Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2005-10581X.pdf [ 13.85MB ]
Metadata
JSON: 831-1.0092261.json
JSON-LD: 831-1.0092261-ld.json
RDF/XML (Pretty): 831-1.0092261-rdf.xml
RDF/JSON: 831-1.0092261-rdf.json
Turtle: 831-1.0092261-turtle.txt
N-Triples: 831-1.0092261-rdf-ntriples.txt
Original Record: 831-1.0092261-source.json
Full Text
831-1.0092261-fulltext.txt
Citation
831-1.0092261.ris

Full Text

Characterization and Treatment of Mouse Models of Huntington Disease by Jeremy Michael Van Raamsdonk B . S c , University of British Columbia, 1997 M . S c , McMaster University, 1999 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Medical Genetics) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A September 2005 © Jeremy Michael Van Raamsdonk, 2005 ABSTRACT Huntington disease (HD) 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 of H D or alter its progression. The major objectives of this thesis were to determine which symptoms of H D are recapitulated in Y A C transgenic mouse models of 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 of 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. To determine the feasibility of genetic modulation of the disease phenotype, we investigated the ability of over-expression of wi ld type htt to prevent striatal neuropathology in Y A C 128 mice based on a putative pro-survival function of wi ld type htt. We demonstrate for the first time that wi ld type htt is neuroprotective in the brain. In Y A C 128 mice, over-expression of wi ld type htt prevented atrophy of striatal neurons(but did not significantly improve striatal volume or striatal neuronal numbers. To determine the feasibility of 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 of 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 of H D recapitulates the progressive motor dysfunction, cognitive deficits and selective neurodegeneration of H D . A s such, these mice can be used for studies of H D pathogenesis and in preclinical therapeutic trials for H D . i i T A B L E O F C O N T E N T S A B S T R A C T ii T A B L E O F C O N T E N T S i i i L I S T O F T A B L E S vi i L I S T O F F I G U R E S v i i i L I S T O F A B B R E V I A T I O N S x P R E F A C E x i i A C K N O W L E D G E M E N T S xvi i 1.0 I N T R O D U C T I O N 1 1.1 Clinical description of Huntington disease 1 1.2. Neuropathology of Huntington disease 2 1.3 Genetics of Huntington disease 4 1.3.1 Huntington disease is caused by a C A G expansion in Huntington disease gene 4 1.3.2 Expression of the Huntington disease gene 5 1.4 Mouse models of Huntington disease 5 1.4.1 Neurotoxin models of Huntington disease 6 1.4.2 Huntington disease mouse models expressing N-terminal fragments of mutant htt 6 1.4.3 Knock-in mouse models of Huntington disease 9 1.4.4 Ful l length transgenic mouse models of Huntington disease 10 1.5 Preclinical therapeutic trials for Huntington disease 12 1.6 Huntingtin function 17 1.6.1 Transcription 19 1.6.2 Transport 20 1.6.3 Neuroprotection 21 1.7 Loss of huntingtin function in Huntington disease 22 1.8 Objectives ....23 2.0 M A T E R I A L S A N D M E T H O D S 26 2.1 Mice .26 i n 2.2 Behavioural assessments 26 2.2.1 Rotarod test of motor coordination 26 2.2.2 Assessment of motor learning on the rotarod 27 2.2.3 Open field activity test 27 2.2.4 Open field habituation test of learning and memory 27 2.2.5 Simple swimming test 28 2.2.6 Pre-pulse inhibition and habituation to acoustic startle 28 2.2.7 Beam crossing test 29 2.2.8 Footprint analysis .....29 2.2.9 Swimming T-maze test with reversal 30 2.3 Physiologic measurements 31 2.3.1 Body weight 31 2.3.2 Organ weights 31 2.3.3 Survival analysis 31 2.3.4 Histological examination of the testis 31 2.4 Neuropathological assessments 32 2.4.1 Volume of brain structures 32 2.4.2 Neuronal numbers 33 2.4.3 Striatal neuronal cross-sectional area 33 2.4.4 Striatal D A R P P - 3 2 expression 34 2.4.5 Nuclear localization of mutant huntingtin 34 2.5 Neurotoxicity 34 2.5.1 Delivery of kainic acid 34 2.5.2 Delivery of quinolinic acid 35 2.5.3 Assessment of hippocampal damage 35 2.5.4 Assessment of striatal damage 36 2.6 Treatment 36 2.6.1 Treatment with over-expression of wi ld type huntingtin 36 2.6.2 Treatment with cystamine 36 2.7 Molecular biology 37 2.7.1 Western blotting 37 2.7.2 Measurement of transglutaminase activity 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 M E A S U R E S 40 3.1 Characterization of the Y A C 7 2 mouse model of Huntington disease 40 3.1.1 Motor dysfunction in Y A C 7 2 mice 40 3.1.2 Neuropathology in Y A C 7 2 mice 43 iv 3.2 Characterization of the Y A C 1 2 8 mouse model of Huntington disease 43 3.2.1 Motor dysfunction in Y A C 1 2 8 mice 43 3.2.2 Cognitive deficits in Y A C 1 2 8 mice 47 3 .2 .2 .1YAC128 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 of 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 of 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 of 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 of mutant huntingtin in Y A C 1 2 8 mice 71 3.2.3.5 Transglutaminase activity is selectively increased in the forebrain of 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 , 80 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 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 O F H U N T I N G T O N D I S E A S E 94 5.1 Genetic treatment of Y A C 1 2 8 mice by over-expression of wi ld type huntingtin 94 5.1.1 Rationale for treatment with wi ld type huntingtin 94 5.1.2 Generation of Y A C 1 2 8 mice that over-express wild type huntingtin 94 5.1.3 Effect of wi ld type huntingtin on neuropathology in Y A C 1 2 8 mice 96 5.2 Pharmacologic treatment of Y A C 1 2 8 mice with cystamine 98 v 5.2.1 Rationale for treatment with cystamine 98 5.2.2 Delivery of therapeutic agent: cystamine 98 5.2.3 Effect of cystamine on neuropathology 99 5.2.4 Effect of cystamine on motor impairment....... 102 6.0 D I S C U S S I O N 105 6.1 The Y A C 7 2 mouse model of Huntington disease 106 6.2 The Y A C 1 2 8 mouse model of 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 of Huntington disease 108 6.2.3 Decreased survival in Y A C 1 2 8 mouse model of Huntington disease 112 6.2.4 Selective degeneration in Y A C 1 2 8 mouse model of Huntington disease 114 6.2.5 Mechanism of selective degeneration in the Y A C 1 2 8 mouse model of Huntington disease 117 6.2.6 Testicular degeneration in the Y A C 1 2 8 mouse model of Huntington disease 120 6.2.7 Comparison of 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 of huntingtin influences weight 128 6.3.3 Possible contribution of wi ld type huntingtin to weight loss in Huntington disease 129 6.4 Treatment of Y A C 1 2 8 mice with the over-expression of wild type huntingtin 131 6.5 Treatment of 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 of Huntington disease.133 6.5.2 Cystamine treatment in the Y A C 1 2 8 mouse model of Huntington disease 134 6.5.3 Mechanism responsible for beneficial effects of cystamine in Huntington disease 136 6.5.4 Implications for treatment of Huntington disease 137 6.6 Contribution of loss of 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 L I S T O F T A B L E S Table 1.1 Mouse models of Huntington disease 7 Table 1.2 Summary of Treatment Trials in Genetic Mouse Models of Huntington Disease .15 Table 1.3 Decreasing wi ld type huntingtin levels results in phenotypic abnormalities 18 Table 3.1 Summary of 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 of Huntington disease 116 Table 6.2 Comparison of Y A C 128 mouse model with human Huntington disease 124 Table 6.3 Criteria based comparison of Y A C 128 mice to other H D mouse models 125 vn L I S T O F F I G U R E S Figure 1.1 Model for the contribution of loss of huntingtin function to Huntington disease. 24 Figure 3.1 Motor function in Y A C 7 2 mice 42 Figure 3.2 Neuropathology in Y A C 7 2 mice 44 Figure 3.3 Motor deficits in Y A C 128 mice 46 Figure 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 Figure 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 Figure 3.8 Y A C 128 mice show cognitive deficits in the reversal phase of the swimming T-maze test 58 Figure 3.9 Cognitive deficits are primarily responsible for increased latencies to reach the platform in swimming tests 60 Figure 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 of Y A C 128 mice 65 Figure 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 Figure 3.15 Down-regulation of striatal D A R P P - 3 2 expression in Y A C 128 mice 70 Figure 3.16 Selective neuropathology in Y A C 128 mice is not correlated with mutant huntingtin expression 72 Figure 3.17 Selective nuclear localization of mutant huntingtin in the brain of Y A C 128 mice 75 Figure 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 v i i i \ Figure 3.20 Expression of mutant huntingtin results in testicular degeneration in Y A C 128 mice 81 Figure 4.1 Over-expression of wi ld type huntingtin protects neurons from kainic acid toxicity in the hippocampus 85 Figure 4.2 Over-expression of w i ld type huntingtin protects neurons from quinolinic acid toxicity in the striatum 87 Figure 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 Figure 4.5 W i l d type huntingtin influences organ weight 93 Figure 5.1 Generation of Y A C 128 mice that over-express wild type huntingtin 95 Figure 5.2 Over-expression of wi ld type huntingtin in Y A C 128 mice results in mild improvements in striatal neuropathology 97 Figure 5.3 Cystamine treatment ameliorates striatal neuropathology in Y A C 128 mice 100 Figure 5.4 Cystamine treatment does not impact motor dysfunction in Y A C 128 mice 103 Figure 6.1 Natural history of abnormalities in Y A C 128 mice 123 ix L I S T O F A B B R E V I A T I O N S Analysis of 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 (CBP) Cytomegalovirus ( C M V ) Decibel (dB) Diaminobenzidine ( D A B ) Dopamine- and cAMP-regulated phosphoprotein, 32 kDa ( D A R P P -Eicosapentaenoic acid (EPA) Ethylenediamine tetra-acetic acid ( E D T A ) Figure (Fig.) Gamma-aminobutyric acid ( G A B A ) Huntingtin (htt) Huntingtin associated protein (HAP) Huntingtin interacting protein (HIP) Huntingtin interacting protein protein interactor (HIPPI) Huntington disease (HD) Hypoxanthine phosphoribosyltransferase gene (HPRT) Kainic acid ( K A ) Kilo-basepair (kb) Kilodalton (KDa) Magnetic resonance imaging (MRI) Magnetic resonance spectroscopy (MRS) Messenger R N A ( m R N A ) N-acetyl aspartate ( N A A ) Neuron restrictive silencer element (NRSE) N-methyl-D-aspartate ( N M D A ) Nuclear Corepressor protein (NCoR) Nuclear export signal (NES) Nuclear localization signal (NLS) Phenyl methyl sulfonyl fluoride (PMSF) Phosphate buffered saline (PBS) Polymerase chain reaction (PCR) Postsynaptic density-95 (PSD-95) Pre-pulse inhibition (PPI) Quinolinic acid (QA) Repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF) Revolutions per minute ( R P M ) R N A interference ( R N A i ) Specificity protein 1 (Spl) Standard error of the mean (SEM) Suberoylanilide hydroxamic acid ( S A H A ) T A T A binding protein (TBP) Tdt-mediated dUTP-biotin nick end labeling ( T U N E L ) Tissue transglutaminase (tTG) Transglutaminase (TG) Trichloroacetic acid ( T C A ) Wild-type (WT) Yeast artificial chromosome ( Y A C ) x i P R E F A C E 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 of the experiments contained in this thesis, analyzed all of the results and composed all of 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 of the hippocampus and cerebellum and performed western blots measuring the levels of huntingtin in mice. Ge L u perfused all of the animals for which neuropathology was assessed and injected quinolinic acid into the striatum of mice. Photographs of hippocampal damage following delivery of 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 Vog l . xn P U B L I C A T I O N S F R O M W O R K I N T H I S T H E S I S 1. V a n Raamsdonk J M , Pearson J, Rogers D , Bissada N , Vog l A W , Hayden M R Leavitt B R (2005). Loss of wi ld type huntingtin influences motor dysfunction and survival in the Y A C 1 2 8 mouse model of Huntington disease. Hum Mol Gen 10(14): 1379-1392. . 2. V a n Raamsdonk J M , Pearson J, Slow EJ, 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 of Huntington's Disease. J. Neurosci 25(16): 4169-80. 3. Slow, EJ , V a n Raamsdonk J M , Rogers D , Coleman S H , Graham R K , Deng Y , Oh 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 of Huntington disease. Hum Mol Genet 12(13): 1555-1567. 4. Pinto JT, V a n Raamsdonk J , Leavitt, B R , Hayden M R , Krasnikov B F , Cooper A J L (2005). Treatment of Y A C 128 Mice and Their Wild-Type Littermates with. Cystamine Does not Lead to Its Accumulation in Plasma or Brain: Implications for the Treatment of Huntington Disease. J. Neurochem. 94(4): 1087-101. 5. V a n Raamsdonk 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 of Huntington's Disease. J. Neurochem. (in press). 6. V a n Raamsdonk J M , Pearson J, Rogers D A , L u G , Hayden M R , Leavitt B R . Ethyl-E P A Treatment Improves Motor Dysfunction, but not Neurodegeneration in H D Mice . Submitted to Experimental Neurology (in press). x i i i M A N U S C R I P T S F R O M W O R K I N T H I S T H E S I S 1. Leavitt B R * , V a n Raamsdonk 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 Raamsdonk J M , Murphy Z , Slow E J , Leavitt B R , Hayden M R Selective Degeneration and Nuclear Localization of Mutant Huntingtin in the Y A C 1 2 8 Mouse Model of Huntington Disease. Submitted to Human Molecular Genetics. 3. V a n Raamsdonk J M , Pearson J, Rogers D A , Bissada N , Hayden M R , Leavitt B R . Over-Expression of W i l d Type Huntingtin Does Not Prevent Striatal Atrophy in the Y A C 1 2 8 Mouse Model of Huntington Disease. Submitted to European Journal of Neuroscience. 4. V a n Raamsdonk J M , Murphy Z , Gibson B , Pearson J, L u G , Leavitt B R , Hayden M R . Effect of 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 THESIS 1. Van Raamsdonk J M , Murphy Z , Slow EJ, Leavitt B R , Hayden M R Selective Degeneration and Nuclear Localization of Mutant Huntingtin in the Y A C 1 2 8 Mouse Model of 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 (HD). International Society of Neurochemistry meeting. Innsbruck, Austria (August, 2005) 3. Van Raamsdonk J M , Pearson J, Rogers D , Bissada N , Vogl A W , Leavitt B R , Hayden M R . Levels of wi ld type huntingtin expression modulate motor dysfunction and survival in the Y A C 1 2 8 mouse model of 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 of mutant huntingtin in human Huntington's disease brain is directly replicated in Y A C 1 2 8 mouse model. American Society of Human Genetics, Toronto, O N , Canada (October, 2004). 5. Van Raamsdonk J M , Pearson J, Slow E , Leavitt B R , Hayden M R , Van Raamsdonk J M . Impaired lifespan and cognitive dysfunction in the Y A C 1 2 8 mouse model of Huntington's disease. Hereditary Disease Foundation Huntington's Disease 2004: Change Advances and Good News ( C A G ) n Huntington's Disease Research Conference, Cambridge, M A , U S A (August, 2004). 6. Van Raamsdonk J M , Rogers D , Pearson J, Lu G , Slow EJ , Hayden M R , Leavitt B R . Pre-clinical trials of experimental therapeutics in the Y A C 1 2 8 transgenic mouse model of Huntington's disease. Hereditary Disease Foundation Huntington's Disease 2004: Change Advances and Good News ( C A G ) n Huntington's Disease Research Conference, Cambridge, M A , U S A (August, 2004). 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 ) n Huntington's Disease Research Conference, Cambridge, M A , U S A (August, 2004). 8. Van Raamsdonk J M , Leavitt B R , Slow E , Hayden M R . Therapeutic Trials in the Y A C 1 2 8 Mouse Model of 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 , Roy S, Raymond L A , Nicholson D W , Hayden M R . Pro-Survival Effects of W i l d Type Huntingtin. Gordon Conference on C A G Triplet Repeat Disorders. Barga, Italy (May, 2003) xv 10. V a n Raamsdonk J M , Leavitt B R , Slow E , Hayden M R . Therapeutic Trials in the Y A C 1 2 8 Mouse Model of Huntington's Disease. Molecular Mechanisms of Neurodegeneration Meeting. Mi lan , Italy (May, 2003) 11. Leavitt B R , V a n Raamsdonk J , Slow E, Hayden M R . Experimental Therapeutics in Transgenic Mouse Models of Human Neurodegenerative Diseases. C I H R new investigators meeting. 12. Slow E , V a n Raamsdonk 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 Model . Hereditary Disease Foundation Huntington's Disease 2002: Change Advances and Good News ( C A G ) n Huntington's Disease Research Conference(Hereditary Disease Foundation General Meeting), Cambridge, M A , U S A (August, 2002). 13. Leavitt B R , V a n Raamsdonk J , Slow E, Devon R S , Simpson E M , Hayden M R . Cautionary "Tails": Genetic Factors Affecting the Behavioral Phenotype of the Y A C Transgenic Mouse Model of Huntington Disease. Huntington's Disease 2002: Change Advances and Good News ( C A G ) n Huntington's Disease Research Conference(Hereditary Disease Foundation General Meeting), Cambridge, M A , U S A (August, 2002). 14. Slow E, V a n Raamsdonk J , Rogers D, Graham R, Bissada N , Oh 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 Model . Huntington Disease Society of America-Coalition for the Cure, Chicago, IL, U S A (Apr i l , 2002) 15. Slow E , V a n Raamsdonk 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 of a Motor Co-ordination Deficit in the Y A C 128 Huntington Disease Mouse Model . Canadian Genetic Diseases Network Meeting, Montreal, Quebec, Canada (Apri l , 2002). xvi A C K N O W L E D G E M E N T S 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 of my manuscripts. I would also like to thank the remainder of 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 of 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 of her assistance. I would like to thank Zoe Murphy, another super-technician, who has helped me considerably in the later stages of my degree by taking care of all of the odds and ends of 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 of 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 of 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 of 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, CJ Opina, Tara Davidson, Y u Deng, Jeff Carroll, Jen Schmidt, Jamie Lepard, Claudia Schwab and Ed 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 of family whenever necessary and also my little sisters, Raeann, Rachelle and Remei, for providing a not so local dose of family. Last, but not least, I would like to thank Maiya Geddes for her love and for making me happy throughout the ups and downs of my thesis work. xvi i D E D I C A T I O N To Maiya for her love, support and the happiness she brings me. xviii 1 . 0 INTRODUCTION Huntington disease (HD) 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 of 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 of 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 of H D is chorea. This excessive, involuntary movement is seen in early phases of 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 of H D is dependent on the presence of specific motor signs on the neurologic exam, persons carrying the disease mutation can demonstrate cognitive dysfunction prior to the onset of 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 Boo 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 of H D has been measured as 53% compared to controls using magnetic resonance imaging (MRI) or post-mortem volume displacement (Rosas et al, 2003; Halliday et al, 1998). Although the magnitude of atrophy is less than in the striatum, significant volume losses of 41% and 23% were reported in the globus pallidus and cortex of 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 of 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 of the hippocampus (Spargo et al, 1993). ln 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 Kowal l , 1990; Hedreen et al, 1991). In addition to cell loss, the striatum shows several markers of inflammation including astrocytosis, microgliosis and complement activation (Singhrao et al, 1999). Microscopically, H D is characterized by the formation of neuronal protein aggregates. A role for aggregate formation in H D pathology was originally proposed based on the appearance of aggregates in a mouse model of H D which preceded the development of neurologic abnormalities (Davies et al, 1997). Re-examination of 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 of aggregates is much higher in the most severe form of H D -juvenile H D (DiFigl ia 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 of 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 HD (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 HD 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 HD 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 HD 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-terminal fragment models 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 Generation Advantages Disadvantages Examples Neurotoxin 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) N-terminal fragment 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) Knock-in 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 5 0 (White et al, 1997) HdhQ 9 2, HdhQ' 1 1 (Wheeler et ai, 2000) CAG71, CAG94 (Levine et al, 1999) CHL2 - Hdh<CAG>150(Lin et al, 2001) CAG140 (Menalled et al, 2003) Full Length 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) The phenotypic similarity of 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 of H D . The R6/1 and R6/2 mouse models were generated through the microinjection of a 1.9 kb genomic fragment of human D N A containing exon 1 from the H D gene with approximately 130 C A G repeats and about 1 kb of the 5' untranslated region (Mangiarini et al, 1996). Initial characterization of R6/2 mice revealed a constant tremor, age onset weight loss, a decreased survival of 10-13 weeks and a non-specific 19% decrease in brain volume in absence of 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 of learning impairments beginning at 3-4 weeks of age which precede the onset of motor deficits at about 5 weeks of age (Carter et al, 1999; Lione et al, 1999). Neuronal intranuclear inclusions are present throughout the brain of 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 of htt with 82 C A G repeats under the control of the prion promoter (Schilling et al, 1999). N171-82Q 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 of a nuclear localization signal (NLS) to the N171-82Q construct did not alter the phenotype of the mice, suggesting that the neuronal dysfunction in these animals is caused by the action of 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 of H D was developed to determine whether cessation of mutant htt expression could reverse the symptoms of the disease. These mice were created to express exon 1 of 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). Mice 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 of 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 of 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 of 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 of human H D . However, changes in the brain are generally not selective, most likely as a result of not expressing the full length protein and missing all or part of the endogenous H D gene promoter. In addition, none of 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 of polyglutamine toxicity but are not ideal for studying H D pathogenesis because the protein context of 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 of H D have been generated through the targeted insertion of 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 of wild-type htt and one copy of mutant htt. The first H D knock-in mouse was generated with 50 C A G repeats - a repeat size at the high end of 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 of mutant htt (Wheeler et al., 2000) and late onset aggregation (Menalled et al., 2002). None of the models showed evidence of 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 of mutant htt, aggregation and astrocytosis (Lin et al., 2001; Menalled et al., 2003). Overall, knock-in models of H D reproduce some of the changes present in H D but the mildness of the phenotypes observed and the absence of neuronal loss in these mice limit their utility in therapeutic trials for H D . Nonetheless, the accurate replication of 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 of 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 of the full length gene. The full length c D N A transgenic mouse model of H D was created to express mutant htt with 89 glutamines under the control of 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 HD that contain a mutant HD gene with 24 kb of sequence 5' of the HD gene transcript (Hodgson et al, 1999). This approach was chosen to facilitate the inclusion of the entire HD 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 HD 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 HD 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 HD 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 HD 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 HD 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. Of 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 YAC72 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 HD 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 RNA. 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 wi l l be delivered, considerations that are critical to the design of therapeutic trials include: when to deliver the treatment; where to deliver treatment and how much treatment to deliver. While the time course of events that lead to neuronal dysfunction and death in H D is currently unknown, it is promising that a recent conditional model of H D suggests that the progression of the disease can be stopped and the deficits reversed by eliminating expression of mutant htt (Yamamoto et al, 2000). The most obvious site for the delivery of 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 of treatment must balance the potential toxicity of excessive treatment with the reduced effect of low levels of treatment. The exact mechanism of pathogenesis in H D is currently unknown. While many of the steps have been elucidated, the order of these steps remains uncertain. The pathogenesis may follow a linear progression, in which case, prevention of 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 of disease phenotypes. The earliest step in H D pathogenesis is the expression of mutant htt which is sufficient to cause H D . Subsequently, the following events are thought to be involved: misfolding of mutant htt, cleavage of 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 of mutant and wild-type htt (Davies et al, 1997; DiFig l ia et al, 1997), nuclear localization of mutant htt (Peters et al, 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, 2002; Gines et al, 2003), increased sensitivity to excitotoxicity (Young et al, 1988; Zeron et al, 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 of neurotrophic factors (Ferrer et al, 2000; Zuccato et al, 2001; Zuccato et al, 2003) and loss of wild-type htt function (Cattaneo et al, 2001). While some of 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 anti-oxidant 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 was riot prevented 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 Mechanism(s) Model Benefits Reference Ascorbate Restore ascrobate levels R 6 / 2 M i l d I motor symptoms Rebec etal., 2003 Paroxetine Antidepressant; inhibits uptake o f serotonin N 1 7 1 - 8 2 Q t surv iva l 10% t body weight , rotarod J. ventr icular enlargement D u a n e / o / . , 2 0 0 4 Envi ronmenta l Enr ichment R6/1 t motor function I c lasp ing | forebrain vo lume 13% V a n D e l l e n e r a/., 2000 Envi ronmenta l Enr ichment R6/1 T rotarod, B D N F , D A R P P - 3 2 Spires etal., 2004 Envi ronmenta l Enr ichment N 1 7 1 - 8 2 Q T rotarod 7 3 % | body weight S c h i l l i n g et al., 2004 C o n g o red Prevent aggregation R 6 / 2 t surv iva l 16% t rotarod, body weight I aggregation, c lasp ing Sanchez et al., 2003 Trehalose Prevent aggregation R 6 / 2 T surv iva l 11% t body weight , brain weight, rotarod J, ventr icular enlargement, aggregates T a n a k a e / a / . , 2 0 0 4 Suberoylani l ide hydroxamic acid ( S A H A ) Restore transcription R 6 / 2 t rotarod H o c k l y etal., 2003 Phenylbutyrate Restore transcription N 1 7 1 - 8 2 Q t surv iva l 2 3 % t cerebral vo lume 2 2 % | neuronal size 115% Gard ian etal., 2004 S o d i u m butyrate Restore transcription R 6 / 2 t surv iva l 2 1 % T rotarod 3 5 % t brain weight 16% t neuronal size 9 8 % Fen-ante et al., 2003 M i t h r a m y c i n Restore transcription (Ant i tumor antibiotic) R 6 / 2 t surv iva l 2 9 % t brain weight 2 3 % | neuronal size 1 3 1 % Ferrante et al, 2004 Dominan t Negat ive Caspase-1 L i m i t apoptosis R6 /2 t surv iva l 2 0 % t rotarod, body weight I aggregation Ona etal., 1999 z V A D - f m k L i m i t apoptosis R 6 / 2 T su rv iva l 2 5 % t rotarod O n a etal., 1999 M i n o c y c l i n e L i m i t apoptosis R 6 / 2 t surv iva l 14% t rotarod C h e n et al., 2000 Y V A D - c m k + D E V D -fmk L i m i t apoptosis R 6 / 2 t surv iva l 12% t rotarod 17% C h e n etal., 2000 B c l - 2 L i m i t apoptosis R 6 / 2 f surv iva l 10% j rotarod Z h a n g etal, 2003 Tauroursodeoxychol ic ac id L i m i t apoptosis An t iox idan t Improve mitochondr ia l function R 6 / 2 I T U N E L posi t ive striatal neurons t striatal vo lume 12% | aggregates t rotarod, act ivi ty K e e n e d a/., 2002 Creat ine Res tor ing cel lular energy levels Inhibits M P T R 6 / 2 t surv iva l 17% t rotarod 7 7 % t brain weight 17% t neuronal size - 5 7 % t body weight I aggregation Ferrante e ra / . , 2000 Creat ine Restor ing cel lular energy levels Inhibits M P T R 6 / 2 t surv iva l 14% t rotarod 2 3 % t neuronal size 6 0 % T body weight 19% t brain weight 17% i aggregation Dedeoglu et al, 2003 Creat ine Res tor ing cel lular energy levels Inhibits M P T N 1 7 1 - 8 2 Q t surv iva l 19% | rotarod, body weight t brain weight 13% I aggregation Andreassen et al, 2001 15 Table 1.2. Summary of Treatment Trials in Genetic Mouse Models of Huntington Disease(continued) Treatment Mechanism(s) Model Benefits Reference Riluzole Anti-excitotoxic R6/2 t survival 10% t body weight J. hyperactivity I size of aggregate Schiefer era/., 2002 Coenzyme QI 0 and remacemide Improve mitochondrial function Anti-excitotoxic N17I-82Q t rotarod 28% Schilling eta!., 2001 Coenzyme Q10 and remacemide Improve mitochondrial function Anti-excitotoxic R6/2 t survival 32% j rotarod 62% j body weight 20% t brain weight 18% t neuronal size 87% J. aggregation Ferrante et al., 2002 Coenzyme Q10 and remacemide Improve mitochondrial function Anti-excitotoxic NI71-82Q f survival 17% f body weight 8% Ferrante et al., 2002 a-Lipoic Acid Anti-oxidant R6/2 t survival 7% Andreassen et al., 2001 a-Lipoic Acid Anti-oxidant NI71-82Q t survival 8% t body weight Andreassen et al., 2001 BN82451 Anti-oxidant R6/2 t survival 15% j striatal volume 22% 1 neuronal size 65% J. aggregation Klivenyi etal., 2003 Essential Fatty Acids Membrane integrity R6/1 M i l d I motor symptoms Clifford etal., 2002 Cystamine Inhibits TG activity Anti-apoptosis Anti-oxidant R6/2 | survival 19% j rotarod 27% t body weight 15% t brain weight 20% f neuronal size 61 % | aggregation Dedeoglu et al, 2002 Cystamine Inhibits TG activity Anti-apoptosis Anti-oxidant R6/2 T survival 12% Mild J, motor symptoms f body weight Karpuj et al., 2002 Cystamine Inhibits TG activity Anti-apoptosis Anti-oxidant R6/2 ] dopamine D2 receptor function I size of aggregates t neuronal morphology Wang et al., 2005 Tissue T G knockout Eliminate tissue TG activity R6/1 t survival 12% T aggregation i degenerating neurons 60-70% t rotarod Mastroberardino et ai, 2002 Tissue T G knockout Eliminate tissue T G activity R6/2 t survival 20% t aggregation 30-35% t rotarod Bailey and Johnson, 2004 Dietary Restriction Decrease ROS Increase B D N F N171-82Q | survival | body weight t rotarod | ventricular enlargement J. aggregation Duan etal., 2003 Striatal transplantation R6/2 Mi ld I motor symptoms Dunnett etal., 1998 Hsp70 Promote proper folding and elimination of mutant htt R6/2 t body weight Hansson et ai, 2003 R N A Interference Decrease mutant htt levels N171-82Q I aggregation t rotarod •T gait Harper etal., 2005 G D N F Neuroprotection R6/2 no benefit Popovic etal., 2005 CGS21680 A 2 A adenosine receptor antagonist R6/2 t rotarod I ventricular enlargement I size of aggregates Chou era/., 2005 Lithium Chloride Limit apoptosis Anti-excitotoxic R6/2 t rotarod 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 Phenotype Reference Heterozygous for targeted inactivation of HD gene 50% Hyperactivity, cognitive defects, neuronal loss Nasire/a/., 1995 O'Kusky et al, 1999 Adult Inactivation of htt in testis and forebrain 10-16% in adult brain Reduced size, clasping, hypoactivity, neuronal degeneration Dragatsis et al, 2000 H d h n e o 5 0 Q h o m o z y g o t e s -33% Gross brain abnormalities, do not survive more than 2 days White etal, 1997 H d h n e o 5 0 Q h e m i z y g o t e s -16% Gross brain abnormalities, embryonic lethal White et al, 1997 Homozygous for the targeted inactivation of the HD gene None Embryonic Lethal 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 wild-type 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 wild-type 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 of extensive apoptosis leading to embryonic lethality in mice homozygous for the targeted inactivation of 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 of 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 of 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 of wild-type htt was thought to be downstream of cytochrome c release from the mitochondria (Rigamonti et al, 2001). These findings of in vitro protection by wild-type htt were extended to show that wild-type htt could protect against the toxicity of polyglutamine expansion. Cells transfected with wild-type htt were shown to be more resistant to cell death caused by the expression of 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 of 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 of 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 of two copies of wild-type htt completely restored testicular morphology in the Y A C 7 2 mice indicating that the expression of 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. HD 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 HD. 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 of 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 of wild-type htt and pharmacologic treatment with cystamine. A genetic treatment and a pharmacologic treatment were selected to demonstrate that both types of 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). Two separate lines of 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 of the University of British Columbia's animal care committee. 2.2 Behavioural assessments Mice were tested during the light phase of 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 of the mice. 2.2.1 Rotarod test of motor coordination Mice were trained at 2 months of age with three trials per day over three days at 24 R P M on the rotarod ( U G O Basile). On 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. Mice were tested bimonthly from 2 to 12 months of 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). Mice were removed from the rotarod after falling or reaching a maximum time of 60 seconds on the fixed speed test or 300 seconds on the accelerating test. The amount of 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 of mice were trained at 2 months, 7 months or 12 months of age with 3 trials per day spaced 2 hours apart for three days. 2 month old mice were trained at a fixed speed of 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 of mice during training. Mice trained at 2 months and 7 months were tested bimonthly and monthly respectively until 12 months of age. A t 12 months of age, the performance of 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). Activi ty was measured as the total number of 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 of 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 of 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 of activity in the first trial. The activity of mice in the open field was assessed in the dark during the light cycle and was measured automatically as the number of photobeam breaks during each trial. Mice that were naive to the open field chamber were given 5 trials of 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. On the first trial, activity was recorded for the duration of the 30 minutes period in order to assess intrasession habituation in 5 minute intervals. The activity during the first 10 minutes of each of 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 of 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 of a linear swimming chamber (76 cm X 13 cm; water depth: 9 cm; platform: 6 cm X 13 cm) facing away from an escape platform. Mice are trained to reach the platform in the shortest amount of time in order to escape from the water. Completion of this task involves learning and remembering the location of the platform or the route followed to reach the platform. On subsequent trials, mice must plan to turn around immediately for the shortest route to the platform. The amount of 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 of 0 while swimming away from the platform was initially given a score of 1. Mice were trained at 2 months of age with 3 pairs of 2 consecutive trials spaced 2 hours apart for 2 days. Mice were tested bimonthly with 3 tests per day spaced 2 hours apart until 12 months of 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 of 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 of learning of memory. If mice remember that there is no biologic consequence associated with a repeated loud sound they wi l l show decreased startle in response. Prior to testing, the sensitivity of the two startle chambers was calibrated using a 28 vibrating standardization unit at 700 volts (San Diego Instruments). Mice 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) of a 40 msec 120 dB noise burst (pulse alone). Subsequently, the mice experienced 8 blocks of 6 trials (48 trials total), each block consisting of 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 dB above background noise). The order of trials within each block of 6 trials was pseudo-randomized and 4 of the 8 blocks contained an extra pulse alone trial. The mice then received another 6 trials (block 10) of 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 of 4 blocks of 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 of 6 trials per pre-pulse as follows: PPI = [(pulse alone startle)-(pre-pulse + pulse startle)]/pulse alone startle. 2.2.7 Beam crossing test Mice were trained at 2 months of age with three sets of 2 consecutive trials. For each trial mice were placed on the beam facing a darkened chamber on the other end of the beam. The lights in the room were turned off 10 minutes prior to testing. A bright light was pointed at the starting end of the pole. The amount of time it took the mouse to reach the darkened chamber was recorded as well as the number of falls, i f any. Mice were tested monthly from 2 to 10 months of 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 of 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 of 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 of 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 of a water-filled T-maze with an escape platform located in the right arm of the maze (T-maze dimensions - arms: 38 cm X 14 cm, water depth: 7 cm, platform: 10 cm X 14 cm). Mice must learn to turn right upon reaching the top of 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 of 0 while swimming left was given a score of 1. Mice received 4 trials per day spaced 45 minutes apart for 3 days. To successfully complete this task, mice must remember either the location of 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 of the maze likely relies on internal rather than external cues. Following one day of rest, a reversal phase to the swimming T-maze test was included to assess the ability of the mice to replace a previously learned strategy. For this test, the platform was switched to the left arm of 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 of 0 while initially swimming away from the platform was given a score of 1. In addition, the total number of arm entries was noted for each mouse. Mice received 4 trials per day spaced 45 minutes apart for 3 days. After 3 days of reversal testing, the swimming speed of the mice was measured by blocking the stem of'the T-maze and measuring the amount of time the mice take to swim the length of the top of the T to reach the platform. 30 Mice 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 Mice were injected with heparin, terminally anesthetized by intraperitoneal injection of 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 PBS 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 of mice were followed up until 12 months of age. The cause of 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 (Log 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 of approximately 1 um were cut and stained with toluidine blue for examination of testicular morphology. 2.4 Neuropathological assessments Brains from mice perfused with 3% paraformaldehyde were infiltrated with sucrose (25% in PBS) 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 of 25 um coronal sections spaced 200 um apart were stained with NeuN for determination of brain structure volumes. Endogenous peroxidase activity was blocked by incubating sections with phenylhydrazine ( lu l /ml PBS) 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, PBS, 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, PBS) 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 cover-slipped using flouromount mounting media (BDH) . Sections were left a minimum of one day for mounting media to harden. 32 The volumes of the striatum, cortex, globus pailidus, hippocampus and cerebellum were determined using Stereo investigator software (Microbrightfield). Striatal volume was measured from the start of the striatum to the start of the hippocampus. The volume of the entire globus pailidus was measured. Cortical volume was measured from the point where the corpus callosum crosses to the start of the hippocampus. Hippocampal volume was measured from the start of the hippocampus to the point where the C A 3 region thickens and descends. The volume of the cerebellum was measured from a coronal series of unstained sections collected directly onto glass slides. To determine the volume of each structure, the perimeter o f the structure was traced using a 2.5X objective in each included section of the coronal series spaced 200 um apart. Subsequently, the total area of the structure in the included sections was multiplied by the distance between the sections (200 urn) to determine the volume of 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 of neurons in the striatum. Neuronal number was calculated as: (total number of neurons counts X total volume of region)/(total number of sites counted X volume of each site). Hippocampal neuronal numbers were estimated by measuring the volume of the hippocampal cellular layer and dividing by the average volume of 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 of all clearly defined neurons within a 550 urn X 550 um grid of 25 um X 25 um counting frames with the 100X objective. . . . 33 2.4.4 Striatal DARPP-32 expression For measurement of 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 Cy3-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Inc., 1:5.00). Pictures of mounted sections were taken using MetaMorph Imaging System and the intensity of the fluorescent stain within the striatum was measured. 2.4.5 Nuclear localization of mutant huntingtin Nuclear localization of mutant htt was examined in a series of coronal sections throughout the brain. Sections were stained with polyclonal E M 4 8 antibody as above at a concentration of 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 of either 2 msec or 63 msec for pictures taken with the 10X or 100X objective respectively. For quantification of 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 of 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 of 25 mg/kg. Mice were weighed immediately before injection in order to calculate the appropriate dose. Mice were monitored for seizures for 2 hours following intra-peritoneal injection of K A . Mice were sacrificed 7 days after injection of K A . 34 2.5.2 Delivery of quinolinic acid Quinolinic acid (QA; Sigma) was stereotaxically injected into the striatum of mice at the coordinates - A P : +0.8 mm from Bregma, M L : ± 1.8 mm from midline, D V : -3.5 mm from skull surface. Each mouse was given 1 ul of 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). Mice were sacrificed 7 days after injection with Q A . 2.5.3 Assessment of hippocampal damage Mice 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 of neuronal damage. Fluorojade staining for degenerating neurons was completed on a series of 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 ml distilled water, 72 ul acetic acid, 0.8 mg fluorojade compound (Histo-Chem; Jefferson, AR)) . Sections were then washed four times with water, dried overnight, cleaned with xylene and mounted with flouromount (BDH) . 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 of alkaline phosphatase conjugated anti-DIG antibody. Detection of antibody binding was accomplished with nitroblue tetrazolium chloride x-phosphate (NBT) 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 NeuN (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 of 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 of the lesion. The number of 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 YAC18/128 mice that express both wild-type and mutant htt transgenes. Successful generation of 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 of 900 mg per litre of water to deliver an approximate dose of 225 mg/kg (Dedeoglu, 2002). Cystamine solution was made fresh weekly. To assess the effect of cystamine on transglutaminase levels, 3 month old Y A C 128 mice were treated with cystamine for a period of 2 weeks prior to sacrifice. Early symptomatic treatment was initiated at 4 months of age while symptomatic treatment began at 7 months of 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 ml , 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 Lowry assay. Proteins were separated by electrophoresis on either 7.5% acrylamide gels or 8% low-bis acrylamide gels when allelic separation was necessary. 100 ug of 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. Al le l i c separation gels were run for a total of 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 (MAB2166 , 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 T P B S before enhanced chemiluminscent ( E C L ) detection and development of the film. Protein levels were quantified by band density measured with Quantity One software (BioRad). 2.7.2 Measurement of transglutaminase activity For measurement of 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 Tr i s -HCl (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 of 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 Tr i s -HCl (pH 7.5), 5 m M D T T , 15 m M 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 (DMC)] in a total volume of 100 ul at 37°C for 1 hr. Nonspecific incorporation of [3H]putrescine was measured by performing the reaction in the absence of CaC12 and in the presence of 5 m M E G T A . The reaction was terminated by the addition of 600 ul of 10% (w/v) ice-cold trichloroacetic acid (TCA) . 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 of 5% (w/v) T C A and centrifuged at 14,000 x g for 20 min at 4°C. 250 ul of 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 of 500 u M GTPyS (Sigma-Aldrich, St. Louis, M O ) . Tissue T G is inhibited by G T P and accounts for the majority of T G activity in mouse brain (Bailey et ai, 2004). 2.8 Statistical Analysis Data are given as the mean ± standard error of the mean (SEM). 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 of age/trial/day/treatment and the interaction of genotype and age/trial/day/treatment as well as between subjects effect of 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 of the Student's t-test, the significance level was adjusted to account for errors of multiple measurements (significance level = 0.05/(number of measurements) eg. 6 trials: significance level = 0.05/6 = 0.008). Simple comparisons of one variable between two 38 genotypes were assessed by Student's t-test with a significance level of 0.05. Comparisons between more than two groups for one outcome measure were assessed by one way A N O V A . Comparisons of categorical data were performed with the Chi-square test. 39 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 M E A S U R E S 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 Characterizat ion of the Y A C 7 2 mouse model of Huntington 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 Motor dysfunction in 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 ( U 3 ) = 0.4, p = 0.5; 24 R P M : 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 ) = 0.1, p = 0.7). Patients with H D generally display an excess of 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 of 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 of beam crosses as a measure of 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,i3 ) = 0.0, p = 0.9). We 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 of 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 of age (Fig. 3.1 panel F ; genotype: F ( U 3 ) = 2.5; p = 0.15). Next, we examined the ability of Y A C 7 2 mice to swim down a linear swimming tank to reach an escape platform. In this test of 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,i3) = 6.3, p = 0.03; W T : 2.7 ± 0.3 seconds, 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 of 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 70 60 50 -{ 40 30 20 10 c ^ _ 1600 to | 1400 6 1200 E m 800 12 R P M 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 Age(Months) 34 R P M 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 Age(Months) 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 Age(Months) 8 I I  6 "rl E — ^ " 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 Age(Months) B D H 1 70 60 50 40 30 20 10 _ 70 o 60 EA 50 1 re 40 r2 30 § 20 2 10 8 20 to E 16 to v m 1 2 CO I 8 o a A CD E 0.30 1 - f 0.25 oo E I f 0-20 Q.T3 £ > 0.15 o > 0.10 • W T Y A C 7 2 24 R P M 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 Age(Months) 40 R P M 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 Age(Months) 2.5 3.5 4.5 5.5 6.5 7.5 8.5 Age(Months) 9.5 2.5 3.5 4 .5 5.5 6.5 7.5 8.5 9.5 Age (Months ) Figure 3.1 Motor function in YAC72 mice. Behavioural analysis was carried out on a cohort of YAC72 mice and WT littermates from 2 to 10 months of age to determine if YAC72 mice exhibit motor dysfunction. In the rotarod test of motor coordination there was no difference in performance between YAC72 and WT mice at 12 RPM (panel A; genotype: F ( M 3 ) = 0.4, p = 0.5), 24 RPM (panel B; genotype: F ( u 3 ) = 0.0, p = 0.9), 34 RPM (panel C; genotype: F ( U 3 ) = 0.1, p = 0.8) or 40 RPM (panel D; genotype: F (i. l 3) = 0.1, p = 0.7). There was also no difference in open field activity (panel E; genotype: F(i, 1 3) = 0.0, p = 0.9) or beam crossing (panel F; genotype: F ( l i | 3 ) f= 2.5; p = 0.15) at any time point. In contrast, YAC72 mice took significantly longer to swim to an escape platform in a linear swimming test (panel G; genotype: F ( 1 j 3 ) = 6.3, p = 0.03; WT: 2.7 ± 0.3 seconds, YAC72: 3.7 ± 0.3 seconds). Walking patterns of YAC72 mice were normal. Quantification of forepaw stride length revealed equal stride length in YAC72 and WT mice (panel H; genotype: F ( 1 , | 3 ) = 0.4, p = 0.6). Thus, overall YAC72 mice have almost equivalent motor function to WT mice except for a mild deficit in swimming. N = 8 WT, 7 YAC72. Error bars indicate SEM. 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; WT : 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; WT : 12.5 ± 0.2 mm 3 , Y A C 7 2 : 11.7 ± 0.3 mm 3 , p = 0.03). Based on the difference and variability in striatal volume, 21 mice would be required in a therapeutic trial to have an 80% chance of detecting a 50% 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 Huntington 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 I * 1 I • WT • YAC72 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 . 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 mm3, YAC72: 11.7 ± 0.3 mm3, 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. _ 1.32E+10 | 1.28E+10 1 1.24E+10 | 1.20E+10 | 1.16E+10 "5 C/D 1.12E+10 1.08E+10 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^is) = 10.7, p = 0.005; accelerating - genotype: F(i j 5 ) = 16.1, p < 0.001). A t 2 months of age, Y A C 128 mice are able to stay on the rotarod as long as W T mice but by 4 months of 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 of performance (24 R P M - age X genotype: F (5 j5 ) = 7.1, p < 0.001; 40 R P M - age X genotype: F ( 5 ; 7 5 ) = 8.7, p < 0.001; accelerating - age X genotype: F ( 5 > 7 5 ) = 10.2, p < 0.001). Thus, at 4 months of 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-hypo-activity pattern observed in H D patients. Initially, Y A C 128 mice at 2, 6 and 12 months of age were tested in a 10 minute open field trial. A t 2 months of 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 of age, there was no difference between the activity of Y A C 1 2 8 and W T mice (WT: 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 (WT: 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 of mice monthly from 7 to 12 months of age. Open field activity was equal at 7 months of age between Y A C 128 and W T mice but beginning at 8 months of 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 of hypoactivity and not differences in transfer arousal, we examined the activity of a cohort of 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 7 0 o 60 m | 50 2 40 o * 30 i 2 0 .1 10 K 0 350 J . 300 | 250 | 200 °£ 150 I 1°° E 50 h 0 B 400 -| 350 >> > 300 -Act 250 200 150 -** ** 24 RPM 4 6 8 10 12 Age(Months) ^ *** *** *** Accelerat ing 6 8 Age(Months) 10 12 8 9 10 11 Age(Months) 12 g 40 I 30 § 2 0 » 10 i i 0 40 RPM 6 8 Age(Months ) 10 12 8. O 4000 • WT —O-• YAC128 — • -Figure 3.3 Motor deficits in YAC128 mice. Motor function in Y A C 128 and WT 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 RPM (panel A), 40 RPM (panel B) and accelerating (panel C). In each case, YAC128 mice show significant motor deficits compared to WT mice (genotype - 24 RPM: F( | j 5 ) = 17.2, p < 0.001; 40 RPM: F ( 1 , 1 5 ) = 10.7, p = 0.005; accelerating: F ( M 5 ) = 16.1, p < 0.001, N = 8 WT, 9 YAC128). There is no difference in motor performance at 2 months of age between WT and Y A C 128 mice. Subsequently, motor coordination in Y A C 128 mice declines steadily from 4 to 12 months while WT mice show little change (age X genotype - 24 RPM: F ( 5. 75) = 7.1, p < 0.001; 40 RPM: F ( 5.7 5) = 8.7, p < 0.001; accelerating: F(5,75) = 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 WT 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 WT, 10 YAC128). At 6 months, there was no difference in activity between YAC128 and WT mice (WT: 328 ± 22 beam breaks, YAC128: 319 ± 15 beam breaks, p = 0.2; N = 17 WT, 14 Y A C 128). At 12 months, Y A C 128 were significantly hypoactive compared to WT mice (WT: 240 ± 9 beam breaks, YAC128: 194 ± 13 beam breaks, p = 0.01; N = 7 WT, 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 ( , I 0 ) = 12.8, p = 0.005; age X genotype: F ( U 0 ) = 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 WT mice (WT: 7662 ± 483 beam breaks, YAC128: 5643 ± 499 beam breaks, p = 0.01, N = 8 WT, 11 YAC128). Error bars show SEM. ** p < 0.01, ***p<0.001. 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 of motor dysfunction. We examined presymptomatic Y A C 128 mice at 2 months of age as well as symptomatic mice at 8-12 months of age. 3.2.2.1 YAC128 mice show impaired motor learning on the rotarod Motor learning was assessed using the rotarod test of motor coordination. Three separate cohorts of mice were trained on the rotarod at 2, 7 or 12 months of age. The scores for the second and third day of training were recorded as well as the score on a subsequent test day. A t 2 months of age, mice were trained and tested on the rotarod at a fixed speed of 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 of 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 , 5 5 ) = 4.4, p = 0.041). Despite receiving equal amounts of training, W T mice are able to stay on the rotarod for 10 seconds longer than Y A C 128 mice on each of 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 of age the motor coordination of 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 of 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. At 7 and 12 months of age, mice were trained and tested in an accelerating rotarod 47 Age 2 Months Day 2 Training Day 3 Training Test Day B „ 300 | ^ 250 a — o 2> 200 X -B c 2 150 O <]> 2 s 100 50 *** *1 Age 7 Months *** in Day 2 Training Day 3 Training U l i U Test Day Age 12 Months Day 2 Training Day 3 Training Test Day ^ 350 300 2 n, 2 5 0 i 2 tn n — o g> O <D CD Q) • WT • YAC 128 100 50 Age 12 Months Trained at 2 months Trained at 7 months Trained at 12 months Figure 3.4 YAC128 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 WT mice learn the rotarod task more rapidly than YAC128 mice (day: F ( 2 , no> = 43.3, p < 0.001; genotype X day: F ( 2 , , i 0 ) = 7.8, p = 0.001; genotype: F ( l j 5 5 ) = 4.4, p = 0.041; N = 27 WT, 30 YAC128). On the test day, Y A C 128 mice can perform as well as WT mice on the rotarod indicating that their motor coordination is equal to WT 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 WT mice during training or on the test day (day: F (2.58) = 10.6, p < 0.001; genotype X day: F (2.58) = 0.9, p = 0.4; genotype: F ( 1 . 2 9 ) = 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 WT mice are still able to learn the task (day: F ( 2 j 4 0) = 7.7, p = 0.001; genotype X day: F(2,4o) = 7.7, p = 0.001; genotype: F ( l i 2 0 ) = 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 - WT: 272 ± 20 seconds, Y A C 128: 139 ± 14 seconds, p < 0.001; Mice trained at 7 months - WT: 180 ± 15 seconds, Y A C 128: 88 ± 14 seconds, p < 0.001; Mice trained at 12 months - WT: 160 ± 18 seconds, YAC128: 7 ± 24 seconds, p < 0.001). Error bars show SEM. ** p < 0.01, *** p < 0.001. 48 test to a maximum score of 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 ( 2,58) = 10.6, p < 0.001; genotype: F(i,29) = 85.1, p < 0.001). Thus, even after 3 days of 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 of 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 of training (day: F ( 2 , i2) = 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 of the cohorts of mice trained at 2, 7 and 12 months of 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 ( 2,n6) = 15.5, p < 0.001). Nonetheless, all three cohorts at 12 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 of 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 of staying on the rotarod at this late age. Thus, the complete failure of previously untrained Y A C 128 mice to maintain their balance on the rotarod at 12 months of age, even after 3 days of 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 of 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). Two month old presymptomatic and eight month old symptomatic Y A C 128 mice were given a total of five 30 minute open field trials that were divided up into 5 minute intervals. At two months of age, the activity of 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 ; 2 2 ) = 1.3, p = 0.3). Similarly, both groups showed equivalent decreases in activity between repeated open field trials (trial: F ( 4 > 8 8 ) = 39.2, p < 0.001; genotype X trial: F(4,88) = 1.9, p = 0.1; genotype: F ( i > 2 2 ) = 1.5, p = 0.2). A s a result, the extent of 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, 2 2) = 0.3, 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 of age. Next we examined symptomatic Y A C 128 mice at 8 months of 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). Activi ty of both the W T and Y A C 128 mice declined in subsequent intervals in the 30 minute open field trial with the activity of W T mice declining more rapidly than Y A C 128 mice (Fig. 3.5 panel A , B ; interval: F( 5 j i5) = 36.4, p < 0.001; genotype X interval: F( 5 i n 5 ) = 4.5, p < 0.001; genotype: F ( i ; 2 3 ) = 8.1, p = 0.009). The initial difference in activity is eliminated and for the last 4 intervals, the activity of 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 ac t i v i t y o f 8 m o n t h o l d m i c e in an open field was tested f o r 30 m inu te s d i v i d e d into 6 f i v e m inu te in te rva l s . A s m i c e b e c o m e f am i l i a r w i t h the open field they s h o w less exp l o r a t o r y ac t i v i t y . A . W h i l e the ac t i v i t y o f bo th W T m i c e and Y A C 128 m i c e decreases du r i n g the 30 m inu te t r ia l , W T ac t i v i t y dec l i nes at a more r ap i d rate ( i n t e rva l : F ( 5 ; n 5 ) = 36.4, p < 0 .001; geno type X in te rva l : F ( 5 i M 5 ) = 4.5, p < 0.001; geno type : F ( l j 2 3 ) = 8.1, p = 0.009) . In i t i a l l y , Y A C 128 m i c e s h o w s i gn i f i c an t h y p o a c t i v i t y c o m p a r e d to W T m i c e ( Interva l 1 - W T : 190 ± 12 beam breaks , Y A C 1 2 8 : 145 ± 7 beam breaks , p = 0.008) but this d i f f e rence is e l im i na t ed as the W T m i c e habi tuate to the open field chamber . B . T o con t ro l fo r d i f f e rences in base l ine a c t i v i t y l eve l s , a c t i v i t y fo r each in te rva l wa s d i v i d e d b y the l eve l o f ac t i v i t y i n the first i n te rva l . W h e n the in i t i a l l eve l o f a c t i v i t y is con t r o l l ed for, W T m i c e s h o w a greater decrease i n a c t i v i t y f r o m the first in te rva l s tar t ing at in te rva l 3 (Percentage o f o r i g i na l a c t i v i t y i n In terva 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 in te rsess ion hab i tua t i on c o m p a r e d to W T m i c e whe re hab i tua t i on is measured as the d i f f e r ence i n a c t i v i t y f r o m the first i n te rva l d i v i d e d b y the ac t i v i t y i n the first i n te rva l ( i n te rva l : F( 4 . 9 2 ) = 8.6, p < 0 .001; geno type X in te rva l : F ( 4 , 9 2 ) = 0.9, p = 0.4; genotype: F(i.23) = 9.7, p = 0.005) . F o l l o w i n g the first 30 m inu te t r i a l , i n te rsess ion open field hab i tua t i on was assessed b y g i v i n g the m i c e 4 add i t i ona l t r ia l s i n w h i c h the ac t i v i t y d u r i n g the f i rst 10 m inu te s wa s measu red . D. B e t w e e n the sess ions, the a c t i v i t y o f W T m i c e de c l i n ed mo r e r a p i d l y than the ac t i v i t y o f Y A C 128 m i c e but both g roups s h o w e d decreased a c t i v i t y ove r repeated t r ia ls ( t r ia l : F ( 4 ; 9 2 ) = 60.8 , p < 0 .001; geno type X t r i a l : F ( 4 , 9 2 ) = 2.5, p = 0.04; genotype: F ( , . 2 3 ) = 4.8, p = 0.04). E. Y A C 1 2 8 m i c e are hypoa c t i v e at 8 mon th s o f age as ind i ca ted b y an in i t i a l 1 8 % d i f f e rence i n a c t i v i t y be tween Y A C 1 2 8 and W T m i c e . W i t h repeated open field t r ia ls , the d i f f e rence i n a c t i v i t y be tween W T and Y A C 128 m i c e p r og r e s s i v e l y dec reased to 5% b y the last t r i a l . C . E x a m i n i n g the rate o f decrease i n a c t i v i t y pe r t r ia l demonst ra tes that W T m i c e s h o w a mo re r ap i d hab i tua t i on than Y A C 1 2 8 m i c e ( W T : 38.8 ± 5.0 beam breaks/ t r ia l , Y A C 1 2 8 : 26 .2 ± 3.2 b e a m breaks/ t r ia l , p = 0.05). N = 12 W T , 13 Y A C 1 2 8 . E r r o r bars s h o w S E M . * p < 0.05, ** p < 0 .01 . 51 initial differences in activity, we calculated habituation as the difference in activity from the first trial divided by the activity of 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 ( 4 )92) = 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( 3 j 6 9) = 0.1, p = 1.0; genotype: F ( i j 2 3) = 3.2, p •= 0.08). Accordingly, the rate of 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 of 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 of age rapidly learned to turn immediately to reach the platform (Fig. 3.6 panel B) . Testing the same cohort of mice at 4 months and 6 months of 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 of age do not impair the swimming ability of the Y A C 128 mice even at 6 months of age. 52 B C 2 4 6 8 Age(Months) 10 12 2 6 8 10 12 Age( Months) - O - W T 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 6 ) = 23.1, p < 0.001). C. The increased time to reach the platform results from the path taken to reach the platform. Mice swimming towards the platform initially were given a score of 0, while mice swimming away from the 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. 5 3 Surprisingly, at 8 months there is a dramatic increase in the amount of 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 of 0 and mice swimming away from the platform a score of 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 of age and persisting to 12 months of age (Fig 3.6 panel C ; Errors in initial swimming direction - W T : 0 of 27 trials, 0 of 9 mice; Y A C 128: 12 of 30 trials, 7 of 10 mice, x2 = 13.7, p < 0.001). Overall, Y A C 128 mice take longer to reach the platform starting at 8 months of age primarily as a result of 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 of age. We developed this test as a simple two-choice test of learning and memory to facilitate rapid training and testing in visually impaired mice. Initially, the escape platform was placed in the right arm of the T-maze. After two trials, both W T and Y A C 1 2 8 mice showed a large decrease in the amount of time required to reach the escape platform (Fig. 3.7 panel A ) . B y the third day, both genotypes had achieved a constant level of 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, 23) = 11.5, p = 0.003; W T : 1.9 ± 0.1 seconds, 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 YAC 128 mice and WT. On the third day, YAC 128 mice take significantly longer to reach the platform (trial: F ( 3 i 6 9 ) = 0.6, p = 0.6; genotype X trial: F ( 3 . 6 9 ) = 0.8, p = 0.5; genotype: F a 2 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; YAC 128: 10 of 52 trials, 6 of 13 mice; x 2 = 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 2 = 4.5 , p = 0.04). N = 12 WT, 13 YAC128. Error bars show SEM. ** p < 0.01. 55 around the average (Fig. 3.7 panel C ; Errant trials greater than 4 seconds - W T : 0 of 44 trials, 0 of 12 mice; Y A C 1 2 8 : 10 of 52 trials, 6 of 13 mice, %2 = 9.4, p = 0.002). To gain insight into the difference, we monitored the direction that each mouse turned upon arrival at the stem of the T. Initially, approximately half of the mice irrespective of genotype turned left (given a score of 1) and the other half turned right (given a score of 0). Upon training, mice learned to turn right to reach the platform and by the third day, all of the W T mice turned right in all of their trials (Fig. 3.7 panel D). In contrast, Y A C 128 mice still turned left in a small number of trials on the third day (WT: 0 of 44 incorrect path trials, 0 of 12 mice; Y A C 1 2 8 : 5 of 52 incorrect path trials, 3 of 13 mice; %2 = 4.5 , p = 0.04). This accounts for only half of the errant trials observed. Observations suggest that the remainder of 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 of 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. Mice 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 of the maze and found the platform. Similar to the W T mice, all of the Y A C 128 mice initially entered the right arm of the T-maze. However, upon discovering that the platform was not present in the right arm of the maze, the majority of the Y A C 1 2 8 mice swam back to the start of the T-maze where they had already been. At this point some of the Y A C 128 mice returned to the right arm of the T-maze while some swam directly to the platform in the left arm. We measured this abnormality by quantifying the number of arms of the maze entered en route to the platform. While all of 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 of 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; %2 = 9.0, p = 0.03) and taking 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 of the T-maze (Fig. 3.8 panels D,E) . Again, on day 3 o f the reversal phase of 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 of performance had not been reached (Fig. 3.8 panels D , E ; genotype: F ( i ; 2 3 ) = 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 of errant trials (not shown), increased pausing and decreased swimming speed (see below). It was not caused by an increased number of arm entries which was equal after day 1 (Fig. 3.8 panel G) . Here, a clear deficit in changing strategy is apparent in the path and time that Y A C 128 mice take to reach the platform in the reversal of the T-maze test which training reduces. The mild cognitive deficit observed in the normal phase of 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 of 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 of age. The stem of the T-maze was blocked off and the time for mice to swim from the right arm of the maze to the platform in the left arm of the maze was measured (Fig. 3.9 panel A ) . Assuming that mice on average swim to the middle of the T before turning, the distance is identical to that traveled in the T-maze tests but differences in the amount of time required for mice to turn around a corner are not determined. In this assessment, the average latency of Y A C 128 mice to reach the platform was significantly longer than that of 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 (WT: 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, %2 = 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 ( 3 . 6 9 ) = 5.9, p = 0.001; genotype X trial: F ( 3 > 6 9 ) = 1.7, p = 0.2; genotype: F„, 2 3 ) = 3.9, p = 0.06; WT: 2.0 ± 0.2 seconds, YAC128: 3.4 ± 0.5 seconds). F. This is partially because YAC 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; x2 = 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. 58 straight swimming time allows estimation of the contribution of 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 of 0.3 seconds only accounts for a small part of the difference between Y A C 128 and W T mice on the final days of either normal phase or reversal phase T-maze testing where the differences were 1.1 and 1.4 seconds respectively. While part of this difference may stem from Y A C 128 mice taking longer to turn the corner (not determined), the remainder of 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 of 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% of 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 of the difference observed (73-95%; Fig. 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 of the overall performance deficit in the simple swimming test and swimming T-maze tests. The remainder of the difference is caused by cognitive dysfunction which is most apparent in the reversal phase of 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 of 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 of 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 of the 12 trials (Fig. 3.10 panel A ) . However, switching the location of the platform to the opposite arm of the maze revealed a difference in changing strategy between 59 B •c CO 3.0 i 2.5 » S c o 0) i5 IE S 2.0 1.5 1.0 0.5 • Cognitive Deficit • Motor Deficit 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. YAC 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 of the swimming T-maze 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 of the 12 W T mice re-entered the stem of the T-maze while 3 of 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). At 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 of 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 of 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 of 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. Mice 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 wi l l startle less than they would for the loud stimulus alone. This PPI is not learned but rather measures sensorimotor gating. At 9 months of 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 of age showed significantly less PPI compared to W T mice indicating a deficit in sensorimotor gating (Fig. 3.11 panel A ; genotype: F(i jg 5) = 62.7, p < 0.001; pre-pulse: F( 3 , 255) = 37.4, p < 0.001). A t 12 months of 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 YAC 128 mice were tested in the swimming T-maze test with reversal. A. During the normal phase of the test, both YAC 128 and WT mice learn to swim to the platform and no differences were observed between YAC 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 ( L J 2 2 ) = 0.0, p = 0.9). B. During the reversal phase, when the location of the platform was switched, YAC 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 YAC 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 (x2 = 6.0, p = 0.05). D. At this age, the swimming speed of YAC 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. 62 m 90% • c I 70% • x 50% -£ 30% 0. *** *** 10% 2 d B 4 d B 8 d B 16 dB Pre-Pulse(above background) B c 50% I 40% S 30% CO * 20% CD 1 10% 5) 0% J L 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 pre-pulse 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 ( 1 ;g 5 ) = 62.7, p < 0.001; prepulse: F ( 3. 2 5 5 ) = 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 ( 2 . 88 ) = 3.9, p = 0.025; genotype X block: F ( 2 > 8 8 ) = 0.3, p = 0.7; genotype: F ( 1, 44) = 7.9, p = 0.008; N = 21 WT, 25 YAC128). Error bars show SEM. * = p < 0.05, ** p<0.01, *** p< 0.001.' 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 ( 2,88) = 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 of other regions including the hippocampus and cerebellum. Here we sought to determine whether atrophy was present in the brains of 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 of the striatum, cortex, globus pailidus, hippocampus and cerebellum in a cohort of 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 of 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 of the brain (Fig. 3.12 panel B ; W T : 12.1 ± 0.1 mm 3 , Y A C 1 2 8 : 10.8 ± 0.2 mm 3 , p < 0.001). We found a similar, 10.8% decrease in the volume of 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 mm 3 , Y A C 1 2 8 : 1.43 ± 0.04 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 mm 3 , Y A C 1 2 8 : 15.1 ± 0.03 mm 3 , p = 0.001). In contrast, the volumes of the hippocampus and cerebellum were unaffected in Y A C 128 mice (Fig. 3.12 panel B ; Hippocampus - W T : 0.0355 ± 0.001 mm 3 , Y A C J 2 8 : 0.0356 ± 0.001 mm 3 , p = 0.9; 64 Cerebellum +2.9% 0.94 p = 0.6 2 . 0 E + 0 6 1 .9E+06 1 .8E+06 1 .7E+06 1 .6E+06 Striatum 9.1%, p 2.4E+06 c 2.3E+06 2 | 2.2E+06 1 2.1E+06 § 2.0E+06 - 1.9E+06 • 0.01 Cortex 8.3%, p = 0.02 „ 1.6E+06 | 1.4E+06 z 1.2E+06 ! 1.0E+06 I 8.0E+05 | 6.0E+05 £ 4.0E+05 Hippocampus 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 mm2, YAC128: 10.8 ± 0.2 mm2, p < 0.001; Globus pallidus - WT: 1.60 ± 0.06 mm2, YAC128: 1.43 ± 0.04 mm2, p = 0.04; Cortex - WT: 16.5 ± 0.02 mm2, YAC 128: 15.1 ± 0.03 mm2, p = 0.001). In contrast, the hippocampus and cerebellum were unaffected in YAC128 mice (Hippocampus - WT: 0.0355 ± 0.001 mm2, YAC128: 0.0356 ± 0.001 mm2, p = 0.9; Cerebellum - WT: 44.3 ± 0.2 mm2, YAC128: 45.6 ± 0.1 mm2, 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. i 65 Cerebellum - W T : 44.3 ± 0.2 mm 3 , Y A C 1 2 8 : 45.6 ± 0 . 1 mm 3 , p = 0.6). Furthermore, examination of 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 (WT: 53.1 ± 0.02 mm , Y A C 1 2 8 : 55.0 ± 0.1 mm 3 , p = 0.5). Thus, the overall decrease in brain weight in Y A C 1 2 8 mice is caused by degeneration in select regions of 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 of 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 of age. The number of 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 mill ion neurons, Y A C 1 2 8 : 1.73 ± 0.03 mill ion neurons, p = 0.01). Examination of the region of cortex above the striatum revealed that there is also neuronal loss in the cortex of 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 mil l ion neurons, p = 0.02). In contrast, Y A C 1 2 8 and W T mice showed no difference in the estimated number of neurons in the cellular layer of the hippocampus (Fig. 3.12 panel E ; W T : 1.42 ± 0.05 mill ion neurons, Y A C 1 2 8 : 1.44 ± 0.03 mill ion 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 of selective degeneration in the striatum and cortex, as well as decreases in brain mass over time to determine the age of onset of 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 of age (Fig. 3.13 panel A ; W T : 12.6 ± 0.5 mm 3 , Y A C 1 2 8 : 12.6 ± 0.5 mm 3 , p - 1.0). A t 9 months, significant striatal volume loss is present in Y A C 1 2 8 mice compared to W T mice (F ig . 3.13 panel A ; W T : 12.5 ± 0.3 mm 3 , Y A C 128: 11.4 ± 0.2 mm 3 , p = 0.009). Similarly, at 12 and 18 months, 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 mm 3 , Y A C 1 2 8 : 11.4 ± 0 . 1 mm 3 , p < 0.001; 18 m o n t h s - W T : 12.1 ± 0.4 mm 3 , Y A C 128: 9.5 ± 0.4 mm 3 , p = 0.006). A two factor A N O V A comparing genotype and age reveals significant differences between Y A C 128 and W T mice and their pattern of 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 ( 3,75) = 7.8, p < 0.001). While the magnitude of 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 mill ion 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 mill ion 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 mill ion neurons, p = 0.001; 18 months - W T : 1.70 ± 0.03 mill ion neurons, Y A C 1 2 8 : 1.46 ± 0.04 mill ion 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: F(ij57) = 15.3, p < 0.001; age X genotype: F ( U 5 2 ) = 2.8, p = 0.04). Cortical volume in Y A C 128 mice is equivalent to W T at 6 and 9 months of age (Fig. 3.13 panel C ; 6 months - W T : 18.2 ± 0.8 mm 3 , Y A C 1 2 8 : 17.9 ± 0.8 mm 3 , p = 0.8; 9 months -W T : 16.9 ± 0.8 mm 3 , Y A C 1 2 8 : 16.4 ± 0.8 mm 3 , p = 0.6). A t 12 and 18 months of age, 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 mm 3 , Y A C 1 2 8 : 14.0 ± 0.4 mm 3 , p = 0.01; 18 months - W T : 18.5 ± 0.7 mm 3 , Y A C 128: 15.1 ± 0.6 mm 3 , p = 0.02). Similarly, cortical neuronal counts were equivalent between Y A C 128 and W T mice until 12 months of 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 mill ion neurons, Y A C 128: 2.62 ± 0 . 1 3 million neurons,"p - 0 . 9 ; 9 months - W T : 2.37 ± 0.12 mill ion 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 WT, 14 YAC128), 9 months (N = 18 WT, 11 YAC128), 12 months (N = 46 WT, 50 YAC128) and 18 months (N = 3 WT, 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 3, YAC128: 11.4 ± 0.2 mm 3, p = 0.009; 12 months: WT: 12.6 ± 0.1 mm 3, YAC128: 11.4 ± 0.1 mm 3, p < 0.001; 18 months: WT: 12.1 ± 0.4 mm 3, YAC128: 9.5 ± 0.4 mm 3, p = 0.006). B. Striatal neuronal loss followed a similar pattern with differences between Y A C 128 and WT 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 WT 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 3, YAC128: 1.40 ± 0.04 mm 3, p = 0.01; 18 months - WT: 1.85 ± 0.07 mm 3, Y A C 128: 1.51 ± 0.06 mm 3, 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 mg,p< 0.001; 18 months-WT: 403 ± 6 mg, YAC128: 380 ± 5 mg, p = 0.04). Error bars show SEM. * = p < 0.05, ** p < 0.01, *** p < 0.001. 68 0.05 mill ion neurons, Y A C 128: 2.06 ± 0.05 million neurons, p = 0.02; 18 months - W T : 2.90 ± 0 . 1 1 mill ion neurons, Y A C 1 2 8 : 2.48 ± 0.09 million neurons, p = 0.03). Examination of brain weight revealed mild atrophy in the brains of Y A C 128 mice. Decreases in brain weight in Y A C 128 mice begin around 9 months of 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 of the brain result in the overall cerebral atrophy observed. Overall, examination of the time course of neuropathological changes in Y A C 128 mice revealed that neuropathology begins around 9 months of 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 of neurons within the striatum. Neurons from a matched coronal section of Y A C 128 and W T brains were stained with NeuN antibody and the perimeter of the cell body was outlined using stereology software which then calculated the cross-sectional area. A t 12 months of 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 um 2 , Y A C 128: 69.8 ± 2.2 um 2 , p = 0.002). Thus, both decreases 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 of 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 of D A R P P - 3 2 expression estimated using the 69 E 100 i ra c (0 80 e < CD "ra 60 • z c ra o 40 ra § Slri ss-S 20 -0 B 1 — * * 1 • WT • Y A C 1 2 8 W T Y A C 1 2 8 Figure 3.14 Striatal neuronal atrophy in YAC128 mice. At 12 months of age striatal neuronal cross-sectional area was measured is YAC128 and WT mice. A. Striatal neuronal size was significantly decreased in YAC128 mice compared to WT mice (WT: 81.6 ± 2.9 um2, YAC128: 69.8 ± 2.2 um2, p = 0.002; N = 46 WT, 50 Y A C 128). Photographs of neurons from WT mice (panel B) and Y A C 128 mice (panel C) demonstrate this difference. Photographs were taken using the 100X objective. Error bars show SEM. ** p < 0.01. YAC 128 Figure 3.15 Down-regulation of striatal DARPP-32 expression in YAC128 mice. Matched coronal sections from Y A C 128 and WT 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 WT mice (pictures taken with 2.5X objective). C,D. At higher power, neurons from WT 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 WT 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 SEM. *** p < 0.001. 70 intensity of D A R P P - 3 2 staining within the striatum. Visual inspection of 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 of the striatum (Fig. 3.15 panels A , B ) . At 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 of the cell body (Fig. 3.15 panels C,D) . Quantification of the overall difference in the intensity of 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). 3.2.3.4 Regional differences in the expression and nuclear localization of mutant huntingtin in Y A C 1 2 8 mice In mouse models of H D , higher levels of 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 of the brain with the highest levels of htt expression are not the most severely affected in the disease (L i et al, 1993; Landwehrmeyer et al, 1995). To determine whether the level of mutant and wild-type htt expression in each region of 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 of 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 of 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 of Y A C 128 mice. Next, we examined regional differences in the nuclear localization of mutant htt. While wi ld-type htt is primarily a cytoplasmic protein (DiFiglia et al, 1995), expansion of 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( 3. 2 5 ) = 9.2, p < 0.001) but that mutant and wild-type htt follow the same pattern of expression ( F ( i . 2 9 ) = 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. 72 polyglutamine tract results in increased nuclear localization of 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 of mutant htt could account for the selective degeneration in Y A C 128 mice, we examined the localization of 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 of htt and has a high affinity for fragments of mutant htt, especially those that have aggregated. We examined 3 month old Y A C 128 mice to determine i f nuclear localization occurred earlier in the more affected regions of the brain as well as 12 month old mice to determine i f the extent of nuclear localization was greater in affected regions. A t 3 months, we found that mutant htt exhibited the greatest degree of 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 of 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 IV 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 of the intensity of nuclear E M 4 8 staining revealed that the nuclear localization of mutant htt was significantly greater in the lateral striatum than in any other region of the brain (Fig. 3.17 U ; region: F(5,17) = 23.8, p < 0.001). A t 12 months of age, nuclear localization of htt was increased in all regions of 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 of 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 of 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 of mutant htt were confirmed by quantification of nuclear E M 4 8 staining intensity (Fig. 2 U ; region: F ^ o ) = 15.5, p < 0.001). Overall, the nuclear localization of 73 N u c l e a r L o ca l i z a t i o n of Mutan t Hunt ingt in o o o -> to ro 01 o oi © o o o 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 of the brain and thus appears to correlate with the amount of cerebral damage. 3.2.3.5 Transglutaminase activity is selectively increased in the forebrain of Y A C 1 2 8 mice Since transglutaminase (TG) activity is increased in H D patients and postulated to contribute to the pathogenesis of 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 of tissue T G (tTG) since tTG 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 of 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 of the non-GTP inhibited T G activity revealed a dramatic increase in the forebrain of Y A C 1 2 8 mice compared to W T mice with a non-significant increase of 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 of 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% of 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 of death of 54 years (calculated from Harper, 1996). Based on the findings of decreased lifespan in human H D 76 A v 160% B • WT • YAC128 Forebrain Hindbrain Forebrain + 30% +18% +620% p = 0.027 p = 0.43 p = 0.029 Hindbrain + 27% p = 0.42 Figure 3.18 YAC128 mice show a forebrain specific increase in transglutaminase activity. Transglutaminase (TG) activity was measured in Y A C 128 and WT littermates by putrescine incorportation assay. A. Total T G activity in the forebrain was increased approximately 30% in YAC128 mice compared to WT 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 WT 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 WT 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 WT, 12 YAC128. Error bars indicate SEM. * 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 of 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 of full length htt. Survival in the Y A C 128 mice was followed prospectively until 12 months of age. Initially we observed a non-significant decrease in survival of 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). Dividing 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 of 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. We examined two of these mice that were sacrificed at 6 months of age for striatal neuropathology but were unable to identify any defects. Similarly, when we examined the rotarod performance and weight of 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 of the brain, the testis expresses the highest levels of htt protein, mutant or wild-type, in the body (Fig. 3.20 panel A ) . We have previously reported that mild expression of mutant htt with 72 glutamines lead to testicular degeneration when the levels of wild-type htt were 78 L h I '— 1 1 Male Mice 100 200 300 400 Age(days) c Sex Genotype Total Mice Deaths Percent Surviving Significance Female WT 30 3 90% p = 0.64 YAC128 55 4 93% Male WT 38 2 95% p = 0.01 YAC128 42 11 74% 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 WT mice beginning around 9 months of age (p = 0.01). C. Data is summarized in table format. D 100% t 90% SJ 80% Q-100 200 300 Age(days) 400 •WT • YAC128 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 of mutant htt or the loss of wild-type htt since expression levels of mutant htt were less than endogenous. The expression of mutant htt with 72 glutamines alone was not sufficient to cause any testicular degeneration as Y A C 7 2 mice expressing two copies of wild-type htt showed normal testicular morphology and sperm production (Leavitt et al, 2001). Thus, to determine whether the expression of mutant htt alone, without the loss of 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 of 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). To 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 of different stages of 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 of cells within the seminiferous tubules was disrupted by large vacuoles. The most striking difference was the observation of cell death and cells being sloughed off into the lumen of the seminiferous tubules. While there were some signs of sperm production, the number of developing sperm was obviously decreased compared to wild-type. Furthermore, few sperm cells developed to the later stages of development and, of those that did, many had abnormal sperm heads. Overall, we observed testicular atrophy and degeneration in the testis o f Y A C 1 2 8 . 3.2.6 Outcome measures for therapeutic trials The goal for the characterization of 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 Y A C 1 2 8 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 Ser tol 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. 8 1 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 of mice required for therapeutic trials (Table 3.1). Based on the biological relevance of striatal volume loss in H D and the robustness of 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 YAC 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 Difference Between YAC128 and WT Significance of Difference Standard Deviation Mice Required to Detect a 50% Rescue Mice Required to Detect a 75% Rescue Striatal volume -10.4% p< 0.001 4.4% 10 5 Striatal neuronal counts -11.0% p = 0.009 5.1% 13 6 Striatal neuronal cell size -18.7% p< 0.001 6.6% 6 3 Striatal DARPP-32 expression -22.2% p< 0.001 9.8% 11 5 Brain weight -4.6% p = 0.07 3.4% 37 16 Rotarod 10 months -51.1% p< 0.001 24.7% 6 3 Open field activity 12 months -22.1% p< 0.001 16.4% 27 12 Swimming T-maze with reversal + 108% p< 0.001 31.0% 23 10 Pre-pulse Inhibition -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 of wild-type htt in vitro has been shown to protect neurons from a variety of toxic stimuli including the expression of an expanded polyglutamine protein (Rigamonti et al, 2000; Ho et al, 2001). We have previously extended these findings in vivo to demonstrate a protective effect of wild-type htt against mutant htt toxicity in the testis. Since the brain is the region of primary pathology in H D , we wanted to determine i f wi ld-type 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, of 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 of 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 of 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 of 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 WT i ' / D Kit • •* t "4\Xz*. YAC 18 t \ WT F YAC 18 Silver Staining T U N E L l j 450 400 1 ? 350 11 2 g 300 ay | 250 ^ S 2 0 0 0 W S p 150 z § 1 » 100 Z 50 • W T • Y A C 18 CA1 CA3 Combined Region of Hippocampus Figure 4.1 Over-expression of wild-type huntingtin protects neurons from kainic acid toxicity in the hippocampus. WT 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 WT 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 WT mice had fragmented D N A and were dying. G. Quantification of the number of degenerating neurons shows significantly more dying neurons in WT mice compared to YAC18 mice in both the CA1 region and CA3 region of the hippocampus (N = 11 WT, 7 Y A C 18). Thus, over-expression of wild-type htt is neuroprotective in the hippocampus. Error bars show SEM. * 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 of decreased numbers of degenerating neurons in Y A C 18 mice compared to W T mice after administration of 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 of 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 of the excitotoxic neurotoxin quinolinic acid (QA), which has been used in neurotoxin models of H D . Following Q A injection, there was no difference in the frequency or duration of 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 NeuN, 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 of 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 mm 3 , Y A C 18: 2.01 ± 0 . 2 2 mm 3 , p = 0.007). In addition to lesion volume, the number of surviving neurons was measured by estimating the number of 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 3.5E+09 3.0E+09 2.5E+09 2.0E+09 1.5E+09 1.0E+09 5.0E+08 O.OE+00 i * * 1 T T 3 • CD C YAC18 1.0E+05 8.0E+04 > 09 6.0E+04 CN 15 c c o o 4.0E+04 OL 3 CD 0- Z OH 2.0E+04 < L J O.OE+00 T T • WT • YAC 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 mm3, 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, over-expression 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. 87 To ensure that the neuroprotection was caused by wild-type htt and was not a site of integration effect, we repeated the experiment using Y A C 18 mice from line B60 (line B60 mice). Both heterozygous and homozygous, line B60 mice express lower levels of 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 of 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 B60, homozygous line B60 and line 212 mice were given intrastriatal injections of quinolinic acid and sacrificed after 7 days. Analysis of the resulting lesion volumes revealed that wild-type htt mediated neuroprotection was dose dependent (genotype: F( 3 ) 7 i ) = 4.5, p = 0.006). W T mice had the largest lesion size followed by heterozygous line B60 mice, homozygous line B60 mice and line 212 mice (Fig. 4.3; W T : 4.62 ± 0.38 mm 3 , B60 +/-: 4.27 ± 0.12 mm 3 , B60 +/+: 3.70 ± 0.16 mm 3 , 212: 3.25 ± 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 B60 mice, heterozygous line B60 mice and W T mice (Fig. 4.3). The difference in overall htt levels result from a difference in the expression levels of transgenic, human htt which can be detected with the human htt specific HD650 antibody (Fig. 4.3). Thus, increasing levels of wild-type htt expression result in increased neuroprotection. This indicates that the protective effect of wild-type htt is dose dependent and that it is independent of site of integration. 4.3 Huntingtin expression influences weight To determine whether the study of 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 B60 mice, homozygous B60 mice and 212 mice from 2 to 12 months of age and found that overall body weight increased with htt expression (genotype: F ( 2 ;36) = 18.2, p < 0.001). Line 212 mice weighed significantly more than line B60 and W T mice from 2 months of age to 12 88 5.5E+09 _ 5.0E+09 m < E 3 O a E _3 o > WT B60+/- B60+/+ 212 • WT • B60 +/-B 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 ( 3 . 7 1 ) = 4.5, p = 0.006; WT: 4.62 ± 0.38 mm3, B60 +/-: 4.27 ± 0.12 mm3, B60 +/+: 3.70 ± 0.16 mm3, 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. 89 months of 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 B60 line showed a trend towards increased weight relative to W T mice beginning at 2 months of age with the differences becoming highly significant by 12 months of age (Fig. 4.4 panel A ; 2 months - W T : 25.5 ± 1.1 g, Line B60: 26.8 ± 0.7 g, p = 0.3; 12 months -W T : 34.5 ± 1.6 g, Line B60: 40.6 ± 1.1 g, p = 0.002). Western blotting for htt expression confirmed that line B60 mice express more htt than W T mice and line 212 mice express more htt than line B60 mice (Fig. 4.4 panel B) . Thus, increased wild-type htt expression results in increased body weight. The demonstration of increased weight in two different lines of 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 of integration and that altered htt levels are causative. Our laboratory has also generated mice heterozygous for the targeted inactivation of the mouse H D gene {Hdh +/- mice). These mice express htt at half of 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 of a cohort of 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 of 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 of age although the difference appeared to be greatest closer to two months of age indicating the difference in weight may stem from altered development in Hdh +/- mice (genotype: F (i i 2o) = 4.2, p = 0.05). Western blotting for htt protein confirmed that Hdh +/- mice express htt at half of 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 ( 2 J 6 ) = 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 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 of 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 of htt expression in each of the organs studied to see i f there was any correlation between the relative increases in weight and the level of htt expression. We found that, as previously reported, expression of 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. We were unable to detect any full length htt in the kidneys. While overall body weight increases with htt expression, this is not universally true of 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 of 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 ; 4 9 ) = 33.6; p < 0.001; 2 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 CD 21 CD 5 150% -140% 130% 120% 110% -100% 9 0 % 8 0 % 7 0 % 6 0 % — *** ** • WT • Y A C 18 Brain Heart Liver Lungs Kidneys Sp leen Test is +1% +24% +33% +15% +26% +36% -6% p = 0.4 p < 0.001 p < 0.001 p = 0.002 p < 0.001 p = 0.002 p = 0.3 B i •g Total wild type htt expression Brain Heart Lner Lungs Kidney Spleen Testis 100% 10% 34% 70% 0% 79% 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 ±0.06 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 MODEL 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 of wild-type htt (Rigamonti et al, 2000; Ho et al, 2001; Leavitt et al, 2001). In this thesis, we have extended these findings to show htt-mediated neuroprotection in the brain in both the hippocampus and the striatum (section 4.1 and 4.2). Since H D patients have decreased levels of wild-type htt, it is plausible that this decrease in htt's neuroprotective function makes neurons more susceptible to the toxicity of 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 of 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 of loss of htt function in H D and whether delivery of wild-type htt would be beneficial in the treatment of 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 of 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 (YAC18/128 mice). YAC18/128 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 of 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 HD650 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 of endogenous htt (Van Raamsdonk et al, 2005). W T mice showed no transgenic expression of htt. Y A C 18 mice expressed only wild-type human htt, while . Y A C 128 mice 94 5 CO CM ST CO CO CO CSI T— O O O < < < >- >- > Mu htt -WT htt-Actin-B 5000 c o 'to tn £ CL X LU 4000 3000 1 2000 c X 1000 • YAC18 • YAC128 • YAC18/128 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 of 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 of wild-type htt did not down-regulate the expression of mutant htt (Fig. 5.1 panel B ; Y A C 1 2 8 : 3389 ± 197 arbitrary units, YAC18/128 : 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 of wild-type htt on the striatal neuropathology observed in Y A C 128 mice. A comparison of YAC18/128 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 mm 3 , YAC18/128 : 11.6 ± 0.2 mm 3 , p = 0.3), striatal neuronal numbers (Fig. 5.2 panel B ; Y A C 128: 1.56 ± 0.03 mill ion neurons, Y A C 18/128: 1.59 ± 0.03 mill ion 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 um 2 , Y A C 18/128: 108 ± 1.9 um 2 , p < 0.001). Moreover, the striatal neuronal cross-sectional area in Y A C 18/128 mice was almost restored to W T (WT: 110 ± 3 um 2 , p = 0.6). Thus, over-expression of wild-type htt resulted in mild improvement of striatal neuronal size but did not improve the hallmark signs of the disease - striatal volume loss and neuronal loss in the striatum. 96 A 1.30E+10 --j- 1.25E+10 -M 1.20E+10 -I _ 1.15E+10 "C » 1.10E+10 -1.05E+10 -B 110% 100% g f 90% » | B0% 1 7 0 % 60% -** *-_ L _ 1750000 „ 1700000 8 1650000 I 1600000 | 1550000 I 1500000 0 5 1450000 1400000 • WT • YAC18 YAC128 YAC 18/128 T H 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 mm2, YAC18/128: 11.6 ± 0.2 mm2, 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 um2, YAC18/128: 108 ± 1.9 um2, 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. 9 7 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 of proteins that interact with htt (Lesort et al, 2002; Chun et al, 2001; Cooper et al, 1997). Cystamine is an inhibitor of 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 of anti-oxidants (Lesort et al, 2003; Fox et al, 2004) . Four previous studies have demonstrated the beneficial effect of either cystamine treatment or decreasing levels of T G in N-terminal fragment mouse models of 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 of 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 of cystamine on striatal neuropathology, including neuronal loss, in these mice. 5.2.2 Delivery of therapeutic agent: cystamine In order to demonstrate delivery of cystamine and the effect of cystamine administration on T G activity, we treated Y A C 128 mice with 225 mg/kg cystamine in drinking water for a period of 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 of 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 of cystamine in drinking water is effective at decreasing T G activity in the brain of Y A C 128 mice. 5.2.3 Effect of cystamine on neuropathology In order to gauge the potential benefit of cystamine treatment in H D , we examined the impact of cystamine treatment on striatal neuropathology in Y A C 128 mice. Based on the significance of striatal volume loss to H D and the robustness, of this finding in Y A C 128 mice, we chose striatal volume as the primary outcome measure. We also examine secondary endpoints of 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 of 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 of 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 of striatal D A R P P - 3 2 expression (unpublished). Examination of striatal volume by A N O V A revealed significant effects of treatment, genotype and the interaction between treatment and genotype (treatment: F ( i j 2 8) = 7.9, p = 0.009; genotype: F ( U 2 8 ) = 14.5, p < 0.001; treatment X genotype: F ( U 2 8 ) = 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 mm 3 , cystamine-treated YAC1.28: 11.8 ± 0.3 mm 3 , p = 0.01). Untreated 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 of 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 mm 3 ; p = 0.96). 99 B ? 1.30E+10 £ f-.1.20E+10 E f 1.10E+10 > TS 1.00E+10 £ 9.00E+09 YAC128 £ 2.00E+06 E: 1.90E+06 CO I 1.80E+06 cu E 1.70E+06 s I 1.60E+06 w W T YAC128 o W T YAC128 D E Si • Untreated • Cystamine k Q: 110% 100% 90% 80% 70% 60% WT YAC128 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 were treated w i t h c y s t am ine b e g i n n i n g at 7 mon ths o f age and neu ropa tho l ogy was assessed at 12 months o f age. A . A s s e s s m e n t o f str iata l v o l u m e revea l ed str iata l v o l u m e loss in Y A C 128 m i c e c o m p a r e d to W T m i c e w h i c h was s i gn i f i c an t l y decreased by treatment w i t h c y s t am ine (treatment: F ( i > 2 g ) = 7.9, p = 0.009; genotype: F ( i . 2 8 ) = 14.5, p < 0 .001; untreated W T : 12.1 ± 0.1 m m 3 , unt reated Y A C 1 2 8 : 10.8 ± 0.2 m m 3 , c y s t am ine treated W T : 12.2 ± 0.2 m m 3 , cys tamine- t rea ted Y A C 1 2 8 : 11.8 ± 0.3 m m 3 ) . B . C y s t a m i n e t reatment p reven ted neurona l loss i n Y A C 1 2 8 m i c e and had no ef fect i n W T m i c e (treatment: F ( 1 2 g ) = 6.4, p = 0.02; genotype: F ( i 2g> = 4.0, p = 0.05; untreated W T : 1.90 ± 0.05 m i l l i o n neurons , untreated Y A C 128: 1.73 ± 0.04 m i l l i o n neurons , cys tamine- t rea ted W T : 1.91 ± 0.04 m i l l i o n neurons, p = 1.0, cys tamine- t rea ted Y A C 1 2 8 : 1.93 ± 0.04 m i l l i o n neurons) . C . S i m i l a r l y , str iata l neu rona l a t rophy in Y A C 128 m i c e was c omp l e t e l y p reven ted b y cy s t am ine and una f fec ted in W T m i c e (treatment: F ( ] . 2 8 ) = 11.0, p = 0.003; genotype: F ( 1 2 8 ) = 6.9, p = 0.014; untreated W T : 97.8 ± 1.5 u m 2 , untreated Y A C 1 2 8 : 78.8 ± 2.1 u m 2 , cys tamine- t rea ted W T : 97.6 ± 3.6 u m 2 , p = 1.0, cys tamine- t rea ted Y A C 1 2 8 : 99.9 ±3.7 u m 2 ) . D . In contrast, c y s t am ine t reatment d i d not s i gn i f i c an t l y i m p r o v e str iata l D A R P P - 3 2 exp ress i on i n Y A C 128 m i c e and had no impac t on D A R P P - 3 2 exp ress i on i n W T m i c e (treatment: F ( i . 2 8 ) = 0.1, p = 0.7; geno type : F { 1 2 8 ) = 20.9 , p < 0 .001; untreated W T : 667 ± 20 in tens i ty uni ts, untreated Y A C 1 2 8 : 519 ± 9 intens i ty uni ts, cys tamine- t rea ted W T : 649 ± 26 in tens i ty uni ts; cys tamine- t rea ted Y A C 1 2 8 : 524 ± 12 intens i ty un i ts) . 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 ind i ca te S E M . * p < 0.05, ** p < 0.01 , * * * p < 0 .001 . 100 Similarly, striatal neuronal numbers were significantly affected by treatment and genotype (Fig. 5.3 panel B ; treatment: F ( ] > 28) = 6.4, p = 0.02; genotype: F ( i ; 2 8 ) = 4.0, p = 0.05; treatment X genotype: F(i > 28) = 3.6, p = 0.07). As with striatal volume, cystamine treatment resulted in a 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 mill ion neurons, cystamine-treated Y A C 1 2 8 : 1.93 ± 0.04 mill ion neurons, p = 0.015). This improvement represents a complete amelioration of 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 mill ion neurons, p = 1.0). Striatal neuronal cross-sectional area was measured using a fluorophore-conjugated NeuN 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 pm 2 , untreated Y A C 128: 78.8 ± 2.1 pm 2 , p = 0.001). Treatment of Y A C 128 mice with cystamine resulted in a complete prevention of 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 of treatment, genotype and the interaction between treatment and genotype (treatment: F ( i , 2 8 ) = 11.0, p = 0.003; genotype: F ( i ; 2 8 ) = 6.9, p = 0.014, treatment X genotype: F ( i , 2 8 ) = 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 2 , p = 1.0). Next, we measured striatal D A R P P - 3 2 expression by the intensity of 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 of treatment despite a clear effect of genotype (treatment: F ( l , 2 8 ) = 0.1, p = 0.7; genotype: F ( l , 2 8 ) = 20.9, p < 0.001; treatment X genotype: F ( i ; 2 8 ) = 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 of 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; WT: p = 1.0). 5.2.4 Effect of cystamine on motor impairment In order to assess the effect of cystamine treatment on motor dysfunction, we followed the performance of cystamine-treated Y A C 128 mice on the rotarod. Mice were trained on the apparatus at 2 months of age and tested bimonthly until 12 months of 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 of treatment or the interaction of treatment and genotype (age: F( 5,i60) = 44.3, p < 0.001; treatment: F ( i ) 3 2 ) = 0.0, p = 1.0; genotype: F ( , j 3 2 ) = 25.6, p < 0.001, treatment X genotype: F ( i j 2 6) = 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 of general health in the treated and untreated mice to determine i f there were any toxic effects of cystamine. The cystamine treated mice were indistinguishable from untreated controls and appeared to be in good health for the duration of the trial. Repeated measures A N O V A revealed no significant effect of cystamine on the weight of W T or Y A C 1 2 8 mice (treatment: F(i ; 3 i) = 2.5, p = 0.12) thereby indicating a lack of obvious toxicity of the cystamine dose used in this experiment. Aged Y A C 128 mice show hypoactivity compared to W T mice beginning between 6 and 8 months of age (section 3.2). A n examination of 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; cystamine-treated W T : 393 ± 25, p = 1.0). 102 A 350 n 300 -to « 250 -200 -§. c 150 -tt) 100 -50 -0 -Trea tment at 7 Mon th s T rea tment at 4 Mon ths B < 350 6 8 Age(Months) —CD- WT WT-Cystamine — • — YAC128 — ± — YAC128-Cystamine D 450 « 400 < 350 300 250 200 W T YAC128 • Unt rea ted • C y s t a m i n e 4 6 Age(Months) WT 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 I 3 2 ) = 0.0, p = 1.0; genotype: F ( i . 3 2 ) = 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, cystamine-treated 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, (1,44) 28.5, p < 0.001; treatment: F ( 1.44) = 2.2, p = 0.15; untreated WT: 200 ± 17 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 4 ) = 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. 103 In the symptomatic trial, we found that cystamine prevented the development of 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 of motor deficits, we treated a cohort of Y A C 128 mice with cystamine beginning at 4 months of 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 of genotype on rotarod performance (genotype: F ( i ) 4 4) = 28.5, p < 0.001), but no beneficial effect of cystamine treatment on rotarod performance in the Y A C 128 mice (Fig. 5.4 panel C ; p = 0.5). Cystamine treatment did not alter the performance of W T mice on the rotarod (5.4 panel C ; p = 0.2). Similarly, there was no effect of cystamine treatment on weight in either Y A C 128 or W T mice (treatment: F(| ; 4 4 ) = 0.0, p = 0.98) suggesting that even long term treatment with 225 mg/kg cystamine is well tolerated. Finally, examination of 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 ;44) = 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 of W T mice (Fig. 5.4 panel D ; untreated W T : 380 ± 22 beams breaks, cystamine-treated 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 of H D . 104 6.0 DISCUSSION Huntington disease is caused by the expression of the htt protein containing an expanded polyglutamine tract. Onset of H D occurs on average at 39 years of age and progresses to death approximately 15 years later (calculated from Harper, 1996). Symptoms of the disease include motor deficits, cognitive dysfunction and psychiatric disturbances. There is currently no treatment that can halt or prevent the progression of this devastating disease. Accordingly, the major goal of this thesis was to characterize a genetically accurate mouse model of H D for use in preclinical therapeutic trials. The ideal mouse model of H D should reproduce the human disease as accurately as possible to increase the likelihood that findings regarding either the pathogenesis of the disease or effective treatments for the disease wi 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 of different treatments can be assessed quantitatively with low numbers of mice. To create a genetically accurate model of 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 of D N A containing the H D gene included 24 kb of upstream D N A in order to include the regulatory elements of the H D gene. This approach led to the appropriate tissue specific expression of 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 of the mouse H D gene from embryonic lethality (Hodgson et al, 1996; Hodgson etal, 1999). 105 6.1 The Y A C 7 2 mouse model of Huntington disease The Y A C 7 2 mouse model of 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 of mutant htt and dark cell degeneration at 12 months (Hodgson et al, 1999). In this thesis, a higher expressing line of Y A C 7 2 mice, line 44, was characterized to determine i f increased expression of wild-type htt results in a more robust phenotype and i f these mice, were suitable for therapeutic trials. Examination of Y A C 7 2 mice with an extensive array of 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 of selective degeneration. The finding of 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 HD-l ike symptoms but these symptoms are limited and mild. Thus, while Y A C 7 2 mice may provide insight into the pathogenesis of the disease they are not ideal for therapeutic trials because there are few outcome measures to assess the efficacy of treatment and large numbers of mice would be required to detect any benefit as a result of 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 of 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 of 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 of 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 of 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 of age which subsides by 6 months of age and is followed by hypoactivity beginning around 8 months of 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 of age and progressively worsens with age. In contrast, increasing age does not affect the rotarod performance of 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 of age. A s the striatum is involved in motor function, the observation of 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 of H D . Rotarod deficits have been reported in models expressing an N-terminal fragment of mutant htt (Carter et al, 1999; Schilling et al, 1999; Yamamoto et al, 2000; Laforet et al, 2001), knock-in mouse models of H D (Lin et al, 2001) and a full length transgenic model of 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 (Lin et al, 2001) and full length transgenic models of 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 of htt exhibit only hypoactivity (Mangiarini et al, 1996; Schilling et al, 1999; Yamamoto et al, 2000) while mice expressing at least one third of 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; Lin 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, 1998A; Lawrence et al, 1998B; Hahn-Barma et al, 1998). These deficits worsen with the advancement of the disease and are also found in symptomatic H D patients (Lawrence et al, 1999; Bamford et al, 1995; Ho 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 of motor coordination (Hyde et al, 2001; McFadyen et al, 2003; Le 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 of 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 of the swimming T-maze test despite swimming as fast as W T mice. At this time point, Y A C 128 mice showed normal cognition in the simple swimming test, the normal phase of the swimming T-maze test and in tests of 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 of 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 of 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 of 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. At 7 months of age, Y A C 128 mice learn the rotarod task but are unable to reach the same level of 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% of 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; Brown et al, 2001). Validation for this test comes from the ability of 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 of 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 mice. 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 of habituation, it is possible that the initial hypoactivity in Y A C 128 mice contributes to their decreased habituation. However, the observation of memory deficits in the simple swimming test and in habituation to acoustic startle supports a memory deficit as the cause of decreased open field habituation in symptomatic Y A C 128 mice. Validation for the use of 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 of 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 of a memory deficit. The use of this test is validated by the ability of 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 of the swimming T-maze test. When the location of 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 of the T-maze after discovering the platform was not present in the right arm. This difference in response strategy provides a clear manifestation of 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; Ho 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. As 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 of 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 of motor symptoms at 5.5 weeks of 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 of H D , a newly generated rat transgenic model of H D that expresses approximately one-third of 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 of H D and is likely caused by a gain of toxic function in mutant htt. Our findings in Y A C 128 mice extend the results obtained in N-terminal fragment models of H D to demonstrate cognitive dysfunction in a mouse model expressing full length mutant htt. The demonstration of cognitive impairment in an accurate genetic model of 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 of potential therapies for H D on the cognitive aspects of the disease. I l l Animal research has demonstrated two distinct forms of learning which are mediated by different systems within the brain. Cognitive learning involves the acquisition of knowledge about the environment, such as the generation of 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 McGaugh, 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 of 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 of death of approximately 54 years (calculated from Harper, 1996). This decreased lifespan has been reproduced in animal models expressing an N-terminal fragment of mutant htt with an expanded C A G tract (Mangiarini et al, 1996; Schilling et al, 1999; Von Horsten et al, 2003). The decreased survival we report in the Y A C 128 mice is the first demonstration of impaired survival in a full length mouse model of H D , as neither the knock-in mouse models of 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; Lin 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 of death in our mice is unknown (Mangiarini et al, 1996; Schilling et al, 1999; Von 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 of mutant htt 112 (Mangiarini et al, 1996; Schilling et al, 1999; Von 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 Age Working Group, 2000; Van 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 of a caspase inhibiting factor (Kolsh and Rao, 2002; Dubai et al, 1999; Zhang et al, 2004). Based on the known involvement of caspases in the pathogenesis of H D , increased caspase inhibitor induction in female mice resulting from higher estrogen levels is in accordance with our finding of 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 of 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 of 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 of 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% of female Y A C 128 mice survived to 12 months but only 74% of male Y A C 1 2 8 mice lived to 12 months (p = 0.01). We 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 of age, 4 of 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 of H D . A t 12 months of age, examination of regional volumes within the brain of these mice revealed volume losses of 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 of selective degeneration in Y A C 128 mice is similar to human H D where volume losses of 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 of the brain again indicating that the global brain atrophy is caused by degeneration of selective regions. We also show here that cell loss of 9.1% and 8.3% is present in the striatum and cortex, respectively, of Y A C 128 mice while 114 neuronal numbers within the hippocampus are spared. Similarly, in human H D , cell loss of 89% and 42% has been reported in the striatum and the motor cortex of end stage patients (MacDonald and Halliday, 2002). While the pattern of selective degeneration is similar between Y A C 128 mice and human H D , the magnitude of change is much greater in human patients. This may stem from marked differences in age of onset (—39 years in humans versus 2-3 months in Y A C 128 mice) and or the duration of 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 of differences between species, animal models of 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 of 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 of the disease then the magnitude of 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 of 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 of common phenotypes (Table 6.1). The most frequently reported observation is the presence of aggregates which in most cases is preceded by the nuclear localization of mutant htt. Interestingly, the knock-in mouse models show very mild striatal neuropathology aside from the nuclear localization of mutant htt. However, this may represent an early step in the pathogenesis of 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 of 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 Type Neuropathology Reference R6/2 N-terminal fragment 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 N171-82Q N-terminal fragment 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 HD100 N-terminal fragment Dystrophic neurites in striatum and cortex only, nuclear localization of mutant htt in affected regions, aggregates, -53% decrease in striatal neuronal cross-sectional area, no neuronal loss Laforet etal, 2001 Hdh4/Q80 Knock-in 10-15% decrease in brain weight, no neuronal loss Shelbourne et al, 1999 Nuclear localization of mutant htt, aggregates Li et al, 2000 CAG94 Knock-in -14% decrease in striatal volume, no neuronal loss, nuclear localization of mutant htt, aggregates Menalled et al, 2002 HdhQ"1 Knock-in 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 YAC128 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 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 of the entire H D gene promoter and or the presence of 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 of 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 of 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 of 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 of 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 of 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, 1993; Trottier et al, 1995; L i et al, 1993; Landwehrmeyer et al, 1995). Thus, the pattern of mutant htt expression is similar in the Y A C 128 mouse model and human H D and in neither case explains the selective degeneration that occurs. 117 In contrast to mutant htt expression, nuclear localization of mutant htt was greatest in the striatum - the region most affected in H D . A t 3 months of age, mutant htt was already present in the nucleus of striatal neurons. This is the age when motor and cognitive deficits are first observed in Y A C 128 mice. While some nuclear localization of mutant htt was present in layer IV of the cortex, in the C A 3 region of the hippocampus and the cellular layer of the cerebellum at 3 months, the amount of mutant htt in the nucleus in striatal neurons was much greater than in any other region of the brain. In addition to the regional specificity of the E M 4 8 staining, we also observed selective nuclear localization of mutant htt within the striatum. The lateral striatum was found to have visually more nuclear localization of mutant htt than the medial striatum. Combined with our previous observation of increased neurodegeneration in the lateral striatum compared to the medial striatum in Y A C models of H D (Hodgson et al, 1999), this supports the possibility that nuclear localization of mutant htt is at least partially responsible for the selective degeneration in H D . Early nuclear localization of 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 of mutant htt compared to the 3 month time point, with the striatum still showing the greatest extent of nuclear localization. In the cortex, all o f the cell dense layers show nuclear localization of htt (II, III, IV and VI) 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 of 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 of 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 (NES) in its carboxy-terminus which can be separated from the amino-terminus o f the protein when htt is cleaved (Xia 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 of the mutant protein (Li et al, 1999; Sapp et al, 1997). This may result from decreased interaction of mutant htt with the nuclear pore translocated promoter region (Tpr) which is thought to transport htt out of the nucleus (Cornett et al, 2005). Many in vitro experiments have demonstrated that nuclear localization of 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 (NLS) 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 of 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 of 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 of this disorder (Katsuno et al, 2002; Katsuno et al, 2003). The adverse effect of 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 of 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 of mutant htt in the nucleus increases expression of caspase 1 and induces activation of caspase 3 and release of cytochrome c thereby leading towards apoptosis (L i et al, 2000). Irrespective of the mechanism of mutant htt toxicity within the nucleus, these findings provide a possible mechanism by which increased nuclear localization of mutant htt leads to increased toxicity as we observed in the striatum of Y A C 128 mice. Early nuclear localization of htt in the striatum has been previously reported in other mouse models of 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 of htt were not examined. In a knock-in model of H D with 140 C A G repeats, nuclear localization of 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 of mutant htt and cerebral damage because of the mild neuropathology in these mice. Staining with E M 4 8 in mouse models expressing N-terminal fragments of mutant htt revealed the presence of nuclear aggregates throughout the brain (Davies et al, 1997; Schilling et al, 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 of 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 of mutant htt occurs to a similar extent in all regions of the brain and this may explain the relatively unselective pathology. The lack of selectivity in R6/1 mice may result from the absence of important regulatory elements controlling the expression of mutant htt and or the absence of the normal protein context for the polyglutamine tract. Overall, the nuclear localization of mutant htt appears to be a common step in the progression of H D and our findings here suggest the possibility that selective nuclear localization of 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 of wild-type htt eliminated the testicular phenotype. Since testicular degeneration only occurred in the presence of 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 of wild-type htt function alone. 120 Here, we show that testicular degeneration can be caused solely by the toxicity of mutant htt. We show testicular atrophy and the disruption of 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 of 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 of the brain (Sharp et al, 1995). In absence of mutant htt, inactivation of wild-type htt in adult mice results in decreased sperm counts and disorganization of the seminiferous tubules (Dragatsis et al, 2000). The importance of htt function for sperm development is indicated by the upregulation of 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 of 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, 1989; Shokeir et al, 1975). However, this is not surprising considering that the average age of 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 of symptoms or degeneration. In our study, we examined the testis of 12 month old Y A C 128 mice. These mice show onset of HD- l ike symptoms at about 2-3 months of 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 of 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 of Y A C 1 2 8 mice is 121 summarized in Figure 6.1. A comparison of the Y A C 128 mouse model with human H D reveals that Y A C 128 mice reproduce many of the symptoms of 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 of 2 copies of the normal H D gene in Y A C 128 mice, it l ikely 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 of 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 of H D . Specifically, Y A C 1 2 8 mice can be used to examine the time course of 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 of various treatments for H D prior to clinical trials. Recently, treatment of 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 of 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 of the Y A C 128 mouse model with other mouse models of H D based on the criteria for an ideal mouse model of H D outlined in section 1.4 reveals that only the Y A C 128 mouse model satisfies all of the criteria (Table 6.3). 6.3 Wild-type huntingtin function The critical importance of wild-type htt function is demonstrated by embryonic lethality in mice that are homozygous for the targeted inactivation of 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 of 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 \ Hypoactivity Decreased Habituation Survival Rotarod Deficit I Cognition in Swimming Tests Deficit I PPI V V V V AGE(Months) 2 8 t Nuclear Localization of Mutant Htt NEUROPATHOLOGY I Brain Weight 4 Striatal Volume Macroaggregates 4 Cortical Volume Neuronal Shrinkage Striatal Neuronal Loss Cortical Neuronal Loss Atrophy of Globus Pallidus DARPP-32 Downregulation Dysfunction ^ 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 T-maze 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. 1 2 3 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. H u m a n H D Y A C 1 2 8 Motor Dysfunction Chorea Hyperkinesia Bradykinesia, gait abnormalities Rotarod deficit Bradykinesia Hypoactivity Cognitive Impairment Procedural learning deficit Deficits in motor learning, simple swimming test, and swimming T-maze Attentional set shifting deficit, perseveration Deficit in strategy shifting Memory deficit Decreased open field habituation Deficit in PPI Deficit in PPI Psychiatric Disturbances Irritability, aggressive outbursts, anxiety and apathy Depression, Not assessed Neuropathology Striatal atrophy 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 Decreased brain weight Gene Expression Express FL mutant htt Express FL mutant htt Tissue specific expression Tissue specific expression Adult onset < 55 CAG repeats -120 CAG repeats 1 copy of WT HD gene 2 copies of WT HD gene 50% mutant htt expression 75% mutant htt expression Onset -40 years 2-3 months Lifespan of unaffected individuals -80 years -2 years Survival 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 Neurotoxin N-terminal fragment Knock-in YAC128 Motor deficits Yes Yes Yes Yes Cognitive Impairment Yes Yes No : Yes -Selective atrophy Yes No Yes Yes Selective neuronal loss Yes No No Yes Progressive phenotype No Yes Yes Yes Full length mutant htt No No Yes Yes Therapeutic trials Yes Yes No •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; Ho 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 of which have been used to model H D . The involvement of excitotoxicity in H D was originally proposed based on the loss of N M D A receptor bearing neurons within the striatum (Young et al, 1988) as well as the induction of striatal neuronal loss using the excitotoxic neurotoxins, kainic acid (Coyle and Schwarcz, 1976; McGeer and McGeer, 1976) and quinolinic acid (Beal et al, 1986; Beal et al, 1991). Excitotoxicity results from an over-stimulation with excitatory amino acids such as glutamate which leads to increased intracellular calcium levels, increased activation of calcium dependent enzymes and signaling pathways and eventual neuronal death (Sattler and Tymianski, 2000). The importance of excitotoxicity to H D is supported by the modulation of age of onset based on variation in the GluR6 kainate receptor (Rubinsztein et al, 1997; MacDonald et al, 1999) and the fact that both human patients and a mouse model of H D were shown to have decreased expression of the glutamate transporter G L T 1 (Arzberger et al, 1997; Lievens et al, 2001). Further, it appears that the expression of mutant htt in H D makes cells more sensitive to glutamate. signals. The expression of 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 of 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 of glutamate resulted in an elevation of 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 of mitochondria (Bezprozvanny and Hayden, 2004; Panov et al, 2002). 126 While mutant htt increases the susceptibility of cells to excitotoxic cell death, the work in this thesis demonstrates that wild-type htt protects against it. A comparison of hippocampal neuronal damage resulting from intraperitoneal injection of 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 over-expression of wild-type htt in Y A C 18 mice resulted in significantly reduced lesion sizes and significantly more surviving neurons after intrastriatal injection of 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 of 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 of mice over-expressing wild-type htt (line B60 and line 212), thereby confirming that the protection does not result from a site of integration effect. Further, we demonstrate that the neuroprotective effect of 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 of 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 of 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; Perez-Navarro et al, 2000) and is thought to at least be partially responsible for the beneficial effects of dietary restriction (Duan et al, 2001). Since wild-type htt increases B D N F expression (Zuccato et al, 2001) and facilitates transport of B D N F (Gauthier et al, 2004), it is plausible that at least part of 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 of the pro-apoptotic protein HIP1 (huntingtin interacting protein 1). Co-expression of 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 of wild-type huntingtin to weight loss in Huntington 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 of striatal neuropathology in Y A C 18/128 mice revealed a mild trend towards improvement of striatal volume, striatal neuronal counts and striatal D A R P P - 3 2 expression, all of which failed to reach significance. There was, however, a significant improvement in striatal neuronal cross-sectional area in YAC18/128 mice compared to Y A C 1 2 8 mice. In parallel with these experiments we examined the effect of 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 of 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 of wild-type htt may be real changes since the removal of wild-type htt results in a mild worsening of these same phenotypes. Previously, two independent studies have compared the phenotype of 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 of wild-type htt has only a small impact on the disease phenotype. Similarly, studies comparing the severity of 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 of 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 of the disease is not always observed, suggests again that the loss of wild-type htt does not have a great impact on the development of H D . A robust finding of this study was that over-expression of 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 of 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 of 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 of 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 of 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 of 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 of 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 of increase in T G activity could be accounted for by GTP-inhibited T G activity, the magnitude of increase in the non-GTP inhibited T G activity was much greater than for total T G activity suggesting the possibility that TGs other than tTG may be important in the pathogenesis of H D (since tTG activity is inhibited by GTP) . The recapitulation of the increased T G activity seen in human patients in the Y A C 128 mouse model lends further support to the use of this model to study the pathogenesis of 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 of the H D . While a correlation exists in vitro between length of polyglutamine expansion and the ability of tTG to cross-link proteins with polyglutamine expansions (Kahlem et al, 1998; Cooper et al, 2002), tTG 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 TG 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 TG 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 TG 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). An 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 TG 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 TG 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 TG 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 TG inhibition, cystamine may be beneficial by inhibiting caspase 3 activity (Lesort et al, 2003) or through by up-regulating 136 the expression of heat shock proteins (Karpuj et al, 2002). A final possibility is that the beneficial effects of 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 of 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 of 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 of mechanisms. 6.5.4 Implications for treatment of Huntington disease Overall, we show that cystamine treatment is neuroprotective and prevents development of striatal neuropathology in the Y A C 1 2 8 mouse model of H D . Since the beneficial effects of cystamine were not beneficial for all symptoms of the disease, it may be necessary to combine cystamine treatment with a compound that can reduce the motor phenotypes of the disease. The positive effects of 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 of this compound for the treatment of 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 Contr ibut ion of loss of huntingtin function to Huntington disease The fact that mutant htt is expressed and that the expression of mutant htt alone, without reductions in wild-type htt levels, is sufficient to cause HD-l ike 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 of function 137 mutation is supported by the fact that deletion of one HD 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 HD (White et al, 1997; Dragatsis et al, 2000; Auerbach et al, 2001). Since mice heterozygous for the targeted inactivation of the mouse HD 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 HD 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 HD 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 HD can be proposed. HD 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 i f 50% protein expression is not sufficient for normal function. In addition, levels of wild-type 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 of wild-type htt with mutant htt (Narain et al, 1999) or an altered localization of 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 of mutant htt these patients would also express decreased levels of 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 of patients examined in all of these studies was few (17 homozygous patients in 4 separate studies), resulting from the limited number of H D homozygotes, more patients w i l l need to be examined to arrive at a definitive conclusion. The generation of knock-in mouse models of H D has provided the opportunity to address this question more fully. In two separate knock-in mouse models of 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 (Lin et al, 2001; Wheeler et al, 2000). This suggests that the conclusions of the latter of 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 of loss of wild-type htt to the exacerbation of phenotype because mutant htt expression was increased simultaneously. Recent studies convincingly demonstrate that increasing the expression of mutant htt results in a more severe phenotype (Graham et al, 2005). The contribution of loss of wild-type htt was directly assessed by eliminating wild-type htt expression in the Y A C 1 2 8 mouse model of H D . In this study, there was a non-significant worsening of striatal volume, striatal neuronal counts and striatal D A R P P - 3 2 expression and a significant exacerbation of 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 of Y A C 1 2 8 mice with the over-expression of wild-type htt results in a mild, non-significant 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 of the loss of wild-type htt function to the striatal neuropathology in H D and suggests that wild-type htt treatment in H D wi l l not be sufficient to prevent striatal neuropathology. 6.7 Conclusions The goals of this thesis were (1) to characterize genetic mouse models of H D (2) to develop a protocol and outcome measures for therapeutic trials in these mice and (3) to demonstrate the efficacy of 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 of H D recapitulates the motor dysfunction, cognitive impairment and selective neurodegeneration of H D . Importantly, this represents the first demonstration of selective and progressive striatal neuronal loss in a mouse model of H D . The phenotypic characterization of 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 D A R P P - 3 2 expression, rotarod performance, open field activity and pre-pulse inhibition. With larger numbers of mice it wi l l also be possible to assess the effect of 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 of 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 of 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 over-express 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 over-expression 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 wild-type 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 of changes that are not part of the disease process. In contrast, Y A C 128 mice can be sacrificed at any time point in the progression of the disease with negligible post mortem interval. Accordingly, the processes involved in H D pathogenesis and more importantly the precise sequence of these events can be determined by analyzing Y A C 128 mice at different time points. In studying the pathogenesis of 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 of 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 of these mice have not been elucidated and warrant further study. Several mouse models of 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 of histone proteins has been found in mouse models of H D and the restoration of histone acetylation has been beneficial in preclinical therapeutic trials (Ferrante et al, 2003; Hockly 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 of 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 of H D and whether the therapeutic efficacy of histone deacetylase inhibitors should be assessed in these mice. Finally, an analysis of protein expression in Y A C 128 mice compared to W T mice may further inform studies of pathogenesis. Axonal transport - Several recent experiments have demonstrated that the expression of mutant htt or the decreased expression of 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 of 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. EM 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 of htt function. Initially, it w i l l be important to determine i f this increase in weight results from an increased number of cells, an increased size of cells or both. It would also be interesting to examine why htt does not effect the weight of the brain and testis despite the fact that these two organs express the highest levels of htt. 4. Mechanism of cystamine neuroprotection in HD While cystamine was shown to be neuroprotective in the Y A C 128 mouse model of H D , it is uncertain the mechanism responsible for this beneficial effect. The possibilities include T G inhibition, caspase inhibition, increased expression of heat shock proteins and increased levels of anti-oxidant. Further experimentation would be necessary to determine i f all o f these mechanisms contribute and which is most important for the effects observed. Determining the exact mechanism may also provide insight into why cystamine was unable to prevent motor dysfunction in Y A C 128 mice. To determine i f increased levels of the anti-oxidant cysteine are responsible (Fox et al, 2004; Pinto et al, 2005), Y A C 1 2 8 mice could be treated with N-acetyl-L-cysteine. 147 7.0 R E F E R E N C E S 1. Agrawal N , Pallos J, Slepko N , Apostol B L , Bodai L , Chang L W , Chiang A S , Thompson L M and Marsh J L (2005). Identification of combinatorial drug regimens for treatment of Huntington's disease using Drosophila. Proc. Natl . Acad. Sci . U . S. A 102(10): 3777-3781. 2. Ahlbom E , Grandison L , Bonfoco E , Zhivotovsky B and Ceccatelli S (1999). Androgen treatment of neonatal rats. decreases susceptibility of cerebellar granule neurons to oxidative stress in vitro. Eur. J. Neurosci 11(4): 1285-1291. 3. A l b i n R L , Reiner A , Anderson K D , Dure L S , Handelin B , Balfour R, Whetsell W O , Jr., Penney JB and Young A B (1992). Preferential loss of striato-external pallidal projection neurons in presymptomatic Huntington's disease. Ann . Neurol. 31(4): 425-430. 4. Altar C A , Cai N , Bl iven T, Juhasz M , Conner J M , Acheson A L , Lindsay R M and Wiegand SJ (1997). Anterograde transport of brain-derived neurotrophic factor and its role in the brain. Nature 389(6653): 856-860. 5. Ambrose C M , Duyao M P , Barnes G , Bates G P , L i n C S , Srinidhi J, Baxendale S, Hummerich H , Lehrach H , Altherr M and . (1994). Structure and expression of the Huntington's disease gene: evidence against simple inactivation due to an expanded C A G repeat. Somat. Cel l M o l . Genet. 20(1): 27-38. 6. Andreassen O A , Ferrante R J , Hughes D B , Kl ivenyi P, Dedeoglu A , Ona V O , Friedlander R M and Beal M F (2000). Malonate and 3-nitropropionic acid neurotoxicity are reduced in transgenic mice expressing a caspase-1 dominant-negative mutant. J. Neurochem. 75(2): 847-852. 7. Andreassen O A , Dedeoglu A , Ferrante R J , Jenkins B G , Ferrante K L , Thomas M , Friedlich A , Browne SE, Schilling G , Borchelt D R , Hersch S M , Ross C A and Beal M F (2001). Creatine increase survival and delays motor symptoms in a transgenic animal model of Huntington's disease. Neurobiol. Dis. 8(3): 479-491. 8. Andreassen O A , Ferrante RJ , Dedeoglu A and Beal M F (2001). Lipoic acid improves survival in transgenic mouse models of Huntington's disease. Neuroreport 12(15): 3371-3373. 9. Andreassen O A , Ferrante R J , Huang H M , Dedeoglu A , Park L , Ferrante K L , Kwon J , Borchelt D R , Ross C A , Gibson G E and Beal M F (2001). Dichloroacetate exerts therapeutic effects in transgenic mouse models of Huntington's disease. Ann. Neurol. 50(1): 112-117. 10. Apostol B L , Kazantsev A , Raffioni S, Illes K , Pallos J, Bodai L , Slepko N , Bear JE, Gertler F B , Hersch S, Housman D E , Marsh J L and Thompson L M (2003). A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila. Proc. Natl. Acad. Sci. U . S. A 100(10): 5950-5955. 148 11. Arrasate M , Mitra S, Schweitzer ES , Segal M R and Finkbeiner S (2004)..Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431(7010): 805-810. 12. Arzberger T, Krampfl K , Leimgruber S and Weindl A (1997). Changes of N M D A receptor subunit (NR1, N R 2 B ) and glutamate transporter (GLT1) m R N A expression in Huntington's disease-an in situ hybridization study. J. Neuropathol. Exp. Neurol. 56(4): 440-454. 13. Auerbach W , Hurlbert M S , Hilditch-Maguire P, Wadghiri Y Z , Wheeler V C , Cohen SI, Joyner A L , Macdonald M E and Turnbull D H (2001). The H D mutation causes progressive lethal neurological disease in mice expressing reduced levels of huntingtin. Hum. M o l . Genet. 10(22): 2515-2523. 14. Backman L , Robins-Wahlin T B , Lundin A , Ginovart N and Farde L (1997). Cognitive deficits in Huntington's disease are predicted by dopaminergic P E T markers and brain volumes. Brain 120 ( Pt 12)2207-2217. 15. Bailey C D , Graham R M , Nanda N , Davies PJ and Johnson G V (2004). Validity of mouse models for the study of tissue transglutaminase in neurodegenerative diseases. M o l . Cel l Neurosci. 25(3): 493-503. 16. Bailey C D and Johnson G V (2005a). Tissue transglutaminase contributes to disease progression in the R6/2 Huntington's disease mouse model via aggregate-independent mechanisms. J. Neurochem. 92(1): 83-92. 17. Bailey C D and Johnson G V (2005b). The protective effects of cystamine in the R6/2 Huntington's disease mouse involve mechanisms other than the inhibition of tissue transglutaminase. Neurobiol. Aging 18. Baldereschi M , D i Carlo A , Rocca W A , Vanni P, Maggi S, Perissinotto E , Grigoletto F, Amaducci L and Inzitari D (2000). Parkinson's disease and parkinsonism in a longitudinal study: two-fold higher incidence in men. I L S A Working Group. Italian Longitudinal Study on Aging. Neurology 55(9): 1358-1363. 19. Bamford K A , Caine E D , Kido D K , Cox C and Shoulson I (1995). A prospective evaluation of cognitive decline in early Huntington's disease: functional and radiographic correlates. Neurology 45(10): 1867-1873. 20. Beal M F , Kowal l N W , Ell ison D W , Mazurek M F , Swartz K J and Martin JB (1986). Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid. Nature 321(6066): 168-171. 21. Beal M F , Ferrante R J , Swartz K J and Kowal l N W (1991). Chronic quinolinic acid lesions in rats closely resemble Huntington's disease. J. Neurosci 11(6): 1649-1659. 149 22. Beal M F , Brouillet E , Jenkins B G , Ferrante R J , Kowal l N W , Mi l le r J M , Storey E , Srivastava R, Rosen B R and Hyman B T (1993). Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J. Neurosci 13(10): 4181-4192. 23. Beal M F (1998). Mitochondrial dysfunction in neurodegenerative diseases. Biochim. Biophys. Acta 1366(1-2): 211-223. 24. Bemelmans A P , Horellou P, Pradier L , Brunet I, Col in P and Mallet J (1999). Brain-derived neurotrophic factor-mediated protection of striatal neurons in an excitotoxic rat model of Huntington's disease, as demonstrated by adenoviral gene transfer. Hum. Gene Ther. 10(18): 2987-2997. 25. Berrios G E , Wagle A C , Markova IS, Wagle S A , Rosser A and Hodges JR (2002). Psychiatric symptoms in neurologically asymptomatic Huntington's disease gene carriers: a comparison with gene negative at risk subjects. Acta Psychiatr. Scand. 105(3): 224-230. 26. Bezprozvanny 1 and Hayden M R (2004). Deranged neuronal calcium signaling and Huntington disease. Biochem. Biophys. Res. Commun. 322(4): 1310-1317. 27. Bhide P G , Day M , Sapp E , Schwarz C, Sheth A , K i m J, Young A B , Penney J, Golden J, Aronin N and DiFigl ia M (1996). Expression of normal and mutant huntingtin in the developing brain. J. Neurosci 16(17): 5523-5535. 28. Bibb J A , Yan Z , Svenningsson P, Snyder G L , Pieribone V A , Horiuchi A , Nairn A C , Messer A and Greengard P (2000). Severe deficiencies in dopamine signaling in presymptomatic Huntington's disease mice. Proc. Natl . Acad. Sci. U . S. A 97(12): 6809-6814. 29. Blackmore L , Simpson S A and Crawford JR (1995). Cognitive performance in U K sample of presymptomatic people carrying the gene for Huntington's disease. J. Med . Genet. 32(5): 358-362. 30. Block-Galarza J, Chase K O , Sapp E , Vaughn K T , Vallee R B , DiF ig l i a M and Aronin N (1997). Fast transport and retrograde movement of huntingtin and H A P 1 in axons. Neuroreport 8(9-10): 2247-2251. 31. Bolivar V J , Caldarone B J , Rei l ly A A and Flaherty L (2000). Habituation of activity in an open field: A survey of inbred strains and FI hybrids. Behav. Genet. 30(4): 285-293. 32. Bolivar V J , Manley K and Messer A (2003). Exploratory activity and fear conditioning abnormalities develop early in R6/2 Huntington's disease transgenic mice. Behav. Neurosci. 117(6): 1233-1242. 33. Bolivar V J , Manley K and Messer A (2004). Early exploratory behavior abnormalities in R6/1 Huntington's disease transgenic mice. Brain Res. 1005(1-2): 29-35. 150 34. Bosher J M and Labouesse M (2000). R N A interference: genetic wand and genetic watchdog. Nat. Cel l B io l . 2(2): E31-E36. 35. Boutell J M , Thomas P, Neal JW, Weston V J , Duce J, Harper PS and Jones A L (1999). Aberrant interactions of transcriptional repressor proteins with the Huntington's disease gene product, huntingtin. Hum. M o l . Genet. 8(9): 1647-1655. 36. Brinkman RR, Mezei M M , Theilmann J, Almqvist E and Hayden M R (1997). The likelihood of being affected with Huntington disease by a particular age, for a specific C A G size. A m . J. Hum. Genet. 60(5): 1202-1210. 37. Brown R G , Redondo-Verge L , Chacon JR, Lucas M L and Channon S (2001). Dissociation between intentional and incidental sequence learning in Huntington's disease. Brain 124(Pt 11): 2188-2202. 38. Browne SE, Ferrante R J and Beal M F (1999). Oxidative stress in Huntington's disease. Brain Pathol. 9(1): 147-163. 39. Buitrago M M , Schulz JB, Dichgans J and Luft A R (2004). Short and long-term motor skill learning in an accelerated rotarod training paradigm. Neurobiol. Learn. Mem. 81(3): 211-216. 40. Carter RJ , Lione L A , Humby T, Mangiarini L , Mahal A , Bates G P , Dunnett SB and Morton A J (1999). Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation. J. Neurosci. 19(8): 3248-3257. 41. Cattaneo E , Rigamonti D , Goffredo D , Zuccato C, Squitieri F and Sipione S (2001). Loss of normal huntingtin function: new developments in Huntington's disease research. Trends Neurosci. 24(3): 182-188. 42. Cha JH (2000). Transcriptional dysregulation in Huntington's disease. Trends Neurosci 23(9): 387-392. 43. Chan E Y , Luthi-Carter R, Strand A , Solano S M , Hanson S A , DeJohn M M , Kooperberg C, Chase K O , DiFig l ia M , Young A B , Leavitt B R , Cha J H , Aronin N , Hayden M R and Olson J M (2002). Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease. Hum. M o l . Genet. 11(17): 1939-1951. 44. Chen M , Ona V O , L i M , Ferrante R J , Fink K B , Zhu S, Bian J, Guo L , Farrell L A , Hersch S M , Hobbs W , Vonsattel JP, Cha J H and Friedlander R M (2000). Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat. Med. 6(7): 797-801. 45. Chou S Y , Lee Y C , Chen H M , Chiang M C , La i H L , Chang H H , W u Y C , Sun C N , Chien C L , Lin Y S , Wang SC, Tung Y Y , Chang C and Chern Y (2005). CGS21680 attenuates symptoms of Huntington's disease in a transgenic mouse model. J. Neurochem. 93(2): 310-320. 151 46. Chun W, Lesort M , Tucholski J, Ross C A and Johnson G V (2001). Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates. J. Cel l B io l . 153(1): 25-34. 47. Clifford JJ, Drago J, Natoli A L , Wong J Y , Kinsella A , Waddington J L and Vaddadi K S (2002). Essential fatty acids given from conception prevent topographies of motor deficit in a transgenic model of Huntington's disease. Neuroscience 109(1): 81-88. 48. Conneally P M (1984). Huntington Disease - Genetics and Epidemiology. American Journal of Human Genetics 36(3): 506-526. 49. Cook M N , Bolivar V J , McFadyen M P and Flaherty L (2002). Behavioral differences among 129 substrains: implications for knockout and transgenic mice. Behav. Neurosci. 116(4): 600-611. 50. Cooper A J , Sheu K R , Burke JR, Onodera O, Strittmatter W J , Roses A D and Blass JP (1997). Transglutaminase-catalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and alpha-ketoglutarate dehydrogenase complex by polyglutamine domains of pathological length. Proc. Natl . Acad. Sci. U . S. A 94(23): 12604-12609. 51. Cooper A J , Jeitner T M , Gentile V and Blass JP (2002). Cross linking of polyglutamine domains catalyzed by tissue transglutaminase is greatly favored with pathological-length repeats: does transglutaminase activity play a role in (CAG)(n)/Q(n)-expansion diseases? Neurochem. Int. 40(1): 53-67. 52. Cornett J, Cao F, Wang C E , Ross C A , Bates G P , L i S H and L i X J (2005). Polyglutamine expansion of huntingtin impairs its nuclear export. Nat. Genet. 37(2): 198-204. 53. Coyle JT and Schwarcz R (1976). Lesion of striatal neurones with kainic acid provides a model for Huntington's chorea. Nature 263(5574): 244-246. 54. Cudkowicz M and Kowal l N W (1990). Degeneration of pyramidal projection neurons in Huntington's disease cortex. Ann. Neurol. 27(2): 200-204. 55. Davies S W , Turmaine M , Cozens B A , DiF ig l ia M , Sharp A H , Ross C A , Scherzinger E , Wanker E E , Mangiarini L and Bates G P (1997). Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the H D mutation. Cel l 90(3): 537-548. 56. de Boo G M , Tibben A , Lanser JB, Jennekens-Schinkel A , Hermans J, Maat-Kievit A and Roos R A (1997). Early cognitive and motor symptoms in identified carriers of the gene for Huntington disease. Arch. Neurol. 54(11): 1353-1357. 57. de Boo G M , Tibben A A , Hermans J A , Jennekens-Schinkel A , Maat-Kievit A and Roos R A (1999). Memory and learning are not impaired in presymptomatic individuals with an increased risk of Huntington's disease. J. C l in . Exp. Neuropsychol. 21(6): 831-836. 152 58. de Cristofaro T, Affaitati A , Cariello L , Avvedimento E V and Varrone S (1999). The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates. Biochem. Biophys. Res. Commun. 260(1): 150-158. 59. DeCoteau W E and Kesner RP (2000). A double dissociation between the rat hippocampus and medial caudoputamen in processing two forms of knowledge. Behav. Neurosci. 114(6): 1096-1108. 60. Dedeoglu A , Kubilus J K , Jeitner T M , Matson SA, Bogdanov M , Kowal l N W , Matson W R , Cooper A J , Ratan RR, Beal M F , Hersch S M and Ferrante R J (2002). Therapeutic effects of cystamine in a murine model of Huntington's disease. J. Neurosci. 22(20): 8942-8950. 61. Dedeoglu A , Kubilus J K , Yang L , Ferrante K L , Hersch S M , Beal M F and Ferrante RJ (2003). Creatine therapy provides neuroprotection after onset of clinical symptoms in Huntington's disease transgenic mice. J. Neurochem. 85(6): 1359-1367. 62. Devan B D and White N M (1999). Parallel information processing in the dorsal striatum: relation to hippocampal function. J. Neurosci. 19(7): 2789-2798. 63. DiFig l ia M , Sapp E , Chase K , Schwarz C, Meloni A , Young C, Martin E , Vonsattel JP, Can-away R, Reeves S A and . (1995). Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14(5): 1075-1081. 64. DiFig l ia M , Sapp E , Chase K O , Davies SW, Bates GP , Vonsattel JP and Aronin N (1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277(5334): 1990-1993. 65. Dixon K T , Cearley J A , Hunter J M and Detloff PJ (2004). Mouse Huntington's disease homolog m R N A levels: variation and allele effects. Gene Expr. 11(5-6): 221-231. 66. Djousse L , Knowlton B , Cupples L A , Marder K , Shoulson I and Myers R H (2002). Weight loss in early stage of Huntington's disease. Neurology 59(9): 1325-1330. 67. Dobkin C, Rabe A , Dumas R, E l Idrissi A , Haubenstock H and Brown W T (2000). F m r l knockout mouse has a distinctive strain-specific learning impairment. Neuroscience 100(2): 423-429. 68. Dorsman JC, Smoor M A , Maat-Schieman M L , Bout M , Siesling S, van Duinen SG, Verschuuren JJ, den Dunnen JT, Roos R A and van Ommen G J (1999). Analysis of the subcellular localization of huntingtin with a set of rabbit polyclonal antibodies in cultured mammalian cells of neuronal origin: comparison with the distribution of huntingtin in Huntington's disease autopsy brain. Philos. Trans. R. Soc. Lond B B i o l . Sci. 354(1386): 1061-1067. 153 69. Dragatsis I, Levine M S and Zeitlin S (2000). Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice! Nat. Genet. 26(3): 300-306. 70. D u L , Bayir H , La i Y , Zhang X , Kochanek P M , Watkins SC, Graham S H and Clark RS (2004). Innate gender-based proclivity in response to cytotoxicity and programmed cell death pathway. J. B i o l . Chem. 279(37): 38563-38570. 71. Duan W , Lee J, Guo Z and Mattson M P (2001). Dietary restriction stimulates B D N F production in the brain and thereby protects neurons against excitotoxic injury. J. M o l . Neurosci 16(1): 1-12. 72. Duan W , Guo Z , Jiang H , Ware M , L i X J and Mattson M P (2003). Dietary restriction normalizes glucose metabolism and B D N F levels, slows disease progression, and increases survival in huntingtin mutant mice. Proc. Natl . Acad. Sci . U . S. A 100(5): 2911-2916. 73. Duan W , Guo Z , Jiang H , Ladenheim B , X u X , Cadet J L and Mattson M P (2004). Paroxetine retards disease onset and progression in Huntingtin mutant mice. Ann . Neurol. 55(4): 590-594. 74. Dubai D B , Wilson M E and Wise P M (1999). Estradiol: a protective and trophic factor in the brain. J. Alzheimers. Dis. 1(4-5): 265-274. 75. Dubois M , Strazielle C, Eyer J and Lalonde R (2002). Sensorimotor functions in transgenic mice expressing the neurofilament/heavy-LacZ fusion protein on two genetic backgrounds. Neuroscience 112(2): 447-454. 76. Duhah A W , Jeong H , Griffin A , K i m Y M , Standaert D G , Hersch S M , Mouradian M M , Young A B , Tanese N and Krainc D (2002). Sp l and TAFII130 transcriptional activity disrupted in early Huntington's disease. Science 296(5576): 2238-2243. 77. Dunnett S B , Carter R J , Watts C, Torres E M , Mahal A , Mangiarini L , Bates G and Morton A J (1998). Striatal transplantation in a transgenic mouse model of Huntington's disease. Exp. Neurol. 154(1): 31-40. 78. Duyao M , Ambrose C, Myers R, Novelletto A , Persichetti F, Frontali M , Folstein S, Ross C, Franz M , Abbott M and . (1993). Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat. Genet. 4(4): 387-392. 79. Duyao M P , Auerbach A B , Ryan A , Persichetti F, Barnes G T , M c N e i l S M , Ge P, Vonsattel JP, Gusella JF, Joyner A L and . (1995). Inactivation of the mouse Huntington's disease gene homolog Hdh. Science 269(5222): 407-410. 80. E l Massioui N , Ouary S, Cheruel F, Hantraye P and Brouillet E (2001). Perseverative behavior underlying attentional set-shifting deficits in rats chronically treated with the neurotoxin 3-nitropropionic acid. Exp. Neurol. 172(1): 172-181. 154 81. Fennema-Notestine C, Archibald S L , Jacobson M W , Corey-Bloom J, Paulsen JS, Peavy G M , Gamst A C , Hamilton J M , Salmon D P and Jernigan T L (2004). In vivo evidence of cerebellar atrophy and cerebral white matter loss in Huntington disease. Neurology 63(6): 989-995. 82. Ferrante R J , Andreassen O A , Jenkins B G , Dedeoglu A , Kuemmerle S, Kubilus J K , Kaddurah-Daouk R, Hersch S M and Beal M F (2000). Neuroprotective effects of creatine in a transgenic mouse model of Huntington's disease. J. Neurosci 20(12): 4389-4397. 83. Ferrante RJ , Andreassen O A , Dedeoglu A , Ferrante K L , Jenkins B G , Hersch S M and Beal M F (2002). Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington's disease. J. Neurosci 22(5): 1592-1599. 84. Ferrante R J , Kubilus J K , Lee J, Ryu H , Beesen A , Zucker B , Smith K , Kowal l N W , Ratan R R , Luthi-Carter R and Hersch S M (2003). Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J. Neurosci 23(28): 9418-9427. 85. Ferrante R J , Ryu H , Kubilus J K , D 'Mel lo S, Sugars K L , Lee J, L u P, Smith K , Browne S, Beal M F , Kristal B S , Stavrovskaya IG, Hewett S, Rubinsztein D C , Langley B and Ratan R R (2004). Chemotherapy for the brain: the antitumor antibiotic mithramycin prolongs survival in a mouse model of Huntington's disease. J. Neurosci 24(46): 10335-10342. 86. Ferrer I, Goutan E , Marin C, Rey M J and Ribalta T (2000). Brain-derived neurotrophic factor in Huntington disease. Brain Res. 866(1-2): 257-261. 87. Fox J H , Barber D S , Singh B , Zucker B , Swindell M K , Norflus F, Buzescu R, Chopra R, Ferrante R J , Kazantsev A and Hersch S M (2004). Cystamine increases L-cysteine levels in Huntington's disease transgenic mouse brain and. in a P C 12 model of polyglutamine aggregation. J. Neurochem. 91(2): 413-422. 88. Gabrieli JD, Stebbins G T , Singh J, Willingham D B and Goetz C G (1997). Intact mirror-tracing and impaired rotary-pursuit skil l learning in patients with Huntington's disease: evidence for dissociable memory systems in skill learning. Neuropsychology. 11(2): 272-281. 89. Gafni J and Ellerby L M (2002). Calpain activation in Huntington's disease. J. Neurosci 22(12): 4842-4849. 90. Gambassi G , Lapane K L , Landi F, Sgadari A , M o r V and Bernabie R (1999). Gender differences in the relation between comorbidity and mortality of patients with Alzheimer's disease. Systematic Assessment of Geriatric drug use via Epidemiology ( S A G E ) Study Group. Neurology 53(3): 508-516. 155 91. Gardian G , Browne SE, Choi D K , Kl ivenyi P, Gregorio J, Kubilus J K , R y u H , Langley B , Ratan R R , Ferrante R J and Beal M F (2005). Neuroprotective effects of phenylbutyrate in the N171-82Q transgenic mouse model of Huntington's disease. J. B i o l . Chem. 280(1): 556-563. 92. Gauthier L R , Charrin B C , Borrell-Pages M , Dompierre JP, Rangone H , Cordelieres FP, De M e y J, Macdonald M E , Lessmann V , Humbert S and Saudou F (2004). Huntingtin controls neurotrophic support and survival of neurons by enhancing B D N F vesicular transport along microtubules. Cel l 118(1): 127-138. 93. Gerlai R, Mi l l en K J , Herrup K , Fabien K , Joyner A L and Roder J (1996). Impaired motor learning performance in cerebellar En-2 mutant mice. Behav. Neurosci. 110(1): 126-133. 94. Gines S, Seong IS, Fossale E , Ivanova E , Trettel F, Gusella JF, Wheeler V C , Persichetti F and Macdonald M E (2003). Specific progressive c A M P reduction implicates energy deficit in presymptomatic Huntington's disease knock-in mice. Hum. M o l . Genet. 12(5): 497-508. 95. Goldberg Y P , Nicholson D W , Rasper D M , Kalchman M A , Koide H B , Graham R K , Bromm M , Kazemi-Esfarjani P, Thornberry N A , Vaillancourt JP and Hayden M R (1996). Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract. Nat. Genet. 13(4): 442-449. 96. Green H (1993). Human genetic diseases due to codon reiteration: relationship to an evolutionary mechanism. Ce l l 74(6): 955-956. 97. Gunawardena S, Her L S , Brusch R G , Laymon R A , Niesman IR, Gordesky-Gold B , Sintasath L , Bonini N M and Goldstein L S (2003). Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron 40(1): 25-40. 98. Gutekunst C A , L i S H , Y i H , Mulroy JS, Kuemmerle S, Jones R, Rye D , Ferrante RJ , Hersch S M and L i X J (1999). Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology. J. Neurosci 19(7): 2522-2534. 99. Hackam A S , Yassa A S , Singaraja R, Metzler M , Gutekunst C A , Gan L , Warby S, Wellington C L , Vaillancourt J, Chen N , Gervais F G , Raymond L , Nicholson D W and Hayden M R (2000). Huntingtin interacting protein 1 induces apoptosis via a novel caspase-dependent death effector domain. J. B i o l . Chem. 275(52): 41299-41308. 100. Hahn-Barma V , Deweer B , Durr A , Dode C, Feingold J, Pi l lon B , A g i d Y , Brice A and Dubois B (1998). Are cognitive changes the first symptoms of Huntington's disease? A study of gene carriers. J. Neurol. Neurosurg. Psychiatry 64(2): 172-177. 101. Halliday G M , McRitchie D A , Macdonald V , Double K L , Trent R J and McCusker E (1998). Regional specificity of brain atrophy in Huntington's disease. Exp. Neurol. 154(2): 663-672. 156 102. Hansson O, Nylandsted J, Castilho RF, Leist M , Jaattela M and Brundin P (2003). Overexpression of heat shock protein 70 in R6/2 Huntington's disease mice has only modest effects on disease progression. Brain Res. 970(1-2): 47-57. 103. Haque N and Isacson O (1997). Antisense gene therapy for neurodegenerative disease? Exp. Neurol. 144(1): 139-146. 104. Harper PS (1996). Huntington's Disease. W . B . Saunders Company Ltd, Toronto. 105. Harper SQ, Staber PD, He X , Eliason S L , Martins IH, Mao Q, Yang L , Kotin R M , Paulson H L and Davidson B L (2005). From the Cover: R N A interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc. Natl . Acad. Sci. U . S. A 102(16): 5820-5825. 106. Haverkamp L J , Appel V and Appel S H (1995). Natural history of amyotrophic lateral sclerosis in a database population. Validation of a scoring system and a model for survival prediction. Brain 118 ( Pt 3)707-719. 107. Hedreen JC, Peyser C E , Folstein SE and Ross C A (1991). Neuronal loss in layers V and V I of cerebral cortex in Huntington's disease. Neurosci. Lett. 133(2): 257-261. 108. Heindel W C , Butters N and Salmon D P (1988). Impaired learning of a motor skill in patients with Huntington's disease. Behav. Neurosci. 102(1): 141-147. 109. Heindel W C , Salmon DP , Shults C W , Walicke P A and Butters N (1989). Neuropsychological evidence for multiple implicit memory systems: a comparison of Alzheimer's, Huntington's, and Parkinson's disease patients. J. Neurosci. 9(2): 582-587. 110. Ho A K , Sahakian BJ , Brown R G , Barker R A , Hodges JR, Ane M N , Snowden J, Thompson J, Esmonde T, Gentry R, Moore J W and Bodner T (2003). Profile of cognitive progression in early Huntington's disease. Neurology 61(12): 1702-1706. 111. Ho L W , Brown R, Maxwell M , Wyttenbach A and Rubinsztein D C (2001). Wild-type Huntingtin reduces the cellular toxicity of mutant Huntingtin in mammalian cell models of Huntington's disease. J. Med. Genet. 38(7): 450-452. 112. Hockly E , Cordery P M , Woodman B , Mahal A , van Dellen A , Blakemore C, Lewis C M , Hannan A J and Bates G P (2002). Environmental enrichment slows disease progression in R6/2 Huntington's disease mice. Ann. Neurol. 51(2): 235-242. 113. Hockly E, Richon V M , Woodman B , Smith D L , Zhou X , Rosa E , Sathasivam K , Ghazi-Noori S, Mahal A , Lowden P A , Steffan JS, Marsh JL , Thompson L M , Lewis C M , Marks P A and Bates G P (2003). Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease. Proc. Natl . Acad. Sci. U . S. A 100(4): 2041-2046. 157 114. Hodgson JG , Smith DJ, McCutcheon K , Koide H B , Nishiyama K , Dinulos M B , Stevens M E , Bissada N , Nasir J, Kanazawa I, Disteche C M , Rubin E M and Hayden M R (1996). Human huntingtin derived from Y A C transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype. Hum. M o l . Genet. 5(12): 1875-1885. 115. Hodgson J G , Agopyan N , Gutekunst C A , Leavitt B R , LePiane F, Singaraja R, Smith D J , Bissada N , McCutcheon K , Nasir J, Jamot L , L i X J , Stevens M E , Rosemond E , Roder JC, Phillips A G , Rubin E M , Hersch S M and Hayden M R (1999). A Y A C mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23(1): 181-192. 116. Holbert S, Denghien I, Kiechle T, Rosenblatt A , Wellington C , Hayden M R , Margolis R L , Ross C A , Dausset J, Ferrante RJ and Neri C (2001). The Gin -Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis. Proc. Natl . Acad. Sci. U . S. A 98(4): 1811-1816. 117. Huang C C , Faber PW, Persichetti F, Mittal V , Vonsattel JP, Macdonald M E and Gusella JF (1998). Amylo id formation by mutant huntingtin: threshold, progressivity and recruitment of normal polyglutamine proteins. Somat. Cel l M o l . Genet. 24(4): 217-233. 118. Huerta JJ, Llamosas M M , Cernuda-Cernuda R and Garcia-Fernandez J M (1999). Spatio-temporal analysis of light-induced Fos expression in the retina of rd mutant mice. Brain Res. 834(1-2): 122-127. 119. Hyde L A , Crnic L S , Pollock A and Bickford P C (2001). Motor learning in Ts65Dn mice, a model for Down syndrome. Dev. Psychobiol. 38(1): 33-45. 120. Ientile R, Campisi A , Raciti G , Caccamo D , Curro M , Cannavo G , L i V G , Macaione S and Vanella A (2003). Cystamine inhibits transglutaminase and caspase-3 cleavage in glutamate-exposed astroglial cells. J. Neurosci. Res. 74(1): 52-59. 121. Igarashi S, Koide R, Shimohata T, Yamada M , Hayashi Y , Takano H , Date H , Oyake M , Sato T, Sato A , Egawa S, Ikeuchi T, Tanaka H , Nakano R, Tanaka K , Hozumi I, Inuzuka T, Takahashi H and Tsuji S (1998). Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated D R P L A protein with an expanded polyglutamine stretch. Nat. Genet. 18(2): 111-117. 122. Jackson WS, Tallaksen-Greene SJ, A l b i n R L and Detloff PJ (2003). Nucleocytoplasmic transport signals affect the age at onset of abnormalities in knock-in mice expressing polyglutamine within an ectopic protein context. Hum. M o l . Genet. 12(13): 1621-1629. 123. Jeitner T M , Bogdanov M B , Matson W R , Daikhin Y , Yudkoff M , Folk JE, Steinman L , Browne SE, Beal M F , Blass JP and Cooper A J (2001). N(epsilon)-(gamma-L-glutamyl)-L-lysine ( G G E L ) is increased in cerebrospinal fluid of patients with Huntington's disease. J. Neurochem. 79(5): 1109-1112. . 158 124. Jenkins B G , Rosas H D , Chen Y C , Makabe T, Myers R, MacDonald M , Rosen BR, Beal M F and Koroshetz W J (1998). 1H N M R spectroscopy studies of Huntington's disease: correlations with C A G repeat numbers. Neurology 50(5): 1357-1365. 125. Jenkins B G , Andreassen O A , Dedeoglu A , Leavitt B R , Hayden M R , Borchelt D, Ross C A , Detloff P, Ferrante RJ and Beal M F (2005). Effects of C A G Repeat Length, H T T Protein Length and Protein Context on Cerebral Metabolism Measured using Magnetic Resonance Spectroscopy in Transgenic Mouse Models of Huntington's Disease. J. Neurochem. 126. Kahlem P, Green H and Djian P (1998). Transglutaminase action imitates Huntington's disease: selective polymerization of Huntingtin containing expanded polyglutamine. M o l . Cel l 1(4): 595-601. 127. Kalchman M A , Koide H B , McCutcheon K , Graham R K , Nichol K , Nishiyama K , Kazemi-Esfarjani P, Lynn F C , Wellington C, Metzler M , Goldberg Y P , Kanazawa I, Gietz R D and Hayden M R (1997). HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat. Genet. 16(1): 44-53. 128. Karpuj M V , Becher M W , Springer JE, Chabas D, Youssef S, Pedotti R, Mitchell D and Steinman L (2002). Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat. Med. 8(2): 143-149. 129. Karpuj M V , Becher M W and Steinman L (2002). Evidence for a role for transglutaminase in Huntington's disease and the potential therapeutic implications. Neurochem. Int. 40(1): 31-36. 130. Katsuno M , Adachi H , Kume A , L i M , Nakagomi Y , N i w a H , Sang C, Kobayashi Y , Doyu M and Sobue G (2002). Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy. Neuron 35(5): 843-854. 131. Katsuno M , Adachi H , Doyu M , Minamiyama M , Sang C, Kobayashi Y , Inukai A and Sobue G (2003). Leuprorelin rescues polyglutamine-dependent phenotypes in a transgenic mouse model of spinal and bulbar muscular atrophy. Nat. Med. 9(6): 768-773. 132. Keene C D , Rodrigues C M , Eich T, Chhabra M S , Steer C J and L o w W C (2002). Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease. Proc. Natl . Acad. Sci. U . S. A 99(16): 10671-10676. 133. Kegel K B , Meloni A R , Y i Y , K i m Y J , Doyle E , Cuiffo B G , Sapp E, Wang Y , Qin Z H , Chen JD, Nevins JR, Aronin N and DiFig l ia M (2002). Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription. J. B i o l . Chem. 277(9): 7466-7476. 159 134. Kenchappa R S , Diwakar L , Annepu J and Ravindranath V (2004). Estrogen and neuroprotection: higher constitutive expression of glutaredoxin in female mice offers protection against MPTP-mediated neurodegeneration. F A S E B J. 18(10): 1102-1104. 135. Kirkwood SC, Siemers E , Stout JC, Hodes M E , Conneally P M , Christian JC and Foroud T (1999). Longitudinal cognitive and motor changes among presymptomatic Huntington disease gene carriers. Arch . Neurol. 56(5): 563-568. 136. Kl ivenyi P, Ferrante R J , Gardian G , Browne S, Chabrier P E and Beal M F (2003). Increased survival and neuroprotective effects of BN82451 in a transgenic mouse model of Huntington's disease. J. Neurochem. 86(1): 267-272. 137. Knopman D and Nissen M J (1991). Procedural learning is impaired in Huntington's disease: evidence from the serial reaction time task. Neuropsychologia 29(3): 245-254. 138. Kolsch H and Rao M L (2002). Neuroprotective effects of estradiol-17beta: implications for psychiatric disorders. Arch. Women Ment. Health 5(3): 105-110. 139. Konno T, M o r i i T, Shimizu H , Oik i S and Dcura K (2005). Paradoxical inhibition of protein aggregation and precipitation by transglutaminase-catalyzed intermolecular cross-linking. J. B i o l . Chem. 140. Konno T, M o r i i T, Hirata A , Sato S, Oik i S and Ikura K (2005). Covalent blocking of fibril formation and aggregation of intracellular amyloidgenic proteins by transglutaminase-catalyzed intramolecular cross-linking. Biochemistry 44(6): 2072-2079. 141. Kremer B , Goldberg P, Andrew SE, Theilmann J, Telenius H , Zeisler J, Squitieri F, L i n B , Bassett A , Almqvist E and . (1994). A worldwide study of the Huntington's disease mutation. The sensitivity and specificity of measuring C A G repeats. N . Engl. J. Med. 330(20): 1401-1406. 142. Kremer H P and Roos R A (1992). Weight loss in Huntington's disease. Arch. Neurol. 49(4): 349-143. Kuemmerle S, Gutekunst C A , Kle in A M , L i X J , L i S H , Beal M F , Hersch S M and Ferrante R J (1999). Huntington aggregates may not predict neuronal death in Huntington's disease. Ann . Neurol. 46(6): 842-849. 144. Laforet G A , Sapp E , Chase K , Mclntyre C, Boyce F M , Campbell M , Cadigan B A , Warzecki L , Tagle D A , Reddy P H , Cepeda C, Calvert C R , Jokel E S , Klapstein G J , Ariano M A , Levine M S , DiFig l ia M and Aronin N (2001). Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington's disease. J. Neurosci 21(23): 9112-9123. 145. L a i TS, Tucker T, Burke JR, Strittmatter W J and Greenberg CS (2004). Effect of tissue transglutaminase on the solubility of proteins containing expanded polyglutamine ; ,'• repeats. J. Neurochem. 88(5): 1253-1260. 160 146. Lalonde R, Bensoula A N and Filal i M (1995). Rotorod sensorimotor learning in cerebellar mutant mice. Neurosci. Res. 22(4): 423-426. 147. Landwehrmeyer G B , M c N e i l S M , Dure L S , Ge P, Aizawa H , Huang Q, Ambrose C M , Duyao M P , Bi rd E D , Bonil la E and . (1995). Huntington's disease gene: regional and cellular expression in brain of normal and affected individuals. Ann . Neurol. 37(2): 218-230. 148. Lawrence A D , Sahakian B J , Hodges JR, Rosser A E , Lange K W and Robbins T W (1996). Executive and mnemonic functions in early Huntington's disease. Brain 119 ( Pt 5)1633-1645. 149. Lawrence A D , Hodges JR, Rosser A E , Kershaw A , ffrench-Constant C, Rubinsztein D C , Robbins T W and Sahakian BJ (1998). Evidence for specific cognitive deficits in preclinical Huntington's disease. Brain 121 ( Pt 7)1329-1341. 150. Lawrence A D , Weeks R A , Brooks D J , Andrews T C , Watkins L H , Harding A E , Robbins T W and Sahakian B J (1998). The relationship between striatal dopamine receptor binding and cognitive performance in Huntington's disease. Brain 121 ( Pt 7)1343-1355. 151. Lawrence A D , Sahakian B J , Rogers R D , Hodge JR and Robbins T W (1999). Discrimination, reversal, and shift learning in Huntington's disease: mechanisms of impaired response selection. Neuropsychologia 37(12): 1359-1374. 152. Lawrence A D , Watkins L H , Sahakian B J , Hodges JR and Robbins T W (2000). Visual object and visuospatial cognition in Huntington's disease: implications for information processing in corticostriatal circuits. Brain 123 ( Pt 7)1349-1364. 153. Le Marec N , Caston J and Lalonde R (1997). Impaired motor skills on static and mobile beams in lurcher mutant mice. Exp. Brain Res. 116(1): 131-138. 154. Leavitt B R , Guttman J A , Hodgson JG , K ime l G H , Singaraja R, Vog l A W and Hayden M R (2001). Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo. A m . J. Hum. Genet. 68(2): 313-324. 155. Leeflang E P , Zhang L , Tavare S, Hubert R, Srinidhi J, Macdonald M E , Myers R H , de Young M , Wexler N S , Gusella JF and . (1995). Single sperm analysis of the trinucleotide repeats in the Huntington's disease gene: quantification of the mutation frequency spectrum. Hum. M o l . Genet. 4(9): 1519-1526. 156. Lesort M , Chun W , Johnson G V and Ferrante R J (1999). Tissue transglutaminase is increased in Huntington's disease brain. J. Neurochem. 73(5): 2018-2027. 157. Lesort M , Chun W , Tucholski J and Johnson G V (2002). Does tissue transglutaminase play a role in Huntington's disease? Neurochem. Int. 40(1): 37-52. 161 158. Lesort M , Lee M , Tucholski J and Johnson G V (2003). Cystamine inhibits caspase activity. Implications for the treatment of polyglutamine disorders. J. B i o l . Chem. 278(6): 3825-3830. 159. Levine M S , Klapstein GJ , Koppel A , Gruen E , Cepeda C, Vargas M E , Jokel ES, Carpenter E M , Zanjani H , Hurst RS , Efstratiadis A , Zeitlin S and Chesselet M F (1999). Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington's disease. J. Neurosci. Res. 58(4): 515-532. 160. L i H , Wyman T, Y u Z X , L i S H and L i X J (2003). Abnormal association of mutant huntingtin with synaptic vesicles inhibits glutamate release. Hum. M o l . Genet. 12(16): 2021-2030. 161. L i S H , Schilling G , Young W S , III, L i X J , Margolis R L , Stine O C , Wagster M V , Abbott M H , Franz M L , Ranen N G and . (1993). Huntington's disease gene (IT15) is widely expressed in human and rat tissues. Neuron 11(5): 985-993. 162. L i S H , Cheng A L , L i H and L i X J (1999). Cellular defects and altered gene expression in PC 12 cells stably expressing mutant huntingtin. J. Neurosci 19(13): 5159-5172. 163. L i S H , Lam S, Cheng A L and L i X J (2000). Intranuclear huntingtin increases the expression of caspase-1 and induces apoptosis. Hum. M o l . Genet. 9(19): 2859-2867. 164. L i S H , Cheng A L , Zhou H , Lam S, Rao M , L i H and L i X J (2002). Interaction of Huntington disease protein with transcriptional activator S p l . M o l . Cel l B i o l . 22(5): 1277-1287. 165. L i S H and L i X J (2004). Huntingtin-protein interactions and the pathogenesis of Huntington's disease. Trends Genet. 20(3): 146-154. 166. Lievens JC, Woodman B , Mahal A , Spasic-Boscovic O, Samuel D , Kerkerian-Le Goff L and Bates GP (2001). Impaired glutamate uptake in the R6 Huntington's disease transgenic mice. Neurobiol. Dis. 8(5): 807-821. 167. L in B , Rommens J M , Graham R K , Kalchman M , MacDonald H , Nasir J, Delaney A , Goldberg Y P and Hayden M R (1993). Differential 3' polyadenylation of the Huntington disease gene results in two m R N A species with variable tissue expression. Hum. M o l . Genet. 2(10): 1541-1545. 168. L in C H , Tallaksen-Greene S, Chien W M , Cearley J A , Jackson W S , Crouse A B , Ren S, L i X J , A l b i n R L and Detloff PJ (2001). Neurological abnormalities in a knock-in mouse model of Huntington's disease. Hum. M o l . Genet. 10(2): 137-144. 169. Lione L A , Carter RJ , Hunt M J , Bates GP , Morton A J and Dunnett SB (1999). Selective discrimination learning impairments in mice expressing the human Huntington's disease mutation. J. Neurosci. 19(23): 10428-10437. 162 170. Lorand L and Graham R M (2003). Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat. Rev. M o l . Cel l B io l . 4(2): 140-156. 171. L u B (2004). R N A interference technologies for understanding and treating neurodegenerative diseases. Neuromolecular. Med. 6(1): 1-12. 172. Luthi-Carter R, Hanson S A , Strand A D , Bergstrom D A , Chun W , Peters N L , Woods A M , Chan E Y , Kooperberg C, Krainc D , Young A B , Tapscott SJ and Olson J M (2002). Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain. Hum. M o l . Genet. 11(17): 1911-1926. 173. Macdonald M E , Ambrose C M , Duyao M P , Myers R H , L i n C, Srinidhi L , Barnes G , Taylor S A , James M , Groot N , Macfarlane H , Jenkins B , Anderson M A , Wexler N S , Gusella JF, Bates GP , Baxendale S, Hummerich H , Ki rby S, North M , Youngman S, Mott R, Zehetner G , Sedlacek Z , Poustka A , Frischauf A M , Lehrach H , Buckler A J , Church D, Doucettestamm L , Odonovan M C , Ribaramirez L , Shah M , Stanton V P , Strobel S A , Draths K M , Wales JL , Dervan P, Housman D E , Altherr M , Shiang R, Thompson L , Fielder T, Wasmuth JJ, Tagle D, Valdes J, Elmer L , Al lard M , Castilla L , Swaroop M , Blanchard K , Collins FS , Snell R, Holloway T, Gillespie K , Datson N , Shaw D and Harper PS (1993). A Novel Gene Containing A Trinucleotide Repeat That Is Expanded and Unstable on Huntingtons-Disease Chromosomes. Cel l 72(6): 971-983. 174. Macdonald M E , Vonsattel JP, Shrinidhi J, Couropmitree N N , Cupples L A , Bi rd E D , Gusella JF and Myers R H (1999). Evidence for the GluR6 gene associated with younger onset age of Huntington's disease. Neurology 53(6): 1330-1332. 175. Macdonald V and Halliday G (2002). Pyramidal cell loss in motor cortices in Huntington's disease. Neurobiol. Dis. 10(3): 378-386. 176. Mahadevan M , Tsilfidis C , Sabourin L , Shutler G , Amemiya C, Jansen G , Neville C , Narang M , Barcelo J, O'Hoy K and . (1992). Myotonic dystrophy mutation: an unstable C T G repeat in the 3' untranslated region of the gene. Science 255(5049): 1253-1255. 177. Mahler JF, Stokes W, Mann PC, Takaoka M and Maronpot R R (1996). Spontaneous lesions in aging F V B / N mice. Toxicol . Pathol. 24(6): 710-716. 178. Mangiarini L , Sathasivam K , Seller M , Cozens B , Harper A , Hetherington C, Lawton M , Trottier Y , Lehrach H , Davies S W and Bates G P (1996). Exon 1 of the H D gene with an expanded C A G repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cel l 87(3): 493-506. 179. Markianos M , Panas M , Kalfakis N and Vassilopoulos D (2005). Plasma testosterone in male patients with Huntington's disease: relations to severity of illness and dementia. Ann. Neurol/57(4): 520-525. 163 180. Mastroberardino P G , Iannicola C, Nardacci R, Bernassola F, De L , V , Melino G , Moreno S, Pavone F, Oliverio S, Fesus L and Piacentini M (2002). Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease. Cel l Death. Differ. 9(9): 873-880. 181. Mastromauro C A , Meissen G J , Cupples L A , Kie ly D K , Berkman B and Myers R H (1989). Estimation of fertility and fitness in Huntington disease in New England. A m . J. Med . Genet. 33(2): 248-254. 182. McDaniel B , Sheng H , Warner DS , Hedlund L W and Benveniste H (2001). Tracking brain volume changes in C57BL/6J and ApoE-deficient mice in a model of neurodegeneration: a 5-week longitudinal micro-MRI study. Neuroimage. 14(6): 1244-1255. 183. McFadyen M P , Kusek G , Bolivar V J and Flaherty L (2003). Differences among eight inbred strains of mice in motor ability and motor learning on a rotorod. Genes Brain Behav. 2(4): 214-219. 184. McGeer E G and McGeer P L (1976). Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acids. Nature 263(5577): 517-519. 185. Melino G , Annicchiarico-Petruzzelli M , Piredda L , Candi E , Gentile V , Davies PJ and Piacentini M (1994). Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells. M o l . Cel l B i o l . 14(10): 6584-6596. 186. Menalled L B , Sison JD, W u Y , Olivieri M , L i X J , L i H , Zeitlin S and Chesselet M F (2002). Early motor dysfunction and striosomal distribution of huntingtin microaggregates in Huntington's disease knock-in mice. J. Neurosci. 22(18): 8266-8276. 187. Menalled L B , Sison JD, Dragatsis I, Zeitlin S and Chesselet M F (2003). Time course of early motor and neuropathological anomalies in a knock-in mouse model of Huntington's disease with 140 C A G repeats. J. Comp Neurol. 465(1): 11-26. 188. Metzler M , Legendre-Guillemin V , Gan L , Chopra V , K w o k A , McPherson PS and Hayden M R (2001). HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2. J. B i o l . Chem. 276(42): 39271-39276. 189. M i n k J W (1996). The basal ganglia: Focused selection and inhibition of competing motor programs. Progress in Neurobiology 50(4): 381-425. 190. Modregger J, DiProspero N A , Charles V , T a g l e D A and Plomann M (2002). P A C S I N 1 interacts with huntingtin and is absent from synaptic varicosities in presymptomatic Huntington's disease brains. Hum. M o l . Genet. 11(21): 2547-2558. 164 191. Morales L M , Estevez J, Suarez H , Villalobos R, Chacin dB and Boni l la E (1989). Nutritional evaluation of Huntington disease patients. A m . J. C l in . Nutr. 50(1): 145-150. 192. Muller U , Cristina N , L i Z W , Wolfer DP , Lipp H P , Rulicke T, Brandner S, Aguzz i A and Weissmanii C (1994). Behavioral and Anatomical Deficits in Mice Homozygous for A Modified Beta-Amyloid Precursor Protein Gene. Cel l 79(5): 755-765. 193. Murphy K P , Carter R J , Lione L A , Mangiarini L , Mahal A , Bates G P , Dunnett SB and Morton A J (2000). Abnormal synaptic plasticity and impaired spatial cognition in mice transgenic for exon 1 of the human Huntington's disease mutation. J. Neurosci 20(13): 5115-5123. 194. Nakao N , Brundin P, Funa K , Lindvall O and Odin P (1995). Trophic and protective actions of brain-derived neurotrophic factor on striatal DARPP-32-containing neurons in vitro. Brain Res. Dev. Brain Res. 90(1-2): 92-101. 195. Narain Y , Wyttenbach A , Rankin J, Furlong R A and Rubinsztein D C (1999). A molecular investigation of true dominance in Huntington's disease. J. Med . Genet. 36(10): 739-746. 196. Nasir J, Floresco SB, O'Kusky JR, Diewert V M , Richman J M , Zeisler J, Borowski A , Marfri JD, Phillips A G and Hayden M R (1995). Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cel l 81(5): 811-823. 197. Norflus F, Nanje A , Gutekunst C A , Shi G , Cohen J, Bejarano M , Fox J, Ferrante R J and Hersch S M (2004). Anti-inflammatory treatment with acetylsalicylate or rofecoxib is not neuroprotective in Huntington's disease transgenic mice. Neurobiol. Dis. 17(2): 319-325. 198. Nucifora F C , Jr., Sasaki M , Peters M F , Huang H , Cooper J K , Yamada M , Takahashi H , Tsuji S, Troncoso J, Dawson V L , Dawson T M and Ross C A (2001). Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity. Science 291(5512): 2423-2428. 199. O'Kusky JR, Nasir J, Cicchetti F, Parent A and Hayden M R (1999). Neuronal degeneration in the basal ganglia and loss of pallido-subthalamic synapses in mice with targeted disruption of the Huntington's disease gene. Brain Res. 818(2): 468-479. 200. Oliverio S, Amendola A , Rodolfo C, Spinedi A and Piacentini M (1999). Inhibition of "tissue" transglutaminase increases cell survival by preventing apoptosis. J. B i o l . Chem. 274(48): 34123-34128. 201. Ona V O , L i M , Vonsattel JP, Andrews L J , Khan SQ, Chung W M , Frey A S , Menon A S , L i X J , Stieg P E , Yuan J, Penney JB, Young A B , Cha J H and Friedlander R M (1999). Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease. Nature 399(6733): 263-267. . . . . . 165 202. Ordway J M , Tallaksen-Greene S, Gutekunst C A , Bernstein E M , Cearley J A , Wiener H W , Dure L S , Lindsey R, Hersch S M , Jope RS , A l b i n R L and Detloff PJ (1997). Ectopically expressed C A G repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell 91(6): 753-763. 203. Packard M G and McGaugh J L (1996). Inactivation of hippocampus or caudate nucleus with lidocaine differentially affects expression of place and response learning. Neurobiol. Learn. Mem. 65(1): 65-72. 204. Panov A V , Gutekunst C A , Leavitt B R , Hayden M R , Burke JR, Strittmatter W J and Greenamyre JT (2002). Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines. Nat. Neurosci 5(8): 731-736. 205. Paulsen JS, Zhao H , Stout JC, Brinkman R R , Guttman M , Ross C A , Como P, Manning C , Hayden M R and Shoulson I (2001). Clinical markers of early disease in persons near onset of Huntington's disease. Neurology 57(4): 658-662. 206. Perez-Navarro E , Alberch J, Neveu I and Arenas E (1999). Brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 differentially regulate the phenotype and prevent degenerative changes in striatal projection neurons after excitotoxicity in vivo. Neuroscience 91(4): 1257-1264. 207. Peters M F , Nucifora F C , Jr., Kushi J, Seaman H C , Cooper J K , Herring W J , Dawson V L , Dawson T M and Ross C A (1999). Nuclear targeting of mutant Huntingtin increases toxicity. M o l . Cel l Neurosci 14(2): 121-128. 208. Petersen A , Mani K and Brundin P (1999). Recent advances on the pathogenesis of Huntington's disease. Exp. Neurol. 157(1): 1-18. 209. Piacentini M , Farrace M G , Piredda L , Matarrese P, Ciccosanti F, Falasca L , Rodolfo C , Giammarioli A M , Verderio E , Griffin M and Malorni W (2002). Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress. J. Neurochem. 81(5): 1061-1072. 210. Pinto JT, Van Raamsdonk J M , Leavitt B R , Hayden M R , Jeitner T M , Thaler H T , Krasnikov B F and Cooper A J L (2005). Treatment of Y A c l 2 8 mice and their wild-type littermates with cystamine does not lead to its accumulation in plasma or brain: implications for the treatment of Huntington disease. J. Neurochem. (in press). 211. Platel A and Porsolt R D (1982). Habituation of exploratory activity in mice: a screening test for memory enhancing drugs. Psychopharmacology (Berl) 78(4): 346-352. 212. Popovic N ; Maingay M , K i r i k D and Brundin P (2005). "Lentiviral gene delivery of G D N F into the striatum of R6/2 Huntington mice fails to attenuate behavioral and neuropathological changes. Exp. Neurol. 193(1): 65-74. 166 213. Pratley R E , Salbe A D , Ravussin E and Caviness J N (2000). Higher sedentary energy expenditure in patients with Huntington's disease. Ann . Neurol. 47(1): 64-70. 214. Pridmore S A and Adams G C (1991). The fertility of HD-affected individuals in Tasmania. Aust. N . Z . J. Psychiatry 25(2): 262-264. 215. Puri B K , Leavitt B R , Hayden M R , Ross C A , Rosenblatt A , Greenamyre JT, Hersch S, Vaddadi K S , Sword A , Horrobin D F and Murck H (2005). Ethyl-eicoasapentanoate in Huntington's disease: A double-blind, randomized placebo-controlled trial. Neurology (in press). 216. Ramsden M , Shin T M and Pike C J (2003). Androgens modulate neuronal vulnerability to kainate lesion. Neuroscience 122(3): 573-578. 217. Rao DS , Chang JC, Kumar P D , Mizukami I, Smithson G M , Bradley S V , Parlow A F and Ross TS (2001). Huntingtin interacting protein 1 Is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors. M o l . Ce l l B i o l . 21(22): 7796-7806. 218. Rebec G V , Barton SJ, Marseilles A M and Collins K (2003). Ascorbate treatment attenuates the Huntington behavioral phenotype in mice. Neuroreport 14(9): 1263-1265. 219. Reddy P H , Will iams M , Charles V , Garrett L , Pike-Buchanan L , Whetsell W O , Jr., Mi l le r G and Tagle D A (1998). Behavioural abnormalities and selective neuronal loss in H D transgenic mice expressing mutated full-length H D c D N A . Nat. Genet. 20(2): 198-202. 220. Reiner A , Del Mar N , Meade C A , Yang H , Dragatsis I, Zeitlin S and Goldowitz D (2001). Neurons lacking huntingtin differentially colonize brain and survive in chimeric mice. J. Neurosci 21(19): 7608-7619. 221. Rigamonti D , Bauer J H , De Fraja C , Conti L , Sipione S, Sciorati C , Clementi E , Hackam A , Hayden M R , L i Y , Cooper J K , Ross C A , Govoni S, Vincenz C and Cattaneo E (2000). Wild-type huntingtin protects from apoptosis upstream of caspase-3. J. Neurosci. 20(10): 3705-3713. 222. Rohrer D , Salmon DP , Wixted JT and Paulsen JS (1999). The disparate effects of Alzheimer's disease and Huntington's disease on semantic memory. Neuropsychology. 13(3): 381-388. 223. Rosas H D , Koroshetz W J , Chen Y I , Skeuse C, Vangel M , Cudkowicz M E , Caplan K , Marek K , Seidman L J , Makris N , Jenkins B G and Goldstein J M (2003). Evidence for more widespread cerebral pathology i n v early H D : an MRI-based morphometric analysis. Neurology 60(10): 1615-1620. 167 224. Rubinsztein D C , Leggo J, Chiano M , Dodge A , Norbury G , Rosser E and Craufurd D (1997). Genotypes at the GluR6 kainate receptor locus are associated with variation in the age of onset of Huntington disease. Proc. Natl . Acad. Sci. U . S. A 94(8): 3872-3876. 225. Sanberg PR, Fibiger H C and Mark R F (1981). Body weight and dietary factors in Huntington's disease patients compared with matched controls. Med . J. Aust. 1(8): 407-409. 226. Sanchez I, X u C J , Juo P, Kakizaka A , Blenis J and Yuan J (1999). Caspase-8 is required for cell death induced by expanded polyglutamine repeats. Neuron 22(3): 623-633. 227. Sanchez 1, Mahlke C and Yuan J (2003). Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature 421(6921): 373-379. 228. Sapp E , Schwarz C, Chase K , Bhide P G , Young A B , Penney J, Vonsattel JP, Aronin N and DiFig l ia M (1997). Huntingtin localization in brains of normal and Huntington's disease patients. Ann . Neurol. 42(4): 604-612. 229. Sapp E , Penney J, Young A , Aronin N , Vonsattel JP and DiFig l ia M (1999). Axonal transport of N-terminal huntingtin suggests early pathology of corticostriatal projections in Huntington disease. J. Neuropathol. Exp. Neurol. 58(2): 165-173. 230. Sattler R and Tymianski M (2000). Molecular mechanisms of calcium-dependent excitotoxicity. J. M o l . Med. 78(1): 3-13. 231. Saudou F, Finkbeiner S, Devys D and Greenberg M E (1998). Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cel l 95(1): 55-66. 232. Schauwecker P E and Steward O (1997). Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc. Natl. Acad. Sci. U . S . A 94(8): 4103-4108. 233. Schiefer J, Landwehrmeyer G B , Luesse H G , Sprunken A , Puis C, Milkereit A , Milkereit E and Kosinski C M (2002). Riluzole prolongs survival time and alters nuclear inclusion formation in a transgenic mouse model of Huntington's disease. M o v Disord. 17(4): 748-757. 234. Schilling G , Sharp A H , Loev SJ, Wagster M V , L i S H , Stine O C and Ross C A (1995). Expression of the Huntington's disease (1T15) protein product in H D patients. Hum. M o l . Genet. 4(8): 1365-1371. 168 235. Schilling G , Becher M W , Sharp A H , Jinnah H A , Duan K , Kotzuk J A , Slunt H H , Ratovitski T, Cooper J K , Jenkins N A , Copeland N G , Price D L , Ross C A and Borchelt D R (1999). Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum. M o l . Genet. 8(3): 397-407. 236. Schilling G , Coonfield M L , Ross C A and Borchelt D R (2001). Coenzyme Q10 and remacemide hydrochloride ameliorate motor deficits in a Huntington's disease transgenic mouse model. Neurosci Lett. 315(3): 149-153. 237. Schilling G , Savonenko A V , Coonfield M L , Morton JL , Vorovich E , Gale A , Neslon C, Chan N , Eaton M , Fromholt D, Ross C A and Borchelt D R (2004). Environmental, pharmacological, and genetic modulation of the H D phenotype in transgenic mice. Exp. Neurol. 187(1): 137-149. 238. Schilling G , Savonenko A V , Klevytska A , Morton JL , Tucker S M , Poirier M , Gale A , Chan N , Gonzales V , Slunt H H , Coonfield M L , Jenkins N A , Copeland N G , Ross C A and Borchelt D R (2004). Nuclear-targeting of mutant huntingtin fragments produces Huntington's disease-like phenotypes in transgenic mice. Hum. M o l . Genet. 13(15): 1599-1610. 239. Schmidtke K , Manner H , Kaufmann R and Schmolck H (2002). Cognitive procedural learning in patients with fronto-striatal lesions. Learn. Mem. 9(6): 419-429. 240. Schmued L C , Albertson C and Slikker W , Jr. (1997). Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res. 751(1): 37-46. 241. Sharp A H , Loev SJ, Schilling G , L i S H , L i X J , Bao J, Wagster M V , Kotzuk J A , Steiner JP, Lo A and . (1995). Widespread expression of Huntington's disease gene (IT15) protein product. Neuron 14(5): 1065-1074. 242. Sharp A H and Ross C A (1996). Neurobiology of Huntington's disease. Neurobiol. Dis. 3(1): 3-15. 243. Shear D A , Dong J, Gundy C D , Haik-Creguer K L and Dunbar G L (1998). Comparison of intrastriatal injections of quinolinic acid and 3-nitropropionic acid for use in animal models of Huntington's disease. Prog. Neuropsychopharmacol. B i o l . Psychiatry 22(7): 1217-1240. 244. Shelbourne PF, Kil leen N , Hevner R F , Johnston H M , Tecott L , Lewandoski M , Ennis M , Ramirez L , L i Z , Iannicola C, Littman D R and Myers R M (1999). A Huntington's disease C A G expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice. Hum. M o l . Genet. 8(5): 763-774. 245. Shokeir M H (1975). Investigation on Huntington's disease in the Canadian Prairies. II. Fecundity and fitness. C l in . Genet. 7(4): 349-353. 169 246. Singaraja RR , Hadano S, Metzler M , Givan S, Wellington C L , Warby S, Yanai A , Gutekunst C A , Leavitt B R , Y i H , Fichter K , Gan L , McCutcheon K , Chopra V , Michel J, Hersch S M , Ikeda JE and Hayden M R (2002). HIP 14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis. Hum. M o l . Genet. 11(23): 2815-2828. 247. Singhrao S K , Neal JW, Morgan B P and Gasque P (1999). Increased complement biosynthesis by microglia and complement activation on neurons in Huntington's disease. Exp. Neurol. 159(2): 362-376. 248. Slow EJ, van Raamsdonk J, Rogers D , Coleman S H , Graham R K , Deng Y , Oh R, Bissada N , Hossain S M , Yang Y Z , L i X J , Simpson E M , Gutekunst C A , Leavitt B R and Hayden M R (2003). Selective striatal neuronal loss in a Y A C 128 mouse model of Huntington disease. Hum. M o l . Genet. 12(13): 1555-1567. 249. Snowden J, Craufurd D, Griffiths H , Thompson J and Neary D (2001). Longitudinal evaluation of cognitive disorder in Huntington's disease. J. Int. Neuropsychol. Soc. 7(1): 33-44. 250. Snowden JS, Craufurd D , Thompson J and Neary D (2002). Psychomotor, executive, and memory function in preclinical Huntington's disease. J. C l i n . Exp. Neuropsychol. 24(2): 133-145. 251. Spargo E, Everall IP and Lantos P L (1993). Neuronal loss in the hippocampus in Huntington's disease: a comparison with H I V infection. J. Neurol. Neurosurg. Psychiatry 56(5): 487-491. 252. Spires T L , Grote H E , Varshney N K , Cordery P M , van Dellen A , Blakemore C and Hannan A J (2004). Environmental enrichment rescues protein deficits in a mouse model of Huntington's disease, indicating a possible disease mechanism. J. Neurosci 24(9): 2270-2276. 253. Squitieri F, Gellera C, Cannella M , Mariotti C, Cislaghi G , Rubinsztein D C , Almqvist E W , Turner D, Bachoud-Levi A C , Simpson S A , Delatycki M , Maglione V , Hayden M R and Donato SD (2003). Homozygosity for C A G mutation in Huntington disease is associated with a more severe clinical course. Brain 126(Pt 4): 946-955. 254. Steffan JS, Kazantsev A , Spasic-Boskovic O, Greenwald M , Zhu Y Z , Gohler H , Wanker E E , Bates G P i Housman D E and Thompson L M (2000). The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc. Natl. Acad. Sci. U . S. A 97(12): 6763-6768. 255. Stine O C , L i S H , Pleasant N , Wagster M V , Hedreen JC and Ross C A (1995). ' Expression of the mutant allele of IT-15 ( the 'HD gene) in striatum and cortex of Huntington's disease patients. Hum. M o l . Genet. 4(1): 15-18. 170 256. Stokin G B , Li l lo C, Falzone T L , Brusch R G , Rockenstein E , Mount S L , Raman R, Davies P, Masliah E, Williams DS and Goldstein L S (2005). Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease. Science 307(5713): 1282-1288. 257. Strauss M E and Brandt J (1990). Are there neuropsychologic manifestations of the gene for Huntington's disease in asymptomatic, at-risk individuals? Arch. Neurol. 47(8): 905-908. 258. Strong T V , Tagle D A , Valdes J M , Elmer L W , Boehm K , Swaroop M , Kaatz K W , Collins FS and Alb in R L (1993). Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nat. Genet. 5(3): 259-265. 259. Sugars K L and Rubinsztein D C (2003). Transcriptional abnormalities in Huntington disease. Trends Genet. 19(5): 233-238. 260. Sun Y , Savanenin A , Reddy P H and L i u Y F (2001). Polyglutamine-expanded huntingtin promotes sensitization of N-methyl-D-aspartate receptors via post-synaptic density 95. J. B io l . Chem. 276(27): 24713-24718. 261. Svenningsson P, Nishi A , Fisone G , Girault J A , Nairn A C and Greengard P (2004). D A R P P - 3 2 : an integrator of neurotransmission. Annu. Rev. Pharmacol. Toxicol . 44269-296. 262. Swerdlow N R , Paulsen J, Braff D L , Butters N , Geyer M A and Swenson M R (1995). Impaired prepulse inhibition of acoustic and tactile startle response in patients with Huntington's disease. J. Neurol. Neurosurg. Psychiatry 58(2): 192-200. 263. Syed V , Gomez E and Hecht N B (1999). m R N A s encoding a von Ebner's-like protein and the Huntington disease protein are induced in rat male germ cells by Sertoli cells. J. B i o l . Chem. 274(16): 10737-10742. 264. Szebenyi G , Morfini G A , Babcock A , Gould M , Selkoe K , Stenoien D L , Young M , Faber P W , Macdonald M E , McPhaul M J and Brady ST (2003). Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport. Neuron 40(1): 41-52. 265. Takano H and Gusella JF (2002). The predominantly H E A T - l i k e motif structure of huntingtin and its association and coincident nuclear entry with dorsal, an N F -kB/Rel/dorsal family transcription factor. B M C . Neurosci 3(1): 15-266. Taketo M , Schroeder A C , Mobraaten L E , Gunning K B , Hanten G , Fox R R , Roderick T H , Stewart C L , L i l l y F, Hansen C T and . (1991). F V B / N : an inbred mouse strain preferable for transgenic analyses. Proc. Natl . Acad. Sci. U . S. A 88(6): 2065-2069. 267. Tallaksen-Greene SJ, Crouse A B , Hunter J M , Detloff PJ and A l b i n R L (2005). Neuronal intranuclear inclusions and neuropil aggregates in HdhCAG(150) knockin mice. Neuroscience 131(4): 843-852. 171 268. Tanaka M , Machida Y , N i u S, Ikeda T, Jana N R , Doi H , Kurosawa M , Nekooki M and Nukina N (2004). Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat. Med. 10(2): 148-154. 269. Tang TS, Slow E , Lupu V , Stavrovskaya IG, Sugimori M , Llinas R, Kristal B S , Hayden M R and Bezprozvanny I (2005). Disturbed Ca2+ signaling and apoptosis of medium spiny neurons in Huntington's disease. Proc. Natl . Acad. Sci . U . S. A 102(7): 2602-2607. 270. Trottier Y , Biancalana V and Mandel J L (1994). Instability of C A G repeats in Huntington's disease: relation to parental transmission and age of onset. J. Med. Genet. 31(5): 377-382. 271. Trottier Y , Devys D , Imbert G , Saudou F, A n I, Lutz Y , Weber C , A g i d Y , Hirsch E C and Mandel J L (1995). Cellular localization of the Huntington's disease protein and discrimination of the normal and mutated form. Nat. Genet.. 10(1): 104-110. 272. Trushina E , Dyer R B , Badger JD, Ure D , Eide L , Tran D D , Vrieze B T , Legendre-Guillemin V , McPherson PS, Mandavil l i B S , Van Houten B , Zeitlin S, McNiven M , Aebersold R, Hayden M , Parisi JE, Seeberg E , Dragatsis I, Doyle K , Bender A , Chacko C and McMurray C T (2004). Mutant huntingtin impairs axonal trafficking in mammalian neurons in vivo and in vitro. M o l . Cell B i o l . 24(18): 8195-8209. 273. Turmaine M , Raza A , Mahal A , Mangiarini L , Bates G P and Davies S W (2000). Nonapoptotic neurodegeneration in a transgenic mouse model of Huntington's disease. Proc. Natl. Acad. Sci. U . S. A 97(14): 8093-8097. 274. Ueki A , Shinjo H , Shimode H , Nakajima T and Morita Y (2001). Factors associated with mortality in patients with early-onset Alzheimer's disease: a five-year longitudinal study. Int. J. Geriatr. Psychiatry 16(8): 810-815. 275. van Dellen A , Welch J, Dixon R M , Cordery P, York D , Styles P, Blakemore C and Hannan A J (2000). N-Acetylaspartate and D A R P P - 3 2 levels decrease in the corpus striatum of Huntington's disease mice. Neuroreport 11(17): 3751-3757. 276. van Dellen A , Blakemore C, Deacon R, York D and Hannan A J (2000). Delaying the onset of Huntington's in mice. Nature 404(6779): 721-722. 277. Van Den Eeden S K , Tanner C M , Bernstein A L , Fross R D , Leimpeter A , Bloch D A and Nelson L M (2003). Incidence of Parkinson's disease: variation by age, gender, and race/ethnicity. A m . J. Epidemiol. 157(11): 1015-1022. 278. Van Raamsdonk J M , Pearson J, Rogers D A , Bissada N , V o g l A W , Hayden M R and Leavitt B R (2005). Loss of wild-type huntingtin influences motor dysfunction and survival in the Y A C 1 2 8 mouse model of Huntington disease. Hum. M o l . Genet. 14(10): 1379-92. 172 279. Van Raamsdonk J M , Pearson J, Rogers D A , L u G , Barakauskas V E , Barr A M , Honer W G , Hayden M R , Leavitt, B R . E thy l -EPA treatment improves motor dysfunction, but not neurodegeneration in the Y A C 128 mouse model of Huntington disease. Exp. Neurol. 280. van Vugt JP, Piet K K , V ink L J , Siesling S, Zwinderman A H , Middelkoop H A and Roos R A (2004). Objective assessment of motor slowness in Huntington's disease: clinical correlates and 2-year follow-up. M o v Disord. 19(3): 285-297. 281. Veldink J H , Bar PR, Joosten E A , Otten M , Wokke J H and van den Berg L H (2003). Sexual differences in onset of disease and response to exercise in a transgenic model of A L S . Neuromuscul. Disord. 13(9): 737-743. 282. von Horsten S, Schmitt I, Nguyen H P , Holzmann C, Schmidt T, Walther T, Bader M , Pabst R, Kobbe P, Krotova J, Stiller D , Kask A , Vaarmann A , Rathke-Hartlieb S, Schulz JB, Grasshoff U , Bauer I, Vieira-Saecker A M , Paul M , Jones L , Lindenberg K S , Landwehrmeyer B , Bauer A , L i X J and Riess O (2003). Transgenic rat model of Huntington's disease. Hum. M o l . Genet. 12(6): 617-624. 283. Vonsattel JP, Myers R H , Stevens TJ , Ferrante RJ , Bird E D and Richardson E P , Jr. (1985). Neuropathological classification of Huntington's disease. J. Neuropathol. Exp. Neurol. 44(6): 559-577. 284. Vonsattel JP and DiFig l ia M (1998). Huntington disease. J. Neuropathol. Exp. Neurol. 57(5): 369-384. 285. Wang J, Gines S, Macdonald M E and Gusella JF (2005). Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation. B M C . Neurosci 6(1): 1-12. 286. Wang X , Sarkar A , Cicchetti F, Y u M , Zhu A , Jokivarsi K , Saint-Pierre M and Brownell A L (2005). Cerebral P E T imaging and histological evidence of transglutaminase inhibitor cystamine induced neuroprotection in transgenic R6/2 mouse model of Huntington's disease. J. Neurol. Sci. 231(1-2): 57-66. 287. Wanker E E , Rovira C, Scherzinger E , Hasenbank R, Walter S, Tait D, Colicel l i J and Lehrach H (1997). HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system. Hum. M o l . Genet. 6(3): 487-495. 288. Watkins L H , Rogers R D , Lawrence A D , Sahakian B J , Rosser A E and Robbins T W (2000). Impaired planning but intact decision making in early Huntington's disease: implications for specific fronto-striatal pathology. Neuropsychologia 38(8): 1112-1125. 289'. 'Wellington C L , Leavitt B R and Hayden M R (2000).'Huntington disease: new insights on the role of huntingtin cleavage. J. Neural Transm. Suppl (58): 1-17. 290. Wellington C L , Ellerby L M , Gutekunst C A , Rogers D , Warby S, Graham R K , Loubser O, van Raamsdonk J, Singaraja R, Yang Y Z , Garni J, Bredesen D , Hersch S M , Leavitt 173 B R , Roy S, Nicholson D W and Hayden M R (2002). Caspase cleavage of mutant, huntingtin precedes neurodegeneration in Huntington's disease. J. Neurosci. 22(18): 7862-7872. 291. Wexler N S , Young A B , Tanzi R E , Travers H , Starosta-Rubinstein S, Penney JB , Snodgrass SR, Shoulson I, Gomez F, Ramos Arroyo M A and . (1987). Homozygotes for Huntington's disease. Nature 326(6109): 194-197. 292. Wheeler V C , Auerbach W , White J K , Srinidhi J, Auerbach A , Ryan A , Duyao M P , Vrbanac V , Weaver M , Gusella JF, Joyner A L and Macdonald M E (1999). Length-dependent gametic C A G repeat instability in the Huntington's disease knock-in mouse. Hum. M o l . Genet. 8(1): 115-122. 293. Wheeler V C , White J K , Gutekunst C A , Vrbanac V , Weaver M , L i X J , L i S H , Y i H , Vonsattel JP, Gusella JF, Hersch S, Auerbach W, Joyner A L and Macdonald M E (2000). Long glutamine tracts cause nuclear localization of a novel form of huntingtin in medium spiny striatal neurons in HdhQ92 and H d h Q l 11 knock-in mice. Hum. M o l . Genet. 9(4): 503-513. 294. White J K , Auerbach W , Duyao M P , Vonsattel JP, Gusella JF, Joyner A L and Macdonald M E (1997). Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease C A G expansion. Nat. Genet. 17(4): 404-410. 295. Wilson R S , Como P G , Garron D C , Klawans H L , Barr A and Klawans D (1987). Memory failure in Huntington's disease. J. C l in . Exp. Neuropsychol. 9(2): 147-154. 296. Wood N I and Morton A J (2003). Chronic lithium chloride treatment has variable effects on motor behaviour and survival of mice transgenic for the Huntington's disease mutation. Brain Res. B u l l . 61(4): 375-383. 297. X i a H , Mao Q, Eliason SL , Harper SQ, Martins IH, Orr H T , Paulson H L , Yang L , Kotin R M and Davidson B L (2004). R N A i suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med. 10(8): 816-820. 298. X i a J, Lee D H , Taylor J, Vandelft M and Truant R (2003). Huntingtin contains a highly conserved nuclear export signal. Hum. M o l . Genet. 12(12): 1393-1403. 299. Yamamoto A , Lucas JJ and Hen R (2000). Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease. Cel l 101(1): 57-66. 300. Young A B , Greenamyre JT, Hollingsworth Z , A l b i n R, D'Amato C , Shoulson I and Penney JB (1988). N M D A receptor losses in putamen from patients with Huntington's disease. Science 241(4868): 981-983. 301. Zainelli G M , Ross C A , Troncoso JC and Muma N A (2003). Transglutaminase cross-links in intranuclear inclusions in Huntington disease. J. Neuropathol. Exp. Neurol. 62(1): 14-24. 174 302. Zainelli G M , Dudek N L , Ross C A , K i m S Y and Muma N A (2005). Mutant huntingtin protein: a substrate for transglutaminase 1, 2, and 3. J. Neuropathol. Exp. Neurol. 64(1): 58-65. 303. Zeitlin S, L i u JP, Chapman D L , Papaioannou V E and Efstratiadis A (1995). Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nat. Genet. 11(2): 155-163. 304. Zeron M M , Chen N , Moshaver A , Lee A T , Wellington C L , Hayden M R and Raymond L A (2001). Mutant huntingtin enhances excitotoxic cell death. M o l . Cel l Neurosci 17(1): 41-53. 305. Zeron M M , Hansson O, Chen N , Wellington C L , Leavitt B R , Brundin P, Hayden M R and Raymond L A (2002). Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease. Neuron 33(6): 849-860. 306. Zhang Y , Tounekti O, Akerman B , Goodyer C G and LeBlanc A (2001). 17-beta-estradiol induces an inhibitor of active caspases. J. Neurosci. 21(20): RC176-307. Zhang Y , L i M , Drozda M , Chen M , Ren S, Mejia Sanchez R O , Leavitt B R , Cattaneo E , Ferrante R J , Hayden M R and Friedlander R M (2003). Depletion of wild-type huntingtin in mouse models of neurologic diseases. J. Neurochem. 87(1): 101-106. 308. Zhang Y , Ona V O , L i M , Drozda M , Dubois-Dauphin M , Przedborski S, Ferrante R J and Friedlander R M (2003). Sequential activation of individual caspases, and of alterations in Bcl-2 proapoptotic signals in a mouse model of Huntington's disease. J. Neurochem. 87(5): 1184-1192. 309. Zuccato C, Ciammola A , Rigamonti D , Leavitt B R , Goffredo D , Conti L , Macdonald M E , Friedlander R M , Silani V , Hayden M R , Timmusk T, Sipione S and Cattaneo E (2001). Loss of huntingtin-mediated B D N F gene transcription in Huntington's disease. Science 293(5529): 493-498. 310. Zuccato C, Tartari M , Crotti A , Goffredo D , Valenza M , Conti L , Cataudella T, Leavitt B R , Hayden M R , Timmusk T, Rigamonti D and Cattaneo E (2003). Huntingtin interacts with R E S T / N R S F to modulate the transcription of NRSE-controlled neuronal genes. Nat. Genet. 35(1): 76-83. 175 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092261/manifest

Comment

Related Items