ) for 2 hours at room temperature. Sections were then washed three times for 10 minutes with P B S and incubated in A B C reagent ( A B C Elite kit, Vector) for 2 hours at room temperature. Finally sections were washed three times for 10 minutes each in P B S and the staining signal was chromogenically detected by incubation in metal-enhanced D A B solution (Pierce) for 3 minutes. Sections were then placed back in P B S before mounting onto slides. After mounting, sections were air-dried overnight, cleaned with xylene and coverslipped using flouromount mounting media ( B D H ) . Sections were left a minimum o f one day for mounting media to harden.  32  The volumes o f the striatum, cortex, globus pailidus, hippocampus and cerebellum were determined using Stereo investigator software  (Microbrightfield).  Striatal volume was  measured from the start o f the striatum to the start o f the hippocampus. The volume o f the entire globus pailidus was measured. Cortical volume was measured from the point where the corpus callosum crosses to the start o f the hippocampus. Hippocampal volume was measured from the start o f the hippocampus to the point where the C A 3 region thickens and descends. The volume o f the cerebellum was measured from a coronal series o f unstained sections collected directly onto glass slides. To determine the volume o f each structure, the perimeter o f the structure was traced using a 2 . 5 X objective in each included section o f the coronal series spaced 200 um apart. Subsequently, the total area o f the structure in the included sections was multiplied by the distance between the sections (200 urn) to determine the volume o f each structure.  2.4.2 Neuronal numbers Striatal and cortical neuron counts were determined using Stereoinvestigator software. Using the same contours traced for volume measurement, neuronal profiles were counted in a 25 urn by 25 urn counting frame with a 550 um by 550 urn grid size. These counts were then extrapolated to estimate the total number o f neurons in the striatum. Neuronal number was calculated as: (total number o f neurons counts X total volume o f region)/(total number o f sites counted X volume o f each site). Hippocampal neuronal numbers were estimated by measuring the volume o f the hippocampal cellular layer and dividing by the average volume o f hippocampal neurons.  2.4.3 Striatal neuronal cross-sectional area To determine neuronal cross-sectional areas, a single matched section from each animal was stained with N e u N antibody either as described above or using an Alexa488-conjugated N e u N antibody ( M A B 3 7 7 X ; Chemicon) with no secondary antibody. Mounted sections were analyzed using Neurolucida software (Microbrightfield) to outline the perimeter o f all clearly defined neurons within a 550 urn X 550 um grid o f 25 um X 25 um counting frames with the 100X objective.  ...  33  2.4.4 Striatal DARPP-32 expression For measurement o f D A R P P - 3 2 expression, sections were blocked in P B S with 5% skim milk powder and 0.1% Triton-X-100 prior to incubation with rabbit anti-DARPP-32 antibody (Chemicon A B 1656, 1:1000). After 3 washes with P B S , sections were incubated in C y 3 conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Inc., 1:5.00). Pictures o f mounted sections were taken using MetaMorph Imaging System and the intensity o f the fluorescent stain within the striatum was measured.  2.4.5 Nuclear localization of mutant huntingtin Nuclear localization o f mutant htt was examined in a series o f coronal sections throughout the brain. Sections were stained with polyclonal E M 4 8 antibody as above at a concentration o f 1:500 followed by an anti-rabbit secondary antibody, A B C amplification and D A B detection. Photographs were taken using Metamorph software with an exposure time o f either 2 msec or 63 msec for pictures taken with the 10X or 100X objective respectively. For quantification o f E M 4 8 staining intensity, pictures from each region were taken using the 100X objective. Subsequently, 20 individual neurons in each picture were outlined and the average intensity was measured by the Metamorph software. The intensity for each region were averaged and subtracted from the average intensity o f a background region containing no cells.  2.5 Neurotoxicity 2.5.1 Delivery of kainic acid Kainic acid ( K A ; Sigma) was delivered by intraperitoneal injection at a dose o f 25 mg/kg. M i c e were weighed immediately before injection in order to calculate the appropriate dose. M i c e were monitored for seizures for 2 hours following intra-peritoneal injection o f K A . M i c e were sacrificed 7 days after injection o f K A .  34  2.5.2 Delivery of quinolinic acid Quinolinic acid ( Q A ; Sigma) was stereotaxically injected into the striatum o f mice at the coordinates - A P : +0.8 m m from Bregma, M L : ± 1.8 mm from midline, D V : -3.5 mm from skull surface. Each mouse was given 1 ul o f 0.04 M Q A in P B S . During the injection mice were anesthetized with isofluorane and injections were performed with stereotaxic equipment (Stoelting). M i c e were sacrificed 7 days after injection with Q A .  2.5.3 Assessment of hippocampal damage M i c e were anestheized deeply with chloroform, brains were extracted and snap frozen in isopentance on dry ice. Coronal sections through the hippocampus were cut on the cryostat and mounted directly onto slides. Sections were then stained for the assessment o f neuronal damage. Fluorojade staining for degenerating neurons was completed on a series o f frozen sections in the hippocampus (Schmued et al,  1997). Sections were post-fixed in 3%  paraformaldehyde for 45 minutes, washed twice with water for 3 minutes then immersed in 100% ethanol for 5 minutes. Sections were then transferred to 70% ethanol for 1 minute, washed twice with water and immersed in 0.06% potassium permanganate for 15 minutes. Excess stain was removed by washing four times with water before staining slides in fluorojade working solution for 30 minutes (working solution: 80 m l distilled water, 72 ul acetic acid, 0.8 mg fluorojade compound (Histo-Chem; Jefferson, A R ) ) . Sections were then washed four times with water, dried overnight, cleaned with xylene and mounted with flouromount ( B D H ) . T U N E L (terminal U T P nick end labeling) was performed on fresh, frozen sections cut on the cryostat. Sections were incubated with digoxigenin (DIG)conjugated U T P and terminal transferase enzyme prior to binding o f alkaline phosphatase conjugated  anti-DIG antibody. Detection o f antibody binding was accomplished with  nitroblue tetrazolium chloride x-phosphate ( N B T ) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrate.  35  2.5.4 Assessment of striatal damage Brains from perfused mice were cut into 25 pm coronal sections and stained with N e u N (1:100; Chemicon), fluorojade or D A R P P - 3 2 (1:500, Chemicon) as described above except D A R P P - 3 2 was detected using a rabbit A B C kit (Vector Labs) and D A B chromogen. Mounted sections were analyzed using Stereoinvestigator  software (Microbrightfield).  Lesion volumes were assessed by outlining the perimeter o f the lesion in D A R P P - 3 2 or fluorojade stained coronal sections spaced 150 pm apart. Lesion area in each section was calculated by the stereology software and extrapolated to estimate the volume o f the lesion. The number o f striatal neurons surviving was determined by counting neurons within 30 pm X 30 pm counting frames spaced evenly throughout matched sections containing the striatum (striatal grid size was 300 pm X 300 pm) using a 100X objective.  2.6 Treatment 2.6.1 Treatment with over-expression of wild-type huntingtin Treatment with wild-type htt was accomplished by crossing Y A C 1 8 mice with Y A C 1 2 8 mice to generate Y A C 1 8 / 1 2 8 mice that express both wild-type and mutant htt transgenes. Successful generation o f Y A C 18/128 mice was confirmed by P C R and Western blotting for htt expression.  2.6.2 Treatment with cystamine Cystamine was delivered orally in drinking water at a concentration o f 900 mg per litre o f water to deliver an approximate dose o f 225 mg/kg (Dedeoglu, 2002). Cystamine solution was made fresh weekly. To assess the effect o f cystamine on transglutaminase levels, 3 month old Y A C 128 mice were treated with cystamine for a period o f 2 weeks prior to sacrifice. Early symptomatic treatment was initiated at 4 months o f age while symptomatic treatment began at 7 months o f age. Both treatments were continued to 12 months.  36  2.7 Molecular biology 2.7.1 Western blotting For Western blots, mice were asphyxiated with carbon dioxide and tissues immediately placed in eppendorf tubes and frozen in isopentane on dry ice. In some cases, brain regions were micro-dissected out prior to freezing. Tissues were homogenized in lysis buffer (0.303 M sucrose, 20 m M Tris p H 7.3, 1 m M magnesium chloride, 0.5 m M E D T A , 1 m M P M S F , 125 (il sodium vanadate/25 m l , 1 protein inhibitor tablet/25 ml) and allowed to lyse for 30 minutes. Following sonication, samples were centrifuged at 4 degrees Celsius for 15 minutes at 14,000 R P M . The supernatant was removed and protein levels were measured by L o w r y assay. Proteins were separated by electrophoresis on either 7.5% acrylamide gels or 8% lowbis acrylamide gels when allelic separation was necessary. 100 ug o f total protein was loaded in each lane. 7.5% gels were run at 100 volts until proteins entered the separating gel and then 200 volts until completion. A l l e l i c separation gels were run for a total o f 600 volt-hours again beginning at  100 volts until  proteins  entered  the  separating  gel. Following  electrophoresis, proteins were transferred from the gel to a membrane at 24 volts for 1.5 hours. Membranes were either probed immediately or dried and then re-wetted with methanol. Membranes were blocked with 5% milk powder in P B S before incubation with primary antibody for 1 hour at room temperature ( M A B 2 1 6 6 , Chemicon, 1:2000; HD650, 1:500). After 3 washes with P B S containing 0.5% Triton-X-100 (TPBS), membranes were incubated with a peroxidase-conjugated secondary antibody for 30 minutes at room temperature.  Finally,  blots  were  washed  six  times  with  chemiluminscent ( E C L ) detection and development o f the  TPBS film.  before  enhanced  Protein levels were  quantified by band density measured with Quantity One software (BioRad).  2.7.2 Measurement of transglutaminase activity For measurement o f transglutaminase activity, mice were asphyxiated with carbon dioxide gas and brains were immediately removed, separated into forebrain and hindbrain and frozen in  isopentane  on dry ice. Tissue was thawed and homogenized (1:5 w/v) in T G  Homogenization Buffer [10 m M T r i s - H C l (pH 7.5) and 1 m M E D T A , supplemented with 2  37  m M P M S F , 20 ug/ml leupeptin, 20 ug/ml peptstatin and 10 ug/ml aprotinin]. Samples were centrifuged at 16,000 x g for 10 min at 4°C and the supernatant was collected. The protein concentration o f each supernatant was determined by the bicinchoninic acid assay using bovine serum albumin as the standard (Pierce, Rockford, 1L, U S A ) . For the T G reaction, 350 ug protein was incubated in T G Assay Buffer [10 m M T r i s - H C l (pH 7.5), 5 m M D T T , 15 mM  N a C l , 400 u M CaC12, 200 u M putrescine, 1 u C i [3H]putrescine (Amersham  Biosciences, Piscataway, N J , U S A ) and 3 mg/ml dimethylcasein ( D M C ) ] in a total volume o f 100 ul at 37°C for 1 hr. Nonspecific incorporation o f [3H]putrescine was measured by performing the reaction in the absence o f CaC12 and in the presence o f 5 m M E G T A . The reaction was terminated by the addition o f 600 ul o f 10% (w/v) ice-cold trichloroacetic acid ( T C A ) . Individual tubes were vortexed and incubated at 4°C for 1 hr. Tubes were centrifuged at 14,000 x g for 20 min at 4 ° C . Pellets were resuspended in 900 ul o f 5% (w/v) T C A and centrifuged at 14,000 x g for 20 min at 4°C. 250 ul o f 0.25 N N a O H was added to each pellet and samples were boiled for 5 min. The radioactivity emitted from bound [3H]putrescine was quantitated by liquid scintillation using a Beckman LS6500 scintillation counter (Beckman Coulter, Inc., Fullerton, C A , U S A ) . For GTP-inhibited T G enzymatic activity, the assay was performed in the presence o f 500 u M GTPyS (Sigma-Aldrich, St. Louis, M O ) . Tissue T G is inhibited by G T P and accounts for the majority o f T G activity in mouse brain (Bailey et ai, 2004).  2.8 Statistical Analysis Data are given as the mean ± standard error o f the mean ( S E M ) . Behavioural measures were analyzed by either repeated measures A N O V A or two factor A N O V A using SPSS software. Within subjects effects o f age/trial/day/treatment and the interaction o f genotype and age/trial/day/treatment as well as between subjects effect o f genotype were assessed. In cases of significant differences between genotypes, post hoc comparisons between genotypes at individual points were assessed with either the Bonferroni method or by Student's t-test. In the case o f the Student's t-test, the significance level was adjusted to account for errors o f multiple measurements (significance level = 0.05/(number of measurements) eg. 6 trials: significance level = 0.05/6 = 0.008). Simple comparisons o f one variable between two  38  genotypes were assessed by Student's t-test with a significance level o f 0.05. Comparisons between more than two groups for one outcome measure were assessed by one way A N O V A . Comparisons o f categorical data were performed with the Chi-square test.  39  3.0 T O W A R D S MEASURES  THERAPEUTIC  TRIALS: MOUSE  MODELS  AND OUTCOME  Therapeutic trials for HD in mice require biologically relevant outcome measures that show quantifiable differences between HD and WT mice. Since the phenotype of the YAC72, line 2511 mice was mild and the observed abnormalities were primarily qualitative (Hodgson et al, 1999), it is not suitable for therapeutic trials. Accordingly, we examined YAC72 , line 44 mice to determine if increasing mutant htt expression would result in a more severe phenotype, with the ultimate goal of identifying quantitative outcome measures for subsequent therapeutic trials. 3.1 Characterization of the Y A C 7 2 mouse model of H u n t i n g t o n disease  YAC72, line 44 mice (YAC72 mice) express mutant htt with 72 glutamines from a YAC transgene that contains all proximal regulatory regions of the HD gene. This line of YAC72 mice expresses higher levels of mutant htt than line 2511 and thus is predicted to have a more severe phenotype. Preliminary analysis of YAC72, line 44 mice revealed signs of dark cell neurodegeneration in the striatum at 6 months of age by toluidine blue staining (B. Leavitt, unpublished). Accordingly, we examined the behaviour of YAC72 mice from 2 to 10 months of age and the neuropathology of YAC72 mice at 10 months. 3.1.1 M o t o r dysfunction i n Y A C 7 2 mice  Since motor impairment is prominent in HD (Harper, 1996), we selected behavioural tests that assess motor function to determine whether motor abnormalities were present in this mouse model. First, we assessed motor coordination on the rotarod. In this test, mice are placed on a rotating rod and the ability of the mouse to stay on the rod is used as a measure of motor coordination and balance. YAC72 mice and WT littermate controls were trained at 2 months of age at 24 RPM and subsequently tested monthly at four different speeds: 12 RPM, 24 RPM, 34 RPM and 40 RPM. At each speed tested, both WT and YAC72 mice could stay on the rotarod for the maximum time of 60 seconds (Fig. 3.1 panels A-D). Repeated measures ANOVA confirmed that there was no difference between YAC72 and  40  W T mice at any speed on the rotarod (genotype - 12 R P M : F F i,i3) = 0.0, p = 0.9; 34 R P M : F {  ( U 3 )  = 0.1, p = 0.8; 40 R P M : F  ( U 3  ( U 3 )  ) = 0.4, p = 0.5; 24 R P M : = 0.1, p =  0.7).  Patients with H D generally display an excess o f involuntary movement early in the disease followed by an increased inability to initiate voluntary movement as the disease progresses (Harper, 1996). Accordingly, we examined the activity o f Y A C 7 2 mice from 2 to 10 months o f age in an automated open field system. In this test, mice are placed in an empty chamber that is lined with photobeams and connected to a computer system which records the total number o f beam crosses as a measure o f activity. In this test, there was no difference in activity between Y A C 7 2 and W T mice at any time point (Fig. 3.1 panel E; genotype: F(i,i = 3)  0.0, p = 0.9).  W e also assessed motor coordination and balance using a beam crossing task. In this test, the time required to cross a narrow beam to a darkened chamber was recorded. A s with the rotarod test o f motor coordination, no motor deficit was apparent in the Y A C 7 2 mice compared to W T mice in this task from 2 to 10 months o f age (Fig. 3.1 panel F ; genotype: F  ( U 3  ) = 2.5; p = 0.15).  Next, we examined the ability o f Y A C 7 2 mice to swim down a linear swimming tank to reach an escape platform. In this test o f motor coordination, mice are motivated by the desire to escape the water. While differences between Y A C 7 2 and W T mice at each time point did not reach significance, repeated measures A N O V A revealed that Y A C 7 2 mice exhibit a mild impairment in this task, as they require significantly more time to reach the platform overall than W T mice (Fig. 3.1 panel G ; genotype: F(i,i ) = 6.3, p = 0.03; W T : 2.7 ± 0.3 seconds, 3  Y A C 7 2 : 3.7 ± 0 . 3 seconds).  To determine whether the gait abnormalities observed in human H D patients are found in the Y A C 7 2 mice, we examined walking patterns o f Y A C 7 2 mice by painting their feet and allowing them to walk along a papered, linear chamber. In this assessment, there was no significant difference between Y A C 7 2 and W T mice from 2 to 10 months (Fig 3.1 panel H ;  41  B 70 60 50 -{ 40 30 20 10  70 60  1  30 20 10  12 R P M 2.5  c  50 40  3.5  4.5  5.5  6.5  7.5  8.5  9.5  24 R P M 2.5  3.5  4.5  6.5  7.5  8.5  9.5  Age(Months)  Age(Months)  ^  5.5  D  _  70  o  60  EA  1  50 40  re 30 r2 34 R P M 2.5  3.5  4.5  5.5  6.5  7.5  8.5  §  20  2  10  9.5  40 R P M 2.5  3.5  4.5  _ 1600 to |  1400  6  1200  E  m  5.5  7.5  8.5  9.5  8  20  E  16  to  v to  m I 8  12  CO  oE  800 2.5  3.5  4.5  5.5  6.5  7.5  8.5  a  9.5  A  2.5  3.5  4.5  CD  H  8  "rl  6.5  7.5  8.5  9.5  0.30  1  oo  6  5.5  Age(Months)  Age(Months)  II  6.5  Age(Months)  Age(Months)  - f 0.25  E  I f  0-20  Q.T3  £ > 0.15 o > 0.10  E —^ " 2.5  3.5  4.5  5.5  6.5  7.5  8.5  9.5  2.5  3.5  4.5  Age(Months)  5.5  6.5  7.5  8.5  9.5  Age(Months) • WT YAC72  Figure 3.1 Motor function in YAC72 mice. Behavioural analysis was carried out on a cohort of YAC72 mice and W T littermates from 2 to 10 months of age to determine if Y A C 7 2 mice exhibit motor dysfunction. In the rotarod test of motor coordination there was no difference in performance between Y A C 7 2 and W T mice at 12 R P M (panel A ; genotype: F = 0.4, p = 0.5), 24 R P M (panel B; genotype: F = 0.0, p = 0.9), 34 R P M (panel C; genotype: F = 0.1, p = 0.8) or 40 R P M (panel D; genotype: F i. ) = 0.1, p = 0.7). There was also no difference in open field activity (panel E ; genotype: F(i, ) = 0.0, p = 0.9) or beam crossing (panel F ; genotype: F | f= 2.5; p = 0.15) at any time point. In contrast, Y A C 7 2 mice took significantly longer to swim to an escape platform in a linear swimming test (panel G ; genotype: F j = 6.3, p = 0.03; WT: 2.7 ± 0.3 seconds, YAC72: 3.7 ± 0.3 seconds). Walking patterns of Y A C 7 2 mice were normal. Quantification of forepaw stride length revealed equal stride length in Y A C 7 2 and W T mice (panel H ; genotype: F , | ) 0.4, p = 0.6). Thus, overall Y A C 7 2 mice have almost equivalent motor function to W T mice except for a mild deficit in swimming. N = 8 WT, 7 Y A C 7 2 . Error bars indicate S E M . ( u 3 )  ( M 3 )  ( U 3 )  (  l3  13  ( l i  3 )  ( 1  3 )  =  (1  3  42  genotype: F(i,i3) = 0.4, p = 0.6). Overall, the motor function of Y A C 7 2 mice was similar to W T mice except for a mild deficit in swimming ability.  3.1.2 Neuropathology in Y A C 7 2 mice Since H D patients exhibit whole brain atrophy and selective degeneration in the striatum (Sharp and Ross, 1996; Vonsattel and DiFiglia, 1998), we examined total brain weight and striatal volume in a cohort of 10 month old Y A C 7 2 mice. There was no significant difference in brain weight between Y A C 7 2 and W T mice (Fig. 3.2; W T : 402 ± 6 mg, Y A C 7 2 : 405 ± 5 mg, p = 0.8). In contrast, we observed a significant decrease in striatal volume in the Y A C 7 2 mice compared to W T mice (Fig. 3.2; W T : 12.5 ± 0.2 m m , Y A C 7 2 : 11.7 ± 0.3 m m , p = 3  3  0.03). Based on the difference and variability in striatal volume, 21 mice would be required in a therapeutic trial to have an 8 0 % chance of detecting a 5 0 % rescue of striatal volume using Y A C 7 2 mice.  3.2 Characterization of the Y A C 1 2 8 mouse model of H u n t i n g t o n disease In order to produce a mouse with a more severe phenotype, we generated Y A C 128 mice. Y A C 128 mice express mutant htt with approximately 128 glutamines from a Y A C transgene. The presence of the regulatory  elements for the H D  gene on the transgene permits  appropriate tissue-specific and temporal expression of mutant htt. Since increased C A G repeat length in human H D patients results in an earlier age of onset (Brinkman et al, 1997), we predicted that by increasing the C A G size from 72 repeats to 128 repeats a more severe phenotype would be produced in the Y A C mice.  3.2.1 M o t o r dysfunction in Y A C 1 2 8 mice Motor dysfunction in the Y A C 128  mice was assessed with the rotarod test of motor  coordination and open field activity testing. The rotarod test was performed at two fixed speeds - 24 R P M and 40 R P M - and in an accelerating rotarod task where the rotation speed was increased from 5 to 40 R P M over 4 minutes. The maximum scores for the rotarod test were 60 seconds for the fixed speed task and 300 seconds for the accelerating task. In all  43  _  1.32E+10  |  1.28E+10  1  1.24E+10  |  1.20E+10  | "5 C/D  I  *  1  I  1.16E+10 1.12E+10 1.08E+10  • WT • YAC72  At 10 months of age, YAC72 and WT mice were sacrificed. A. Comparing brain weights between YAC72 and WT mice revealed no difference (WT: 402 ± 6 mg, YAC72: 405 ± 5 mg, p = 0.8). B. In contrast, YAC72 mice showed a significant decrease in striatal volume compared to WT mice (WT: 12.5 ± 0.2 mm , YAC72: 11.7 ± 0.3 mm , p = 0.03). Combined this suggests that only select regions of the brain are affected in YAC72 mice. N = 8 WT, 7 YAC72. Error bars indicate SEM. * p < 0.05. F i g u r e 3.2 N e u r o p a t h o l o g y i n Y A C 7 2 m i c e .  3  3  44  three tests, Y A C 128 mice perform significantly worse than W T mice indicating a clear motor deficit (Fig 3.3 panels A - C ; 24 R P M - genotype: F  ( U 5 )  = 17.2, p < 0.001; 40 R P M -  genotype: F ^ i s ) = 10.7, p = 0.005; accelerating - genotype: F(ij ) = 16.1, p < 0.001). A t 2 5  months o f age, Y A C 128 mice are able to stay on the rotarod as long as W T mice but by 4 months o f age exhibit a trend towards decreased rotarod performance. With increased age, the motor coordination o f Y A C 128 mice on the rotarod worsens while W T mice maintain a relatively constant level o f performance (24 R P M - age X genotype: 40 R P M - age X genotype:  F(  5 ; 7  5)  F(5j5)  = 7.1, p < 0.001;  = 8.7, p < 0.001; accelerating - age X genotype:  F(5  >75  )  =  10.2, p < 0.001). Thus, at 4 months o f age, Y A C 128 mice develop a deficit in motor coordination on the rotarod that progressively worsens with age.  Next, we examined open field activity in the Y A C 128 mice using an automated open field activity apparatus to determine whether Y A C 128 mice recapitulate the biphasic hyper-hypoactivity pattern observed in H D patients. Initially, Y A C 128 mice at 2, 6 and 12 months o f age were tested in a 10 minute open field trial. A t 2 months o f age, Y A C 128 mice exhibited increased activity compared to W T mice (Fig. 3.3 panel D ; W T : 292 ± 1 7 beam breaks, Y A C 128: 351 ± 14 beam breaks, p = 0.02). A t 6 months o f age, there was no difference between the activity o f Y A C 1 2 8 and W T mice ( W T : 328 ± 22 beam breaks, Y A C 128: 319 ± 15 beam breaks, p = 0.2). A t 12 months, Y A C 128 mice were shown to be significantly hypoactive compared to W T mice ( W T : 240 ± 9 beam breaks, Y A C 128: 194 ± 13 beam breaks, p = 0.01).  To determine when Y A C 128 mice develop hypoactivity, we monitored a cohort o f mice monthly from 7 to 12 months o f age. Open field activity was equal at 7 months o f age between Y A C 128 and W T mice but beginning at 8 months o f age Y A C 128 mice become progressively more hypoactive compared to W T mice (Fig. 3.3 panel E ; genotype: F(i,io) = 12.8, p = 0.005; age X genotype: F^in) = 6.5, p < 0.001). To ensure that the differences observed in a 10 minute open field trial were indicative o f hypoactivity and not differences in transfer arousal, we examined the activity o f a cohort o f 12 month old Y A C 128 mice in an 8 hour open field trial. A s with the 10 minute open field trials, Y A C 128 mice at 12 months  45  B  70  |  o m  2 o  **  60 50 40  **  g 40  I  * 30  i  §  20  .1 10  2  0  » 10  24 RPM  0  K  30  ii  4  6  8  10  0  12  6  Age(Months)  J. | | °£  I °° 1  E h  ^  350 300 250 200 150  40 RPM 8  10  12  Age(Months)  *** *** ***  Accelerating  50 0  6  8  10  12  Age(Months) 4 0 0 -|  Act  350 >> > 300 250 8. O 4000  200 150 8  9  10  12  11  Age(Months) • •  WT — O YAC128 — • -  Figure 3.3 Motor deficits in YAC128 mice. Motor function in Y A C 128 and W T mice was assessed from 2 to 12 months of age using the rotarod test of motor coordination and an automated open field activity test. Rotarod testing was performed at 24 R P M (panel A), 40 R P M (panel B) and accelerating (panel C). In each case, YAC128 mice show significant motor deficits compared to W T mice (genotype - 24 R P M : F ( | j = 17.2, p < 0.001; 40 R P M : F , = 10.7, p = 0.005; accelerating: F = 16.1, p < 0.001, N = 8 WT, 9 YAC128). There is no difference in motor performance at 2 months of age between W T and Y A C 128 mice. Subsequently, motor coordination in Y A C 128 mice declines steadily from 4 to 12 months while W T mice show little change (age X genotype - 24 RPM: F . 5) = 7.1, p < 0.001; 40 R P M : F .7 ) = 8.7, p < 0.001; accelerating: F , ) = 10-2, p < 0.001). D. Open field activity testing at 2, 6 and 12 months of age revealed a biphasic hyperactive-hypoactive pattern of activity in Y A C 128 mice compared to W T mice. Y A C 128 mice were hyperactive at 2 months of age (WT: 292 ± 17 beam breaks, YAC128: 351 ± 14 beam breaks, p = 0.02; N = 7 W T , 10 YAC128). At 6 months, there was no difference in activity between YAC128 and W T mice (WT: 328 ± 22 beam breaks, YAC128: 319 ± 15 beam breaks, p = 0.2; N = 17 W T , 14 Y A C 128). At 12 months, Y A C 128 were significantly hypoactive compared to W T mice (WT: 240 ± 9 beam breaks, YAC128: 194 ± 13 beam breaks, p = 0.01; N = 7 W T , 11 YAC128). E . Following the development of hypoactivity in YAC128 mice reveal that YAC128 mice begin to show hypoactivity at 8 months of age and become increasingly hypoactive with age thereafter (genotype: F , = 12.8, p = 0.005; age X genotype: F = 6.5, p<0.001;N = 5 WT, 7 Y A C 128). F. YAC128 mice also show significant hypoactivity in an 8 hour open field activity trial compared to W T mice (WT: 7662 ± 483 beam breaks, YAC128: 5643 ± 499 beam breaks, p = 0.01, N = 8 W T , 11 YAC128). Error bars show S E M . ** p < 0.01, ***p<0.001. 5)  ( 1  1 5 )  ( M 5 )  (5  7  (5  5  (5  75  (  I 0 )  ( U 0 )  46  show significant hypoactivity compared to W T mice (Fig. 3.3 panel F ; W T : 7662 ± 483 beam breaks, Y A C 1 2 8 : 5643 ± 499 beam breaks, p = 0.01).  3.2.2 Cognitive deficits in YAC128 mice In addition to motor dysfunction, H D patients exhibit cognitive impairment which is present prior to clinical onset and progressively worsens with age (Paulsen et al., 2001). To determine whether cognitive function was impaired in Y A C 128 mice, we adapted or developed multiple behavioural tests which could be used to specifically assess cognitive abilities in the presence o f motor dysfunction. W e examined presymptomatic Y A C 128 mice at 2 months o f age as well as symptomatic mice at 8-12 months o f age.  3.2.2.1 YAC128 mice show impaired motor learning on the rotarod Motor learning was assessed using the rotarod test o f motor coordination. Three separate cohorts o f mice were trained on the rotarod at 2, 7 or 12 months o f age. The scores for the second and third day o f training were recorded as well as the score on a subsequent test day. A t 2 months o f age, mice were trained and tested on the rotarod at a fixed speed o f 24 R P M . In this test, the maximum score is 60 seconds and normal mice are able to stay on the rotarod for the full duration o f the test on most trials. While both W T and Y A C 128 mice learn the rotarod task, W T mice learn the task more rapidly than Y A C 128 mice (Fig. 3.4 panel A ; genotype: F(i, ) = 4.4, p = 0.041). Despite receiving equal amounts o f training, W T mice are 55  able to stay on the rotarod for 10 seconds longer than Y A C 128 mice on each o f the training days (Fig. 3.4 panel A ; Training Day 2 - W T : 43.7 ± 3.0 seconds, Y A C 1 2 8 : 31.5 ± 2.8 seconds, p = 0.005). However, on the test day Y A C 128 mice perform equally to W T mice on the rotarod (Fig. 3.4 panel A ; p = 0.54) indicating that once trained they are physically able to stay on the rod as long as W T mice. Thus, at 2 months o f age the motor coordination o f Y A C 128 mice on the rotarod is normal, but there is a mild motor learning deficit whereby Y A C 128 mice require more training to reach the same level o f performance as W T mice.  Next we examined older Y A C 128 mice to determine i f the motor learning deficit worsened with age. A t 7 and 12 months o f age, mice were trained and tested in an accelerating rotarod  47  B  Age 2 Months  Age 7 Months „  300  | ^ 250 a — o 2> 200  X -B  c 2 150 O <]>  2 s 100 50  2  Day Training  Day 3 Training  ***  in  ***  *1  UliU Day 2 Training  Test Day  Day 3 Training  Age 12 Months  Test Day  Age 12 Months ^  350 300  2 tn n — 250 > 2o gn, O <D CD Q)  i  100  50  Day 2 Training  Day 3 Training  Trained at 2 months  Test Day  Trained at 7 months  Trained at 12 months  • WT • YAC 128  Figure 3.4 Y A C 1 2 8 mice show deficits in motor learning on the rotarod. Three cohorts of mice were trained on the rotarod at 2, 7 or 12 months of age. Training consisted of three trials per day for three days. The scores for the second day and third day were recorded. The mice were subsequently given three trials on each test day and the scores averaged. A . At two months of age, both wild-type and Y A C 128 mice learn the rotarod task but W T mice learn the rotarod task more rapidly than YAC128 mice (day: F , no> = 43.3, p < 0.001; genotype X day: F , , i = 7.8, p = 0.001; genotype: F = 4.4, p = 0.041; N = 27 W T , 30 YAC128). On the test day, Y A C 128 mice can perform as well as W T mice on the rotarod indicating that their motor coordination is equal to W T but their motor learning is impaired (WT: 53.7 ± 2.2 seconds, YAC128: 55.6 ± 2.1 seconds, p = 0.54). B. At 7 months of age, Y A C 128 mice learn the rotarod task but cannot perform as well as W T mice during training or on the test day (day: F . ) = 10.6, p < 0.001; genotype X day: F .58) 0.9, p = 0.4; genotype: F . = 85.1, p < 0.001; p < 0.001; N = 16 Y A C 128, 15 WT). C. At 12 months of age, previously untrained Y A C 128 mice fail to learn the rotarod task while W T mice are still able to learn the task (day: F ) = 7.7, p = 0.001; genotype X day: F , o) = 7.7, p = 0.001; genotype: F = 32.8, p < 0.001; N = 8 YAC128^ 14 WT). D. YAC128 mice that have been trained at an earlier time point can run on the rotarod indicating that 12 month YAC128 mice fail to run on the rotarod because of a learning deficit (Mice trained at 2 months - W T : 272 ± 20 seconds, Y A C 128: 139 ± 14 seconds, p < 0.001; Mice trained at 7 months - W T : 180 ± 15 seconds, Y A C 128: 88 ± 14 seconds, p < 0.001; Mice trained at 12 months - W T : 160 ± 18 seconds, YAC128: 7 ± 24 seconds, p < 0.001). Error bars show S E M . ** p < 0.01, *** p < 0.001. (2  (2  0 )  ( l j 5 5 )  =  (2  58  (2  (1  29)  (2j40  (2 4  ( l i 2 0 )  48  test to a maximum score o f 300 seconds. Seven month old mice from both genotypes learn the rotarod task but Y A C 128 mice perform significantly worse than W T mice during training and on the test day (Fig. 3.4 panel B ; day:  F ,58) (2  = 10.6, p < 0.001; genotype:  F(i,29)  = 85.1, p  < 0.001). Thus, even after 3 days o f training, Y A C 128 mice are unable to maintain their balance on the rotarod as well as W T mice. Similarly, Y A C 128 mice trained at 12 months o f age perform significantly worse than W T mice during training and testing (Fig. 3.4 panel C ; genotype: F(i,20) = 32.8, p < 0.001). A t this age the differences in rotarod performance are dramatic as the Y A C 128 mice fail to demonstrate any improvement in performance with training and thereby show a complete inability to stay on the rotarod even after three days o f training (day:  F ,i2) (2  = 0.5, p = 0.6).  To determine whether the severe rotarod impairment in Y A C 128 mice at 12 months stemmed from a motor deficit or an inability to learn, we compared the performance o f the cohorts o f mice trained at 2, 7 and 12 months o f age at the 12 month time point. There was a clear effect of age on rotarod performance indicating that either earlier training or more training results in improved performance (age: F ,n6) = 15.5, p < 0.001). Nonetheless, all three cohorts at 12 (2  months demonstrate a clear rotarod deficit in the Y A C 128 mice compared to W T mice (Fig. 3.4 panel D ; genotype: F^sg) = 70.5, p < 0.001). Examining the performance o f mice trained prior to 12 months reveals that Y A C 128 mice trained at earlier time points are able to appropriately perform the rotarod task at 12 months ( Y A C 128 mice trained at 2 months: 139 ± 14 seconds; Y A C 1 2 8 mice trained at 7 months: 88 ± 14 seconds; Y A C 1 2 8 mice trained at 12 months: Y A C 1 2 8 : 7 ± 24 seconds). This indicates that Y A C 1 2 8 mice are physically capable o f staying on the rotarod at this late age. Thus, the complete failure o f previously untrained Y A C 128 mice to maintain their balance on the rotarod at 12 months o f age, even after 3 days o f training, cannot be solely accounted for by deficiencies in motor coordination. In addition to motor impairment, Y A C 128 mice also have a substantial learning deficit at 12 months.  49  3.2.2.2 YAC128 mice show impaired memory in a test of open field habituation Habituation is the decrease in response following repeated exposure to the same stimulus and can be used as a simple test o f learning and memory. Accordingly, we used an open field habituation test to determine whether the memory deficits in H D patients are also present in Y A C 128 mice. Open field habituation is measured by the decrease in exploratory activity with increased exposure to the open field chamber within one trial (intrasession) or over multiple trials (intersession).  T w o month  old presymptomatic  and eight month old  symptomatic Y A C 128 mice were given a total o f five 30 minute open field trials that were divided up into 5 minute intervals.  A t two months o f age, the activity o f Y A C 128 mice did not differ from W T mice (WT: 163 ± 4 beam breaks, Y A C 1 2 8 : 170 ± 4 beam breaks, p = 0.18). Within a 30 minute open field trial both genotypes showed a decrease in activity between intervals but the decreases in activity were equal (interval: F(5, no) = 48.3, p < 0.001; genotype X interval: F ^ no) = 1.0, p = 0.4; genotype: F(i ) = 1.3, p = 0.3). Similarly, both groups showed equivalent decreases in ;22  activity between repeated open field trials (trial: F 4 ) = 39.2, p < 0.001; genotype X trial: (  F(4,88) = 1.9, p = 0.1; genotype: F i (  > 2 2 )  >88  = 1.5, p = 0.2). A s a result, the extent o f intrasession  and intersession habituation was not different between Y A C 128 and W T mice (Intrasession habituation - genotype: F ( i  ; 2 2 )  = 1.8, p = 0.2; Intersession habituation - genotype: F(i, ) = 0.3, 22  p = 0.57). Thus, presymptomatic Y A C 1 2 8 mice do not exhibit a learning and memory deficit in open field habituation at 2 months o f age.  Next we examined symptomatic Y A C 128 mice at 8 months o f age. A t this time, Y A C 128 mice showed significant hypoactivity compared to W T mice (Fig. 3.5 panel A ; Interval 1 W T : 190 ± 11 beam breaks, 145 ± 7.4 beam breaks, p = 0.008). Activity o f both the W T and Y A C 128 mice declined in subsequent intervals in the 30 minute open field trial with the activity o f W T mice declining more rapidly than Y A C 128 mice (Fig. 3.5 panel A , B ; interval: F( j i5) = 36.4, p < 0.001; genotype X interval: F( n ) = 4.5, p < 0.001; genotype: F i ) = 8.1, 5  5i  5  (  ;23  p = 0.009). The initial difference in activity is eliminated and for the last 4 intervals, the activity o f W T and Y A C 128 mice does not differ (Fig. 3.5 panel A ) . To account for the  50  F i g u r e 3.5 Y A C 1 2 8 m i c e s h o w d e c r e a s e d o p e n  field  h a b i t u a t i o n . T h e activity o f 8 month o l d mice in an  open  field  w a s tested f o r 3 0 m i n u t e s d i v i d e d into 6 f i v e minute intervals. A s m i c e b e c o m e f a m i l i a r w i t h the  open  field  t h e y s h o w less e x p l o r a t o r y a c t i v i t y . A . W h i l e t h e a c t i v i t y o f b o t h W T m i c e a n d Y A C 128 m i c e  d e c r e a s e s d u r i n g t h e 3 0 m i n u t e t r i a l , W T a c t i v i t y d e c l i n e s at a m o r e r a p i d rate ( i n t e r v a l : F 0.001; genotype X interval: F  ( 5 i M 5 )  = 4.5, p < 0.001; g e n o t y p e : F  ( l j 2 3 )  ( 5 ;  n5) = 36.4, p <  = 8 . 1 , p = 0 . 0 0 9 ) . I n i t i a l l y , Y A C 128 m i c e  s h o w s i g n i f i c a n t h y p o a c t i v i t y c o m p a r e d t o W T m i c e ( I n t e r v a l 1 - W T : 190 ± 12 b e a m b r e a k s , Y A C 1 2 8 : 145 ± 7 b e a m b r e a k s , p = 0 . 0 0 8 ) b u t t h i s d i f f e r e n c e i s e l i m i n a t e d a s t h e W T m i c e h a b i t u a t e t o t h e o p e n field c h a m b e r . B. T o control for differences in baseline activity levels, activity for each interval w a s d i v i d e d b y the level o f a c t i v i t y i n t h e first i n t e r v a l . W h e n t h e i n i t i a l l e v e l o f a c t i v i t y i s c o n t r o l l e d f o r , W T m i c e s h o w a g r e a t e r d e c r e a s e i n a c t i v i t y f r o m t h e first i n t e r v a l s t a r t i n g at i n t e r v a l 3 ( P e r c e n t a g e o f o r i g i n a l a c t i v i t y i n I n t e r v a l 3 - W T : 6 6 % , Y A C 1 2 8 : 8 9 % , p = 0.004). C . A s a result, Y A C 1 2 8 m i c e s h o w decreased intersession habituation c o m p a r e d to WT  m i c e w h e r e h a b i t u a t i o n i s m e a s u r e d a s t h e d i f f e r e n c e i n a c t i v i t y f r o m t h e first i n t e r v a l d i v i d e d b y t h e  a c t i v i t y i n t h e first i n t e r v a l ( i n t e r v a l : F ( . ) = 8.6, p < 0 . 0 0 1 ; g e n o t y p e X i n t e r v a l : F , ) = 0.9, p = 0.4; g e n o t y p e : 4  F(i.23)  =  92  (4  92  9.7, p = 0 . 0 0 5 ) . F o l l o w i n g t h e first 3 0 m i n u t e t r i a l , i n t e r s e s s i o n o p e n field h a b i t u a t i o n w a s a s s e s s e d b y  g i v i n g t h e m i c e 4 a d d i t i o n a l t r i a l s i n w h i c h t h e a c t i v i t y d u r i n g t h e f i r s t 10 m i n u t e s w a s m e a s u r e d . D . B e t w e e n the s e s s i o n s , t h e a c t i v i t y o f W T m i c e d e c l i n e d m o r e r a p i d l y t h a n t h e a c t i v i t y o f Y A C 128 m i c e b u t b o t h g r o u p s s h o w e d decreased activity over repeated trials (trial: F 0.04; genotype: F ,. (  23)  ( 4 ; 9 2 )  = 60.8, p < 0.001; genotype X trial: F , ( 4  9 2 )  = 2.5, p =  = 4 . 8 , p = 0 . 0 4 ) . E . Y A C 1 2 8 m i c e a r e h y p o a c t i v e at 8 m o n t h s o f a g e as i n d i c a t e d b y a n  i n i t i a l 1 8 % d i f f e r e n c e i n a c t i v i t y b e t w e e n Y A C 1 2 8 a n d W T m i c e . W i t h r e p e a t e d o p e n field t r i a l s , t h e d i f f e r e n c e i n a c t i v i t y b e t w e e n W T a n d Y A C 128 m i c e p r o g r e s s i v e l y d e c r e a s e d t o 5 % b y t h e last t r i a l . C . E x a m i n i n g t h e rate o f d e c r e a s e i n a c t i v i t y p e r t r i a l d e m o n s t r a t e s that W T m i c e s h o w a m o r e r a p i d h a b i t u a t i o n t h a n Y A C 1 2 8 m i c e ( W T : 3 8 . 8 ± 5.0 b e a m b r e a k s / t r i a l , Y A C 1 2 8 : 2 6 . 2 ± 3.2 b e a m b r e a k s / t r i a l , p = 0 . 0 5 ) . N = 12 W T , 13 Y A C 1 2 8 . E r r o r b a r s s h o w S E M . * p < 0 . 0 5 , ** p < 0 . 0 1 .  51  initial differences in activity, we calculated habituation as the difference in activity from the first trial divided by the activity o f the first trial. This calculation revealed decreased intrasession habituation in the Y A C 128 mice compared to W T mice (Fig. 3.5 panel C ; interval:  F(4 92) ;  = 8.6, p < 0.001; genotype X interval:  F(4,92>  = 0.9, p = 0.4; genotype:  F(i,23) —  9.7, p = 0.005).  In subsequent trials on the following two days, both Y A C 128 and W T mice demonstrated intersession habituation (Fig. 3.5 panel D ; trial: F ^ ^ ) = 60.8, p < 0.001; genotype X trial: F 92) (4)  = 2.5, p = 0.04; genotype:  F(i,23)  = 4.8, p = 0.04). Again, in the intersession comparison,  Y A C 128 mice were hypoactive in the first trial with the difference in activity between the W T and Y A C 128 mice declining with repeated trials (Fig. 3.5 panel E ) . This results in a trend towards decreased intersession habituation in Y A C 128 mice compared to W T mice (trial:  F(3,69)  = 39.9, p < 0.001; genotype X trial:  F(  3j6  9)  = 0.1, p = 1.0; genotype:  F i 3) (  j 2  = 3.2, p  •= 0.08). Accordingly, the rate o f decline in activity per trial was significantly less in Y A C 128 mice compared to W T mice indicating a deficit in intersession habituation (Fig. 3.5 panel F ; p = 0.05). Overall, symptomatic Y A C 128 mice show decreased intrasession and intersession habituation compared to W T mice suggesting a deficit in memory.  3.2.2.3 YAC128 mice show cognitive deficits in a simple swimming test A simple swimming test was designed to assess the ability o f mice to learn and remember how to reach an escape platform. In this test mice are placed in a linear swimming chamber facing away from the escape platform and learn to turn around and swim to the platform (Fig. 3.6 panel A ) . Both W T and Y A C 128 mice trained at 2 months o f age rapidly learned to turn immediately to reach the platform (Fig. 3.6 panel B ) . Testing the same cohort o f mice at 4 months and 6 months o f age revealed no differences in the time or path taken to reach the platform between Y A C 128 and W T mice. Here, it appears that the motor deficits detected on the rotarod starting at 4 months o f age do not impair the swimming ability o f the Y A C 128 mice even at 6 months o f age.  52  C  B  2  4  6  8  Age(Months)  10  12  2  6  10  8  12  Age( Months) -O-WT YAC128|  Figure 3.6 YAC128 mice show a cognitive deficit in a simple swimming test. Mice are placed at the start position facing away from the platform and the time and path taken to reach the platform are recorded. The apparatus is shown in panel A. At 2 months of age, all mice learn to turn immediately and swim directly to the platform and there is no significant difference in the amount of time required to reach the platform (WT: 0.9 ± 0.1 seconds, 1.7 ± 0.4 seconds, p = 0.1). B. The amount of time needed to reach the platform remains equal between the YAC 128 and WT mice until 8 months of age when YAC 128 mice take significantly longer to reach the platform (8 months - WT: 1.0 ± 0.3 seconds, YAC 128: 3.7 ± 0.5 seconds, p < 0.001). Overall, YAC 128 mice take significantly longer to reach the platform than WT mice (genotype: F i.i = 23.1, p < 0.001). C. The increased time to reach the platform resultsfromthe path taken to reach the platform. Mice swimming towards the platform initially were given a score of 0, while mice swimming awayfromthe platform initially were given a score of 1. Starting at 8 months of age, YAC 128 mice show a number of errors in the path chosen to reach the platform likely resulting from a memory deficit (Errors in initial swimming direction - WT: 0 of 27 trials, 0 of 9 mice; YAC128: 12 of 30 trials, 7 of 10 mice, = 13.7, p < 0.001). N = 9 WT, 9 YAC128. Error bars show SEM. ** p < 0.01, *** p < 0.001. (  6)  53  Surprisingly, at 8 months there is a dramatic increase in the amount o f time Y A C 128 mice take to reach the platform compared to W T animals (Fig. 3.6 panel B ; W T : 1.0 ± 0.3 seconds, Y A C 1 2 8 : 3.7 ± 0.5 seconds, p < 0.001). To ascertain whether the increased latency to reach the platform stemmed from a cognitive deficit, we examined the path taken to the platform. In contrast to W T mice, Y A C 128 mice often fail to swim directly to the platform. To quantify this observation, we arbitrarily gave mice swimming towards the platform a score o f 0 and mice swimming away from the platform a score o f 1. Using this measurement, the Y A C 128 mice show a significant deficit in initiating the task in the correct direction, first detected at 8 months o f age and persisting to 12 months o f age (Fig 3.6 panel C ; Errors in initial swimming direction - W T : 0 o f 27 trials, 0 o f 9 mice; Y A C 128: 12 o f 30 trials, 7 o f 10 mice, x = 13.7, p < 0.001). Overall, Y A C 128 mice take longer to reach the platform starting 2  at 8 months o f age primarily as a result o f choosing the incorrect direction to swim at the outset (Time to reach platform: genotype: F(i i6) >  =  23.1, p < 0.001). A motor deficit that  decreases swimming speed also contributes to the time difference (see section 3.2.2.5).  3.2.2.4 YAC128 mice show multiple cognitive deficits in swimming T-maze test A swimming T-maze test was used to test procedural and spatial learning in symptomatic Y A C 128 mice at 8.5 months o f age. We developed this test as a simple two-choice test o f learning and memory to facilitate rapid training and testing in visually impaired mice. Initially, the escape platform was placed in the right arm o f the T-maze. After two trials, both W T and Y A C 1 2 8 mice showed a large decrease in the amount o f time required to reach the escape platform (Fig. 3.7 panel A ) . B y the third day, both genotypes had achieved a constant level o f performance with Y A C 128 mice taking significantly longer than W T mice to reach the platform (Fig 3.7 panel B ; genotype: F i, 3) = 11.5, p = 0.003; W T : 1.9 ± 0.1 seconds, (  2  Y A C 1 2 8 : 3.0 ± 0 . 3 seconds).  To determine whether the increased latency to reach the platform resulted from a motor or cognitive deficit, we examined the path taken to reach the platform and individual swim times for the third day. This analysis revealed that Y A C 128 mice have several "errant" trials :  that are much slower than the average, while the times for W T mice are' tightly bunched  54  Figure 3.7 YAC128 mice show cognitive and motor deficits in swimming T-maze. Eight and a half month old mice were trained to swim to a platform located in the right arm of a swimming T-maze. Mice received three days of testing with four trials per day. The time and path to reach the platform were recorded. For the path to the platform, right was given a score of 0 and left was given a score of 1. A. After two trials, both YAC 128 and WT mice showed a marked decrease in the time required to reach the platform with minor improvements in time thereafter. B. Over the first two days there is no difference in the time required to reach the platform between Y A C 128 mice and WT. On the third day, Y A C 128 mice take significantly longer to reach the platform (trial: F = 0.6, p = 0.6; genotype X trial: F . = 0.8, p = 0.5; genotype: F 3 ) = 11.5, p = 0.003; WT: 1.9 ± 0.1 seconds, YAC128: 3.0 ± 0.3 seconds). C. Closer inspection of the latency to reach the platform on day three reveals that WT mice show very little variation while the YAC 128 group have several errant trials (Errant trials greater than 4 seconds - WT: 0 of 44 trials, 0 of 12 mice; Y A C 128: 10 of 52 trials, 6 of 13 mice; x = 9.4, p = 0.002). D. Some of these errant trials are caused by choosing an indirect path to the platform. Initially, both groups demonstrate a chance level of selecting the correct arm to swim down to reach the platform. By the third day, WT mice always swim down the correct arm while YAC128 mice have some trials where the wrong arm is chosen (Trials swimming down incorrect arm - WT: 0 of 44 trials, 0 of 12 mice; YAC128: 5 of 52 trials, 3 of 13 mice; x = 4.5 , p = 0.04). N = 12 WT, 13 YAC128. Error bars show SEM. ** p < 0.01. (3i69)  (3  69)  a 2  2  2  55  around the average (Fig. 3.7 panel C ; Errant trials greater than 4 seconds - W T : 0 o f 44 trials, 0 o f 12 mice; Y A C 1 2 8 : 10 o f 52 trials, 6 o f 13 mice, % = 9.4, p = 0.002). T o gain insight into 2  the difference, we monitored the direction that each mouse turned upon arrival at the stem o f the T. Initially, approximately half o f the mice irrespective o f genotype turned left (given a score o f 1) and the other half turned right (given a score o f 0). Upon training, mice learned to turn right to reach the platform and by the third day, all o f the W T mice turned right in all o f their trials (Fig. 3.7 panel D). In contrast, Y A C 128 mice still turned left in a small number o f trials on the third day ( W T : 0 o f 44 incorrect path trials, 0 o f 12 mice; Y A C 1 2 8 : 5 o f 52 incorrect path trials, 3 o f 13 mice; %  2 =  4.5 , p = 0.04). This accounts for only half o f the  errant trials observed. Observations suggest that the remainder o f the difference in average time to reach the platform is caused by increased pausing and decreased swimming speed (see section 3.2.2.5). Nonetheless, the decision to take inappropriate paths to the platform indicates that a cognitive deficit contributed to the increased latency to reach the platform observed in Y A C 128 mice.  To assess the ability o f Y A C 128 mice to change strategy, we incorporated a reversal phase into the swimming T-maze test by switching the platform from the right arm to the left arm of the maze. M i c e that were trained to swim directly to the right arm would now have to change their strategy to find the shortest path to the platform. In the first trial after switching the platform to the left arm, there was a remarkably clear difference between the Y A C 128 and W T mice. A l l o f the W T mice initially swam down the right arm and upon discovering that the platform was no longer present immediately swam down the unvisited left arm o f the maze and found the platform. Similar to the W T mice, all o f the Y A C 128 mice initially entered the right arm o f the T-maze. However, upon discovering that the platform was not present in the right arm o f the maze, the majority o f the Y A C 1 2 8 mice swam back to the start of the T-maze where they had already been. A t this point some o f the Y A C 128 mice returned to the right arm o f the T-maze while some swam directly to the platform in the left arm.  W e measured this abnormality by quantifying the number o f arms o f the maze entered en route to the platform. While all o f the W T mice showed 2 arm entries, Y A C 128 mice showed anywhere from 2 to 5 arm entries to reach the platform (Fig. 3.8 panel A ) . This pattern o f  56  response resulted in the Y A C 128 mice having more arm entries than W T mice (Fig. 3.2.6 panel B ; W T : 2 arm entries, Y A C 1 2 8 : 2.9 ± 0.3 arm entries; % = 9.0, p = 0.03) and taking 2  twice as long to reach the platform as W T mice (Fig. 3.8 panel C ; W T : 7.5 ± 0.4 seconds, Y A C 128: 15.6 ± 1.9 seconds, p < 0.001). With additional trials, both the W T and Y A C 128 mice learn to swim directly to platform in the left arm and by the last trial swim times were similar to the last trial for the normal phase o f the T-maze (Fig. 3.8 panels D , E ) . Again, on day 3 o f the reversal phase o f the T-maze testing, Y A C 128 mice took longer than W T mice to reach the platform but this difference did not reach significance possibly because a constant level o f performance had not been reached (Fig. 3.8 panels D , E ; genotype: F i 3 ) = (  ;2  3.9, p = 0.06; W T : 2.0 ± 0.2 seconds, Y A C 1 2 8 : 3.4 ± 0.5 seconds). The difference in time to reach the platform on the third day resulted from choosing the incorrect arm more frequently (Fig. 3.8 panel F ; not significant), an increased number o f errant trials (not shown), increased pausing and decreased swimming speed (see below). It was not caused by an increased number o f arm entries which was equal after day 1 (Fig. 3.8 panel G ) . Here, a clear deficit i n changing strategy is apparent in the path and time that Y A C 128 mice take to reach the platform in the reversal o f the T-maze test which training reduces. The mild cognitive deficit observed in the normal phase o f the T-maze test is also observed here.  3.2.2.5  Deficits of Y A C 1 2 8 mice in swimming tests are primarily cognitive  To determine the relative contributions o f cognitive and motor dysfunction to the increased latency to reach the platform in our swimming tests, we measured swimming speed in mice at 8.5 months o f age. The stem o f the T-maze was blocked off and the time for mice to swim from the right arm o f the maze to the platform in the left arm o f the maze was measured (Fig. 3.9 panel A ) . Assuming that mice on average swim to the middle o f the T before turning, the distance is identical to that traveled in the T-maze tests but differences in the amount o f time required for mice to turn around a corner are not determined. In this assessment, the average latency o f Y A C 128 mice to reach the platform was significantly longer than that o f W T mice (WT: 1.6 ± 0.1 seconds, Y A C 1 2 8 : 1.9 ± 0.1 seconds, p = 0.03) indicating that Y A C 1 2 8 mice have a slower swimming speed then W T mice likely due to a deficit in motor coordination ( W T : 48.2 ± 2.7 cm/second, Y A C 1 2 8 : 39.1 ± 2.0 cm/second, p = 0.01). The difference in  57  Figure 3.8 YAC128 mice show cognitive deficits in the reversal phase of the swimming T-maze test. Eight and a half month old mice were trained to swim to a platform located in the right arm of a swimming T-maze. After three days of four trials each day, the platform was switched to the left arm of the maze. The time to reach the platform, the path to the platform and the total number of arm entries were recorded. A. On the first trial of the reversal phase, all WT mice swam right initially then swam straight to the platform in the left arm for a total of 2 arm entries. In contrast, most YAC 128 mice swam back to the start of the maze before either retracing their steps or exploring the left arm of the maze leading to anywhere from 2 to 5 arm entries before reaching the platform. B. This resulted in YAC 128 mice showing a greater number of arm entries compared to WT mice (WT: 2 arm entries, YAC128: 2.9 ± 0.3 arm entries, % = 9.0, p = 0.03). C. YAC128 mice also show a greater latency to reach the platform on the first trial of the reversal phase despite all mice choosing the same initial swimming direction (WT: 7.5 ± 0.4 seconds, YAC128: 15.6 ± 1.9 seconds, p < 0.001). D,E. In subsequent trials, both groups improve their latency to reach the platform as they learn to swim directly to the platform in the left arm of the T-maze. On the third day, there is a trend towards an increased latency to reach the platform in YAC128 mice compared to WT mice (trial: F . = 5.9, p = 0.001; genotype X trial: F = 1.7, p = 0.2; genotype: F„, = 3.9, p = 0.06; WT: 2.0 ± 0.2 seconds, YAC128: 3.4 ± 0.5 seconds). F. This is partially because Y A C 128 mice continue to choose indirect paths to the platform in the last two trials while all WT mice swam directly to the platform (Trials swimming down the incorrect arm - WT: 0 of 22 trials, 0 of 12 mice; YAC128: 5 of 26 trials, 3 of 13 mice; x = 4.7, p = 0.03). G. The difference in latency to reach the platform on the third day did not result from an increased number of arm entries which were equal after day 1. N = 12 WT, 13 YAC128. Error bars show SEM.** p < 0.01, *** p < 0.001. 2  (3  69)  (3>69)  23)  2  58  straight swimming time allows estimation o f the contribution o f motor deficits to the overall increased latency to reach the platform in Y A C 128 mice. A s predicted, the difference in straight swimming time o f 0.3 seconds only accounts for a small part o f the difference between Y A C 128 and W T mice on the final days o f either normal phase or reversal phase Tmaze testing where the differences were 1.1 and 1.4 seconds respectively. While part o f this difference may stem from Y A C 128 mice taking longer to turn the corner (not determined), the remainder o f the difference is most likely accounted for by cognitive dysfunction.  Similarly, we used the swimming speed calculated in the T-maze apparatus, to estimate the relative contributions o f motor and cognitive deficits to the differences observed in the simple swimming test. We calculated the predicted difference in latency to reach the platform in the simple swimming test based on motor dysfunction alone as 0.2 seconds. This accounts for only 5% o f the actual difference observed in the time to reach the platform at 8 months. Comparing the difference in latency to reach the platform between Y A C 128 and W T mice in the three swimming tests reveals that cognitive factors account for the majority o f the difference observed (73-95%; F i g . 3.9 panel B ) . Thus, by measuring swimming speed in a non-cognitive test, we show that motor dysfunction is present at this age but only accounts for a small proportion o f the overall performance deficit in the simple swimming test and swimming T-maze tests. The remainder o f the difference is caused by cognitive dysfunction which is most apparent in the reversal phase o f the swimming T-maze test where a change in strategy is required.  3.2.2.6 Presymptomatic YAC128 mice show difficulty in shifting strategy Since presymptomatic H D patients show mild cognitive symptoms that worsen as the disease progresses, we tested a cohort o f 2 month old presymptomatic Y A C 128 mice in the swimming T-maze to determine i f the cognitive deficits in symptomatic Y A C 128 mice are present at this young age. In the normal phase o f the test, both Y A C 128 and W T mice rapidly learn to swim to the platform and there is no difference in the time taken to reach the platform in any o f the 12 trials (Fig. 3.10 panel A ) . However, switching the location o f the platform to the opposite arm o f the maze revealed a difference in changing strategy between  59  •c CO  3.0  B i  »S  • Cognitive Deficit  2.5  • Motor Deficit  2.0  c o 0) i5 1.5 IE S  1.0 0.5  Simple Swimming Test  Normal T-maze Test  Reversal T-maze Test  Figure 3.9 Cognitive deficits are primarily responsible for increased latencies to reach the platform in  swimming tests. A. Swimming speed of the mice was assessed by measuring how long the mice take to swim linearly to the platform along the top of the T in the swimming T-maze. Y A C 128 mice swim slower than WT mice indicating a deficit in motor coordination at 8.5 months of age (WT: 48.2 ± 2.7 cm/second, YAC128: 39.1 ± 2.0 cm/second, p = 0.01, N = 12 WT, 13 YAC128). B. Using swimming speed to calculate the difference in latency to reach the platform predicted by motor deficit alone and comparing this to the actual difference observed reveals that motor dysfunction only accounts for a small percentage of the difference. Most of the difference in the latency to reach the platform results from cognitive deficits. Error bars show SEM.  60  the Y A C 1 2 8 and W T mice even at this age. While the difference was not as dramatic as we observed in symptomatic Y A C 128 mice, 2 month old Y A C 128 mice took significantly longer than W T mice to reach the platform during the reversal phase o f the swimming Tmaze test (Fig. 3.10 panel B ; W T : 4.9 ± 0.4 seconds, Y A C 1 2 8 : 9.5 ± 1.2 seconds, p = 0.003). Again, none o f the 12 W T mice re-entered the stem o f the T-maze while 3 o f the 12 Y A C 128 mice retraced their path before reaching the platform (Fig. 3.10 panel C ) . With additional trials, Y A C 128 mice are able to reach the platform as fast as W T mice (not shown). A t this age, there was no significant difference in swimming speed between the Y A C 128 and W T mice (Fig. 3.10 panel D ; W T : 45 ± 6 cm/second, Y A C 128: 45 ± 6 cm/second, p = 0.99). Overall, this demonstrates that presymptomatic Y A C 128 mice have difficulty in changing strategy but do not show any cognitive deficit in the normal phase o f the swimming T-maze test. A s with the rotarod test at this age, no deficit in motor coordination was detected in measuring swimming speed.  3.2.2.7 YAC128 mice show decreased sensorimotor gating in pre-pulse inhibition test Based on observations o f decreased PPI in human H D patients (Swerdlow et al., 1995), we tested symptomatic Y A C 1 2 8 mice at 9 and 12 months o f age to determine i f this difference was evident in the mouse model. Using the same apparatus, we also assessed habituation to acoustic startle. Acoustic startle is a fast response to a loud noise stimulus which can be measured by movement sensitive apparatus. M i c e exposed to repeated high intensity sounds learn to startle less when they recognize the sound and remember that there is no harmful event associated with the sound. This habituation to acoustic startle can be used to assess learning and memory. When mice are exposed to a quieter sound before the loud stimulus, they w i l l startle less than they would for the loud stimulus alone. This PPI is not learned but rather measures sensorimotor gating. A t 9 months o f age, there was no difference between W T and Y A C 128 mice in PPI or habituation to acoustic startle (data not shown).  Y A C 128 mice tested at 12 months o f age showed significantly less P P I compared to W T mice indicating a deficit in sensorimotor gating (Fig. 3.11 panel A ; genotype: F(i g ) = 62.7, p j  < 0.001; pre-pulse:  F( , 55) 3  2  5  = 37.4, p < 0.001). A t 12 months o f age, Y A C 128 mice also show  61  WT  YAC128  Figure 3.10 Presymptomatic YAC128 mice show cognitive deficit in strategy shifting. Two month old  Y A C 128 mice were tested in the swimming T-maze test with reversal. A. During the normal phase of the test, both Y A C 128 and WT mice learn to swim to the platform and no differences were observed between Y A C 128 and WT mice (trial: F i,,242) = 1 1.0, p < 0.001; genotype X trial: F i,242) 0-3, p = 1.0; genotype: F ) = 0.0, p = 0.9). B. During the reversal phase, when the location of the platform was switched, Y A C 128 mice required significantly more time to find the platform than WT mice (WT: 4.9 ± 0.4 seconds, YAC 128: 9.5 ± 1.2 seconds, p = 0.003). C. After the platform was switched to the opposite arm of the T-maze, none of the WT mice swam down the stem of the T-maze. In contrast, 3 of the 12 Y A C 128 mice retraced their steps to the starting position before swimming to the platform. This resulted in the YAC128 mice exhibiting more arm entries en route to the platform (x = 6.0, p = 0.05). D. At this age, the swimming speed of Y A C 128 mice is equal to that of WT mice indicating that motor coordination is normal at this time point (WT: 45 ± 6 cm/second, YAC128: 45 ± 6 cm/second, p = 0.99). N = 12 WT, 12 YAC128. Error bars show SEM. ** p < 0.01. =  (  ()  (LJ22  2  62  m  ***  90% •  c  I  B  ***  c 50%  I  70% •  J L  40%  S 30% CO  x 50% -  *  £ 30%  20%  CD  0.  1 5)  10% 2dB  4dB  8dB  16 dB  10% 0%  Pre-Pulse(above background)  Block  Figure 3.11 YAC128 mice show decreased pre-pulse inhibition and decreased habituation to acoustic startle. A. Pre-pulse inhibition was measured as the percentage decrease in the amount of startle with a prepulse compared to the amount of startle with no pre-pulse. At 9 months of age there was not a significant difference between WT and YAC128 mice (N = 11 WT, 13 YAC128; not shown). At 12 months of age, YAC 128 mice show a deficit in pre-pulse inhibition at 2 dB, 4 dB, 8 dB and 16 dB above background (genotype: F g = 62.7, p < 0.001; prepulse: F 3 . = 37.4, p < 0.001; N = 39 WT, 48 YAC128). B. At this age, YAC 128 mice also show a decreased habituation to acoustic startle compared to WT mice (block: F . 8 8 ) 3.9, p = 0.025; genotype X block: F = 0.3, p = 0.7; genotype: F , 4) = 7.9, p = 0.008; N = 21 WT, 25 YAC128). Error bars show SEM. * = p < 0.05, ** p<0.01, *** p< 0.001.' (1;  5)  (  255)  =  ( 2  (2>88)  (1  4  63  decreased habituation to acoustic startle. With repeated pulses, both W T and Y A C 128 mice show a decreased response but the habituation is greater in W T mice than in Y A C 128 mice (Fig. 3.11 panel B ; block:  F 2,88) (  = 3.9, p = 0.025; genotype X block:  F ,88) (2  = 0.3, p = 0.7;  genotype: F(i,44) = 7.9, p = 0.008). The decreased habituation to acoustic startle reflects a deficit in learning and memory while the decreased PPI suggests a difficulty in inhibiting the motor response to the sound. In both cases the acoustic startle observed in Y A C 128 mice may be influenced by emotional state as this is known to impact startle response magnitude and emotional changes are common in patients with H D .  3.2.3 Neuropathology in YAC128 mice 3.2.3.1 Selective atrophy and neuronal loss in YAC128 brain Huntington disease is characterized by selective degeneration in the basal ganglia and cortex with relative sparing o f other regions including the hippocampus and cerebellum. Here we sought to determine whether atrophy was present in the brains o f Y A C 128 mice and, i f so, whether it was limited to regions affected in H D . To this end, we examined the volume o f the striatum, cortex, globus pailidus, hippocampus and cerebellum in a cohort o f 12 month old Y A C 128 mice and W T littermates.  Overall brain weight was decreased 4% in Y A C 1 2 8 mice compared to W T mice (Fig. 3.12 panel A ; W T : 411 ± 6 mg, Y A C 1 2 8 : 394 ± 4 mg, p = 0.05). The volume o f the striatum in Y A C 128 mice was found to be 10.4% less than the striatal volume in W T mice suggesting that the small global changes in brain weight may be explained by larger changes in select regions o f the brain (Fig. 3.12 panel B ; W T : 12.1 ± 0.1 m m , Y A C 1 2 8 : 10.8 ± 0.2 m m , p < 3  3  0.001). We found a similar, 10.8% decrease in the volume o f the globus pailidus in Y A C 128 mice compared to W T mice (Fig. 3.12 panel B ; W T : 1.60 ± 0.06 m m , Y A C 1 2 8 : 1.43 ± 0.04 3  mm , p = 0.04). Cortical volume was also decreased in Y A C 128 mice compared to W T mice but to a lesser extent than the striatum or globus pailidus (Fig. 3.12 panel B ; decreased 8.6%; W T : 16.5 ± 0.02 m m , Y A C 1 2 8 : 15.1 ± 0.03 m m , p = 0.001). In contrast, the volumes o f the 3  3  hippocampus and cerebellum were unaffected  in Y A C 128 mice (Fig. 3.12 panel B ;  Hippocampus - W T : 0.0355 ± 0.001 m m , Y A C J 2 8 : 0.0356 ± 0.001 m m , p = 0.9; 3  3  64  Cerebellum +2.9% 0.94  p = 0.6  2.4E+06  2.0E+06  „ c 2  1.9E+06 1.8E+06  2.3E+06  |  2.2E+06  1  2.1E+06  §  2.0E+06  •  1.7E+06 1.6E+06 Striatum 9.1%, p  -  1.9E+06  1.4E+06  z  1.2E+06  !  1.0E+06  I  8.0E+05  | £  Cortex  0.01  1.6E+06  |  6.0E+05 4.0E+05 Hippocampus  8.3%, p = 0.02  1.5%, p = 0.72  Figure 3.12 Selective degeneration in the brains of YAC128 mice. At 12 months of age, YAC128 mice and  WT littermate controls were perfused. Brain weight was measured prior to stereological assessment of regional volumes within the brain. A. Brain weight was significantly decreased in YAC 128 mice compared to WT controls (WT: 411 ± 6 mg, YAC128: 394 ± 4 mg, p = 0.05). B. The overall decrease in brain weight resulted from atrophy of specific regions of the brain. The striatum, globus pallidus and cortex all showed significant atrophy in YAC128 mice compared to WT mice (Striatum - WT: 12.1 ± 0.1 mm , YAC128: 10.8 ± 0.2 mm , p < 0.001; Globus pallidus - WT: 1.60 ± 0.06 mm , YAC128: 1.43 ± 0.04 mm , p = 0.04; Cortex - WT: 16.5 ± 0.02 mm , YAC 128: 15.1 ± 0.03 mm , p = 0.001). In contrast, the hippocampus and cerebellum were unaffected in YAC128 mice (Hippocampus - WT: 0.0355 ± 0.001 mm , YAC128: 0.0356 ± 0.001 mm , p = 0.9; Cerebellum - WT: 44.3 ± 0.2 mm , YAC128: 45.6 ± 0.1 mm , p = 0.6). Neuronal loss was also selective in YAC128 mice. C. There was significant cell loss in the striatum of YAC128 mice compared to WT controls (WT: 1.90 ± 0.05 million neurons, YAC128: 1.73 ± 0.03 million neurons, p = 0.01). D. Similarly, less neurons were present in the cortex of YAC128 mice compared to WT mice (WT: 2.25 ± 0.05 million neurons, YAC128: 2.06 ± 0.05 million neurons, p = 0.02). E. In contrast, there was no difference in the estimated number of hippocampal neurons between YAC128 and WT mice (WT: 1.42 ± 0.05 million neurons, YAC128: 1.44 ± 0.03 million neurons, p = 0.72). N = 8 WT, 8 YAC128. Error bars show SEM. * = p < 0.05, ** p < 0.01. 2  2  2  2  2  2  2  2  2  2  i  65  Cerebellum - W T : 44.3 ± 0.2 m m , Y A C 1 2 8 : 45.6 ± 0 . 1 m m , p = 0.6). Furthermore, 3  3  examination o f cerebellar volume in 18 month Y A C 128 mice and W T controls revealed that even at this late age there is no cerebellar atrophy in Y A C 128 mice ( W T : 53.1 ± 0.02 m m , Y A C 1 2 8 : 55.0 ± 0.1 m m , p = 0.5). Thus, the overall decrease in brain weight in Y A C 1 2 8 3  mice is caused by degeneration in select regions o f the brain and the regional specificity in Y A C 128 mice is remarkably similar to that seen in patients with H D .  To determine whether the pattern o f neuronal loss in H D is also recapitulated in the Y A C 128 mouse model, we estimated neuronal numbers within the striatum, cortex and hippocampus at 12 months o f age. The number o f striatal neurons was significantly decreased in Y A C 128 mice compared to W T mice (Fig. 3.12 panel C ; W T : 1.90 ± 0.05 million neurons, Y A C 1 2 8 : 1.73 ± 0.03 million neurons, p = 0.01). Examination o f the region o f cortex above the striatum revealed that there is also neuronal loss in the cortex o f Y A C 1 2 8 mice (Fig. 3.12 panel D ; W T : 2.25 ± 0.05 million neurons, Y A C 1 2 8 : 2.06 ± 0.05 million neurons, p = 0.02). In contrast, Y A C 1 2 8 and W T mice showed no difference in the estimated number o f neurons in the cellular layer o f the hippocampus (Fig. 3.12 panel E ; W T : 1.42 ± 0.05 million neurons, Y A C 1 2 8 : 1.44 ± 0.03 million neurons, p = 0.72). Thus, as in human H D , Y A C 128 mice exhibit selective neuronal loss.  3.2.3.2  Progression of neuropathology in Y A C 1 2 8 mice  Next we examined the time course o f selective degeneration in the striatum and cortex, as well as decreases in brain mass over time to determine the age o f onset o f degeneration and whether the changes in Y A C 128 mice are progressive. For this purpose, brains from cohorts of mice at 6, 9, 12 and 18 months were analyzed.  Striatal volume in Y A C 128 mice was not different than W T at 6 months o f age (Fig. 3.13 panel A ; W T : 12.6 ± 0.5 m m , Y A C 1 2 8 : 12.6 ± 0.5 m m , p - 1.0). A t 9 months, significant 3  3  striatal volume loss is present in Y A C 1 2 8 mice compared to W T m i c e ( F i g . 3.13 panel A ; W T : 12.5 ± 0.3 m m , Y A C 128: 11.4 ± 0.2 m m , p = 0.009). Similarly, at 12 and 18 months, 3  3  striatal volume in Y A C 1 2 8 mice is significantly less than in W T mice (Fig. 3.13 panel A ; 12  66  m o n t h s - W T : 12.6 ± 0 . 1 m m , Y A C 1 2 8 : 11.4 ± 0 . 1 m m , p < 0.001; 18 m o n t h s - W T : 12.1 3  3  ± 0.4 m m , Y A C 128: 9.5 ± 0.4 m m , p = 0.006). A two factor A N O V A comparing genotype 3  3  and age reveals significant differences between Y A C 128 and W T mice and their pattern o f change over time (genotype:  F(i,i ) 5 7  = 44.6, p < 0.001; age X genotype: F p , ^ ) = 9.4, p <  0.001). This results from a progressive decline in striatal volume in Y A C 128 mice (age: F ,75) (3  = 7.8, p < 0.001).  While the magnitude o f the changes are more subtle, striatal neuronal counts follow the same pattern as striatal volume. A t 6 months, there is no striatal neuronal loss (Fig. 3.13 panel B ; W T : 1.55 ± 0.16 million neurons, Y A C 128: 1.52 ± 0.14 million neurons, p = 0.2). A t 9 months, there is a trend towards decreased striatal neuronal numbers in Y A C 128 mice compared to W T mice (Fig. 3.13 panel B ; W T : 1.60 ± 0.09 millions neurons, Y A C 1 2 8 : 1.36 ± 0 . 1 1 million neurons, p = 0.1). Striatal neuronal loss becomes significant at 12 months (Fig. 3.2.11 panel B ; 1 2 m o n t h s - W T : 1.79 ± 0 . 0 3 millions neurons, Y A C 1 2 8 : 1.64 ± 0 . 0 3 million neurons, p = 0.001; 18 months - W T : 1.70 ± 0.03 million neurons, Y A C 1 2 8 : 1.46 ± 0.04 million neurons, p = 0.006). Striatal neuronal counts in Y A C 128 mice also progressively decrease with age (age:  F 3 (  ; 7 5  )  = 21.4, p < 0.001) and there are significant differences between  genotype and the interaction between age and genotype (genotype: age X genotype: F  ( U 5 2  F(ij57)  = 15.3, p < 0.001;  ) = 2.8, p = 0.04).  Cortical volume in Y A C 128 mice is equivalent to W T at 6 and 9 months o f age (Fig. 3.13 panel C ; 6 months - W T : 18.2 ± 0.8 m m , Y A C 1 2 8 : 17.9 ± 0.8 m m , p = 0.8; 9 months 3  3  W T : 16.9 ± 0.8 m m , Y A C 1 2 8 : 16.4 ± 0.8 m m , p = 0.6). A t 12 and 18 months o f age, 3  3  Y A C 1 2 8 mice show decreased cortical volume compared to W T mice (Fig. 3.2.11 panel C ; 12 months - W T : 16.0 ± 0.4 m m , Y A C 1 2 8 : 14.0 ± 0.4 m m , p = 0.01; 18 months - W T : 3  3  18.5 ± 0.7 m m , Y A C 128: 15.1 ± 0.6 m m , p = 0.02). Similarly, cortical neuronal counts 3  3  were equivalent between Y A C 128 and W T mice until 12 months o f age when Y A C 128 mice began to exhibit cortical neuronal loss (Fig. 3.13 panel D ; 6 months - W T : 2.64 ± 0.13 million neurons, Y A C 128: 2.62 ± 0 . 1 3 million neurons,"p - 0 . 9 ; 9 months - W T : 2.37 ± 0.12 million neurons, Y A C 1 2 8 : 2.13 ± 0.12 million neurons, p = 0.3; 12 months - W T : 2.25 ±  67  A  B  |  80%  o  70%  -I  ,  ,  6  ,  9  12  18  Age(Months)  Figure 3.13 Progressive neuropathology in YAC128 mice. Striatal and cortical neuropathology was assessed in cohorts of YAC128 mice sacrificed at 6 months (N = 13 W T , 14 YAC128), 9 months (N = 18 W T , 11 YAC128), 12 months (N = 46 W T , 50 YAC128) and 18 months (N = 3 W T , 4 Y A C 128). A . Differences in striatal volume first became apparent at 9 months of age and progressively worsened with age (9 months: WT: 12.5 ± 0.3 mm , YAC128: 11.4 ± 0.2 mm , p = 0.009; 12 months: WT: 12.6 ± 0.1 mm , YAC128: 11.4 ± 0.1 mm , p < 0.001; 18 months: WT: 12.1 ± 0.4 mm , YAC128: 9.5 ± 0.4 mm , p = 0.006). B. Striatal neuronal loss followed a similar pattern with differences between Y A C 128 and W T becoming significant at 12 months (12 months - WT: 1.79 ± 0.03 millions neurons, YAC128: 1.64 ± 0.03 million neurons, p = 0.001; 18 months WT: 1.70 ± 0.03 million neurons, YAC128: 1.46 ± 0.04 million neurons, p = 0.006). C. Cortical volume was similar between Y A C 128 and W T mice at 6 and 9 months of age but showed significant atrophy in Y A C 128 mice starting at 12 months (12 months - WT: 1.60 ± 0.04 mm , YAC128: 1.40 ± 0.04 mm , p = 0.01; 18 months - WT: 1.85 ± 0.07 mm , Y A C 128: 1.51 ± 0.06 mm , p = 0.02). D. Cortical neuronal loss also became significant at 12 months of age (12 months - WT: 2.25 ± 0.05 million neurons, YAC128: 2.06 ± 0.05 million neurons, p = 0.02; 18 months - WT: 2.90 ± 0.11 million neurons, YAC128: 2.48 ± 0.09 million neurons, p = 0.03). E . Examination of brain weight in Y A C 128 mice revealed small differences from wild-type beginning at 9 months of age and becoming significant at 12 months (6 months - WT: 388 ± 9 mg, YAC128: 388 ± 8 mg, p = 1.0; 9 months - WT: 385 ± 5 mg, YAC128: 379 ± 6 mg, p = 0.5; 12 months - WT: 398 ± 3 mg, YAC128: 382 ± 3 m g , p < 0.001; 18 m o n t h s - W T : 403 ± 6 mg, YAC128: 380 ± 5 mg, p = 0.04). Error bars show S E M . * = p < 0.05, ** p < 0.01, *** p < 0.001. 3  3  3  3  3  3  3  3  3  3  68  0.05 million neurons, Y A C 128: 2.06 ± 0.05 million neurons, p = 0.02; 18 months - W T : 2.90 ± 0 . 1 1 million neurons, Y A C 1 2 8 : 2.48 ± 0.09 million neurons, p = 0.03).  Examination o f brain weight revealed mild atrophy in the brains o f Y A C 128 mice. Decreases in brain weight in Y A C 128 mice begin around 9 months o f age and become significant at 12 months (Fig. 3.13 panel E ; 6 months - W T : 388 ± 9 mg, Y A C 128: 388 ± 8 mg, p = 1.0; 9 months - W T : 385 ± 5 mg, Y A C 128: 379 ± 6 mg, p = 0.5; 12 months - W T : 398 ± 3 mg, Y A C 1 2 8 : 382 ± 3 mg, p < 0.001; 18 months - W T : 403 ± 6 mg, Y A C 1 2 8 : 380 ± 5 mg, p = 0.04). Again the changes in brain weight are less than the changes observed in striatal or cortical volume indicating that atrophy in select regions o f the brain result in the overall cerebral atrophy observed. Overall, examination o f the time course o f neuropathological changes in Y A C 128 mice revealed that neuropathology begins around 9 months o f age and progressively worsens with age.  3.2.3.3 Striatal neuronal atrophy and DARPP-32 down-regulation in YAC128 mice To determine whether a decrease in striatal neuronal size contributed to the decreased striatal volume in Y A C 128 mice, we examined the cross-sectional area o f neurons within the striatum. Neurons from a matched coronal section o f Y A C 128 and W T brains were stained with N e u N antibody and the perimeter o f the cell body was outlined using stereology software which then calculated the cross-sectional area. A t 12 months o f age, striatal neuronal size was significantly decreased in Y A C 128 mice compared to W T mice (Fig. 3.14 panels A - C ; W T : 81.6 ± 2.9 u m , Y A C 128: 69.8 ± 2.2 um , p = 0.002). Thus, both decreases 2  2  in striatal neuronal number and striatal neuronal size contribute to the overall decrease in striatal volume.  A s D A R P P - 3 2 expression has been found to be decreased in mouse models o f H D (Bibb et al. 2000; van Dellen et ai, 2000) and D A R P P - 3 2 is known to be important for dopaminergic neurotransmission (Svenningsson et al, 2004), we examined striatal D A R P P - 3 2 expression in Y A C 128 mice. Matched sections were stained with D A R P P - 3 2 antibody, detected with a fluorescent secondary antibody and the level o f D A R P P - 3 2 expression estimated using the  69  B E  100  i  ra (0 c  80  CD "ra  60 •  e< z  c rao  Slri  ss-S  ra§  1 — * *  1  40 20 0  WT  YAC128  • WT •  YAC128  Figure 3.14 Striatal neuronal atrophy in YAC128 mice. At 12 months of age striatal neuronal cross-sectional area was measured is YAC128 and W T mice. A . Striatal neuronal size was significantly decreased in YAC128 mice compared to W T mice (WT: 81.6 ± 2.9 um , YAC128: 69.8 ± 2.2 um , p = 0.002; N = 46 W T , 50 Y A C 128). Photographs of neurons from W T mice (panel B) and Y A C 128 mice (panel C) demonstrate this difference. Photographs were taken using the 100X objective. Error bars show S E M . ** p < 0.01. 2  2  Y A C 128  Figure 3.15 Down-regulation of striatal DARPP-32 expression in YAC128 mice. Matched coronal sections from Y A C 128 and W T mice were stained with DARPP-32 and detected using a fluorescent secondary antibody. A , B . Examination of stained sections reveals visually less DARPP-32 staining in Y A C 128 mice compared to W T mice (pictures taken with 2.5X objective). C , D . At higher power, neurons from W T mice show more intense staining than Y A C 128 neurons and there is also more staining outside of the cell body (pictures taken with 40X objective). E . Quantification of the average staining intensity confirms that there is significantly less striatal DARPP-32 expression in YAC128 mice compared to W T mice (WT: 667 ± 20 intensity units, YAC128: 549 ± 26 intensity units, p < 0.001; N = 10 WT, 10 Y A C 128). Error bars show S E M . *** p < 0.001.  70  intensity o f D A R P P - 3 2 staining within the striatum. Visual inspection o f D A R P P - 3 2 stained sections revealed more intense D A R P P - 3 2 staining in W T mice compared to Y A C 128 mice especially in the lateral region o f the striatum (Fig. 3.15 panels A , B ) . A t high power, D A R P P - 3 2 staining in W T mice show more intense neuronal staining than in Y A C 128 mice and also show more staining outside o f the cell body (Fig. 3.15 panels C , D ) . Quantification of the overall difference in the intensity o f D A R P P - 3 2 staining within the striatum revealed significantly decreased striatal D A R P P - 3 2 expression in Y A C 128 mice compared to W T mice (Fig. 3.15 panel E ; W T : 667 ± 20 intensity units, Y A C 128: 549 ± 26 intensity units, p < 0.001).  Regional differences in the expression and nuclear localization of mutant huntingtin in Y A C 1 2 8 mice  3.2.3.4  In mouse models o f H D , higher levels o f mutant htt expression consistently result in greater toxicity (Mangiarini et al, 1996; Reddy et al, 1998; Schilling et al, 1999). However, in H D , the regions o f the brain with the highest levels o f htt expression are not the most severely affected in the disease ( L i et al, 1993; Landwehrmeyer et al, 1995). T o determine whether the level o f mutant and wild-type htt expression in each region o f the brain could explain the observed selectivity in atrophy and neuronal loss in Y A C 128 mice, we examined htt expression in the striatum, cortex, hippocampus and cerebellum. We found that mutant htt expression was highest in the cerebellum, approximately equal in the hippocampus and cortex and least in the striatum (Fig. 3.16 panels A , B ; Cerebellum: 130 ± 18 arbitrary units, Cortex: 100 ± 10 arbitrary units, Striatum: 84 ± 9 arbitrary units, Hippocampus: 108 ± 8 arbitrary units). Wild-type htt followed the same pattern o f expression as mutant htt (Fig. 3.16 panels A , B ; Cerebellum: 124 ± 12 arbitrary units, Cortex: 100 ± 7 arbitrary units, Striatum: 80 ± 6 arbitrary units, Hippocampus: 99 ± 5 arbitrary units). Overall there were significant differences in htt expression between different regions o f the brain (region:  F(3 25) ;  = 9.2, p < 0.001) but, as in patients with H D , the regional differences in mutant htt expression levels does not explain the selective degeneration in the brain o f Y A C 128 mice.  Next, we examined regional differences in the nuclear localization o f mutant htt. While w i l d type htt is primarily a cytoplasmic protein (DiFiglia et al,  1995), expansion o f the  71  Cerebellum  Cortex  Striatum  Hippocampus  Figure 3.16 Selective neuropathology in YAC128 mice is not correlated with mutant huntingtin expression. Mutant and wild-type htt expression was examined in the striatum, cortex, cerebellum and hippocampus of 12 month old Y A C 128 mice. A . Western blotting with a htt specific MAB2166 antibody shows highest expression of htt in the cerebellum and lowest htt expression in the striatum. Both wild-type and mutant htt follow the same pattern of expression indicating that the transgenic mutant htt is expressed appropriately. B. Quantification of the band densities as a measure of protein level reveals significant differences between brain regions (F( . = 9.2, p < 0.001) but that mutant and wild-type htt follow the same pattern of expression ( F i . = 0.6, p = 0.4). For both mutant and wild-type protein, htt expression was highest in the cerebellum and least in the striatum (Cerebellum: 130 ± 18 arbitrary units, Cortex: 100 ± 10 arbitrary units, Striatum: 84 ± 9 arbitrary units, Hippocampus: 108 ± 8 arbitrary units). Thus, the regional specificity of mutant htt toxicity is not a result of increased mutant htt expression in the affected regions of the brain. N = 4 Y A C 128 mice. Error bars indicate SEM. 3  25)  (  2 9 )  72  polyglutamine tract results  in increased nuclear localization o f htt and it has  been  demonstrated that mutant htt is more toxic in the nucleus than the cytoplasm (Peters et al, 1999; L i et al, 1999; Saudou et al, 1998). In order to determine whether differences in the nuclear localization o f mutant htt could account for the selective degeneration in Y A C 128 mice, we examined the localization o f mutant htt in Y A C 128 mice by staining with E M 4 8 antibody (Gutekunst et al, 1999; Wheeler et al, 2000). This antibody was raised against the first 256 amino acids o f htt and has a high affinity for fragments o f mutant htt, especially those that have aggregated. W e examined 3 month old Y A C 128 mice to determine i f nuclear localization occurred earlier in the more affected regions o f the brain as well as 12 month old mice to determine i f the extent o f nuclear localization was greater in affected regions.  A t 3 months, we found that mutant htt exhibited the greatest degree o f nuclear localization in the striatum (Fig. 3.17 panels A,I) which was visibly greater in the lateral striatum compared to the medial striatum (Fig. 3.17 panels A , Q ) . Mutant htt was also localized to the nucleus in parts o f the cortex (Fig. 3.17 panels B,J), hippocampus (Fig. 3.17 panels C , H ) and cerebellum (Fig. 3.17 panels D,I). In the cortex, layer I V shows the most nuclear localization of mutant htt at this age (Fig. 3.17 panels B ) . In the hippocampus, nuclear E M 4 8 staining is darkest in the dentate gyrus, detectable in the C A 3 regions and absent from the C A 1 region (Fig. 3.17 panels C , H , R ) . Quantification o f the intensity o f nuclear E M 4 8 staining revealed that the nuclear localization o f mutant htt was significantly greater in the lateral striatum than in any other region o f the brain (Fig. 3.17 U ; region: F(5,17) = 23.8, p < 0.001).  A t 12 months o f age, nuclear localization o f htt was increased in all regions o f the brain compared to the 3 month time point (age: F(\^$) = 86.7, p < 0.001). A s at 3 months, the striatum shows more nuclear localization o f mutant htt than is present in other regions of the brain (Fig. 3.17 panels E,J). In the cortex, layers 11 and III show the most intense nuclear E M 4 8 staining (Fig. 3.17 panels F , K ) . In the hippocampus, the dentate gyrus still shows the greatest nuclear localization o f mutant htt, with more nuclear E M 4 8 staining in the C A 3 region than the C A 1 region (Fig. 3.17 panels G , L , S , T ) . Again, differences in nuclear localization o f mutant htt were confirmed by quantification o f nuclear E M 4 8 staining intensity (Fig. 2 U ; region: F ^ o ) = 15.5, p < 0.001). Overall, the nuclear localization o f  73  N u c l e a r L o c a l i z a t i o n of M u t a n t H u n t i n g t i n  o  -> © o  01  o o  to o o o  ro oi o o  S t r (Lat)  J  -  ;  Ti  Figure 3.17 Selective nuclear localization of mutant huntingtin in the brain of YAC128 mice. Matched coronal sections from 3 month and 12 month old YAC 128 mice were stained with EM48 for visualization of mutant htt localization within the cell. At 3 months of age, the striatum (A) shows more nuclear localization of htt than the cortex (B), hippocampus (C) or cerebellum (D). Under high power striatal neurons (I) show strong EM48 staining indicating high amounts of mutant htt in the nucleus. Layer IV of the cortex (J), the CA3 regions of the hippocampus (K), and the cellular layer of the cerebellum (L) show limited nuclear localization of htt. At this time point, the lateral striatum (I) shows more intense staining than the medial striatum (Q). In the hippocampus, the dentate gyrus (R) is more intensely stained than the CA3 region (K) and there is no staining in the CA1 region. At 12 months of age, the striatum, cortex and hippocampus all show more nuclear localization than at 3 months. The striatum (E) is still more intensely stained than the cortex (F), hippocampus (G) or cerebellum (H). Within the cortex, layers II and 111 show the greatest degree of nuclear localization of mutant htt. As at 3 months, the order of staining within the hippocampus from most to least is dentate gyrus, CA3 region then CA1 region. At high power, there are clearly more intensely labeled cells in the striatum (L), cortex (M: layer II/III) and hippocampus (N: CA3 region, S: dentate gyrus, T: CA1 region) while the staining in the cerebellum (O) appears similar to 3 months. Photographs A-H were taken using the 10X objective (scale bar = 500 um). Photographs I-T were taken using the 100X objective (scale bar = 50 um). U. Quantification of nuclear EM48 staining intensity as a measure of the amount of mutant htt in the nucleus, confirms these observations and indicates that there is selective and progressive nuclear localization of mutant htt in YAC128 mice. Error bars indicate SEM.  75  mutant htt is regionally selective. It occurs earlier and to a greater extent in the striatum than in other regions o f the brain and thus appears to correlate with the amount o f cerebral damage.  3.2.3.5  mice  Transglutaminase activity is selectively increased in the forebrain of  YAC128  Since transglutaminase ( T G ) activity is increased in H D patients and postulated to contribute to the pathogenesis o f the disease (Lesort et al, 1999), we examined the brains of Y A C 1 2 8 mice to determine i f T G activity was also increased in our model. Fresh frozen brains from Y A C 128 mice and W T littermate controls were split into forebrain and hindbrain quarters prior to T G assay to determine whether any observed increase in T G activity showed regional specificity. Both total and non-GTP inhibited T G activity were measured in order to estimate the relative contribution o f tissue T G (tTG) since t T G is inhibited by G T P . Y A C 128 mice show a 30% increase in total T G activity in the forebrain compared to W T mice (Fig. 3.18 panel A ; W T : 240 ± 22 pmol putrescine incorporated/mg protein/hour, Y A C 1 2 8 : 313 ± 21 pmol putrescine incorporated/mg protein/hour, p = 0.03). Total T G activity was also increased in the hindbrain o f the Y A C 128 mice but this 18% increase did not reach significance  (Fig. 3.18  panel  A ; W T : 361  ±  54 pmol putrescine  incorporated/mg  protein/hour, Y A C 1 2 8 : 425 ± 59 pmol putrescine incorporated/mg protein/hour, p = 0.4). A n examination o f the non-GTP inhibited T G activity revealed a dramatic increase in the forebrain o f Y A C 1 2 8 mice compared to W T mice with a non-significant increase o f lesser magnitude observed in the hindbrain (Fig. 3.18 panel B ; forebrain: 620%) increase, p = 0.03; hindbrain: 27% increase, p = 0.4). However, the majority o f T G activity within the brain is inhibited by G T P and thus, even though the percentage increase in non-GTP inhibited T G activity was much greater than for total T G activity, this difference accounted for only 13% o f the difference in total T G activity.  3.2.4  Decreased survival in Y A C 1 2 8 mice  Patients with H D have a decreased lifespan with an average age o f death o f 54 years (calculated from Harper, 1996). Based on the findings o f decreased lifespan in human H D  76  A  v  B  160%  • •  Figure 3.18  WT YAC128  Forebrain  Hindbrain  Forebrain  Hindbrain  + 30% p = 0.027  +18% p = 0.43  +620% p = 0.029  + 27% p = 0.42  YAC128  mice show a forebrain specific  increase  in transglutaminase  activity.  Transglutaminase (TG) activity was measured in Y A C 128 and W T littermates by putrescine incorportation assay. A . Total T G activity in the forebrain was increased approximately 30% in YAC128 mice compared to W T mice (WT: 240 ± 22 pmol/mg/hr, 313 ± 21 pmol/mg/hr, p = 0.027). Total T G activity in the hindbrain was increased by 18% in the YAC128 mice compared to W T mice (WT: 361 ± 54 pmol/mg/hr, YAC128: 425 ± 59 pmol/mg/hr, p = 0.43). B. GTP inhibits tissue T G which accounts for most of the T G activity in mouse brain. The non-GTP inhibited T G activity was increased by 620% in the forebrain of Y A C 128 mice compared to W T mice (WT: 1.5 ± 0.7 pmol/mg/hr, YAC128: 10.8 ± 3.7 pmol/mg/hr, p = 0.029) but showed no significant difference in the hindbrain (WT 5.9 ± 1.1 pmol/mg/hr; Y A C 128 7.5 ± 1.6 pmol/mg/hr, p = 0.42). N = 10 W T , 12 YAC128. Error bars indicate S E M . * p < 0.05.  77  patients, we determined whether this survival deficit was recapitulated in the Y A C 128 mouse model. Previously, decreased survival has only been demonstrated in animals expressing a fragment o f htt with an expanded polyglutamine tract and not in mice expressing full length mutant htt (Mangiarini et al, 1996; Schilling et al 1999; V o n Horsten et al, 2003). Thus, it was also important to determine i f early death is observed in the context o f full length htt.  Survival in the Y A C 128 mice was followed prospectively until 12 months o f age. Initially we observed a non-significant decrease in survival o f the Y A C 128 mice compared to W T mice (Mice surviving to 12 months: W T = 93%, N = 68; Y A C 128 = 85%, N = 97; p = 0.14). D i v i d i n g the cohort by sex, we discovered that there was no difference in survival between female Y A C 1 2 8 and W T mice (Fig. 3.19 panels A , C ; Female mice surviving to 12 months: W T = 90%, N = 30; Y A C 1 2 8 = 93%, N = 55; p = 0.64). In contrast, there was a clear and significant decrease in the survival o f male Y A C 128 mice compared to male W T mice or female Y A C 128 mice that entirely accounted for the overall survival differences between Y A C 128 and W T mice (Fig. 3.19 panels B , C ; Male mice surviving to 12 months: W T = 95%, N = 38; Y A C 1 2 8 = 74%, N = 42; p = 0.01).  In most cases, deaths were sudden and unexpected as regular cage inspections did not reveal any outward abnormality in the mice prior to death. In a couple of cases, mice appeared unwell and staff in our animal facility concluded that the mice would not survive and should be sacrificed for ethical reasons. W e examined two o f these mice that were sacrificed at 6 months o f age for striatal neuropathology but were unable to identify any defects. Similarly, when we examined the rotarod performance and weight o f mice that did not survive to 12 months before they passed away, rotarod performance was no different than mice that survive to 12 months and there was no obvious change in body weight.  3.2.5 YAC128 mice exhibit atrophy and degeneration in the testis Outside o f the brain, the testis expresses the highest levels o f htt protein, mutant or wild-type, in the body (Fig. 3.20 panel A ) . W e have previously reported that mild expression o f mutant htt with 72 glutamines lead to testicular degeneration when the levels o f wild-type htt were  78  D 100%  t SJ Q-  L  90%  h  80%  I  '— 1  1  Male Mice 100  200  400  300  Age(days)  c  Sex  Female  Male  100 •WT • YAC128  200  300  400  Age(days)  Percent  Genotype  Total Mice  Deaths  WT  30  3  90%  YAC128  55  4  93%  WT  38  2  95%  YAC128  42  11  74%  Surviving  Significance  p = 0.64  p = 0.01  Figure 3.19 YAC128 mice show a male specific deficit in survival. The survival of Y A C 128 and WT mice was monitored up until 12 months of age. Kaplan-Meier survival plots are shown where each step represents the death of one mouse. A . There were no differences in survival observed among female mice. B. In contrast, male Y A C 128 mice showed a significantly decreased lifespan compared to W T mice beginning around 9 months of age (p = 0.01). C. Data is summarized in table format.  79  decreased (Leavitt et al, 2001). However, in our previous experiment, it was not possible to determine whether the testicular pathology was caused by the expression o f mutant htt or the loss o f wild-type htt since expression levels o f mutant htt were less than endogenous. The expression o f mutant htt with 72 glutamines alone was not sufficient to cause any testicular degeneration as Y A C 7 2 mice expressing two copies o f wild-type htt showed normal testicular morphology and sperm production (Leavitt et al,  2001). Thus, to determine  whether the expression o f mutant htt alone, without the loss o f wild-type htt expression, could result in testicular degeneration we compared testis from Y A C 128 mice and W T mice. Since Y A C 128 mice express mutant htt protein with a larger polyglutamine tract than Y A C 7 2 mice, a more severe testicular phenotype is predicted.  In contrast to Y A C 7 2 mice, Y A C 128 mice expressing normal levels o f wild-type htt show testicular atrophy compared to W T mice (Fig. 3.20 panel A ; W T : 158 ± 3 mg, Y A C 1 2 8 : 126 ± 6 mg, p < 0.001). T o determine whether the testicular atrophy we observed resulted from testicular degeneration, we performed a histological examination of toluidine blue stained sections. Testis from W T mice were fully spermatogenic as indicated by he presence o f different stages o f spermatogenesis in cross sections (Fig. 3.20 panels C , E ) . In contrast, testis from Y A C 128 mice appeared  visually abnormal (Fig. 3.20 panels D , F ) . The  organization o f cells within the seminiferous tubules was disrupted by large vacuoles. The most striking difference was the observation o f cell death and cells being sloughed off into the lumen o f the seminiferous tubules. While there were some signs o f sperm production, the number o f developing sperm was obviously decreased compared to wild-type. Furthermore, few sperm cells developed to the later stages o f development and, o f those that did, many had abnormal sperm heads. Overall, we observed testicular atrophy and degeneration in the testis ofYAC128.  3.2.6 Outcome measures for therapeutic trials The goal for the characterization o f the Y A C 128 mice -was to develop a protocol for therapeutic trials for H D including biologically relevant, quantifiable outcome measures. Table 3.1 summarizes the differences we observed between Y A C 128 and W T mice. While  80  YAC128  Figure 3.20 Expression of mutant huntingtin results in testicular degeneration in YAC128 mice. A . Outside of the brain, the highest levels of mutant htt expression occur in the testis as shown by Western blot using the 1C2 antibody for expanded polyglutamines. B . Examination of testicular weight for signs of atrophy revealed that the testis of Y A C 128 mice weighed significantly less than WT testis indicating a toxic effect of mutant htt (WT: 158 ± 3 mg, YAC128: 126 ± 6 mg, p < 0.001, N = 20 WT, 15 YAC128). Sections of testis were stained with toluidine blue and examined for degeneration. C,E. Seminiferous tubules from WT mice show a stratified organization and are spermatogenic. D,F. Seminiferous tubules of Y A C 128 mice show a reduced number of developing sperm, disorganization of the S e r t o l i cell layer and sloughing off of cells into the lumen. Large vacuoles are also present. Photographs in C and D were taken with 40X objective. Photographs in E and F were taken with 100X objective. Scale bar represents 5 um. Error bars show S E M . *** p < 0.001.  81  the percentage differences between Y A C 1 2 8 and W T mice were greatest for the behavioural outcome measures, the variability for these tests was also high. In contrast, neuropathological differences in the striatum show very little variability. Power calculations with these results were performed to determine the number o f mice required for therapeutic trials (Table 3.1). Based on the biological relevance o f striatal volume loss in H D and the robustness o f this finding in Y A C 128 mice, this outcome measure was chosen as the primary outcome measure for therapeutic trials.  82  Table 3.1 Summary of phenotypic difference between YAC128 and WT mice that can be used as outcome measures for therapeutic trials. Difference between Y A C 128 and WT was calculated as (YAC 128 value WT value)/WT value. Standard deviation is expressed as a percentage of WT value (standard deviation/WT value). Power calculations were performed to determine the number of mice to have an 80% chance of detecting either a 50% or 75% rescue of the phenotype.  Outcome Measure Striatal volume Striatal neuronal counts Striatal neuronal cell size Striatal DARPP32 expression Brain weight Rotarod 10 months Open field activity 12 months Swimming Tmaze with reversal Pre-pulse Inhibition  Difference Between YAC128 and WT  Significance of Difference  Standard Deviation  Mice Required to Detect a 50% Rescue  Mice Required to Detect a 75% Rescue  -10.4%  p< 0.001  4.4%  10  5  -11.0%  p = 0.009  5.1%  13  6  -18.7%  p< 0.001  6.6%  6  3  -22.2%  p< 0.001  9.8%  11  5  -4.6%  p = 0.07  3.4%  37  16  -51.1%  p< 0.001  24.7%  6  3  -22.1%  p< 0.001  16.4%  27  12  + 108%  p< 0.001  31.0%  23  10  -26.9%  p = 0.001  21.0%  27  12  83  4.0 WILD-TYPE HUNTINGTIN FUNCTION 4.1 Wild-type huntingtin is neuroprotective in the hippocampus The over-expression o f wild-type htt in vitro has been shown to protect neurons from a variety o f toxic stimuli including the expression o f an expanded polyglutamine protein (Rigamonti et al, 2000; H o et al, 2001). We have previously extended these findings in vivo to demonstrate a protective effect o f wild-type htt against mutant htt toxicity in the testis. Since the brain is the region o f primary pathology in H D , we wanted to determine i f wildtype htt is neuroprotective in brain. Y A C 18, line 212 mice express wild-type htt with 18 glutamines from a Y A C transgene (Hodgson et al,  1996). Expression, o f wild-type htt in  these mice is approximately 2-3 times endogenous and htt is expressed in an appropriate tissue-specific manner.  Since excitotoxicity is thought to be involved in the neuronal death in H D , we examined the ability of wild-type htt to protect against hippocampal neurodegeneration caused by kainic acid ( K A ) , an excitotoxic neurotoxin. Y A C 18 and W T mice were injected intraperitoneally with 25 mg/kg K A and sacrificed after 7 days. Monitoring the mice after K A injection revealed no difference in the severity o f seizures caused by K A between Y A C 18 and W T mice. The damage induced by K A was assessed by fluorojade staining, silver staining and T U N E L . Fluorojade and silver staining both detect degenerating neurons, while TLTNEL detects dying cells containing fragmented D N A .  Visual inspection o f fluorojade stained sections revealed clearly more fluorojade-positive degenerating neurons in W T mice than Y A C 18 mice after exposure to K A (Fig. 4.1 panels A , B ) . Similarly, silver staining in sections from W T mice revealed numerous degenerating neurons (stained black, see arrows) but almost none in Y A C 18 sections (Fig. 4.1 panels C,D). T U N E L was also much greater in sections from W T mice than sections from Y A C 18 mice, confirming that the hippocampal cells were dying (Fig. 4.1 panels E,F). Quantification of the number o f fluorojade positive degenerating neurons revealed that there was significantly more degenerating neurons in W T mice than Y A C 18 mice after injection with K A (Fig. 4.1 panel G ; C A 1 region - W T : 205 ± 58 degenerating neurons/section, Y A C 1 8 :  84  A  B  Fluorojade Staining  WT  YAC 18  c  D Kit • • *  t "4\Xz*.  Silver Staining  WT  i  ' /  t  YAC 18 F  \ WT  TUNEL  YAC 18  lj  450  1?  350  400  11 2 g 300 ay  | 250  ^  S  0  2 0 0 W  •  WT  •  Y A C 18  S p 150  z§ 1 Z  » 100 50  CA1  CA3  Combined  Region of Hippocampus  Figure 4.1 Over-expression of wild-type huntingtin protects neurons from kainic acid toxicity in the hippocampus. W T and Y A C 18 mice were given intraperitoneal injections of the excitotoxic neurotoxin kainic acid (KA) to determine if over-expression of wild-type htt was neuroprotective in vivo. Seven days after K A injection, matched coronal sections from the hippocampus were analyzed for degeneration. A , B . Fluorojade staining for degenerating neurons revealed numerous degenerating neurons in the hippocampus of WT mice but few or none in Y A C 18 mice. C , D . Detection of degenerating neurons by silver staining revealed an identical pattern, with many dying neurons in W T mice but few in Y A C 18 mice. E,F. T U N E L was used to show that the degenerating neurons in the W T mice had fragmented D N A and were dying. G . Quantification of the number of degenerating neurons shows significantly more dying neurons in W T mice compared to Y A C 1 8 mice in both the CA1 region and CA3 region of the hippocampus (N = 11 W T , 7 Y A C 18). Thus, over-expression of wildtype htt is neuroprotective in the hippocampus. Error bars show S E M . * p < 0.05, ** p < 0.01.  85  0.3 ± 0.1 degenerating neurons/section, p = 0.01; C A 3 region - W T : 192 ± 60 degenerating neurons/section, Y A C 18: 6.5 ± 3.6 degenerating neurons/section, p = 0.02; all regions - W T : 326 ± 85 degenerating neurons/section, Y A C 18: 6.8 ± 3.6 degenerating neurons/section, p = 0.003). The difference between Y A C 18 and W T mice was dramatic as almost no degenerating neurons were detected in the Y A C 18 mice. The finding o f decreased numbers of degenerating neurons in Y A C 18 mice compared to W T mice after administration o f K A suggests that wild-type htt is neuroprotective and limits excitotoxic cell death.  4.2 Wild-type huntingtin is neuroprotective in the striatum Since the primary region o f pathology in H D is the striatum, we next investigated whether wild-type htt was also neuroprotective in the striatum. Y A C 18 and W T mice were given intrastriatal injections o f the excitotoxic neurotoxin quinolinic acid ( Q A ) , which has been used in neurotoxin models o f H D . Following Q A injection, there was no difference in the frequency or duration o f seizures between Y A C 18 and W T mice. After 7 days, mice were sacrificed and damage to the striatum was assessed with fluorojade and by staining with N e u N , which is specific for neuronal nuclei, or anti-DARPP-32 antibody. In each case the lesion size in W T mice was visually larger than the lesion in Y A C 18 mice (Fig. 4.2 panels A C). To quantify this difference, the volume o f the entire lesion in each mouse was assessed by stereology. This analysis revealed that the lesion volume in W T mice was 62% greater than the lesion volume in Y A C 18 mice (Fig. 4.2 panel D ; W T : 3.26 ± 0.35 m m , Y A C 18: 3  2.01 ± 0 . 2 2 m m , p = 0.007). 3  In addition to lesion volume, the number o f surviving neurons was measured by estimating the number o f neurons in matched sections from W T and Y A C 18 mice. In this assessment, sections from Y A C 18 mice were found to have 30% more neurons remaining than sections from W T mice (Fig. 4.2 panel E; W T : 68.5 ± 6.7 thousand neurons, Y A C 18: 88.6 ± 4.0 thousand neurons, p = 0.027). Combined with the differences in lesion volume, this indicates that wild-type htt is neuroprotective in the striatum.  86  D 4.5E+09 4.0E+09  i  3.5E+09  T  1  * *  3.0E+09 2.5E+09 T  2.0E+09 1.5E+09 1.0E+09 5.0E+08 O.OE+00  3 •  CD  1.0E+05 T  8.0E+04  T  >  C YAC18 CN co OL  0-  OH  <  LJ  09 6.0E+04 15 c  o 4.0E+04  3 CD  Z  2.0E+04 O.OE+00  • WT •  Y A C 18  Figure 4.2 Over-expression of wild-type huntingtin protects neurons from quinolinic acid toxicity in the striatum. WT mice and YAC 18, line 212 mice were given intrastriatal injections of the excitotoxic neurotoxin quinolinic acid (QA). After 7 days, the size of lesion resulting from QA delivery was assessed by staining with fluorojade (panel A), NeuN (panel B) and DARPP-32 (panel C). In each case, both WT and YAC 18 mice developed striatal lesions in response to QA injection but the size of the lesion in YAC18 mice was smaller than lesions in WT mice. D. Quantification of lesion volume revealed that lesions in YAC 18 mice were significantly smaller than lesions in WT mice indicating a protective effect of wild-type htt (WT: 3.26 ± 0.35 mm , YAC18: 2.01 ± 0.22 mm , p = 0.007). E. Matched sections were used to estimate the number of neurons remaining after QA injection. In this estimation, YAC 18 mice were shown to have significantly more neurons remaining than WT mice (WT: 68.5 ± 6.7 thousand neurons, YAC 18: 88.6 ± 4.0 thousand neurons, p = 0.027). Here, overexpression of wild-type htt is neuroprotective in the striatum, the region most affected in HD. N = 13 WT, 24 YAC 18. Error bars show SEM. * p < 0.05, ** p < 0.01. 3  87  To ensure that the neuroprotection was caused by wild-type htt and was not a site o f integration effect, we repeated the experiment using Y A C 18 mice from line B60 (line B60 mice). Both heterozygous and homozygous, line B 6 0 mice express lower levels o f wild-type htt than Y A C 18, line 212 mice (line 212 mice) which were used in the initial experiment. Combined with W T mice, the varying levels o f wild-type htt expression also allowed us to determine whether wild-type htt mediated neuroprotection occurs in a dose-dependent manner. W T , heterozygous line B 6 0 , homozygous line B 6 0 and line 212 mice were given intrastriatal injections o f quinolinic acid and sacrificed after 7 days.  Analysis o f the resulting lesion volumes revealed that wild-type htt mediated neuroprotection was dose dependent (genotype: F( i) = 4.5, p = 0.006). W T mice had the largest lesion size 3)7  followed by heterozygous line B 6 0 mice, homozygous line B 6 0 mice and line 212 mice (Fig. 4.3; W T : 4.62 ± 0.38 m m , B 6 0 +/-: 4.27 ± 0.12 m m , B60 +/+: 3.70 ± 0.16 m m , 212: 3.25 ± 3  3  3  0.28 mm ). Western blotting with M A B 2 1 6 6 antibody, which recognizes both mouse and human htt, confirmed that line 212 mice express the most wild-type htt protein followed by homozygous line B 6 0 mice, heterozygous line B 6 0 mice and W T mice (Fig. 4.3). The difference in overall htt levels result from a difference in the expression levels o f transgenic, human htt which can be detected with the human htt specific H D 6 5 0 antibody (Fig. 4.3). Thus, increasing levels o f wild-type htt expression result in increased neuroprotection. This indicates that the protective effect o f wild-type htt is dose dependent and that it is independent o f site o f integration.  4.3 Huntingtin expression influences weight To determine whether the study o f Y A C 18 mice could provide any additional insight into htt function, we examined Y A C 18 mice for overt phenotypes resulting from the over-expression of wild-type htt. Visual inspection revealed that aged Y A C 18 mice appear larger than W T mice. To quantify this difference we monitored body weight in W T mice, heterozygous B 6 0 mice, homozygous B60 mice and 212 mice from 2 to 12 months o f age and found that overall body weight increased with htt expression (genotype:  F 36) (2;  = 18.2, p < 0.001). Line 212  mice weighed significantly more than line B 6 0 and W T mice from 2 months o f age to 12  88  5.5E+09 _  m < E  • • B •  5.0E+09  3  WT B60 +/B60 +/+ 212  O  a  E o >  _3  WT  B60+/-  B60+/+  212 NT htt (transgenic) W T htt (total) Actin  Figure 4.3 Dose dependent wild-type huntingtin mediated neuroprotection in the striatum. WT mice, heterozygous YAC 18, line B60 mice (B60 +/-), homozygous YAC 18, line B60 mice (B60 +/+) and YAC 18, line 212 mice were given intrastriatal injections of quinolinic acid (QA) and the size of the resulting lesion was compared with total wild-type htt expression. WT mice showed the largest lesion size followed by B60 +/mice, B60 +/+ mice and line 212 mice (genotype: F . = 4.5, p = 0.006; WT: 4.62 ± 0.38 mm , B60 +/-: 4.27 ± 0.12 mm , B60 +/+: 3.70 ± 0.16 mm , 212: 3.25 ± 0.28 mm3; N = 22 WT, 11 B60 +/-, 14 B60 +/+, 32 line 212). Total wild-type htt expression was greatest in line 212 mice followed by B60 +/+ mice, B60 +/- mice and WT mice. The differences in total htt expression stem from differences in the amount of trangenically expressed human htt as seen by Western blot with the human specific HD650 antibody. The demonstration of neuroprotection in two lines of YAC 18 mice indicates that wild-type htt is neuroprotective independent of the site of integration of the htt transgene. More importantly, increased wild-type htt expression results in a smaller lesion size showing that htt mediated neuroprotection is dose dependent. Error bars show SEM. * p < 0.05, ** p < 0.01. 3  (3  3  71)  3  89  months o f age (Fig. 4.4 panel A ; 2 months - W T : 25.5 ± 1.1 g, Line 212: 30.0 ± 1.0 g, p = 0.006; 12 months - W T : 34.5 ± 1.6 g, Line 212: 46.5 ± 1.4 g, p < 0.001). The lower expressing B 6 0 line showed a trend towards increased weight relative to W T mice beginning at 2 months o f age with the differences becoming highly significant by 12 months o f age (Fig. 4.4 panel A ; 2 months - W T : 25.5 ± 1.1 g, Line B 6 0 : 26.8 ± 0.7 g, p = 0.3; 12 months W T : 34.5 ± 1.6 g, Line B 6 0 : 40.6 ± 1.1 g, p = 0.002). Western blotting for htt expression confirmed that line B 6 0 mice express more htt than W T mice and line 212 mice express more htt than line B 6 0 mice (Fig. 4.4 panel B ) . Thus, increased wild-type htt expression results in increased body weight. The demonstration o f increased weight in two different lines o f Y A C 18 mice and the dose dependent increase that is observed indicates that the enlarged weight is not a result of the site o f integration and that altered htt levels are causative.  Our laboratory has also generated mice heterozygous for the targeted inactivation o f the mouse H D gene {Hdh +/- mice). These mice express htt at half o f W T levels (Nasir et  al,  1995). To determine i f a decrease in htt levels resulted in lower body weight, we examined the weight o f a cohort o f W T and Hdh +/- mice from 2 to 11 months. We found that Hdh +/mice weigh significantly less than W T mice beginning before 2 months o f age (Fig 4.4 panel C ; 2 months - W T : 24.1 ± 0.4 g, Hdh +/-: 21.0 ± 0.8 g, p = 0.003). The difference was maintained until 11 months o f age although the difference appeared to be greatest closer to two months o f age indicating the difference in weight may stem from altered development in Hdh +/- mice (genotype: F i o) = 4.2, p = 0.05). Western blotting for htt protein confirmed (  i2  that Hdh +/- mice express htt at half o f wild-type levels (Fig. 4.4 panel D ) . In combination with our findings in mice over-expressing wild-type type htt, it is clear that expression levels of wild-type htt influence overall body weight.  In order to determine whether the increase in body weight in Y A C 18 mice resulted from increased organ weights or just an increase in fat accumulation, we measured organ weights in 12 month old Y A C 18 line 212 mice. We found that organ weight was significantly increased in Y A C 18 mice compared to W T mice in the heart, liver, lungs, kidneys and spleen (Fig. 4.5 panel A ; Heart - W T : 187 ± 4 mg, Y A C 1 8 : 231 ± 7 mg, p < 0.001; Liver - W T : 1.94 ± 0.06 g, Y A C 1 8 : 2.59 ± 0 . 1 1 g, p < 0.001; Lungs - W T : 420 ± 12 mg, Y A C 18: 485 ±  90  Figure 4.4 Wild-type huntingtin influences body weight. A. Measurement of body weight of YAC 18 and WT mice from 2 to 12 months of age revealed a significant effect of wild-type htt expression on body weight (genotype: F = 18.2, p < 0.001). Both YAC 18, line B60 mice and YAC 18, line 212 mice weighed significantly more than WT mice (12 months - WT: 34.5 ± 1.6 g, Line 212: 46.5 ± 1.4 g, Line B60: 40.6 ±1.1 g, p < 0.001, N = 9 WT, 14 Line B60, 16 Line 212). B. Examination of total htt expression confirmed that increased expression of htt resulted in a dose dependent increase in body weight as line B60 mice express more htt than WT mice and line 212 mice express more htt than line B60 mice. C. To determine if reductions in htt expression also affected body weight, we examined mice heterozygous for the targeted inactivation of the mouse HD gene {Hdh +/- mice) and found that Hdh +/- mice weighed significantly less than WT mice (2 months - WT: 24.1 ± 0.4 g, Hdh +/-: 21.0 ± 0.8 g, p = 0.003, N = 12 WT, 10 Hdh +/-). D. Western blotting confirmed that Hdh +/- mice have reduced levels of wild-type htt expression. Thus, wild-type htt has a clear effect on body weight which is not eliminated by CAG expansion. Error bars show SEM ( 2 J 6 )  91  15 mg, p = 0.002; Kidneys - W T : 673 ± 13 mg, Y A C 18: 848 ± 26 mg, p < 0.001; Spleen W T : 108 ± 2 mg, Y A C 18: 147 ± 11, p = 0.002). In contrast to all o f the other organs tested, the brain and testis show no increase in weight (Fig. 4.5 panel A ; Brain - 403 ± 3 mg, Y A C 1 8 : 4 0 7 ± 4 m g , p = 0.4; T e s t i s - W T : 161 ± 4 mg, Y A C 18: 152 ± 7 mg, p = 0.3),  Next, we examined the level o f htt expression in each o f the organs studied to see i f there was any correlation between the relative increases in weight and the level o f htt expression. We found that, as previously reported, expression o f full length htt was highest in the brain and testis (Fig. 4.5 panel C ; Brain: 100 ± 9 arbitrary units, Heart: 29 ± 6, Liver: 48 ± 12; Lungs: 77 ± 16; Kidney: 22 ± 2; Spleen: 84 ± 13; Testis: 108 ± 7). Among the remaining peripheral tissues htt expression was highest in the lungs, which showed very little increase in weight, and the spleen which showed the largest change in weight in male mice. W e were unable to detect any full length htt in the kidneys. While overall body weight increases with htt expression, this is not universally true o f organs within the body, as the two organs with the highest htt expression are not enlarged with increased htt.  To determine i f the effect o f htt on body and organ weight is disrupted by polyglutamine expansion, we examined body weight and organ weight in mice that transgenically express mutant htt from a Y A C transgene. A s with mice over-expressing wild-type htt, these mice showed increased body weight compared to W T mice (genotype: F i ) = 33.6; p < 0.001; 2 (  ;49  months: +10%, p = 0.001; 12 months: +29%, p < 0.001). The organ weights at 12 months in mice transgenically expressing mutant htt were also increased compared to W T mice except in the brain and testis where atrophy was present (Brain: -4%, p < 0.001; Testis - -22%, p < 0.001; Heart: +18%, p < 0.001; Liver: +23%, p < 0.001; Kidneys: +23%, p < 0.001; Spleen: +24%), p < 0.001). Although it is uncertain whether mutant htt is as efficient as wild-type htt at modulating weight, it is clear that polyglutamine expansion does not markedly disrupt htt's effect on body and organ weight.  92  150% 140%  ***  **  130% 120% 110% 100%  CD  • WT  90%  2  80%  5  60%  1  CD  •  Y A C 18  70% — Brain +1% p = 0.4  Heart +24% p < 0.001  Liver +33% p < 0.001  Lungs +15% p = 0.002  Kidneys +26% p < 0.001  Spleen +36% p = 0.002  Testis -6% p = 0.3  B  i  •g  Total wild type htt expression Brain 100%  Heart 10%  Lner 34%  Lungs 70%  Kidney Spleen 0% 79%  Testis 110%  Figure 4.5 Wild-type huntingtin influences organ weight. Organs were collected from 12 month old YAC 18, line 212 and WT mice and weighed. A. Organ weight in the YAC 18 mice was greater than WT mice for the heart, liver, lungs, kidneys and spleen (Fig. 2A; Heart - WT: 187 ± 4 mg, YAC18: 231 ± 7 mg, p < 0.001; Liver - W T : 1.94 ± 0 . 0 6 g, YAC18: 2.59 ± 0.11 g, p < 0.001; Lungs - WT: 420 ± 12 mg, YAC 18: 485 ± 15 mg, p = 0.002; Kidneys - WT: 673 ± 13 mg, YAC 18: 848 ± 26 mg, p < 0.001; Spleen - WT: 108 ± 2 mg, YAC 18: 147 ± 11, p = 0.002). In contrast, over-expression of wild-type htt did not increase the weight of the brain or testis (Brain - 403 ± 3 mg, YAC 18: 407 ± 4 mg, p = 0.4; Testis - WT: 161 ± 4 mg, YAC18: 152 ± 7 mg, p = 0.3). N = 34 WT, 21 YAC18. B,C. Quantification of total htt expression in organs of YAC18 mice revealed that htt expression was highest in the brain and testis, the two organs that did not show increased weight in the YAC 18 mice. Among the other organs, the spleen and lungs had the highest level of htt expression (Brain: 100 ± 9 arbitrary units, Heart: 29 ± 6, Liver: 48 ± 12; Lungs: 77 ± 16; Kidney: 22 ± 2; Spleen: 84 ± 13; Testis: 108 ± 7; N = 4). Error bars show SEM. ** p < 0.01 *** p < 0.001.  93  5.0 PRE-CLINICAL THERAPEUTIC TRIALS IN THE YAC128 MOUSE M O D E L OF HUNTINGTON DISEASE 5.1 Genetic treatment of YAC128 mice by over-expression of wild-type huntingtin 5.1.1 Rationale for treatment with wild-type huntingtin Wild-type htt is essential for embryonic survival and normal function in adulthood (Nasir et al,  1995; Duyao et al, 1995; Zeitlin et al,  1995, Dragatsis et al, 2000). Recent work has  demonstrated a neuroprotective effect o f wild-type htt (Rigamonti et al, 2000; H o et  al,  2001; Leavitt et al, 2001). In this thesis, we have extended these findings to show httmediated neuroprotection in the brain in both the hippocampus and the striatum (section 4.1 and 4.2). Since H D patients have decreased levels o f wild-type htt, it is plausible that this decrease in htt's neuroprotective function makes neurons more susceptible to the toxicity o f mutant htt. The reduction in wild-type htt levels may also impact gene expression and transport as htt has been shown to be involved in both o f the processes (Zuccato et al, 2001; Zuccato et al, 2003; Gauthier et al, 2004; Trushina et al, 2004). Thus, in order to assess the role o f loss o f htt function in H D and whether delivery o f wild-type htt would be beneficial in the treatment o f H D , we treated Y A C 128 mice with wild-type htt.  5.1.2 Generation of YAC128 mice that over-express wild-type huntingtin In order to ensure delivery o f wild-type htt to all affected cells in the brain, we crossed Y A C 128 mice with Y A C 1 8 to generate Y A C 1 2 8 mice that over-express wild-type htt ( Y A C 1 8 / 1 2 8 mice). Y A C 1 8 / 1 2 8 mice were generated in equal proportions to W T , Y A C 18 and Y A C 128 mice indicating normal embryonic survival. The presence o f both transgenes was confirmed by P C R (not shown) and htt expression was examined by Western blot for human htt (Fig. 5.1 panel A ) . Since the Y A C transgenes used to generate Y A C 18 and Y A C 128 mice express human htt, we probed the Western blot with H D 6 5 0 antibody which is specific for human htt. In this way, only the htt expressed from Y A C transgenes was detected since we have previously shown that transgenically expressed htt does not affect  the  expression o f endogenous htt (Van Raamsdonk et al, 2005). W T mice showed no transgenic expression o f htt. Y A C 18 mice expressed only wild-type human htt, while . Y A C 128 mice  94  CO CM  ST  5 Mu htt -  CO  CO  O  O  < >-  CO CSI  T—  <>  B  O  < >  c  o  tn £  CL  X  3000  1 2000 c  Actin-  5000  'to 4000 LU  WT htt-  • YAC18 • YAC128 • YAC18/128  1000  X Wild Type Htt  Mutant Htt  Figure 5.1 Generation of YAC128 mice that over-express wild-type huntingtin. To determine whether the over-expression of wild-type htt could ameliorate the HD-like symptoms in YAC 128 mice, YAC 18/128 mice were generated by crossing YAC 18 and YAC 128 mice. A. An examination of protein expression using the human specific HD650 antibody indicates that YAC18/128 mice express both wild-type htt and mutant htt from YAC transgenes. As expected WT mice express no human htt, YAC 18 mice express only wild-type human htt and YAC 128 mice express only mutant human htt. B. Quantification of htt protein expression levels reveals that YAC18/128 mice express wild-type human htt at the same level as YAC18 mice (YAC18: 3873 ± 26 arbitrary units, YAC18/128: 3923 ± 145 arbitrary units, p = 0.8) and express mutant human htt at the same level as YAC128 mice (YAC128: 3389 ± 197 arbitrary units, YAC18/128: 3303 ± 316 arbitrary units, p = 0.8). Error bars indicate SEM.  95  expressed only mutant human htt. Y A C 18/128 mice expressed both wild-type and mutant human htt. Importantly, the level o f mutant htt expression was equal between the Y A C 1 2 8 and Y A C 18/128 mice indicating that the over-expression o f wild-type htt did not downregulate the expression o f mutant htt (Fig. 5.1 panel B ; Y A C 1 2 8 : 3389 ± 197 arbitrary units, Y A C 1 8 / 1 2 8 : 3303 ± 316 arbitrary units, p = 0.8). Similarly, the level of wild-type htt expression was equal between Y A C 18/128 and Y A C 18 mice indicating that the expression of mutant htt did not affect wild-type htt expression (Fig. 5.1 panel B ; Y A C 1 8 : 3873 ± 26 arbitrary units, Y A C 18/128: 3923 ± 145 arbitrary units, p = 0.8).  5.1.3 Effect of wild-type huntingtin on neuropathology in YAC128 mice Since wild-type htt was shown to be neuroprotective (section 4.1, 4.2), we specifically examined the effect o f wild-type htt on the striatal neuropathology observed in Y A C 128 mice. A comparison o f Y A C 1 8 / 1 2 8 mice and Y A C 1 2 8 mice revealed a slight trend towards improvement in striatal volume (Fig. 5.2 panel A ; Y A C 1 2 8 : 11.3 ± 0.2 m m , Y A C 1 8 / 1 2 8 : 3  11.6 ± 0.2 m m , p = 0.3), striatal neuronal numbers (Fig. 5.2 panel B ; Y A C 128: 1.56 ± 0.03 3  million neurons, Y A C 18/128: 1.59 ± 0.03 million neurons, p = 0.4) and striatal D A R P P - 3 2 expression (Fig. 5.2 panel C ; Y A C 128: 828 ± 28 arbitrary units, Y A C 18/128: 868 ± 22 arbitrary units, p = 0.3) with increased wild-type htt expression which failed to reach significance. In contrast, increasing wild-type htt expression in Y A C 128 mice resulted in a significant reduction in striatal neuronal atrophy. Y A C 18/128 mice had significantly larger striatal neuronal cross-sectional areas than Y A C 1 2 8 mice (5.2 panel D ; Y A C 1 2 8 : 96.2 ± 1.6 u m , Y A C 18/128: 108 ± 1.9 u m , p < 0.001). Moreover, the striatal neuronal cross-sectional 2  2  area in Y A C 18/128 mice was almost restored to W T ( W T : 110 ± 3 u m , p = 0.6). Thus, over2  expression o f wild-type htt resulted in mild improvement o f striatal neuronal size but did not improve the hallmark signs o f the disease - striatal volume loss and neuronal loss in the striatum.  96  A  B  1.30E+10 -  1750000 „ 1700000  -j- 1.25E+10 -  8 1650000 M 1.20E+10 -  I 1600000  I _  |  1.15E+10  "C  »  T  1550000  I 1500000 0 5  1.10E+10 -  1450000  H  1400000 1.05E+10 110%  -***_ L _  100%  gf  90%  » |  B0%  1  70%  60%  • WT  • YAC18  YAC128 YAC 18/128  Figure 5.2 Over-expression of wild-type huntingtin in YAC128 mice results in mild improvements in striatal neuropathology. Comparison of striatal phenotypes between YAC 128 and YAC 18/128 mice revealed that over-expression of wild-type htt resulted in non-significant increases in striatal volume (panel A: YAC 128: 11.3 ± 0.2 mm , YAC18/128: 11.6 ± 0.2 mm , p = 0.3), striatal neuronal counts (panel B: YAC128: 1.56 ± 0.03 million neurons, YAC 18/128: 1.59 ± 0.03 million neurons, p = 0.4) and striatal DARPP-32 expression (panel C: YAC 128: 828 ± 28 arbitrary units, YAC 18/128: 868 ± 22 arbitrary units, p = 0.3). In contrast, over-expression of wild-type htt resulted in a significant improvement in striatal neuronal cross-sectional area (panel D: YAC128: 96.2 ± 1.6 um , YAC18/128: 108 ± 1.9 um , p < 0.001). For each outcome measure, YAC128 mice show a significant deficit compared to WT mice. N = 17 WT, 17 YAC 128, 14 YAC 18, 17 YAC 18/128. Error bars show SEM. **p<0.01.***p< 0.001. 2  2  2  2  97  5.2 Pharmacologic treatment of YAC128 mice with cystamine 5.2.1 Rationale for treatment with cystamine H D patients have been shown to have increased transglutaminase (TG) activity (Lesort et al, 1999; Karpuj et al, 2002) and we demonstrate here that there is a forebrain specific increase in T G activity in Y A C 1 2 8 mice (section 3.2.3.5). Increased T G activity may be harmful in H D by cross-linking mutant htt leading to potentially toxic aggregation intermediates (de Cristofaro et al, 1999; Kahlem et al, 1998), by increasing sensitivity to apoptosis (Melino et al,  1994; Piacentini et al,  2002; Oliverio et al,  1999) or through  inappropriate  modifications o f proteins that interact with htt (Lesort et al, 2002; Chun et al, 2001; Cooper et al, 1997). Cystamine is an inhibitor o f T G activity which may also be beneficial through inhibiting caspase-3 activity (Lesort et al, 2003) increasing expression o f heat shock proteins (Karpuj et al, 2002) or increasing the levels o f anti-oxidants (Lesort et al, 2003; Fox et al, 2004) . Four previous studies have demonstrated the beneficial effect o f either cystamine treatment or decreasing levels o f T G in N-terminal fragment mouse models o f H D (Dedeoglu et al, 2002; Karpuj et al, 2002; Mastroberardino et al, 2002; Bailey and Johnson, 2004). However, in these experiments it was not possible to assess the effect o f cystamine treatment on the striatal neuronal loss which characterizes the neuropathology in human H D . Accordingly, we treated Y A C 128 mice with cystamine to determine the effect o f cystamine on striatal neuropathology, including neuronal loss, in these mice.  5.2.2 Delivery of therapeutic agent: cystamine In order to demonstrate delivery o f cystamine and the effect o f cystamine administration on T G activity, we treated Y A C 128 mice with 225 mg/kg cystamine in drinking water for a period o f two weeks based on the protocol used previously in R6/2 mice (Dedeoglu et al, 2002). Untreated Y A C 128 mice were given normal drinking water. Frozen brains from cystamine-treated and untreated mice were split into hemispheres for measurement  of  cystamine levels and T G activity. While cystamine was not detectable in brain, a small increase in cysteamine was apparent as well as a significant increase in cysteine (Pinto et al, 2005) . Nonetheless, cystamine treatment reduced T G activity in the forebrain o f Y A C 128  98  mice. Three month old Y A C 128 mice treated with cystamine for 2 weeks show a 16% reduction in T G activity compared to Y A C 1 2 8 mice given normal drinking water (untreated Y A C 1 2 8 : 405 ± 1 1 pmol/mg/hour; cystamine-treated Y A C 1 2 8 : 348 ± 18 pmol/mg/hour; p = 0.02). This indicates that oral delivery o f cystamine in drinking water is effective at decreasing T G activity in the brain o f Y A C 128 mice.  5.2.3 Effect of cystamine on neuropathology In order to gauge the potential benefit o f cystamine treatment in H D , we examined the impact o f cystamine treatment on striatal neuropathology  in Y A C 128 mice. Based on  the  significance o f striatal volume loss to H D and the robustness, o f this finding in Y A C 128 mice, we chose striatal volume as the primary outcome measure. W e also examine secondary endpoints o f striatal neuronal numbers, striatal neuronal cross-sectional area, striatal D A R P P 32 expression, rotarod performance and open field activity. This study was powered to have an 80%o chance o f detecting a 33%> rescue in striatal volume (Slow et al., 2003) with a lesser ability to detect significant rescue in secondary outcome measures. Treatment was begun in symptomatic Y A C 128 mice at 7 months o f age to model potential therapeutic trials in H D patients. A t this age, Y A C 128 mice show a deficit in motor coordination on the rotarod (section 3.2) and down-regulation o f striatal D A R P P - 3 2 expression (unpublished).  Examination o f striatal volume by A N O V A revealed significant effects o f treatment, genotype and the interaction between treatment and genotype (treatment: F i 8) = 7.9, p = (  0.009; genotype:  F  ( U 2  8)  = 14.5, p < 0.001; treatment X genotype:  F  ( U 2  8)  j2  = 5.0, p = 0.03). Post-  hoc comparison revealed that cystamine-treated Y A C 128 mice showed a significant 9.5% increase in striatal volume compared to untreated Y A C 128 mice (Fig. 5.3 panel A ; untreated Y A C 1 2 8 : 10.8 ± 0.2 m m , cystamine-treated YAC1.28: 11.8 ± 0.3 m m , p = 0.01). Untreated 3  3  Y A C 1 2 8 mice showed a 10.4% decrease in striatal volume from untreated W T mice (Fig. 5.4 panel A ; untreated W T : 12.1 ± 0.1 mm , p = 0.002) and thus the improvement with cystamine treatment represents an 82% rescue o f our primary outcome measure, striatal volume loss. Cystamine treatment did not affect striatal volume in W T mice (Fig. 5.3 panel A ; cystamine treated W T : 12.2 ± 0.2 m m ; p = 0.96). 3  99  B ? £  1.30E+10  £  f-.1.20E+10  E: 1.90E+06 CO  E f 1.10E+10 > TS 1.00E+10 £  2.00E+06  I 1.80E+06 cu E  1.70E+06  s  9.00E+09  I w  YAC128  1.60E+06  WT  YAC128  D 110%  100% E Si  90%  k  70%  80%  60%  Q:  o  WT  WT  YAC128  • •  YAC128  Untreated Cystamine  F i g u r e 5.3 C y s t a m i n e t r e a t m e n t a m e l i o r a t e s s t r i a t a l n e u r o p a t h o l o g y i n Y A C 1 2 8 m i c e . Y A C 1 2 8 m i c e w e r e t r e a t e d w i t h c y s t a m i n e b e g i n n i n g at 7 m o n t h s o f a g e a n d n e u r o p a t h o l o g y w a s a s s e s s e d at 12 m o n t h s o f a g e . A . A s s e s s m e n t o f s t r i a t a l v o l u m e r e v e a l e d s t r i a t a l v o l u m e l o s s i n Y A C 128 m i c e c o m p a r e d t o W T m i c e w h i c h w a s s i g n i f i c a n t l y d e c r e a s e d b y t r e a t m e n t w i t h c y s t a m i n e ( t r e a t m e n t : F i g ) = 7.9, p = 0 . 0 0 9 ; g e n o t y p e : F i . (  (  >2  2 8 )  = 14.5,  p < 0 . 0 0 1 ; u n t r e a t e d W T : 12.1 ± 0.1 m m , u n t r e a t e d Y A C 1 2 8 : 10.8 ± 0.2 m m , c y s t a m i n e t r e a t e d W T : 12.2 ± 3  0.2 m m , c y s t a m i n e - t r e a t e d Y A C 1 2 8 : 3  3  11.8 ± 0.3 m m ) . B . C y s t a m i n e t r e a t m e n t p r e v e n t e d n e u r o n a l l o s s i n 3  Y A C 1 2 8 m i c e a n d h a d n o effect i n W T m i c e (treatment: F  ( 1 2  g ) = 6.4, p = 0 . 0 2 ; g e n o t y p e : F i g> = 4 . 0 , p = 0 . 0 5 ; (  2  u n t r e a t e d W T : 1.90 ± 0 . 0 5 m i l l i o n n e u r o n s , u n t r e a t e d Y A C 1 2 8 : 1.73 ± 0 . 0 4 m i l l i o n n e u r o n s , c y s t a m i n e - t r e a t e d WT:  1.91 ± 0 . 0 4 m i l l i o n n e u r o n s , p = 1.0, c y s t a m i n e - t r e a t e d Y A C 1 2 8 :  1.93 ± 0 . 0 4 m i l l i o n n e u r o n s ) . C .  S i m i l a r l y , s t r i a t a l n e u r o n a l a t r o p h y i n Y A C 128 m i c e w a s c o m p l e t e l y p r e v e n t e d b y c y s t a m i n e a n d u n a f f e c t e d i n W T m i c e (treatment: F . ( ]  2 8 )  = 11.0, p = 0 . 0 0 3 ; g e n o t y p e : F  = 6.9, p = 0 . 0 1 4 ; u n t r e a t e d W T : 9 7 . 8 ± 1.5 u m , 2  ( 1 2 8 )  u n t r e a t e d Y A C 1 2 8 : 7 8 . 8 ± 2.1 u m , c y s t a m i n e - t r e a t e d W T : 9 7 . 6 ± 3.6 u m , p = 1.0, c y s t a m i n e - t r e a t e d Y A C 1 2 8 : 2  2  99.9 ±3.7 u m ) . D . I n contrast, c y s t a m i n e treatment d i d not s i g n i f i c a n t l y i m p r o v e striatal D A R P P - 3 2 e x p r e s s i o n 2  i n Y A C 128 m i c e a n d h a d n o i m p a c t o n D A R P P - 3 2 e x p r e s s i o n i n W T m i c e ( t r e a t m e n t : F i . (  genotype: F  { 1 2 8 )  2 8 )  = 0.1, p = 0.7;  = 20.9, p < 0.001; untreated W T : 6 6 7 ± 2 0 intensity units, untreated Y A C 1 2 8 : 5 1 9 ± 9 intensity  u n i t s , c y s t a m i n e - t r e a t e d W T : 6 4 9 ± 2 6 i n t e n s i t y u n i t s ; c y s t a m i n e - t r e a t e d Y A C 1 2 8 : 5 2 4 ± 12 i n t e n s i t y u n i t s ) . N = 8 W T , 7 W T - C y s t a m i n e , 8 Y A C 1 2 8 , 9 Y A C 1 2 8 - C y s t a m i n e . E r r o r bars i n d i c a t e S E M . * p < 0 . 0 5 , ** p < 0 . 0 1 , * * * p < 0.001.  100  Similarly, striatal neuronal numbers were significantly affected by treatment and genotype (Fig. 5.3 panel B ; treatment: F  (]>2  8) = 6.4, p = 0.02; genotype: F i 8 ) = 4.0, p = 0.05; treatment (  ;2  X genotype: F(i 8) = 3.6, p = 0.07). A s with striatal volume, cystamine treatment resulted in a >2  significant 11.4% increase in striatal neuronal numbers in cystamine-treated Y A C 128 mice compared to untreated Y A C 1 2 8 mice (Fig. 5.3 panel B ; untreated Y A C 1 2 8 : 1.73 ± 0.04 million neurons, cystamine-treated Y A C 1 2 8 : 1.93 ± 0.04 million neurons, p = 0.015). This improvement represents a complete amelioration o f the neuronal loss present in Y A C 128 mice compared to W T mice (Fig. 5.3 panel B ; untreated W T : 1.90 ± 0.05 million neurons, p = 0.046). There was no difference in neuronal numbers between cystamine-treated and untreated W T mice (Fig. 5.3 panel B ; cystamine-treated W T : 1.91 ± 0.04 million neurons, p = 1.0).  Striatal neuronal cross-sectional area was measured using a fluorophore-conjugated N e u N antibody to precisely define neuronal cell bodies. Using this approach we demonstrate a highly significant 19%> decrease in neuronal cross-sectional area in Y A C 128 mice compared to W T mice (Fig. 5.3 panel C ; untreated W T : 97.8 ± 1.5 p m , untreated Y A C 128: 78.8 ± 2.1 2  pm , 2  p = 0.001). Treatment o f Y A C 128 mice with cystamine resulted in a complete  prevention o f the striatal neuronal atrophy (Fig. 5.3 panel C ; cystamine-treated Y A C 1 2 8 : 99.9 ± 3.7 pm , p < 0.001). Overall, there were significant effects o f treatment, genotype and the interaction between treatment and genotype (treatment: F i , ) = 11.0, p = 0.003; (  genotype:  F i (  ; 2 8 )  = 6.9, p = 0.014, treatment X genotype: F i , (  2 8 )  28  = 7.2, p = 0.012). Cystamine  treatment did not affect neuronal size in W T mice (Fig. 5.3 panel C ; cystamine-treated W T : 97.6 ± 3.6 p m , p = 1.0). 2  Next, we measured striatal D A R P P - 3 2 expression by the intensity o f D A R P P - 3 2 staining within the striatum in fluorescently labeled coronal sections. In contrast to the other striatal phenotypes, D A R P P - 3 2 expression showed no effect o f treatment despite a clear effect o f genotype (treatment: F , ) = 0.1, p = 0.7; genotype: F , (l  genotype: F i (  ; 2 8 )  28  ( l  2 8 )  = 20.9, p < 0.001; treatment X  = 0.0, p = 0.8). Y A C 128 mice showed a significant 22% decrease in striatal  D A R P P - 3 2 expression compared to W T mice (Fig. 5.3 panel D ; untreated W T : 667 ± 20 intensity units, untreated Y A C 1 2 8 : 519 ± 9 intensity units, p = 0.001). However, there is no  101  impact o f cystamine treatment on D A R P P - 3 2 expression in Y A C 128 or W T mice (Fig. 5.3 panel D ; Y A C 1 2 8 : p - 1.0; W T : p = 1.0).  5.2.4 Effect of cystamine on motor impairment In order to assess the effect o f cystamine treatment on motor dysfunction, we followed the performance o f cystamine-treated Y A C 128 mice on the rotarod. M i c e were trained on the apparatus at 2 months o f age and tested bimonthly until 12 months o f age. Cystamine treatment began at 7 months in symptomatic Y A C 128 mice. In the rotarod test, there was a significant effect o f age and genotype but no effect o f treatment or the interaction o f treatment and genotype (age:  F( ,i60) 5  = 44.3, p < 0.001; treatment:  genotype: F , ) = 25.6, p < 0.001, treatment X genotype: (  j32  F i 6) (  j 2  F(i  ) 3 2  )  = 0.0, p = 1.0;  = 0.2, p = 0.7). Y A C 128  mice demonstrated a significant rotarod impairment compared to W T mice which began at 4 months and worsened with age (Fig. 5.4 panel A ; p = 0.003). Cystamine treatment did not ameliorate this deficit or alter rotarod performance in W T mice (Fig. 5.4 panel A ; Y A C 128: p = 1.0, W T : p = 1.0).  We also monitored body weight as a measure o f general health in the treated and untreated mice to determine i f there were any toxic effects o f cystamine. The cystamine treated mice were indistinguishable from untreated controls and appeared to be in good health for the duration o f the trial. Repeated measures A N O V A revealed no significant effect o f cystamine on the weight o f W T or Y A C 1 2 8 mice (treatment: F(i i) = 2.5, p = 0.12) thereby indicating a ;3  lack o f obvious toxicity o f the cystamine dose used in this experiment.  A g e d Y A C 128 mice show hypoactivity compared to W T mice beginning between 6 and 8 months o f age (section 3.2). A n examination o f open field activity at 12 months revealed no improvement in cystamine-treated Y A C 1 2 8 mice compared to untreated Y A C 1 2 8 mice (Fig. 5.4 panel B ; untreated Y A C 1 2 8 mice: 328 ± 11 beam breaks, cystamine-treated Y A C 1 2 8 : 295 ± 12 beam breaks, p = 0.7). Similarly, cystamine treatment did not affect open field activity in W T mice (Fig. 5.4 panel B ; untreated W T : 400 ± 38 beam breaks; cystaminetreated W T : 393 ± 25, p = 1.0).  102  A  350  n  T r e a t m e n t at 7 M o n t h s  T r e a t m e n t at 4 M o n t h s  300 to  250 -  «  200 -  §.  150 -  c tt)  100 50 06  8  Age(Months)  4 —CD—•—  — ± —  WT WT-Cystamine YAC128 YAC128-Cystamine  B < 350  D  450  «  400  <  350  6 Age(Months)  300 250 200  YAC128  WT  WT  •  Untreated  •  Cystamine  YAC128  Figure 5.4 Cystamine treatment does not impact motor dysfunction in YAC128 mice. Symptomatic treatment with cystamine was begun at 7 months of age, as indicated (black triangle with C). A. YAC 128 mice began showing a deficit on the rotarod at 4 months of age and this impairment was not prevented by treatment with cystamine (treatment: F I ) = 0.0, p = 1.0; genotype: F i . ) 25.6, p < 0.001; untreated WT: 226 ± 18 seconds, untreated YAC128: 131 ± 16 seconds, cystamine-treated WT: 217 ± 18 seconds, cystamine-treated YAC128: 138 ± 16 seconds). B. Treatment with cystamine did not prevent hypoactivity in YAC128 mice at 12 months (untreated WT: 400 ± 38 beam breaks, untreated YAC128 mice: 328 ± 11 beam breaks, cystaminetreated WT: 393 ± 25, cystamine-treated YAC128: 295 ± 12 beam breaks) N = 8 WT, 8 WT-Cystamine, 10 YAC128, 10 YAC128-Cystamine. C. To determine if treatment with cystamine at the time of motor onset could prevent the rotarod deficit or hypoactivity, YAC 128 mice were treated with cystamine beginning at 4 months of age. Even with early treatment with cystamine, there was no improvement in rotarod performance in the YAC 128 mice (genotype: F, 28.5, p < 0.001; treatment: F (1.44) = 2.2, p = 0.15; untreated WT: 200 ± 17 (1,44) seconds; untreated YAC128: 134 ± 12 seconds; cystamine-treated WT: 229 ± 14 seconds; cystamine-treated YAC128: 146 ± 13 seconds). D. Similarly, treatment with cystamine beginning at 4 months of age did not improve activity in YAC128 mice at 12 months of age (genotype: F 3 , = 4.5, p = 0.008; untreated WT: 380 ± 22 beams breaks, untreated YAC128 mice: 313 ± 15 beam breaks, cystamine-treated WT: 366 ± 11 beam breaks, cystamine-treated YAC 128 mice: 323 ± 13 beam breaks). N = 8 untreated WT, 15 untreated YAC 128 mice, 12 cystamine-treated WT, 13 cystamine-treated YAC 128 mice. Error bars indicate SEM. * p < 0.05. =  (  I32  (  32  (  (  4 4 )  103  In the symptomatic trial, we found that cystamine prevented the development o f striatal neuropathology but was unable to reverse already present motor dysfunction. To determine whether earlier cystamine treatment would have a greater effect on the development o f motor deficits, we treated a cohort o f Y A C 128 mice with cystamine beginning at 4 months o f age around the time when difficulties on the rotarod first become significant (section 3.2). A s in the symptomatic trial there was a significant effect o f genotype on rotarod performance (genotype: F i 4 ) = 28.5, p < 0.001), but no beneficial effect o f cystamine treatment on (  )4  rotarod performance in the Y A C 128 mice (Fig. 5.4 panel C ; p = 0.5). Cystamine treatment did not alter the performance o f W T mice on the rotarod (5.4 panel C ; p = 0.2). Similarly, there was no effect o f cystamine treatment on weight in either Y A C 128 or W T mice (treatment: F(| ) = 0.0, p = 0.98) suggesting that even long term treatment with 225 mg/kg ;44  cystamine is well tolerated. Finally, examination o f open  field  activity revealed that  cystamine was unable to prevent the age onset hypoactivity in Y A C 128 mice (Fig. 5.4 panel D ; genotype:  F(3 4 ) ;  4  = 4.5,  p = 0.008; untreated Y A C 1 2 8 mice: 313 ± 15 beam breaks,  cystamine-treated Y A C 128 mice: 323 ± 13 beam breaks, p = 0.9) and did not affect the activity o f W T mice (Fig. 5.4 panel D ; untreated W T : 380 ± 22 beams breaks, cystaminetreated W T : 366 ± 11 beam breaks; p = 0.9). Thus, earlier treatment with cystamine was still unable to prevent motor dysfunction in the Y A C 128 mouse model o f H D .  104  6.0 DISCUSSION Huntington disease is caused by the expression o f the htt protein containing an expanded polyglutamine tract. Onset o f H D occurs on average at 39 years o f age and progresses to death approximately 15 years later (calculated from Harper, 1996). Symptoms o f the disease include motor deficits, cognitive dysfunction and psychiatric disturbances. There is currently no treatment that can halt or prevent the progression o f this devastating disease. Accordingly, the major goal o f this thesis was to characterize a genetically accurate mouse model o f H D for use in preclinical therapeutic trials.  The ideal mouse model o f H D should reproduce the human disease as accurately as possible to increase the likelihood that findings regarding either the pathogenesis o f the disease or effective treatments for the disease w i l l be applicable to H D patients. Thus, the ideal mouse model should express full length mutant htt from its endogenous regulatory elements and should exhibit motor dysfunction, cognitive impairment and psychiatric  disturbances.  Importantly, the ideal mouse model o f H D should reproduce the selective neurodegeneration of H D . Furthermore, the behavioural and neuropathological abnormalities should be progressive.  In order to use the mice for therapeutic trials it is also necessary that the  phenotypes in the H D mice are robust and quantifiable such that the potential benefits o f different treatments can be assessed quantitatively with low numbers o f mice.  To create a genetically accurate model o f H D , the entire human H D gene with either 72 or 128 C A G repeats was incorporated into a Y A C and used to generate transgenic mice. The genomic fragment o f D N A containing the H D gene included 24 kb o f upstream D N A in order to include the regulatory elements o f the H D gene. This approach led to the appropriate tissue specific expression o f mutant htt (Hodgson et al, 1999). This is supported by the fact that htt expressed from a Y A C transgene could rescue mice homozygous for the targeted inactivation o f the mouse H D gene from embryonic lethality (Hodgson et al, 1996; Hodgson etal,  1999).  105  6.1 The  The Y A C 7 2 mouse model of Huntington disease Y A C 7 2 mouse model o f H D expresses mutant htt with 72 glutamines. The low  expressing line 2511 was found  to have abnormal electrophysiology at 6 months,  hyperactivity beginning at 7 months and nuclear localization o f mutant htt and dark cell degeneration at 12 months (Hodgson et al, 1999). In this thesis, a higher expressing line o f Y A C 7 2 mice, line 44, was characterized to determine i f increased expression o f wild-type htt results in a more robust phenotype and i f these mice, were suitable for therapeutic trials. Examination o f Y A C 7 2 mice with an extensive array o f behavioural tests that were focused on assessing motor function revealed only a subtle deficit in swimming ability. A t 10 months, these mice showed decreased striatal volume compared to W T mice but no change in brain weight, indicating the occurrence o f selective degeneration. The finding o f decreased striatal volume despite relatively normal motor function suggests the possibility that separate processes lead to neuronal dysfunction and neuropathology in these mice and possibly in H D . Overall, Y A C 7 2 mice exhibit H D - l i k e symptoms but these symptoms are limited and mild. Thus, while Y A C 7 2 mice may provide insight into the pathogenesis o f the disease they are not ideal for therapeutic trials because there are few outcome measures to assess the efficacy of treatment and large numbers o f mice would be required to detect any benefit as a result o f the mild differences from W T mice.  6.2  The Y A C 1 2 8 mouse model of Huntington disease  The Y A C 128 mouse model o f H D was generated to produce a mouse with a more severe phenotype and earlier onset. Y A C 128 mice express more mutant htt than Y A C 7 2 mice and have a larger C A G repeat. Since larger C A G repeat sizes are associated with an early onset in humans (Trottier et al, 1994; Brinkman et al, 1997) and increased expression o f mutant htt results in a more severe phenotype in mice (Mangiarini et al, 1996; Hodgson et al, 1999), Y A C 128 mice were predicted to have a more severe phenotype than Y A C 7 2 mice.  106  6.2.1  Motor dysfunction in the Y A C 1 2 8 mouse model of Huntington disease  In human H D , motor dysfunction is the most obvious symptom and often heralds the onset o f the disease. Early hyperkinetic movement known as chorea is normally superseded by increasing rigidity, dystonia and bradykinesia as the disease progresses. A similar biphasic pattern o f activity is observed in Y A C 128 mice with open field activity testing. In this test, Y A C 128 mice show hyperkinesia at 2 months o f age which subsides by 6 months o f age and is followed by hypoactivity beginning around 8 months o f age. The hypoactivity worsens with age to 12 months of age (oldest age tested). Y A C 128 mice also show a clear deficit in motor coordination and balance in the rotarod task. This deficit develops between 2 and 4 months o f age and progressively worsens with age. In contrast, increasing age does not affect the rotarod performance o f W T mice. Finally, Y A C 128 mice show a motor deficit at 8 months in linear swimming which is not present at 2 months o f age.  A s the striatum is involved in motor function, the observation o f motor deficits in Y A C 128 mice is in accordance with the observed striatal pathology. Motor deficits have previously been demonstrated in other mouse models o f H D . Rotarod deficits have been reported in models expressing an N-terminal fragment o f mutant htt (Carter et al, 1999; Schilling et al, 1999; Yamamoto et al, 2000; Laforet et al, 2001), knock-in mouse models o f H D ( L i n et al,  2001) and a full length transgenic model o f H D (Reddy et al,  1998). Similarly,  alterations in activity have been reported in N-terminal fragment models (Carter et al, 1999; Schilling et al, 1999; Yamamoto et al, 2000; Laforet et al, 2001), knock-in mouse models of H D ( L i n et al, 2001) and full length transgenic models o f H D including the Y A C 7 2 mouse model (Reddy et al, 1998; Hodgson et al.., 1999). Interestingly, mice expressing only a small N-terminal fragment  o f htt exhibit only hypoactivity (Mangiarini et al,  1996;  Schilling et al, 1999; Yamamoto et al, 2000) while mice expressing at least one third o f the full length htt protein exhibit hyperactivity followed by hypoactivity as in the Y A C 128 mice (Laforet et al, 2001; Menalled et al, 2002; Menalled et al, 2003; Reddy et al,  1998).  Changes in gait and clasping have also commonly been reported in H D mice (Mangiarini et al, 1996; Yamamoto et al, 2000; Schilling et al, 1999; Laforet et al, 2001; Menalled et al, 2003; Wheeler et al, 2000; L i n et al, 2001) but were not assessed in Y A C 1 2 8 mice. Overall,  107  it appears that motor dysfunction is a common symptom among H D mouse models. Since motor symptoms are present prior to neuropathology, they likely result from neuronal dysfunction rather than neuronal loss.  6.2.2 Cognitive deficits in the YAC128 mouse model of Huntington disease Presymptomatic H D patients show mild cognitive deficits in procedural learning, memory, planning and shifting strategy (Snowden et al, 2002; Lawrence et al, al,  1998B; Hahn-Barma et al,  1998A; Lawrence et  1998). These deficits worsen with the advancement o f the  disease and are also found in symptomatic H D patients (Lawrence et al, al,  1999; Bamford et  1995; H o et al, 2003). We tested for presymptomatic cognitive deficits in 2 month old  Y A C 128 mice and found a learning deficit on the rotarod test o f motor coordination (Hyde et al, 2001; McFadyen et al, 2003; L e Marec and Lalonde, 1997; Lalonde et al, 1995; Gerlai et al, 1996; Dubois et al, 2002). A t this age, Y A C 128 mice did not have a motor deficit as trained Y A C 128 mice perform as well as W T mice. Since Y A C 128 mice required more training to reach the same level o f performance, it is clear that they learn slower than W T mice. Two month old Y A C 128 mice also showed a deficit in strategy shifting whereby they took longer to reach the platform in the reversal phase o f the swimming T-maze test despite swimming as fast as W T mice. A t this time point, Y A C 128 mice showed normal cognition in the simple swimming test, the normal phase o f the swimming T-maze test and in tests o f habituation.  Thus, it appears that cognitive dysfunction occurs early in the development of the disease in Y A C 128 mice and is present well before neuropathology is detected. Similar findings have been reported in human H D and the R6/2 mouse model (Snowden et al, 2002; Lawrence et al, 1998A; Lawrence et al, 1998B; Hahn-Barma et al, 1998). Since H D is characterized by motor dysfunction, it is not surprising that cognitive tasks involving motor  function  deteriorate first especially when H D patients show deficits in several paradigms o f skill learning (Heindel et al,  1988; Heindel et al,  1989; Gabrieli et al,  1997; Schmidtke et  al,  2002; Knopmann and Nissen, 1991). Similarly, difficulty in changing strategy is one o f the earliest reported cognitive symptoms in H D (Lawrence et al, 1998A).  108  Symptomatic H D patients show widespread cognitive dysfunction (Backman et al, 1997). In our assessment o f symptomatic Y A C 128 mice, we examined cognitive abilities deficient in both presymptomatic and symptomatic H D patients, namely procedural learning, memory, and strategy shifting (Snowden et al, 2002; Lawrence et al, 1998A; Lawrence et al, 1998B; Hahn-Barma et al,  1998). The rotarod was used to assess motor learning. A t 7 months o f  age, Y A C 128 mice learn the rotarod task but are unable to reach the same level o f performance as W T mice. B y 12 months, the motor learning deficit originally detected at 2 months, progresses into a severe learning deficit that completely prevents Y A C 128 mice from learning the rotarod task. A t this age, previously untrained Y A C 128 mice cannot perform the rotarod task, while mice trained earlier are physically able to run on the rotarod. The fact that learning on the rotarod is thought to result from changes in motor strategy and not increased motor function (Buitrago et al, 2004) suggests that the reason that Y A C 128 mice trained at 7 months perform better than Y A C 128 mice trained at 12 months is because of differences in cognitive rather than physical ability.  In the simple swimming test, Y A C 128 mice take longer to reach the platform than W T mice beginning at 8 months. Only 5% o f this difference is accounted for by differences in swimming speed. The remainder results from cognitive deficits which predispose  the  Y A C 128 mice to choose an indirect path to the platform. Since these mice are unable to rely on visual cues for navigation (Taketo et al, 1991; Huerta et al, 1999), this is likely a deficit in procedural rather than spatial memory. Memory deficits have also been reported in human H D (Wilson et al  1987; Rohrer et al,  1999; Lawrence et al, 2000; B r o w n et al, 2001).  Validation for this test comes from the ability o f W T mice to learn to swim directly to the platform and remember this skill in subsequent trials.  Symptomatic Y A C 128 mice also show deficits in open field habituation - a simple test o f learning and memory (Gerlai et al,  1996; Bolivar et al, 2000; Cook et al, 2002). A t 8  months, Y A C 1 2 8 m i c e . are hypoactive compared to W T mice. While Y A C 1 2 8 mice demonstrate intrasession and intersession habituation, in both cases W T mice show a more rapid decline in activity. Accordingly, the difference in activity between W T and Y A C 128 mice diminishes with repeated trials. While we have attempted to control for the initial  109  difference in activity in our calculation o f habituation, it is possible that the initial hypoactivity in Y A C 128 mice contributes to their decreased habituation. However, the observation o f memory deficits in the simple swimming test and in habituation to acoustic startle supports a memory deficit as the cause o f decreased open field habituation in symptomatic Y A C 128 mice. Validation for the use o f open field habituation for memory testing comes from experiments showing more rapid habituation in rodents treated with drugs prescribed for memory disorders (Platel and Porsolt, 1982).  We also demonstrate cognitive deficits in a swimming T-maze test at 8.5 months o f age where trained Y A C 128 mice take longer to reach the platform than W T mice. A motor deficit in swimming speed is present at this age but does not account for the difference observed. While W T mice all swim directly to the platform on the third day, Y A C 128 mice show multiple errant trials where they swim down the wrong arm possibly as a result o f a memory deficit. The use o f this test is validated by the ability o f W T mice to improve their latency to reach the platform and choose the correct arm in every trial. Similarly, cognitive function has been previously assessed in a water plus-maze (Dobkin et al, 2000).  Symptomatic Y A C 1 2 8 mice show a dramatic cognitive deficit in the reversal phase o f the swimming T-maze test. When the location o f the platform is changed, W T mice all rapidly adapt to the new task, reaching the platform without retracing their path. In contrast, most Y A C 128 mice returned to the start o f the T-maze after discovering the platform was not present in the right arm. This difference in response strategy provides a clear manifestation o f cognitive dysfunction in Y A C 128 mice. Remarkably, not a single W T mouse had difficultly in changing strategy, while difficulty was the norm in Y A C 128 mice. Interestingly, difficulties in changing strategy are present in H D patients (Lawrence et al, 1999; H o et al, 2003).  A t 12 months, Y A C 128 mice have decreased PPI compared to W T . This represents a decreased ability to inhibit a motor response. A s motor inhibition is thought to be mediated by the striatum, this finding is in line with the striatal pathology reported in Y A C 128 mice (Mink, 1996). Decreased PPI has been found in human H D patients and in the R6/2 mouse  110  model (Swerdlow et al,  1995; Carter et al,  1999). We also demonstrate decreased  habituation to acoustic startle in 12 month old Y A C 128 mice providing additional evidence for impaired memory. Since 9 month old Y A C 128 mice have an equivalent motor deficit on the rotarod as 12 month old Y A C 128 mice and do not show deficits in PPI or habituation to acoustic startle, the deficits in 12 month animals are likely cognitive and not related to the motor dysfunction at this age.  Cognitive dysfunction is also present in other animal models o f H D . R6/2 mice have been reported to have deficits in learning and reversal learning in a two choice swim test, alternation learning in a T-maze test and in spatial learning in the Morris water maze (Lione et al,  1999; Murphy et al, 2000). Cognitive impairments in R6/2 mice begin to develop  prior to the onset o f motor symptoms at 5.5 weeks o f age (Carter et al,  1999). Similar to  Y A C 128 mice and human H D , R6/2 mice show deficient sensorimotor gating in an acoustic PPI test (Carter et al, 1999). However, R6/2 mice show normal open field habituation while the less severely affected R6/1 line have been shown to display less open field habituation than W T mice (Bolivar et al,  2003; Bolivar et al,  2004). While there have been no  published reports examining cognitive function in other mouse models o f H D , a newly generated rat transgenic model o f H D that expresses approximately one-third o f the mutant htt protein has been shown to have cognitive deficits in the radial maze test for spatial memory (von Horsten et al,  2003). Combined, these results suggest that cognitive  dysfunction is an important aspect o f H D and is likely caused by a gain o f toxic function in mutant htt.  Our findings in Y A C 128 mice extend the results obtained in N-terminal fragment models o f H D to demonstrate cognitive dysfunction in a mouse model expressing full length mutant htt. The demonstration o f cognitive impairment in an accurate genetic model o f H D provides us with the opportunity to examine the relationship between cognitive impairment, motor dysfunction and the underlying biochemical changes and neuropathology throughout the entire disease progression. These findings are also important for preclinical therapeutic trials as it is now possible to specifically assess the impact o f potential therapies for H D on the cognitive aspects o f the disease.  Ill  Animal research has demonstrated two distinct forms o f learning which are mediated by different systems within the brain. Cognitive learning involves the acquisition o f knowledge about the environment, such as the generation o f a spatial map, which is then used to influence subsequent actions. Stimulus-response learning involves procedural learning in response to stimulus. Lesions involving the lateral striatum eliminate stimulus-response learning while those involving the medial striatum or hippocampus affect cognitive learning (De Coutea and Kesner, 2000; Devan and White, 1999; Packard and M c G a u g h , 1996). Studies involving striatal damage caused by the mitochondrial toxin 3NP have shown perseveration in a lever pressing task and a T-maze alteration task as well as a spatial learning deficit in lesioned animals (El Massioui et al, 2001; Shear et al, 1998). Thus, the demonstration o f procedural learning deficits and perseveration in Y A C 128 mice is in accordance with the observed striatal pathology in these mice since similar deficits have been achieved in animals with striatal lesions.  6.2.3  Decreased survival in Y A C 1 2 8 mouse model of Huntington disease  Patients with H D show a decreased survival with an average age o f death o f approximately 54 years (calculated from Harper, 1996). This decreased lifespan has been reproduced in animal models expressing an N-terminal fragment o f mutant htt with an expanded C A G tract (Mangiarini et al,  1996; Schilling et al,  1999; V o n Horsten et al, 2003). The decreased  survival we report in the Y A C 128 mice is the first demonstration o f impaired survival in a full length mouse model o f H D , as neither the knock-in mouse models o f H D nor a transgenic mouse model expressing a htt c D N A with 89 glutamines were reported to have a shortened lifespan (Reddy et al, 1998; L i n et al, 2001; Levine et al, 1999; Menalled et al, 2002; Menalled et al, 2003; White et al, 1997; Wheeler et al, 1999; Wheeler et al, 2000; Shelbourne et al, 1999). A s with the other animal models, the cause o f death in our mice is unknown (Mangiarini et al,  1996; Schilling et al,  1999; V o n Horsten et al,  2003).  However, unlike these models, we did not observe any significant weight loss prior to death.  The survival deficit was observed only in male Y A C 128 mice and not in females. This finding was not reported in animal models expressing an N-terminal fragment o f mutant htt  112  (Mangiarini et al, 1996; Schilling et al, 1999; V o n Horsten et al, 2003). Furthermore, there have been no reports documenting differences in lifespan, incidence or phenotypic severity between the sexes in human H D . In contrast, differences between the sexes have been reported in other neurodegenerative diseases and their corresponding animal models (Italian Longitudinal Study on A g e Working Group, 2000; V a n Den Eeden et al, 2003; Gambassi et al, 1999; Ueki et al, 2001; Kenchappa et al, 2004; Haverkamp et al, 1995; Veldink et al, 2003) .  Estradiols have been shown to be protective in the brain partially through the induction o f a caspase inhibiting factor (Kolsh and Rao, 2002; Dubai et al,  1999; Zhang et al, 2004).  Based on the known involvement o f caspases in the pathogenesis o f H D , increased caspase inhibitor induction in female mice resulting from higher estrogen levels is in accordance with our finding o f a less severe survival phenotype in female Y A C 128 mice (Wellington et al, 2000; Wellington et al, 2002). It is also possible that decreased testosterone levels contribute to the survival deficit in male Y A C 128 mice. Testosterone has been shown to protect neurons from death caused by oxidative stress (Ahlbom et al, 1999) and excitotoxicity (Ramsden et al, 2003), both o f which are proposed to be involved in H D pathogenesis. Since testosterone has recently been shown to be decreased in male H D patients (Markianos et al, 2005), it is possible that a similar reduction in testosterone in male Y A C 128 mice results in a decrease in testosterone mediated neuroprotection in the brain and predisposes these mice to early death.  The difference in survival between the sexes may also stem from differential susceptibility to cell death in male and female cells. Recent in vitro studies have shown that cells cultured from male rats are more sensitive to excitotoxic cell death than cells from female rats and that the male cells are less able to maintain levels o f the anti-oxidant glutathione (Du et al, 2004) . This may be particularly relevant to this study as the Y A C 128 mice are maintained on the F V B / N strain background which are sensitive to excitotoxicity (Shauwecker and Steward, 1999). Thus, it is possible that increased susceptibility to excitotoxicity and oxidative damage in male Y A C 128 mice contributes to the early death observed in these mice especially since both excitotoxicity and oxidative stress have been implicated in the pathogenesis o f H D (Petersen etal,  1999).  113  Under normal conditions, both male and female F V B / N mice show a 60% survival rate at 24 months (Mahler et al, 1996). Similarly, our study shows equivalent survival between male and female W T mice at 12 months with a 95% survival rate in males and a 90% survival rate in females (p = 0.47). However, when the mutant htt transgene is introduced a marked difference in survival became apparent where 93% o f female Y A C 128 mice survived to 12 months but only 74% o f male Y A C 1 2 8 mice lived to 12 months (p = 0.01). W e have previously observed that male F V B / N mice are less able to survive stressful conditions than females. When placed on a harsh caloric restriction diet from 2 to 12 months o f age, 4 o f 4 calorie-restricted, male mice died while all o f the 5 calorie-restricted female mice and all o f the males and females in the normally fed group survived to 12 months (unpublished). Thus, it appears that male F V B / N mice are more susceptible to premature death under stressful conditions which may explain the differences in Y A C 128 survival that we observe.  6.2.4 Selective degeneration in YAC128 mouse model of Huntington disease In this thesis we demonstrate regionally selective degeneration and neuronal loss in the Y A C 128 mouse model o f H D . A t 12 months o f age, examination o f regional volumes within the brain o f these mice revealed volume losses o f 10.4%, 10.8% and 8.6% in the striatum, globus pailidus and cortex respectively with no change in hippocampal or cerebellar volume. A s brain weight was only decreased 4% in Y A C 128 mice compared to W T mice, it is apparent that degeneration in select regions is responsible for the overall brain atrophy. The pattern o f selective degeneration in Y A C 128 mice is similar to human H D where volume losses o f approximately 50%, 40% and 25% are reported in the striatum (caudate and putamen), globus pailidus and cortex respectively with little or no volume changes in the hippocampus and cerebellum (Fennema-Notestine et al, 2004; Rosas et al, 2003; Halliday et al,  1998; Vonsattel and Difiglia, 1998). In the human disease, brain weight in advanced  cases can be reduced by up to 30% (Sharp and Ross, 1996). This is less than the volume changes observed in the most affected regions o f the brain again indicating that the global brain atrophy is caused by degeneration o f selective regions. We also show here that cell loss of 9.1% and 8.3% is present in the striatum and cortex, respectively, o f Y A C 128 mice while  114  neuronal numbers within the hippocampus are spared. Similarly, in human H D , cell loss o f 89% and 42% has been reported in the striatum and the motor cortex o f end stage patients (MacDonald and Halliday, 2002).  While the pattern o f selective degeneration is similar between Y A C 128 mice and human H D , the magnitude o f change is much greater in human patients. This may stem from marked differences in age o f onset (—39 years in humans versus 2-3 months in Y A C 128 mice) and or the duration o f disease progression (-15 years in humans versus 9 months in Y A C 128 mice at the 12 month time point). Perhaps for this reason, or because o f differences between species, animal models o f H D have repeatedly demonstrated that mutant htt with C A G repeat sizes comparable to those seen in adult onset H D result in mild phenotypes, i f any, in mice (Hodgson etal, 1999; White etal., 1997; Shelbourne etal., 1999; Levine et al.,. 1999). In the Y A C 128 mouse model we have increased the expression o f mutant htt and used a C A G length that would produce a severe case of juvenile onset H D in humans in order to observe a clear phenotype within a limited timeframe. It is possible that i f mice were permitted to live to the end stages o f the disease then the magnitude o f atrophy and cell loss would be more similar to that observed in human H D . Alternately, i f disease pathogenesis occurs in linear time, rather than in time relative to organismal lifespan, then it may be the case that mice simply don't live long enough to manifest the severe symptoms observed in human H D . Another possibility is that i f transport deficits contribute significantly to H D , then perhaps the increased axonal lengths in humans amplify the phenotypic consequences o f impaired transport.  In summarizing the neuropathological phenotypes observed in all o f the mouse models o f H D , there appear to be a number o f common phenotypes (Table 6.1). The most frequently reported observation is the presence o f aggregates which in most cases is preceded by the nuclear localization o f mutant htt. Interestingly, the knock-in mouse models show very mild striatal neuropathology aside from the nuclear localization o f mutant htt. However, this may represent an early step i n the pathogenesis o f H D which precedes other abnormalities in brain. Decreases in overall brain weight, brain volume and neuronal cross-sectional area have also been reported in multiple models, though in the case o f N-terminal models o f the  115  Table 6.1 Neuropathological phenotypes among mouse models of HD. Nuclear localization of mutant htt is the earliest and most common phenotype. Mouse Model  R6/2  Type  N-terminal fragment  N171-82Q  N-terminal fragment  HD100  N-terminal fragment  Hdh4/Q80  Knock-in  CAG94  Knock-in  HdhQ"  Knock-in  1  Neuropathology  Reference  Brain volume decreased 19% (non-specific), no neuronal loss, no astrocytosis  Mangiarini et al, 1996  Aggregates in striatum, cortex, cerebellum, spinal cord  Davies etal., 1997  ~ 20% decrease in brain weight, -40% decrease in neuronal cross-sectional area  Ferrante et al, 2000, 2004  Dark cell degeneration  Turmaine etal., 1999  Decreased DARPP-32 expression  van Dellen et ai, 2000  Widespread aggregates (ctx, hip, cer, str), no neuronal loss, no astrocytosis  Schilling etal, 1999  -16% decrease in brain weight, -37% decrease in neuronal cross-sectional area  Andreassen et ai, 2001  Dystrophic neurites in striatum and cortex only, nuclear localization of mutant htt in affected regions, aggregates, -53% decrease in striatal neuronal crosssectional area, no neuronal loss  Laforet etal, 2001  10-15% decrease in brain weight, no neuronal loss  Shelbourne et al, 1999  Nuclear localization of mutant htt, aggregates  Li et al, 2000  -14% decrease in striatal volume, no neuronal loss, nuclear localization of mutant htt, aggregates  Menalled et al, 2002  Nuclear localization of mutant htt, aggregates  Wheeler et al, 2000  Dark cell degeneration in some mice  Wheeler et al, 2002  CAG140  Knock-in  Nuclear localization of mutant htt, aggregates  Menalled et al, 2003  CHL2(150 CAG)  Knock-in  Reactive astrocytosis, no neuronal loss, aggregates  Lin et al, 2001  HD89  Full length transgenic  Neuronal loss in the striatum (-20%), cortex, hippocampus, thalamus, not cerebellum, reactive gliosis, TUNEL positive neurons, aggregates  Reddy et al, 1998  YAC72  Full length transgenic  Nuclear localization of mutant htt, aggregates, dark cell degeneration  Hodgson etal, 1999  Full length transgenic  -4% decrease in brain weight, -10% decrease in striatal volume, -9% decrease in striatal neuronal numbers, -14% decrease in striatal neuronal cross-sectional area, -22% decrease in striatal DARPP-32 expression, nuclear localization of mutant htt, aggregates  Section 3.2 Slow et al, 2003  YAC128  disease, these changes are generally not selective. In contrast, Y A C 128 mice show selective atrophy and degeneration in a pattern similar to H D . This may result from the presence o f the entire H D gene promoter and or the presence o f the complete H D gene. O f all the H D mouse models only the Y A C 128 mouse model and a full length c D N A transgenic model (Reddy et al, 1998) demonstrated quantifiable neuronal cell loss. While it is uncertain why these two models demonstrate cell loss and others do not, it may result from the expression o f full length htt or stem from the fact that both models are maintained on the F V B / N background strain, which is particularly susceptible to excitotoxicity (Shauwecker and Steward, 1999). It is also possible that rapid disease progression in the N-terminal fragment models result in the mice dying before neuronal death occurs and, conversely, in the knock-in models that the progression is mild enough that the mice do not live long enough to exhibit neuronal loss. Despite the absence o f detectable neuronal loss, neurons undergoing dark cell degeneration were found in some models (Turmaine et al,  1999; Hodgson et al,  1999; Wheeler et  al,  2002) but it is uncertain whether these cells are eventually lost.  6.2.5 Mechanism of selective degeneration in the YAC128 mouse model of Huntington disease To investigate the mechanism underlying the selective degeneration in Y A C 128 mice, we examined the regional expression o f mutant htt within the brain. Mutant htt expression in Y A C 128 mice was found to be highest in cerebellum, moderate in the hippocampus and cortex and lowest in the striatum o f the regions we examined. Thus, it seems that the regions with highest htt expression are those with the greatest neuronal density (Petersen et al, 1999) which follows from the fact that htt is more highly expressed in neurons than glia (Strong et al, 1993). Similarly, in studies o f mutant htt R N A or protein levels in humans, it has been found that htt expression is higher in the cerebellum, hippocampus and cortex than in the striatum (Sharp et al,  1995; Strong et al,  Landwehrmeyer et al,  1995). Thus, the pattern o f mutant htt expression is similar in the  Y A C 128 mouse  1993; Trottier et al,  model and human H D and in neither  1995; L i et al,  case explains the  1993;  selective  degeneration that occurs.  117  In contrast to mutant htt expression, nuclear localization o f mutant htt was greatest in the striatum - the region most affected in H D . A t 3 months o f age, mutant htt was already present in the nucleus o f striatal neurons. This is the age when motor and cognitive deficits are first observed in Y A C 128 mice. While some nuclear localization o f mutant htt was present in layer I V o f the cortex, in the C A 3 region o f the hippocampus and the cellular layer o f the cerebellum at 3 months, the amount o f mutant htt in the nucleus in striatal neurons was much greater than in any other region o f the brain. In addition to the regional specificity o f the E M 4 8 staining, we also observed selective nuclear localization o f mutant htt within the striatum. The lateral striatum was found to have visually more nuclear localization o f mutant htt than the medial striatum. Combined with our previous observation o f increased neurodegeneration in the lateral striatum compared to the medial striatum in Y A C models o f H D (Hodgson et al, 1999), this supports the possibility that nuclear localization o f mutant htt is at least partially responsible for the selective degeneration  in H D . Early nuclear  localization o f mutant htt may result in neuronal dysfunction that manifests  in the  behavioural abnormalities observed in Y A C 128 mice at this age. This may be mediated by changes in gene expression (Kegel et al, 2002; Steffan et al, 2000; L i et al, 1999).  A t 12 months, all regions o f the brain show increased nuclear localization o f mutant htt compared to the 3 month time point, with the striatum still showing the greatest extent o f nuclear localization. In the cortex, all o f the cell dense layers show nuclear localization o f htt (II, III, I V and V I ) which is greatest in layers II and III. In the hippocampus, the dentate gyrus shows the most intense nuclear E M 4 8 staining followed by the C A 3 region and the C A 1 region. Overall, the nuclear localization o f htt is greatest in the striatum, which may explain why this region is most affected in H D . This supports the possibility that regionally selective nuclear localization o f mutant htt may contribute to the selective atrophy and cell loss in H D .  Wild-type htt is primarily a cytoplasmic protein which is also detected in the nucleus (Sapp et al, 1997; Dorsman et al, 1999). Htt contains a nuclear export signal ( N E S ) in its carboxyterminus which can be separated from the amino-terminus o f the protein when htt is cleaved ( X i a et al, 2003). Thus, it is thought that wild-type htt functions in the cytoplasm and  118  nucleus and can shuttle between them. Mutant htt also shows cytoplasmic and nuclear localization but shows increased nuclear localization compared to wild-type htt, especially N terminal fragments o f the mutant protein ( L i et al, 1999; Sapp et al, 1997). This may result from decreased interaction o f mutant htt with the nuclear pore translocated promoter region (Tpr) which is thought to transport htt out o f the nucleus (Cornett et al, 2005).  M a n y in vitro experiments have demonstrated that nuclear localization o f mutant htt is important for its toxicity (Peters et al, 1999; Saudou et al, 1998; L i et al, 1999). Further, the demonstration that a mutant htt fragment linked to a nuclear localization signal ( N L S ) causes the same phenotype in mice as the fragment alone suggests that the mutant htt fragment without the N L S is primarily localized to the nucleus and that it already exerts its toxic effects in the nucleus (Schilling et al, 2004). This is supported by the acceleration o f behavioural onset in mice expressing H P R T with 150 C A G repeats when an N L S signal is added and a later onset when an N E S is added (Jackson et al, 2003). In addition, preventing the nuclear localization o f the mutant androgen receptor protein in spinal and bulbar muscular atrophy ( S B M A ) resulted in a complete amelioration of the disease symptoms suggesting that nuclear localization is essential for the pathogenesis o f this disorder (Katsuno et al, 2002; Katsuno et al, 2003).  The adverse effect o f mutant htt in the nucleus on cell function and survival may result from alterations in gene expression, as mutant htt has been found to interact with a number o f transcription factors within the nucleus (Kegel et al, 2002; Steffan et al, 2000; L i et  al,  1999). It has also been demonstrated that the presence o f mutant htt in the nucleus increases expression o f caspase 1 and induces activation o f caspase 3 and release o f cytochrome c thereby leading towards apoptosis ( L i et al, 2000). Irrespective o f the mechanism o f mutant htt toxicity within the nucleus, these findings provide a possible mechanism by which increased nuclear localization o f mutant htt leads to increased toxicity as we observed in the striatum o f Y A C 128 mice.  Early nuclear localization o f htt in the striatum has been previously reported in other mouse models o f H D (Wheeler et al, 2000; Menalled et al, 2002; Menalled et al, 2003; Laforet et  119  al, 2001; L i n et al, 2001; L i et al, 2000; Tallaksen-Greene et al, 2005). In most cases regional differences in nuclear localization o f htt were not examined. In a knock-in model o f H D with 140 C A G repeats, nuclear localization o f htt appeared greatest in the striatum, less in the cortex and hippocampus and least in the cerebellum (Menalled et al, 2004). However, in these mice it was not possible to examine the relationship between nuclear localization o f mutant htt and cerebral damage because o f the mild neuropathology in these mice.  Staining with E M 4 8 in mouse models expressing N-terminal fragments  o f mutant htt  revealed the presence o f nuclear aggregates throughout the brain (Davies et al, Schilling et al,  1997;  1999). In these mice, aggregation and volume loss do not appear to be  selective (Mangiarini et al, 1996; Schilling et al, 1999). Pilot studies in our lab indicate that nuclear localization o f mutant htt and volume loss in R6/1 mice are unselective (Van Raamsdonk, unpublished). In contrast to the Y A C 1 2 8 mouse model, nuclear localization o f mutant htt occurs to a similar extent in all regions o f the brain and this may explain the relatively unselective pathology. The lack o f selectivity in R6/1 mice may result from the absence o f important regulatory elements controlling the expression o f mutant htt and or the absence o f the normal protein context for the polyglutamine tract. Overall, the nuclear localization o f mutant htt appears to be a common step in the progression o f H D and our findings here suggest the possibility that selective nuclear localization o f mutant htt may contribute to the selective degeneration in H D .  6.2.6  Testicular degeneration in the Y A C 1 2 8 mouse model of Huntington disease  Our laboratory has previously demonstrated that mutant htt with 72 glutamines causes testicular degeneration when wild-type htt levels are reduced by half (Leavitt et al, 2001). Further decreases in wild-type htt lead to more severe testicular pathology. Conversely, normal expression levels o f wild-type htt eliminated the testicular phenotype. Since testicular degeneration only occurred in the presence o f mutant htt when overall htt expression was below normal, it was not possible in that experiment to attribute the testicular pathology to mutant htt toxicity or loss o f wild-type htt function alone.  120  Here, we show that testicular degeneration can be caused solely by the toxicity o f mutant htt. We show testicular atrophy and the disruption o f seminiferous tubule structure and function in Y A C 128 mice. Since both increased mutant htt expression and increased C A G expansion lead to a more severe phenotype (Reddy et al, 1998), we expected the toxicity o f mutant htt in the Y A C 128 mice to be significantly worse than in Y A C 7 2 mice.  The testis shows the highest htt expression outside o f the brain (Sharp et al,  1995). In  absence o f mutant htt, inactivation o f wild-type htt in adult mice results in decreased sperm counts and disorganization o f the seminiferous tubules (Dragatsis et al,  2000). The  importance o f htt function for sperm development is indicated by the upregulation o f htt transcription in germ cells that are co-cultured with Sertoli cells (Syed et al,  1999).  Interestingly, mice that are homozygous for the targeted inactivation o f HIP1 (huntingtin interacting protein 1) also show testicular degeneration (Rao et al, 2001). Since mutant htt shows decreased HIP1 binding with increased C A G size, the interaction between htt and HIP1 may be important for normal testicular function.  In human H D , fertility has been reported as either equivalent to or greater than fertility among unaffected individuals (Pridmore and Adams, 1991; Mastromauro et al, Shokeir et al,  1989;  1975). However, this is not surprising considering that the average age o f  onset for H D is 39.2 years (calculated from Harper, 1996) and H D patients are therefore likely to reproduce prior to the development o f symptoms or degeneration. In our study, we examined the testis o f 12 month old Y A C 128 mice. These mice show onset o f H D - l i k e symptoms at about 2-3 months o f age and, thus, we observe testicular degeneration well after onset. To our knowledge, testicular degeneration or fertility has not been examined in symptomatic H D patients. However, testosterone and luteinizing hormone levels have been shown to be decreased in male H D patients (Markianos et al, 2005).  6.2.7 Comparison of YAC128 mouse model with human Huntington disease The accurate temporal and spatial expression o f full length mutant htt in the Y A C 128 mouse model results in a phenotype that is similar to H D . The natural history o f Y A C 1 2 8 mice is  121  summarized in Figure 6.1. A comparison o f the Y A C 128 mouse model with human H D reveals that Y A C 128 mice reproduce many o f the symptoms o f the human  disease  (summarized in Table 6.2). The C A G expansion mutation in Y A C 128 mice is more than twice as large as is normally found in H D patients, yet H D patients develop a more severe phenotype. While this may be due in part to the presence o f 2 copies o f the normal H D gene in Y A C 128 mice, it likely results primarily from differences between species particularly with respect to lifespan. It is uncertain whether the disease progresses according to linear time or is related to relative time in the aging process. Nonetheless, the progressive motor dysfunction, cognitive impairment and selective degeneration o f human H D are recapitulated in the Y A C 128 mouse model. Accordingly, these mice can be used as a tool for studying the pathogenesis o f H D . Specifically, Y A C 1 2 8 mice can be used to examine the time course o f symptom development and to determine the relationship between the underlying biochemical and neuropathological changes and the motor and cognitive symptoms. In addition, Y A C 128 mice can be used for experimental therapeutics to assess the efficacy o f various treatments for H D prior to clinical trials. Recently, treatment o f H D patients with ethyl-eicosapentaenoic acid (ethyl-EPA) was shown to result in mild improvements in motor function in those patients which remained on protocol (Puri et al, 2005). The fact that treatment o f Y A C 1 2 8 mice with ethyl-EPA also resulted in mild motor benefit suggests that results obtained in the Y A C 128 mouse model may be predictive for human clinical trials (Van Raamsdonk et al, submitted). Further, a comparison o f the Y A C 128 mouse model with other mouse models o f H D based on the criteria for an ideal mouse model o f H D outlined in section 1.4 reveals that only the Y A C 128 mouse model satisfies all o f the criteria (Table 6.3).  6.3 Wild-type huntingtin function The critical importance o f wild-type htt function is demonstrated by embryonic lethality in mice that are homozygous for the targeted inactivation o f the mouse H D gene (Duyao et al, 1995; Nasir et al, 1995; Zeitlin et al, 1995). This notion is further supported by phenotypic • abnormalities in mice with levels o f wild-type htt that are 50% or less o f wild-type levels (Nasir et al, 1995; White et al, 1997; Auerbach et al, 2001). The continued importance o f wild-type htt function in adult brain is indicated by the neurological phenotype that develops  122  PRESYMPTOMATIC  SYMPTOMATIC  BEHAVIOUR Hyperkinesia 4 Motor Learning J Shifting Strategy  \  AGE(Months)  Hypoactivity Rotarod Deficit  Decreased Habituation I Cognition in Swimming Tests  V  2  V  Survival Deficit  I PPI  V  V  8  t Nuclear Localization of Mutant Htt  I Brain Weight 4 Striatal Volume  4 Cortical Volume Neuronal Shrinkage Striatal Neuronal Loss Cortical Neuronal Loss Atrophy of Globus Pallidus D A R P P - 3 2 Downregulation  NEUROPATHOLOGY  Dysfunction  Macroaggregates  ^  Degeneration  Figure 6.1 Natural history of abnormalities in YAC128 mice. At 2 months of age, YAC 128 mice show motor learning deficits on the rotarod and strategy shifting deficits in the reversal phase of the swimming Tmaze test. At this age motor function on the rotarod is normal. Also at 2 months of age, YAC 128 mice exhibit hyperkinesia. At 4 months, YAC 128 mice begin to show motor deficits on the rotatod. Between 8 and 9 months, YAC 128 mice show cognitive and motor deficits in the simple swimming test and swimming T-maze test. Also at this time point, YAC 128 mice exhibit decreased activity and have deficits in open field habituation. In the brain, nuclear localization of mutant htt is detected at 3 months - long before striatal neuropathology becomes apparent. At 9 months, YAC 128 mice exhibit significant decreases in striatal volume and brain weight compared to WT mice. At 12 months, YAC 128 mice show atrophy in other regions of the brain, including the cortex and globus pallidus, and show striatal and cortical neuronal loss. At this time point, YAC 128 mice show decreased PPI and habituation to acoustic startle. Finally, at 18 months macroaggregates become apparent in the YAC 128 mice.  123  Table 6.2 Comparison of YAC128 mouse model with human Huntington disease. The accurate expression of mutant htt in the YAC 128 mouse model of HD results in the recapitulation of the essential features of human HD.  Motor Dysfunction  Human H D  YAC128  Chorea  Hyperkinesia  Bradykinesia, gait abnormalities  Rotarod deficit  Bradykinesia  Hypoactivity Deficits in motor learning, simple swimming test, and swimming Tmaze  Procedural learning deficit Cognitive Impairment  Psychiatric Disturbances  Neuropathology  Gene Expression  Onset  Attentional set shifting deficit, perseveration Memory deficit Deficit in PPI Irritability, aggressive outbursts, anxiety and apathy Depression, Striatal atrophy  Deficit in strategy shifting Decreased open field habituation Deficit in PPI Not assessed Striatal atrophy  Cortical atrophy  Cortical atrophy  Striatal neuronal atrophy  Striatal neuronal atrophy  Atrophy of Globus pallidus  Atrophy of Globus pallidus  Striatal neuronal loss  Striatal neuronal loss  Cortical neuronal loss  Cortical neuronal loss  Not assessed  DARPP-32 down-regulation  Decreased brain weight Express FL mutant htt Tissue specific expression Adult onset < 55 CAG repeats 1 copy of WT HD gene 50% mutant htt expression -40 years  Decreased brain weight Express FL mutant htt Tissue specific expression -120 CAG repeats 2 copies of WT HD gene 75% mutant htt expression 2-3 months  Lifespan of unaffected individuals Survival  -80 years  -2 years  Early death in both males and females  Decreased lifespan in males only  Peripheral phenotypes  Decreased testosterone  Not assessed  Not assessed  Testicular degeneration  124  Table 6.3 Criteria based comparison of YAC128 mice to other HD mouse models. The YAC 128 mouse model of HD was compared with the other categories of HD mouse models based on the criteria set forth for the ideal mouse model of HD. Neurotoxin mouse models do not reproduce the genetic defect in HD and do not exhibit a progressive phenotype. N-terminal fragment models of HD also do not reproduce the genetic defect in HD and do not exhibit selective neuropathology. These two models can be used for therapeutic trials but are not well suited for studies of HD pathogenesis. Knock-in mouse models reproduce the genetic defect in HD and thus can be used for studies of HD pathogenesis but the mild phenotype in these mice limits their use in therapeutic trials. YAC 128 mice reproduce the genetic defect in HD and show quantifiable abnormalities similar to HD and thus can be used both for studies of pathogenesis and therapeutic trials.  Human HD Motor deficits Cognitive Impairment Selective atrophy Selective neuronal loss Progressive phenotype Full length mutant htt Therapeutic trials  Neurotoxin  N-terminal fragment  Knock-in  Yes Yes Yes Yes No No Yes  Yes Yes No No Yes No Yes  Yes No Yes No Yes Yes No  YAC128 :  Yes Yes Yes Yes Yes Yes •Yes  -  125  when htt levels are reduced by 84-90% in adult mouse forebrain (Dragatsis et al, 2000). Wild-type htt function appears to have a role in transcription (Zuccato et al, 2003), transport (Gauthier et al, 2004) and neuroprotection (Rigamonti et al, 2000; H o et al, 2001).  6.3.1 Wild-type huntingtin is neuroprotective in the brain Here, we demonstrate that wild-type htt is neuroprotective in brain, both in the hippocampus and in the striatum, against the excitotoxic neurotoxins kainic acid and quinolinic acid, respectively, both o f which have been used to model H D . The involvement o f excitotoxicity in H D was originally proposed based on the loss o f N M D A receptor bearing neurons within the striatum (Young et al, 1988) as well as the induction o f striatal neuronal loss using the excitotoxic neurotoxins, kainic acid (Coyle and Schwarcz, 1976; M c G e e r and McGeer, 1976) and quinolinic acid (Beal et al, 1986; Beal et al, 1991). Excitotoxicity results from an overstimulation with excitatory amino acids such as glutamate which leads to increased intracellular calcium levels, increased activation o f calcium dependent enzymes signaling pathways  and  and eventual neuronal death (Sattler and Tymianski, 2000). The  importance o f excitotoxicity to H D is supported by the modulation o f age o f onset based on variation in the G l u R 6 kainate receptor (Rubinsztein et al,  1997; MacDonald et al,  1999)  and the fact that both human patients and a mouse model o f H D were shown to have decreased expression o f the glutamate transporter G L T 1 (Arzberger et al, 1997; Lievens et al, 2001). Further, it appears that the expression o f mutant htt in H D makes cells more sensitive to glutamate. signals. The expression o f full length mutant htt in cultured neurons resulted in increased excitotoxic cell death upon exposure to N M D A compared to expression of a control protein (Zeron et al, 2001). Similarly, intrastriatal injection o f quinolinic acid caused significantly more damage in Y A C 7 2 mice than in W T mice (Zeron et al, 2002). In cultured neurons from Y A C 128 mice, it was shown that addition o f glutamate resulted in an elevation o f intracellular calcium that was not observed in cells from W T mice (Tang et al, 2005). Based on these findings, a model for excitotoxicity in H D has been proposed where mutant htt increases calcium influx via N R 2 B N M D A receptors, increases intracellular calcium release via IP3 receptors and reduces the calcium buffering capacity o f mitochondria (Bezprozvanny and Hayden, 2004; Panov et al, 2002).  126  While mutant htt increases the susceptibility o f cells to excitotoxic cell death, the work in this thesis demonstrates that wild-type htt protects against it. A comparison o f hippocampal neuronal damage resulting from intraperitoneal injection o f kainic acid revealed that there were dramatically less fluorojade-positive, degenerating neurons in Y A C 18 mice than in W T mice. This result was confirmed with silver staining and T U N E L . Similarly, the overexpression o f wild-type htt in Y A C 18 mice resulted in significantly reduced lesion sizes and significantly more surviving neurons after intrastriatal injection o f quinolinic acid. This is in contrast to Y A C 7 2 mice which demonstrated an increased lesion size compared to W T mice after intrastriatal injection with quinolinic acid (Zeron et al, 2002). Clearly, wild-type htt functions to protect neurons from excitotoxic cell death and this function is disrupted by C A G expansion. In support o f htt's role in counteracting excitotoxicity, there is a decrease in sensitivity to excitotoxicity beginning at day P6-P7 which corresponds to increasing htt levels from day P7 to P30 (Bhide et al,  1996). Importantly, we demonstrate htt mediated  neuroprotection in two separate lines o f mice over-expressing wild-type htt (line B 6 0 and line 212), thereby confirming that the protection does not result from a site o f integration effect. Further, we demonstrate that the neuroprotective effect o f wild-type htt is gene dose dependent since increasing htt expression resulted in increased protection against quinolinic acid toxicitiy.  While the mechanism responsible for the neuroprotective effect o f wild-type htt in unknown, it may be mediated through B D N F . B D N F is delivered from the cortex to the striatum and promotes the survival o f striatal neurons (Alter et al, 1997; Nakao et al, 1995). B D N F has been shown to protect neurons from excitotoxicity in vivo (Bemelmans et al, 1999; PerezNavarro et al, 2000) and is thought to at least be partially responsible for the beneficial effects o f dietary restriction (Duan et al,  2001). Since wild-type htt increases  BDNF  expression (Zuccato et al, 2001) and facilitates transport o f B D N F (Gauthier et al, 2004), it is plausible that at least part o f htt's neuroprotective effect is mediated by B D N F . Recent work suggests that wild-type htt may inhibit caspase 3 activity or expression and, thus, this may also contribute to htt mediated neuroprotection (Zhang et al, submitted). Alternately, the protective effect may be mediated by sequestration o f the pro-apoptotic protein HIP1 (huntingtin interacting protein 1). Co-expression o f wild-type htt and HTP1 in vitro resulted  127  in significantly less cell death than expression of HIP 1 and a control protein (Hackam et al, 2000) suggesting that over-expressing wild-type htt may be protective be reducing the apoptotic signal generated by HIP1. The fact that wild-type htt has been shown to protect against polyglutamine toxicity in the testis (Leavitt et al, 2001) suggests that BDNF may not be the only mechanism of htt mediated neuroprotection. 6.3.2 Novel function of huntingtin influences weight  In addition to the neuroprotective effect of wild-type htt, we demonstrate that htt acts to increase body weight and organ weight. Both YAC 18, line B60 and YAC 18, line 212 mice show increased body weight. This suggests that the increase in body weight in these mice results from htt over-expression and not a site of integration effect. Further, htt's effect on body weight is dose dependent as the higher expressing line 212 mice weighed significantly more than the lower expressing B60 line. Support for the role of htt in modulating body weight comes from the observation that mice heterozygous for the targeted inactivation of the mouse HD gene show decreased weight compared to WT mice. Thus, htt has a clear dose-dependent effect on body weight. Examination of individual organs in YAC 18, line 212 mice revealed that the over-expression of wild-type htt also resulted in significant increases in the weight of most organs including liver, spleen, kidney, heart and lungs. In contrast, increased htt expression did not affect brain weight and there was a trend toward decreased testicular weight in mice over-expressing wild-type htt. This is interesting considering that these two organs express the highest levels of htt (Sharp et al, 1995). It is possible that the high levels of htt present in the brain and testis are already sufficient for maximum growth and that additional htt has little impact. Alternately, it may be the case that the high levels of htt present indicate the importance of htt function in these two organs and that if htt expression exceeds a certain threshold there is a functional overdose of htt. In addition, after suturing has occurred the skull likely limits the amount the brain can grow in response to increased htt expression.  128  It is currently uncertain how htt expression leads to increased body weight or organ weight. It is possible that an anti-apoptotic effect of htt permits cells to survive longer than normal cells perhaps leading to increased cell numbers or cell size and, in turn, larger organs and increased total body weight. Another possibility is that cell over-expressing htt are more proliferative than wild-type cells. This possibility is supported by preliminary results in our laboratory indicating that cells transfected with wild-type htt exhibit increased proliferation (Simon Warby, unpublished). Nonetheless, further investigation will be required to unravel the mechanism behind htt's effect on body and organ weight. In contrast to the effect of htt on weight defined here, the activity of wild-type htt in transcription (Zuccato et al, 2001; Zuccato et al, 2003), transport (Gauthier et al, 2004) and neuroprotection (Rigamonti et al, 2000) are all disrupted by polyglutamine expansion. Since many of the functions of wild-type htt have been defined by their loss in mutant htt, it follows that most known functions of wild-type htt are disrupted by polyglutamine expansion. The fact that mutant htt can rescue mice homozygous for the targeted inactivation of the mouse HD gene from embryonic lethality (Hodgson et al 1999; Leavitt et al, 2001; Van Raamsdonk et al. 2005) suggests that the most critical functions of wild-type htt must be ^ maintained in mutant htt. Perhaps the effect of htt on weight that we demonstrate here represents one of these critical functions. In further defining the functions of wild-type htt it will be important to carefully characterize mice over-expressing wild-type htt in addition to defining those functions that are lost through polyglutamine expansion.  6.3.3 Possible contribution o f wild-type huntingtin to weight loss in H u n t i n g t o n disease  The cause of weight loss in HD patients is currently unknown. Decreased body mass index is reported in the early stages of the disease with obvious thinning occurring in later stages (Djousse et al, 2000). While some researchers suggested that hypothalamic cell loss may alter the regulation of food intake (Kremer and Roos, 1992), studies have shown that HD • patients consume more calories than unaffected individuals (Morales et al, 1989; Sanberg et- < al, 1981). Others have proposed that the increased energy expenditure associated with chorea results in the decrease in weight. However, weight loss appears to worsen with the  129  progression of the disease while chorea can subside. Furthermore, while sedentary energy expenditure is increased in HD patients, overall energy expenditure is not increased because HD patients engage in less voluntary physical activity (Pratley et al, 2000). It has also been suggested that mitochondrial dysfunction in HD leads to increased energy expenditure and decreased weight (Beal, 1998). However, sleeping metabolic rates of HD patients and unaffected individuals were not different suggesting that mitochondrial defects do not impact overall energy expenditure in HD patients (Pratley etal, 2000). Our study suggests the possibility that loss of htt function contributes to weight loss in HD. We show that htt has a dose dependent effect on body weight. Thus, it is plausible that loss of this function in HD patients leads to decreased weight as HD patients have a 50% genetic reduction of wild-type htt. Examination of weight in a knock-in mouse model of HD revealed that homozygous knock-in mice show greater weight loss than heterozygous knock-in mice suggesting the possibility that polyglutamine expansion reduces the ability of htt to increase weight (Lin et al, 2001). Even if mutant htt had an impact on weight equal to wild-type htt, the fact that mRNA levels from an HD gene allele expressing mutant htt were found to be decreased compared to a wild-type HD gene allele indicates that the overall impact of weight will be reduced in HD patients (Dixon et al, 2004). Furthermore, wild-type htt levels are thought to be decreased during the progression of HD and this has been demonstrated in a mouse model of the disease (Ona et al, 1999; Zhang et al, 2003). In HD, both mutant and wild-type htt are sequestered into aggregates, both show increased cleavage resulting from increased activation of proteases such as caspases and htt exhibits altered sub-cellular localization. It has also previously been demonstrated the htt levels are decreased in the striatum of HD patients (Schilling et al, 1995). Our work and previous studies in mouse models support a role of htt in determining body weight. Here we demonstrate that a 50% reduction in wild-type htt level resulted in a decrease in body weight. Although this difference was not reported in two other heterozygous htt knockout mice, no data on weight was presented (Duyao et ^al, 1995; Zeitlin et al, 1995). Mice with severely reduced htt levels in the adulthood show a 20% decrease in body mass with proportional decreases in organ weight (Dragatsis et al, 2000).  130  Similarly, chimeric mice, in which 20-75% of cells do not express wild-type htt, show decreases in body mass as great as 40% (Reiner et al, 2001). Mice expressing only 10-20% of endogenous htt levels have also been shown to have markedly decreased weight compared to controls (Auerbach et al, 2001). Thus, it is clear that decreases in htt expression are associated with decreased body weight. Weight loss has also been reported in mouse models of HD. Two mouse models expressing a small N-terminal fragment of mutant htt have demonstrated progressive weight loss that precedes premature death (Mangiarini et al, 1996; Schilling et al, 1999). In one of these models, the transgenic expression of a mutant htt fragment was shown to result in the depletion of full length wild-type htt beginning at 7 weeks of age which is followed closely by a decrease in body weight at 8 weeks (Zhang et al, 2003). Weight loss has also been demonstrated in a knock-in model of HD with 150 CAG repeats (Lin et al, 2001). Thus, it is clear that expression of full length htt influences body weight and the loss of this function may contribute to weight loss in HD. 6.4 Treatment of YAC128 mice with the over-expression of wild-type huntingtin Based on our findings demonstrating a protective effect of wild-type htt against excitotoxicity in the striatum, we investigated whether over-expression of wild-type htt could reduce the selective striatal neuropathology in the YAC 128 mouse model of HD. YAC 128 mice were crossed with YAC 18 mice to generate YAC 128 mice that over-express wild-type htt (YAC18/128 mice). YAC18/128 mice were found to express mutant htt at the same level as YAC 128 mice and wild-type htt at the same level as YAC 18 mice. This suggests that there is no feedback mechanism controlling the level of htt protein within the cell and that expression of htt from one transgene does not affect expression from another transgene or expression of the endogenous gene. It has previously been shown that htt mRNA levels are determined by the number of copies of the HD gene present and not affected by the overall level of mRNA in the cell (Dixon et al, 2004). Similarly, htt protein levels are determined by the number of copies of the HD gene present independent of the level of htt protein, mutant of wild-type, present in the cell (Van Raamsdonk et al, 2005).  131  Examination o f striatal neuropathology in Y A C 18/128 mice revealed a mild trend towards improvement o f striatal volume, striatal neuronal counts and striatal D A R P P - 3 2 expression, all o f which failed to reach significance. There was, however, a significant improvement in striatal neuronal cross-sectional area in Y A C 1 8 / 1 2 8 mice compared to Y A C 1 2 8 mice. In parallel with these experiments we examined the effect o f eliminating wild-type htt expression in Y A C 128 mice and found that there was a trend towards decreased striatal volume, striatal neuronal counts and striatal D A R P P - 3 2 expression (Van Raamsdonk et al, 2005). Similar to this experiment, the change in striatal neuronal size, caused in this case by the loss o f wild-type htt, was significant. Taken together, these results suggest that the mild, non-significant benefits we observe in Y A C 128 mice with over-expression o f wild-type htt may be real changes since the removal o f wild-type htt results in a mild worsening o f these same phenotypes.  Previously, two independent studies have compared the phenotype o f heterozygous and homozygous H D knock-in mice. In both cases, homozygous H D knock-in mice exhibited a more severe phenotype than heterozygous H D knock-in mice, but the differences were mild (Lin et al, 2001; Wheeler et al, 2000). The fact that homozygous H D knock-in mice have more mutant htt expression and no wild-type htt expression and still are only mildly more affected than heterozygous H D knock-in mice is in line with our findings that increasing levels o f wild-type htt has only a small impact on the disease phenotype. Similarly, studies comparing the severity o f patients homozygous and heterozygous for mutations in the H D gene have been in conflict as some studies report similar phenotypes while others indicate that homozygous patients have a more severe form o f the disease (Squitieri et al, 2003; Wexler et al, 1987; Myers et al, 1989; Kremer et al, 1994; Durr et al, 1999). The fact that a clear increase in the severity o f the disease is not always observed, suggests again that the loss o f wild-type htt does not have a great impact on the development o f H D .  A robust finding o f this study was that over-expression o f wild-type htt in Y A C 128 mice restored striatal neuronal size. Similarly, we have found that decreasing wild-type htt levels in Y A C 128 mice results in decreased neuronal size (Van Raamsdonk et al, 2005). This suggests that loss o f wild-type htt in H D may contribute to the striatal neuronal atrophy  132  observed in the disease. A n alternate possibility is that striatal neuronal size is more responsive to mildly beneficial effects o f treatments as this measure has been shown to exhibit the most dramatic improvements in therapeutic trials in H D mice (Gardian et  al,  2004; Ferrante et al, 2003; Ferrante et. al., 2004). The effect o f wild-type htt on neuronal size may be related to htt's ability to increase B D N F transcription (Zuccato et al, 2001) and transport (Gauthier et al, 2004) since B D N F promotes the survival and differentiation o f striatal neurons.  6.5 Treatment of YAC128 mice with cystamine 6.5.1 Transglutaminase activity in the YAC128 mouse model of Huntington disease Patients with H D have increased T G activity in the striatum - the primary region o f neuropathology in H D (Karpuj et al, 2002; Lesort et al, 1999). In this thesis, we show that T G activity is increased in the forebrain o f Y A C 128 mice. T G activity was increased to a lesser extent in the hindbrain although not significantly. Thus, increases in T G activity appear to be correlated with pathology as the region with the greatest increase in T G activity includes the striatum where we observe selective degeneration (section 3.2.3.1). While the majority o f increase in T G activity could be accounted for by GTP-inhibited T G activity, the magnitude o f increase in the non-GTP inhibited T G activity was much greater than for total T G activity suggesting the possibility that T G s other than t T G may be important in the pathogenesis o f H D (since t T G activity is inhibited by G T P ) . The recapitulation o f the increased T G activity seen in human patients in the Y A C 128 mouse model lends further support to the use o f this model to study the pathogenesis o f H D .  T G activity in the R6/2 mouse model was shown to be increased by 34% (Dedeoglu et al, 2002) - slightly greater than the 30%> observed in the Y A C 128 forebrains. However, in a recent study no increase was observed in T G activity in the R6/2 mice at both 6 and 10 weeks (Bailey and Johnson, 2005a). It is uncertain if, or how, increases in T G activity contribute to the pathogenesis  o f the H D . While a correlation exists in vitro between  length o f  polyglutamine expansion and the ability o f t T G to cross-link proteins with polyglutamine expansions (Kahlem et al,  1998; Cooper et al,  2002), t T G does not, contribute to  133  aggregation cell culture models (Chun et al, 2001). Furthermore, recent work suggests that macroaggregates are not toxic but possibly neuroprotective (Arrasate et al, 2004; Saudou et al, 1998, Kuemmerle et al, 1999). In the YAC 128 mouse model, macroaggregates are not observed until 18 months of age - a time point well after striatal neurodegeneration is present (Slow et al, 2003). Furthermore, inhibiting TG activity in the R6/1 and R6/2 model of HD resulted in increased aggregation in combination with an amelioration of disease phenotypes (Mastroberardino et al, 2002; Bailey and Johnson, 2005a). These data suggest that if increased TG activity plays a role in HD pathogenesis, it likely does not contribute to pathology through catalysis of aggregation. Increased TG activity may contribute to HD by cross-linking mutant htt monomers to generate toxic oligomers or aggregation intermediates. It has also been suggested that TG could catalyze intramolecular cross-links which prevent aggregation (Lai et al, 2004; Konno et al, 2005a; Konno et al, 2005b). In this case, if aggregates are in fact protective, the prevention of aggregation and the maintenance of mutant htt in a toxic monomer or oligomer state may contribute to the disease. Increased TG activity may also contribute to HD through deleterious modifications of other proteins within the cell which lead to their inactivation (Chun et al, 2001; Cooper et al, 1997) or through increasing the susceptibility of cells to apoptosis (Melino et al., 1994; Oliverio et al, 1999; Piacentini et al, 2002). 6.5.2 Cystamine treatment in the YAC128 mouse model of Huntington disease  In this thesis, we use the YAC 128 mouse model of HD to examine the effect of cystamine treatment on striatal neuronal loss - a hallmark feature of HD. We show that cystamine treatment prevents neuronal loss in YAC 128 mice. Cystamine also prevented striatal neuronal atrophy and limited volume loss within the striatum. Our findings are in line with the neuropathological benefits of cystamine demonstrated in the R6/2 mouse model where cystamine treatment resulted in a 73% rescue of brain weight decrease, a visible reduction of ventricular enlargement and a 46% rescue of neuronal atrophy (Dedeoglu et al, 2002). Eliminating tTG expression in R6/1 mice resulted in similar beneficial effects namely a 40% rescue of brain weight and about a 65%> rescue of degenerating neurons (Mastroberardino et  134  al, 2002). R6/2 mice that were homozygous for the targeted inactivation of tTG also showed a trend towards improved striatal volume compared to R6/2 mice expressing normal levels of tTG (Bailey and Johnson, 2005a).  In contrast to the pathological improvement, treating Y A C 128 mice with cystamine beginning at 7 months of age resulted in no reduction in motor dysfunction as assessed by rotarod performance and open field activity. Y A C 128 mice begin to have difficulty on the rotarod between 2 and 4 months of age and develop hypoactivity between 6 and 8 months (section 3.2.1). Thus, at the time of treatment YAC128 mice already showed a severe deficit in this task and cystamine was unable to improve their performance. By completing an additional trial where cystamine treatment began at 4 months of age, we show that even when cystamine is delivered earlier, it was unable to prevent motor deficits on the rotarod or hypoactivity. In contrast, treatment of R6/2 mice with cystamine or the elimination of tTG expression in R6/1 or R6/2 mice all resulted in improvement in rotarod performance (Dedeoglu et al, 2002; Mastroberardino et al, 2002; Bailey and Johnson, 2005a). In each case the degree of T G inhibition (34%-70%) was greater than we achieved in our mice (16%) and may explain why we observed no benefit on the rotarod. It is also possible that treating Y A C 128 mice with cystamine earlier than 4 months could result in improved rotarod performance.  Cystamine treatment also failed to restore striatal DARPP-32 expression in Y A C 128 mice. DARPP-32 expression is critical for dopaminergic neurotransmission (Svenningsson et al, 2004) and its down-regulation in Y A C 128 mice beginning prior to 6 months of age (unpublished) may be a biochemical marker of neuronal dysfunction.  Overall, it appears that cystamine treatment in Y A C 128 mice was neuroprotective but was unable to prevent or reverse neuronal dysfunction. Y A C 128 mice exhibit motor deficits, cognitive dysfunction and DARPP-32 down-regulation months before striatal atrophy and neuronal loss are detected. Thus, the expression of mutant htt initially results in neuronal dysfunction with cell death occurring at a later time point. If the process leading to neuronal death involves neuronal dysfunction then our results suggest that the beneficial effect of  135  cystamine is downstream of neuronal dysfunction. Alternatively, there may be separate processes that lead to neuronal dysfunction and neuronal death in which case, cystamine appears to prevent the process leading to cell death but not cell dysfunction.  6.5.3 Mechanism responsible for beneficial effects of cystamine in Huntington disease The mechanism by which cystamine is beneficial in Y A C 128 mice and R6/2 mice is uncertain. The fact that elimination of tissue T G expression in R6/1 and R6/2 mice results in improvement in survival and motor performance suggests that inhibition of TG activity is at least partially responsible (Mastroberardino et al, 2002; Bailey and Johnson, 2005a). Decreasing T G activity was originally thought to be beneficial through a decrease in aggregate formation and associated toxicity that has been shown in vitro (Igarashi et al., 1998; de Cristofaro et al, 1999). However, cystamine has been shown to be beneficial here and in the R6/2 mouse model without any affect on aggregation (Karpuj et al, 2002). Furthermore, eliminating tissue T G expression in R6/1 and R6/2 mice, lead to improvements in the disease phenotype despite increased aggregation (Mastroberardino et al, 2002; Bailey and Johnson, 2005a). A n alternative possibility is that cystamine mediated decrease in TG activity is beneficial through a decreased susceptibility to cell death (Melino et ai, 1994; Piacentini etal, 2002; Oliverio etal, 1999).  The fact that we observed only a modest 16% reduction in T G activity suggests that other actions of cystamine may be responsible for its neuroprotective effect. While it is possible that the degree of TG inhibition by cystamine was greater in older Y A C 128 mice, a genetic reduction of total T G activity by 40%> in R6/2 mice resulted in no significant phenotypic improvement compared to R6/2 mice (Bailey and Johnson, 2005a). In contrast, reducing total T G levels by 30% with cystamine resulted in improvements in motor function, survival and neuropathology, suggesting that mechanisms other TG inhibition contribute to cystamine mediated neuroprotection (Bailey and Johnson, 2005b). This conclusion is supported by the fact that cystamine treatment improves lifespan and motor function in R6/2 mice that do not express tTG (Bailey and Johnson, 2005b). Aside from T G inhibition, cystamine may be beneficial by inhibiting caspase 3 activity (Lesort et al, 2003) or through by up-regulating  136  the expression o f heat shock proteins (Karpuj et al, 2002). A final possibility is that the beneficial effects o f cystamine result from cystamine-mediated increases in anti-oxidants within the cell. Cystamine treatment has been shown to increase glutathione in vitro (Lesort et al, 2003) and cysteine levels in vivo (Fox et al, 2004; Pinto et al, 2005) - both o f which act as anti-oxidants.  The fact that cystamine treatment in Y A C 128 mice resulted in decreased T G activity and neuroprotection despite no detectable increase in brain cystamine levels (Pinto et al, 2005) suggests that an active metabolite o f cystamine may be responsible for the beneficial effects in brain. For example, cystamine is converted to cysteamine in the body which can cross the blood brain barrier and subsequently can act as an anti-oxidant (Gentile and Cooper, 2004). Regardless, cystamine treatment likely exerts its beneficial effects  in H D through a  combination o f mechanisms.  6.5.4 Implications for treatment of H u n t i n g t o n disease Overall, we show that cystamine treatment is neuroprotective and prevents development o f striatal neuropathology in the Y A C 1 2 8 mouse model o f H D . Since the beneficial effects o f cystamine were not beneficial for all symptoms o f the disease, it may be necessary to combine cystamine treatment with a compound that can reduce the motor phenotypes o f the disease. The positive effects o f cystamine in this model are in accordance to previous studies in the R6/1 and R6/2 mouse models and lend support to further study o f this compound for the treatment o f H D (Dedeoglu et al, 2002; Karpuj et al, 2002). Importantly, we extend these previous findings, to show that cystamine can prevent striatal neuronal cell death.  6.6 C o n t r i b u t i o n of loss of huntingtin function to Huntington disease The fact that mutant htt is expressed and that the expression o f mutant htt alone, without reductions in wild-type htt levels, is sufficient to cause H D - l i k e symptoms in cells and in mouse models (Mangiarini et al, 1996; Schilling et al, 1999; Reddy et al, 1998; Laforet et al, 2001; Hodgson etui, 1999; Slow et al, 2003) suggests that H D is caused by a toxic gain of function that results from polyglutamine expansion in the htt. protein. A gain o f function  137  mutation is supported by the fact that deletion of one H D gene allele in either humans or mice does not result in HD (Nasir et al, 1995; Duyao et al, 1995; Zeitlin et al, 1995). On the other hand, decreasing levels of wild-type htt from conception or in adult brain does result in neurological abnormalities suggesting that loss of wild-type htt function may also contribute to H D (White et al, 1997; Dragatsis et al, 2000; Auerbach et al, 2001). Since mice heterozygous for the targeted inactivation of the mouse H D gene show abnormal behaviour and neuronal loss (Nasir et al, 1995; O'Kusky et al, 1999), it indicates that decreasing htt by just 50% is detrimental and suggests that H D patients should show symptoms resulting from decreased wild-type htt unless mutant htt is able to replace the function of wild-type htt.  While mutant htt is capable of rescuing mice homozygous for the targeted inactivation of the H D gene from embryonic lethality (Hodgson et al,  1999; Leavitt et al, 2001; Van  Raamsdonk et al, 2005), many of the assayable functions of wild-type htt are disrupted by C A G expansion (Rigamonti et al, 2000; Zuccato et al, 2001; Gauthier et al, 2004) and most of htt's protein-protein interactions are altered as well (Li and L i , 2004). Furthermore, preliminary data showing that mice expressing only low levels of wild-type htt survive better than mice expressing equivalent low levels of mutant htt indicates that mutant htt is not as effective as wild-type htt at rescuing mice homozygous for the targeted inactivation of the mouse HD gene from embryonic lethality (Rona Graham, personal communication).  Based on the neuroprotective function of wild-type htt that we demonstrate here and htt's role in transport and transcription within the cell, a simple model for the contribution of loss of htt function to the pathogenesis of H D can be proposed. H D patients express both mutant htt and decreased levels of wild-type htt. Mutant htt expression results in neuronal toxicity while decreased wild-type htt expression results in decreased neuroprotection. Consequently, the loss of wild-type htt would make cells more susceptible to the toxicity of mutant htt. Further, decreased wild-type htt levels may impact transport and transcription within the cell if 50% protein expression is not sufficient for normal function. In addition, levels of wildtype htt within the cell can be further reduced during the progression of the disease (Ona et al, 1999; Zhang et al, 2003). This could occur through increased proteolysis of wild-type htt  138  (Wellington et al, 2000), aggregation o f wild-type htt with mutant htt (Narain et al, 1999) or an altered localization o f htt.  Based on this model, it would be predicted that patients who are homozygous for the disease mutation would show a more severe phenotype than patients that are heterozygous because in addition to increased expression o f mutant htt these patients would also express decreased levels o f wild-type htt. While early reports regarding patients homozygous for the disease mutation suggested that the disease was no more severe than in heterozygotes (Wexler et al, 1987; Myers et al, 1989; Durr et al, 1999), recent work suggests that patients homozygous for the H D mutation have a more severe disease progression compared to patients that are heterozygous (Squitieri et al, 2003). A s the numbers o f patients examined in all o f these studies was few (17 homozygous patients in 4 separate studies), resulting from the limited number o f H D homozygotes, more patients w i l l need to be examined to arrive at a definitive conclusion.  The generation o f knock-in mouse models o f H D has provided the opportunity to address this question more fully. In two separate knock-in mouse models o f H D with 111 and 150 C A G repeats inserted into the mouse H D gene, it was shown that homozygous knock-in mice exhibit a more severe phenotype than heterozygous knock-in mice ( L i n et al, 2001; Wheeler et al, 2000). This suggests that the conclusions o f the latter o f the human H D homozygote studies are correct and that homozygosity for the H D mutation leads to a more severe clinical course than heterozygosity. However, in these experiments it was not possible to determine the relative contribution o f loss o f wild-type htt to the exacerbation o f phenotype because mutant  htt  expression  was  increased  simultaneously.  Recent  studies  convincingly  demonstrate that increasing the expression o f mutant htt results in a more severe phenotype (Graham et al, 2005).  The contribution o f loss o f wild-type htt was directly assessed by eliminating wild-type htt expression in the Y A C 1 2 8 mouse model o f H D . In this study, there was a non-significant worsening o f striatal volume, striatal neuronal counts and striatal D A R P P - 3 2 expression and a significant exacerbation o f striatal neuronal atrophy (Van Raamsdonk et al, 2005). The  139  results from this study are in accordance with the results here demonstrating that the treatment o f Y A C 1 2 8 mice with the over-expression o f wild-type htt results in a mild, nonsignificant improvement in striatal volume, striatal neuronal counts and striatal D A R P P - 3 2 expression with a more robust, significant improvement in striatal neuronal size. These two studies combined suggest only a mild contribution o f the loss o f wild-type htt function to the striatal neuropathology in H D and suggests that wild-type htt treatment in H D w i l l not be sufficient to prevent striatal neuropathology.  6.7 Conclusions The goals o f this thesis were (1) to characterize genetic mouse models o f H D (2) to develop a protocol and outcome measures for therapeutic trials in these mice and (3) to demonstrate the efficacy o f performing preclinical therapeutic trials for H D in these mice. A l l three objectives were met. This thesis demonstrates that the Y A C 128 mouse model o f H D recapitulates the motor  dysfunction,  cognitive  impairment  and  selective  neurodegeneration  of H D .  Importantly, this represents the first demonstration o f selective and progressive striatal neuronal loss in a mouse model o f H D . The phenotypic characterization o f Y A C 128 mice revealed several biologically relevant, quantifiable differences from W T mice that can be used as outcome measures for therapeutic trials including: striatal volume, striatal neuronal loss,  striatal  neuronal  cross-sectional  area,  striatal  DARPP-32  expression,  rotarod  performance, open field activity and pre-pulse inhibition. With larger numbers o f mice it will also be possible to assess the effect o f potential treatments on survival in male mice and cognitive function.  In this thesis, two therapeutic trials were completed in Y A C 128 mice: genetic treatment with over-expression o f wild-type htt and pharmacologic treatment with cystamine. In each case, differences between Y A C 128 and W T mice were reproduced in the trial setting. Both treatments improved striatal neuropathology in Y A C 128 mice with cystamine proving to be more neuroprotective than wild-type htt as cystamine was able to limit striatal volume loss and neuronal loss in addition to striatal neuronal atrophy. These represent the first therapeutic trials completed in the Y A C 128 mice model and demonstrate the feasibility o f experimental  140  therapeutics in this model. The cystamine trial is also the first time a treatment has been shown to prevent selective neuronal loss in a genetic mouse model of HD. In addition to work on the YAC128 mouse model of HD, studies of YAC18 mice that overexpress wild-type htt provided insight into wild-type htt function. The work in this thesis provides the first in vivo evidence that wild-type htt is neuroprotective in brain. In addition, examination of YAC 18 mice revealed a novel function of htt protein whereby overexpression of htt results in a dose-dependent increase in body and organ weight. Thus, YAC 18 mice provide a valuable tool for investigations of wild-type htt function and this information may inform future studies of HD pathogenesis. 6.8 Future directions The work presented in this thesis has provided insight into the pathogenesis of HD, has improved understanding of wild-type htt function and importantly has provided a standardized protocol for experimental therapeutics for HD in the YAC 128 mouse model. Nonetheless, several questions remain unanswered. There is still no treatment that can halt the progression of HD, the pathogenesis of HD is incompletely understood and the functions of wild-type htt have only been partially defined. The following are suggestions of future avenues of research that may shed light on these areas. 1. Treatment of Huntington Disease Combination therapies - In this thesis, cystamine was shown to be neuroprotective but did not improve motor function in YAC 128 mice. Similarly, over-expression of wild-type htt improved striatal neuronal size but was not beneficial in other outcome measures. We have recently demonstrated a mild motor benefit resulting from treatment with ethyl-EPA which occurred in absence of neuroprotection (Van Raamsdonk et al, 2005). In developing a treatment for HD, it may be necessary to use combinations of therapies with complementary effects. This can be accomplished by combining treatments based on benefits observed singly or by selecting therapies that target different mechanisms in the pathogenesis of HD. j  141  Encouragingly, it has recently been shown that treatments used in combination (SAHA, cystamine and creatine) can exhibit significant protection at doses where none of the treatments are effective individually (Agrawal et al, 2005). R N A Interference - Since the order of events in the pathogenesis of HD is unknown, it is difficult to target an early step in the pathogenesis. The genetic defect in HD leads to increased expression of mutant htt and decreased expression of wild-type htt. A therapy that could directly counteract the genetic defect would prevent all harmful downstream events. Recent work suggests that while the loss of wild-type htt does play a minor role in HD pathogenesis, the HD phenotype appears to be primarily determined by the expression of mutant htt (Van Raamsdonk et al, 2005). This suggests that, without gaining any further insight into the disease pathogenesis, a treatment designed to reduce mutant htt expression would be effective in HD. This could be accomplished by RNA interference (Bosher and Labouesse, 2000; Lu, 2004). This approach was recently shown to be efficacious in the treatment of a mouse model of another polyglutamine disorder (Xia et al, 2004). Further, the effectiveness of decreasing mutant htt levels in the treatment of HD is supported by the reversal of phenotypes when mutant htt expression was stopped in a conditional mouse model of HD (Yamamoto et al, 2000). Finally, the demonstration that complete loss of wildtype htt results in only a modest worsening of most disease phenotypes suggests that even a non-specific RNAi approach that reduces levels of both mutant and wild-type htt may be beneficial (Van Raamsdonk et al, 2005). It has recently been demonstrated in the N171-82Q mouse model of HD that RNAi could reduce mutant htt expression by 50% resulting in improvement in motor function and decreased aggregation (Harper et al, 2005).  2. Y A C 1 2 8  mouse model  Pathogenesis of Huntington Disease - This thesis demonstrates that the YAC 128 mouse model of HD reproduces many of the clinical findings in human HD. Accordingly, this model can be used in studies of disease pathogenesis. Tissue samples from human HD patients are limited to cells outside of the brain prior to death. As such, samples of brain tissue prior to onset or during the disease progression are rare and lengthy post-mortem  142  intervals can lead to the detection o f changes that are not part o f the disease process. In contrast, Y A C 128 mice can be sacrificed at any time point in the progression o f the disease with  negligible post  mortem  interval. Accordingly,  the  processes  involved  in H D  pathogenesis and more importantly the precise sequence o f these events can be determined by analyzing Y A C 128 mice at different time points.  In studying the pathogenesis o f H D in these mice a more complete understanding could be obtained by completing biochemical analyses,  examining gene expression  and  also  examining protein expression. The analyses o f the Y A C 128 mice in this thesis have primarily been at a gross level. Aside from demonstrating decreased transglutaminase activity, the underlying biochemical changes in the brains o f these mice have not been elucidated and warrant further study. Several mouse models o f H D exhibit changes in gene expression including the Y A C 7 2  mouse model (Luthi-Carter et al,  2002; Chan et al,  2002).  Furthermore, hypoacetylation o f histone proteins has been found in mouse models o f H D and the restoration o f histone acetylation has been beneficial in preclinical therapeutic trials (Ferrante et al, 2003; H o c k l y et al, 2003). Since changes in gene expression may contribute to H D pathogenesis in Y A C 128 mice, gene expression should be assessed at multiple time points in disease progression to determine what genes are changed and when these changes occur during the pathogenesis o f the disease. Furthermore, histone acetylation should be assessed in the Y A C 128 mice to determine whether histone hypoacetylation is present in a full length mouse model o f H D and whether the therapeutic efficacy o f histone deacetylase inhibitors should be assessed in these mice. Finally, an analysis o f protein expression in Y A C 128 mice compared to W T mice may further inform studies o f pathogenesis.  Axonal transport - Several recent experiments have demonstrated that the expression o f mutant htt or the decreased expression o f wild-type htt result in disruptions in fast axonal transport (Szebenyi et al, 2003; Gunawardena et al, 2003; Gauthier et al, 2004; Trushina et al, 2004). To determine whether axonal transport is affected in Y A C 128 mice, the number o f labeled cells in the striatum could be assessed following fluorogold injections into the substantia nigra (Trushina et al, 2004). In addition, axonal fibers could be examined for unusually large varicosities (Stokin et al, 2005). If axonal transport deficits were present in  143  YAC 128 mice then the contribution of these deficits to HD pathogenesis could be assessed by genetically modulating transport and examining the effect of phenotypic severity in YAC 128 mice. Mechanism of selective degeneration - In this thesis, YAC 128 mice are shown to reproduce the pattern of selective degeneration that occurs in human HD. As in the human disease, this pattern was not explained by increased mutant htt expression in affected regions but there was some correlation between nuclear localization of mutant htt and pathology. If selective nuclear localization of mutant htt is responsible for the regional specificity of damage in HD, then the question becomes why are there selective increases in nuclear localization of mutant htt in affected regions of the brain? If cleavage of full length htt is required for its entry into the nucleus then processes leading to caspase or calpain activation may be responsible. Regardless, YAC 128 mice provide the opportunity to experimentally assess mechanisms that can potentially explain the selective degeneration in HD. Furthermore, the YAC 128 mouse model can be used to assess whether compounds that can prevent the nuclear localization of mutant htt (Wang et al, 2005) may be beneficial in treating HD. Assessment of brain phenotypes in live mice - In this thesis, striatal volume and striatal neuronal numbers were assessed only after sacrifice and comparing separate cohorts of mice from different time points indicated the progression of these phenotypes. In future therapeutic trials in these mice it would be beneficial to be able to monitor the striatal volume decrease in live mice by magnetic resonance imaging (MRI). This would allow us to assess the benefit of treatments at multiple time points and determine not just if neuropathology is prevented but also if it is significantly delayed. Previous studies have demonstrated the feasibility of assessing brain volumes with MRI (McDaniel et al, 2001). To use this approach in YAC 128 mice, it would need to have sufficient resolution to detect the small differences in striatal volume that we observe. Magnetic Resonance Spectroscopy (MRS) has shown decreases in N-acetyl aspartate (NAA) levels in both human HD patients and mouse models of HD (Jenkins et al, 2000; van Dellen  144  et al, 2000; Jenkins et al, 1998). Most recently NAA levels have been shown to be decreased in the YAC72 mouse model of HD (Jenkins et al, 2005). NAA is thought to be a marker of neuronal health and thus monitoring NAA levels during the progression of the disease could provide another means of assessing brain phenotypes in live mice during therapeutic trials. Again, preliminary studies would need to be undertaken to determine if differences are robustly detected in YAC 128 mice compared to WT mice. Mechanism of sex specificity of survival deficit - YAC 128 mice were shown to have a male specific survival deficit. To determine if the cause of this discrepancy results from the protective effects of estrogen, the survival of male YAC 128 mice treated with estrogen could be compared to untreated YAC 128 mice. To determine if the difference results from a decrease in testosterone, the survival of male YAC 128 mice treated with testosterone could be compared to untreated YAC 128 mice. As a preliminary step, it would be important to compare testosterone levels in WT and YAC 128 mice. The possible contribution of loss of testosterone to the phenotype in YAC 128 mice could also be assessed by castrating male YAC128mice. Morphological analyses in YAC128 mice - Dystrophic dendrites have been reported in HD patients as well as mouse models of HD (DiFiglia et al, 1998; Laforet et al, 2001). Golgi staining could be used to examine dendritic morphology in YAC 128 mice. Additional morphological analysis of the Y A C 128 mice should include examination of striatal neurons at the electron microscope (EM) level. E M analysis of striatal neurons in YAC72 mice revealed condensed cytoplasm, mitochondrial swelling and abnormalities in the Golgi apparatus (Hodgson et al, 1999). Additional behavioural characterization of YAC128 mice - In this thesis, YAC128 mice are shown to reproduce the motor and cognitive dysfunction of HD. However, psychiatric disturbances were not assessed. Since psychiatric problems such as depression are common in HD patients, it will be important to assess these phenotypes in YAC 128 mice with specially designed and validated behavioural tests.  145  As one of the current limitations of behavioural testing in YAC 128 mice is the fact that they are maintained on the FVB/N background strain which develops retinal degeneration (Taketo et al, 1991), back-crossing YAC128 mice onto a strain with normal vision would allow for a wider variety of behavioural analyses with increased complexity. To this end, we have begun breeding YAC 128 mice onto a 129 strain background since these mice show a similar susceptibility to excitotoxicity as FVB/N mice (Shauwecker and Steward, 1997) but have normal vision. 3. Wild-type huntingtin Additional functions of wild-type huntingtin - Aside from the effect of htt on body weight and organ weight, all of the currently defined functions of wild-type htt have been identified by their loss in HD. Accordingly, the function of wild-type htt in transcription (Zuccato et al., 2003), transport (Gauthier et al., 2004) and neuroprotection (Rigamonti et al., 2000) are all disrupted by polyglutamine expansion. Nonetheless, mutant htt is able to rescue mice that are homozygous for the targeted inactivation of htt from embryonic lethality (Hodgson et al., 1999; Leavitt et al., 2001; Van Raamsdonk et al., 2005), indicating that some of the most critical functions of htt are maintained in the presence of CAG expansion and have yet to be defined. A closer analysis of deficiencies in mice heterozygous for the targeted inactivation of the mouse HD gene as well as further characterization of mice that over-express wild-type htt may reveal additional functions of wild-type htt. Mechanism of neuroprotective effect of wild-type huntingtin - While a clear neuroprotective effect of wild-type htt has been demonstrated here in Y A C 18 mice, the mechanism of neuroprotection is uncertain. A plausible explanation is that the protection is mediated through BDNF as htt has been shown to increase both transcription and transport of BDNF (Zuccato'ef al, 2001; Gauthier et al, 2004). To test this hypothesis, anti-BDNF antibodies or RNAi for BDNF could be used to specifically decrease BDNF levels in YAC 18 mice or cultured cells that over-express wild-type htt (Duan et al, 2001). If BDNF expression is reduced to normal in the presence of htt over-expression then it would be  146  possible to determine whether htt acts through B D N F . Htt mediated neuroprotection may also act through HIP1 sequestration, caspase 3 inhibition or an alternate mechanism.  Mechanism of htt mediated weight gain - In this thesis, htt over-expression was shown to increase body weight and organ weight, but not in brain and testis. Further investigation into the mechanism behind these changes may increase our knowledge o f htt function. Initially, it w i l l be important to determine i f this increase in weight results from an increased number o f cells, an increased size o f cells or both. It would also be interesting to examine why htt does not effect the weight o f the brain and testis despite the fact that these two organs express the highest levels o f htt.  4. Mechanism of cystamine neuroprotection in H D While cystamine was shown to be neuroprotective in the Y A C 128 mouse model o f H D , it is uncertain the mechanism responsible for this beneficial effect. 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