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Characterization and use of the YAC128 mouse model of HD Slow, Elizabeth Jane 2005

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Characterization and Use of the YAC128 Mouse Model of HD By Elizabeth Jane Slow B.Sc., The University of Guelph, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Genetics) T H E UNIVERSITY OF BRITISH COLUMBIA January 2005 © Elizabeth Jane Slow, 2005 Abstract Huntington disease (HD) is caused by a C A G expansion in the gene encoding the protein huntingtin and manifests clinically with motor and cognitive impairments and psychiatric disturbances. A n accurate animal model is crucial for elucidation of the underlying mechanisms which lead to pathogenesis and also for testing experimental therapeutics. The yeast artificial chromosome ( Y A C ) mouse model expresses full-length human huntingtin with 128 C A G repeats (YAC128) from an endogenous promoter. The Y A C 128 model accurately recapitulates the age-dependent brain atrophy including cortical and striatal atrophy accompanied by striatal neuronal loss characteristic of H D . The onset of impairments in motor co-ordination, pre-pulse inhibition and cognition, along with a biphasic activity profile composed of initial hyperactivity and late hypoactivity in the Y A C 128 mice recapitulates the clinical manifestation of the human disease. The accurate recapitulation of the human disorder in the Y A C 1 2 8 mouse allows the utilization of this model to identify the primary events which lead to pathogenesis and target those events for therapeutics. The phenotypes in the Y A C 1 2 8 are robust on multiple background strains, reproducible and demonstrate low inter-animal variability, indicating the Y A C 1 2 8 model is highly suited for use in therapeutic trials. Huntingtin inclusions or aggregates are present late in the natural history of disease in the Y A C 128 mouse model, after the onset of both neuronal dysfunction indicated by rotarod deficit and neuronal degeneration, indicated by brain and striatal atrophy. The onset of neuronal degeneration in the Y A C 128 mice on the B6 strain background is delayed, yet the time course and severity of huntingtin inclusion formation is unaltered. The shortstop mouse expresses a short fragment of huntingtin with the identical C A G size, tissue distribution and level of transgenic protein expression as the full-length Y A C 1 2 8 model. Huntingtin inclusions form earlier and are more prevalent both in number and tissue distribution in the shortstop compared to the full-length Y A C 1 2 8 model. Despite widespread inclusion formation, the shortstop mouse does not manifest the neuronal dysfunction or degeneration present in the full-length Y A C 128 model. Together, these results demonstrate that huntingtin aggregates are not the direct cause of toxicity in H D and are likely a benign by-product of polyglutamine expansion. T A B L E OF C O N T E N T S ABSTRACT II TABLE OF CONTENTS Ill LIST OF FIGURES VI LIST OF TABLES Vlll LIST OF ABBREVIATIONS MX ACKNOWLEDGEMENTS XI CHAPTER 1: INTRODUCTION 1 1.1 Clinical findings 2 1.2 Neuropathology of HD 2 1.3 Huntington disease gene 3 1.4 Age of onset correlation with CAG repeat length 3 1.5 The huntingtin protein 5 1.6 Huntingtin functions 5 1.6.1 Cellular survival 5 1.6.2 Transcriptional regulation 7 1.6.3 Endocytosis and cellular transport 7 1.7 Huntingtin proteolysis 8 1.8 Huntingtin aggregation 12 1.9 Mouse models of Huntington disease 13 1.9.1 Cell culture and invertebrate organism models of HD 13 1.9.2 Truncated mouse models 15 1.9.3 Full-length mouse models 17 1.9.4 YAC mouse model 18 1.10 Objectives : 21 CHAPTER 2: MATERIALS AND METHODS 23 2.1 YAC mutagenesis and generation of mice 24 2.2 Copy number, RNA and protein analysis 24 iii 2.2.1 Protein analysis 24 2.2.2 RNA analysis :25 2.3 Morphological analysis 25 2.4 Quantitative analysis 25 2.5 Assessment of aggregates 26 2.5.1 Light microscopy 26 2.5.2 Electron microscopy 26 2.5.3 Aggregation foci 27 2.6 Behavioural assessment 27 2.6.1 Rotarod analysis 28 2.6.2 Open-field analysis 28 2.6.3 Locomotor activity 29 2.6.4 Pre-pulse inhibition and habituation 29 2.7 Statistics 29 2.8 PCR Characterization of YAC Shortstop Truncation Sites 30 2.9 Bioinformatics on Shortstop 31 2.10 CAG Repeat Sizing 31 CHAPTER 3: SELECTIVE STRIATAL NEURONAL LOSS IN A YAC128 MOUSE MODEL OF HUNTINGTON DISEASE..... 33 3.1 Introduction 35 3.2 Results 37 3.2.1 Establishment of YAC 128 mice 37 3.2.2 Brain weight decrease in YAC128, line 53 38 3.2.3 Striatal and cortical volume is decreased in YAC128 mice 39 3.2.4 Neuronal loss and dysfunction in YAC128 mice 40 3.2.5 Behavioural assessment 42 3.2.6 Rotarod performance correlates with severity of neuronal loss 43 3.2.7 Presence of aggregates 45 3.2.8 Low variability in the phenotypes of YAC128 mice 46 3.3 Discussion 48 CHAPTER 4: A YAC MOUSE EXPRESSING A TRUNCATED FRAGMENT OF HUNTINGTIN (SHORTSTOP) DISPLAYS WIDESPREAD NEURONAL INCLUSIONS WITHOUT NEURONAL DYSFUNCTION OR DEGENERATION 53 4.1 Introduction 55 4.2 Results 57 4.2.1 Discovery of the shortstop mouse 57 4.2.2 Characterization of the shortstop 3' YAC truncation site and insertion site 58 4.2.3 Shortstop and YAC128 mice have identical promoter, regulatory elements and CAG sizes 60 iv 4.2.4 Shortstop and YAC128 mice exhibit an identical pattern of transgenic protein expression 60 4.2.5 Inclusion formation in shortstop mice 62 4.2.6 Aggregation foci in shortstop and YAC128 mice 64 4.2.7 Shortstop does not exhibit a rotarod deficit 66 4.2.8 Shortstop does not exhibit neuronal degeneration 66 4.3 Discussion 69 CHAPTER 5: THE YAC128 EXHIBITS DEFICITS IN PPI, COGNITION AND NEURONAL DEGENERATION ON THE B6 STRAIN 77 5.1 Introduction 79 5.2 Results 82 5.2.1 The YAC 128 mice on a B6 background exhibit selective brain weight loss 82 5.2.2 Striatal volume and neuronal number are decreased in B6-YAC128 83 5.2.3 Rotarod and home cage activity in B6-YAC128 compared to FVB-YAC128 84 5.2.4 YAC 128 on B6 and FVB exhibit similar deficits in pre-pulse inhibition and habituation 85 5.2.5 Inclusions are similar in the B6-YAC128 and FVB-YAC128 mice 88 5.2.6 Later onset of neurodegeneration in B6-YAC 128 compared to FVB-YAC 128 89 5.3 Discussion 92 CHAPTER 6: DISCUSSION AND FUTURE DIRECTIONS 97 6.1 The YAC128 mouse model accurately recapitulates Huntington disease 98 6.2 The YAC128 mouse model is highly suited for use in therapeutic trials 100 6.2.1 Significance of YAC128 model for the HD field 102 6.3 Huntingtin inclusions (aggregates) and their precursors are not toxic 105 6.3.1 Significance of aggregate findings for the HD field 107 6.4 The shortstop mouse suggests there is specificity of the proteolytic fragment for the toxic fragment hypothesis 108 6.5 Future directions 109 6.6 Conclusions 114 CHAPTER 7: REFERENCES 116 v List of Figures Figure 1.1: Huntingtin proteolysis 10 Figure 1.2: The toxic fragment hypothesis 11 Figure 3.1: Characterization of Y A C 1 2 8 transgenic mouse: D N A 37 Figure 3.2: Characterization of Y A C 1 2 8 transgenic mouse: R N A and protein 38 Figure 3.3: Brain weight is selectively decreased in Y A C 1 2 8 mice 39 Figure 3.4: Striatal and cortical volume are decreased in Y A C 1 2 8 mice 40 Figure 3.5: Striatal neuron surface area is decreased in Y A C 1 2 8 mice 41 Figure 3.6: Rotarod deficit in Y A C 1 2 8 mice 42 Figure 3.7: Y A C 1 2 8 s initially exhibit hyper- followed by hypokinetic behaviour 43 Figure 3.8: Impairment in rotarod activity is correlated with striatal neuronal loss 44 Figure 3.9: E M 4 8 immunoreactivity in the striatum of 12 (A and D) and 18 (B, C, E and F) month old Y A C 1 2 8 mice 46 Figure 3.10: Comparison of the natural history of H D in the Y A C 1 2 8 mouse model and in human patients 49 Figure 4.1: Protein discovery and shortstop localization in mouse chromosome 4 57 Figure 4.2: Comparison of shortstop with Y A C 1 2 8 , line 53 61 Figure 4.3: Huntingtin inclusions in shortstop and Y A C 1 2 8 mice at 18 months 62 Figure 4.4: Aggregation foci in shortstop and Y A C 1 2 8 mice 65 Figure 4.5: The shortstop mouse does not manifest the neuronal dysfunction or degeneration of the full-length Y A C 1 2 8 model at 12 months of age 67 Figure 4.6 Shortstop mice do not exhibit decreases in brain weight or striatal volume at 18 months of age 68 Figure 5.1: Brain weight is selectively decreased in B 6 - Y A C 1 2 8 mice 82 Figure 5.2: Striatal volume is decreased in B 6 - Y A C 1 2 8 mice 83 Figure 5.3 Rotarod deficit is dependent on strain in Y A C 1 2 8 mice 84 Figure 5.4: Y A C 1 2 8 mice exhibit hypoactivity at 12 months of age 85 Figure 5.5: Startle magnitude in Y A C 1 2 8 on an F V B and B6 background 86 Figure 5.6: Impairments in pre-pulse inhibition and habituation in Y A C 1 2 8 on the B6 and F V B background 87 Figure 5.7: B 6 - Y A C 1 2 8 exhibit huntingtin inclusions 89 vi Figure 6.1: Natural history of H D in the Y A C 1 2 8 mouse model 100 vi i List of Tables Table 1.1: Polyglutamine (polyQ) repeat disorders with differential sites of disease pathogenesis 4 Table 1.2: Summary of the design, behavioural and neuropathological findings in FID mouse models 16 Table 1.3: Results from studies using H D Y A C mice to investigate different pathways involved in the pathogenesis of H D 20 Table 3.1: Striatal neuron number in Y A C 1 2 8 mice 41 Table 3.2: Overlap and power analysis for quantitative phenotypes in individual Y A C 128 mice 47 Table 4.1: Nuclear localization of htt is accelerated in the shortstop mice and exhibits regional differences compared to the full-length Y A C 128 mouse 63 Table 4.2: Huntingtin inclusion formation is accelerated and more widespread in the shortstop mice compared to the full-length Y A C 1 2 8 mouse 64 Table 5.1: Neuronal decrease in B 6 - Y A C 1 2 8 mice at 12 months of age 83 Table 5.2: Summary of the neuropathological and behavioural phenotypes exhibited in the F V B - Y A C 1 2 8 and B 6 - Y A C 1 2 8 mice at 12 months of age 90 Table 6.1: Y A C 128 collaborations 104 v i i i List of Abbreviations a-arnino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( A M P A ) Adaptor protein 2 (Ap2) Adenosine triphosphate (ATP) Amyotrophic Lateral Sclerosis (ALS) Amino acids (aa) Analysis of Variance ( A N O V A ) Base pair (bp) Brain-derived neurotrophic factor ( B D N F ) C57B1/6 (B6) Cytomegalovirus ( C M V ) Cil iary neurotrophic factor (CNTF) cAMP-response element binding protein ( C R E B ) Diaminobenzidine ( D A B ) Dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32) Dentate gyrus (DG) Dentatorubral-pallidoluysian atrophy ( D R P L A ) Deoxynucleotide triphosphate (dNTP) Figure (Fig.) Glutamine (Q) Heat shock protein (HSP) Histone deacetylase ( H D A C ) Huntingtin (htt) Huntingtin associated protein (Hap) Huntingtin interacting protein (Hip) Huntington disease (HD) Hypoxanthine phosphoribosyltransferase gene (Hprt) Kilo-basepair (kb) Knockout (KO) Left Y A C arm ( L Y A ) Long-term potentiation (LTP) Mammalian target of rapamycin (mTOR) Medium spiny neurons (MSNs) National Center for Biotechnology Information (NCBI) Neuron restrictive silencer element (NRSE) Neuron-specific enolase (NSE) N-methyl-D-aspartate (NMDA) Phosphate buffered saline (PBS) Polyglutamine (polyQ) Polymerase chain reaction (PCR) Pre-pulse inhibition (PPI) Quantitative trait loci (QTL) Repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF) Reverse transcription (RT) Revolutions per minute (rpm) Right Y A C arm (RYA) Serum- and glucocorticoid-induced kinase (SGK) Shortstop (SS) Single nucleotide polymorphism (SNP) Small interfering RNA (siRNA) Specificity protein 1 (Spl) Spinal and Bulbar Muscular Atrophy (SBMA) Spinocerebellar ataxia (SCA) Standard deviation (SD) Standard error of the mean (SEM) Suberoylanilide hydroxamic acid (SAHA) Tdt-mediated dUTP-biotin nick end labeling (TUNEL) Transgenic (Tg) Truncated huntingtin (mtt) Wild-type (WT) Yeast artificial chromosome (YAC) Inositol (l,4,5)-triphosphate receptor (InsP3Rl) x Acknowledgements There are a number of individuals who have contributed to this thesis whom I would like to thank. First, I would like to thank my supervisor, Dr. Michael Hayden for his support and guidance throughout the course of my Ph.D. studies and also in my plans for the future. He has given me unique opportunities and experiences throughout my degree which have proved both challenging and educational and he has provided a learning environment of incredibly talented and generous co-workers. Thank you for always encouraging me to aim higher. This thesis has benefited enormously from my interactions with other members of the H D group. Fwould like to especially thank Rona Graham for a very fruitful collaboration over the past two years and for her great friendship and personal generosity. I would like to thank Blair Leavitt and Jeremy van Raamsdonk for their collaborative efforts with the Y A C 1 2 8 model. I would like to acknowledge wonderful technical support and friendship from Nagat Bissada and Y u (Deborah) Deng. Fwould also like to thank Rebecca, Brendan, Ed , Simon, Jamie, Susie, Erick and Jen for good advice and good memories over the years. I would like to acknowledge the input and the contributions of collaborators including Alex Osmand, Claire-Anne Gutekunst and Beth Simpson and her lab members for all of their help. I would also like to thank my thesis committee members Dr. C . Brown, Dr. E . Simpson and Dr. L . Clarke for their advice and very enjoyable meetings over the years. Last, but certainly not least, I would like to acknowledge the loving support of my parents Lynne and Robert and my brother Matthew. It would be impossible to have accomplished this without their incredible encouragement, support and generosity over the years. Finally, I would like to thank Bryan for his unfailing support and enthusiastic and unwavering encouragement over the past four years. x i Chapter 1: Introduction 1.1 Clinical findings Huntington disease (HD) is a devastating neurodegenerative disorder, affecting approximately 1/10000 people of European descent (Hayden, 1981). The disease was first described by George Huntington in 1872, whose name the illness now bears (Huntington, 2003), but clinical features of the disease have been recorded at early as the 1500s, when the term "chorea" from the Latin for choreus or dancing was first used to describe the involuntary, uncoordinated, rapid, jerky type of movement which is one of the characteristic features of the disorder. Age of onset typically occurs in middle age and usually presents with motor disturbances including mild chorea (Harper et al., 2002). Juvenile onset H D is defined as those patients who experience onset of the illness before the age of 20 and comprise a subset of 5-10% of H D cases (Hayden, 1981). Onset in these patients is typically marked by rigidity and spasticity and the disease follows a different clinical course from adult onset H D . While motor abnormalities including chorea are characteristic of the adult onset disease, psychiatric disturbances including irritability and depression are also common and can be the first sign of onset in some patients (Harper et al., 2002). Cognitive impairment is one of the cardinal features of H D and includes memory and judgment decline, disorientation and intellectual impairment (Harper et al., 2002). Initial presentation of the illness involves uncontrolled movements, but as the disease progresses, the patient experiences rigidity (Harper et al., 2002). During the course of the disease's progression, which typically result in death 15-25 years after the initial onset of symptoms, every aspect of brain function is afflicted, from motor to cognitive to psychiatric, earning the description "one of the most dreadful diseases that man is liable to" (Davenport and Muncey, 1916). Despite 500 years of H D awareness and description, there is still no cure to offer patients. 1.2 Neuropathology of HD The pathology of Huntington disease is restricted to the brain and involves selective neuronal loss. This selective neuronal loss occurs primarily in the striatum (caudate nucleus and putamen) and it is this region which exhibits the first pathological changes in the illness (Hayden, 1981;Harper et al., 2002). The cells which appear to be most susceptible are the G A B A - e r g i c medium spiny neurons in the striatum (Vonsattel et 2 al., 1985). These cells receive excitatory (glutamatergic) impulses from the neocortex and send inhibitory projections to the globus pallidus and substantia nigra (Albin et al., 1990). The cerebral cortex is also affected, with layers V through III demonstrating neuronal loss (Harper et al., 2002). In late stages of the disease, the degeneration becomes more generalized and can affect other brain regions including the cerebellum, globus pallidus, thalamus and substantia nigra (Vonsattel et al., 1985). The loss of neurons is accompanied by an overall atrophy of the brain, which can weigh 20-30% less than control brains in advanced stages of the illness (Harper et al., 2002). 1.3 Huntington disease gene Huntington disease exhibits an autosomal dominant mode of inheritance (Hayden, 1981). A huge step forward in the understanding and potential for therapeutics arrived with the cloning of the gene which underlies H D in 1993 (1993). H D results from an expansion in the C A G tract in exon 1 of the gene encoding the protein huntingtin (1993). The gene is located on the short arm of chromosome 4 and consists of 67 exons spanning over 200 kb (Ambrose et al., 1994). The sequence of the H D gene is highly conserved across human, mouse, rat and fugu, with overall homology to human huntingtin ranging from 90% in fugu to 95% in mouse and 100% homology across all 3 species for the first 15 amino acids of huntingtin before the polyglutamine repeat (Huq et al., 1998). A huntingtin homolog is also present in the Drosophila melanogaster genome, but there are no homologs in Caenorhabditis elegans or Saccharomyces cerevisiae (Takano and Gusella, 2002). 1.4 Age of onset correlation with CAG repeat length The C A G repeat length in the normal population is highly polymorphic, ranging from 10-35 with a median of 18 (Kremer et al., 1994). In H D , the C A G repeat length can range from 36-121 (Andrew et al., 1993), with juvenile onset H D patients typically resulting from a C A G repeat of over 50. The C A G tract length demonstrates the fascinating property of being inversely related to age of onset (i.e. the longer the C A G tract, the earlier the age of onset) (1993;Duyao et al., 1993;Snell et al., 1993;Brinkman et al., 1997;Langbehn et al., 2004). A detailed analysis of a large database of patients revealed incomplete penetrance of the disease for repeat sizes of 36-41, while a repeat 3 size of 42 and above was completely penetrant (Leavitt et al., 1999). The length of the G A G repeat is estimated to account for 70% of the variability in the age of onset of the disease, suggesting that other genetic and/or environmental factors may play a role in the age of onset of H D (Rosenblatt et al., 2001). H D is one of 9 polyglutamine disorders previously described (Table 1.1). Table 1.1: Polyglutamine (polyQ) repeat disorders with differential sites of disease pathogenesis. Disease Protein PolyQ (normal) PolyQ (pathogenic) Site of Pathology References Huntington disease Huntingtin 8-35 36-121 1) Caudate putamen 2) Cortex (1993) Spinocerebellar ataxia type 1 Ataxin-1 6-44 40-82 1) Cerebellum (Purkinje cells) 2) Spinocerebellar tracts (Orr et al., 1993) Spinocerebellar ataxia type 2 Ataxin-2 14-32 33-77 1) Cerebellum (Purkinje cells) 2) Inferior olive 3) Substantia nigra (Imbert et al., 1996;Pulstet al., 1996;Sanpei et al., 1996) Spinocerebellar ataxia type 3 or Machado-Joseph disease Ataxin-3 12-40 55-86 1) Basal ganglia 2) Brainstem 3) Cerebellum 4) Spinal Cord (Kawaguchi et al., 1994) Spinocerebellar ataxia type 6 A-1A voltage-dependent calcium channel 4-18 21-30 1) Cerebellum (Purkinje cells) 2) Inferior olive (Zhuchenko et al., 1997) Spinocerebellar ataxia type 7 Ataxin-7 7-17 38-200 1) Retinal degeneration 2) Cerebellum (Purkinje cells) (David et al., 1997) Spinocerebellar ataxia type 17 TATA-binding protein 27-44 47-55 1) Cerebellum (Purkinje cells) (Tsuji, 2004) Dentatorubral-pallidoluysian atrophy Atrophin 3-36 49-88 1) Dentate nucleus 2) Globus pallidus (Koide et al., 1994) Spinal and bulbar muscular atrophy Androgen receptor 9-36 38-62 motor neurons in 1) Spinal cord 2) Brainstem (La Spada et al., 1991) Although the underlying mutation, an expanded polyglutamine repeat, is identical in all of these disorders, the site of pathology varies for each of the diseases. The differences in pathology are thought to primarily occur due to the protein context of the mutation i.e. the particular properties of the protein in which the polyglutamine tract resides. 4 1.5 The huntingtin protein Huntingtin is 3144 amino acids in length (for a polyglutamine tract length of 23) with an approximate molecular mass of over 350 kDa. It is ubiquitously expressed with highest expression in the brain and testes and lower expression in the peripheral tissues (Huq et al., 1998). Recent research has located a nuclear export signal in the C-terminus of huntingtin (Xia et al., 2003), and bioinformatics analysis has revealed numerous H E A T repeats throughout the protein, proposed to facilitate protein-protein interactions (Andrade and Bork, 1995;Takano and Gusella, 2002). In an attempt to discern the function of huntingtin, which had no known function when the gene was originally cloned in 1993, three different groups disrupted murine huntingtin and assessed the resulting phenotype (Duyao et al., 1995;Nasir et al., 1995;Zeitlin et al., 1995). The huntingtin knock-out animals were embryonic lethal due to increased apoptosis (Zeitlin et al., 1995), revealing both the first hint at the function of huntingtin along with the fact that huntingtin is an essential protein required for appropriate development. 1.6 Huntingtin functions Experimental evidence indicates huntingtin is a multifunctional protein that plays a variety of roles in the cell. Alterations in any of these functions may be responsible for the neurodegeneration displayed in H D patients. 1.6.1 Cellular survival The first evidence for huntingtin's potential role in cellular survival was indicated by the increased apoptosis in embryos lacking endogenous huntingtin (-/-), indicating huntingtin may play a role in anti-apoptotic, or cell survival processes (Zeitlin et al., 1995). In one of the huntingtin knock-out models, heterozygous mice expressing fifty percent of endogenous huntingtin levels exhibited behavioural abnormalities and neuronal loss in the basal ganglia (Nasir et al., 1995;0'Kusky et al., 1999). Studies with mice expressing less than 50% of endogenous huntingtin protein levels due to insertion of a neo cassette demonstrated aberrant brain development and perinatal lethality (White et al., 1997). Together, these results reveal the necessity of a specific, minimum level of huntingtin expression for appropriate brain development and neuronal survival. 5 Additional evidence for huntingtin's role in cell survival was demonstrated in a conditional knock-out huntingtin mouse, where expression in brain and testes was selectively reduced in adult mice (Dragatsis et al., 2000). These mice demonstrated progressive neuronal and testicular degeneration upon inactivation of huntingtin, revealing the necessity of huntingtin for cellular survival in adult mice (Dragatsis et al., 2000). In huntingtin knock-out mice rescued with a mutant huntingtin transgene (72 CAGs), the testes undergo massive apoptosis (Leavitt et al., 2001). This apoptosis is reduced by increasing the level of endogenous, wild-type huntingtin (Leavitt et al., 2001), further supporting a role for wild-type huntingtin in pro-survival pathways. Direct evidence supporting a role for huntingtin in cellular survival was demonstrated in a study of striatal cells which over-expressed wild-type huntingtin. These cells were resistant to cellular toxicity caused by serum deprivation and 3-nitropropionic acid, a mitochondrial toxin (Rigamonti et al., 2000). Similar studies in vivo using mice over-expressing wild-type huntingtin in a Y A C transgenic model discovered that these mice were resistant to neuronal degeneration caused by ischemia (Zhang et al., 2003), and excitotoxicity caused by quinolinic acid and kainic acid injection (Leavitt and Hayden, submitted) suggesting overexpression of wild-type huntingtin is neuroprotective. Evidence for a potential mechanism underlying this pro-survival function came from studies that demonstrated wild-type huntingtin's ability to up-regulate transcription of brain-derived neurotrophic factor (BDNF) both in vitro and in a mouse overexpressing wild-type huntingtin (Zuccato et al., 2001). BDNF is trophic factor necessary for striatal neuronal survival (Mizuno et al., 1994;Ventimiglia et al., 1995). Subsequent studies demonstrated that wild-type huntingtin regulates transcription of BDNF by cytoplasmic sequestration of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), a transcription factor which binds to a neuron restrictive silencer element (NRSE) in the promoter of BDNF (Zuccato et al., 2003). The sequestration of this repressor element by wild-type huntingtin leads to less repression and therefore increased transcription of BDNF. Further evidence supporting a functional role for huntingtin in cellular survival was indicated by the revelation that huntingtin is a substrate for phosphorylation by Akt, 6 a potent cell survival kinase which exerts its effects through phosphorylation of its substrates (Humbert et al., 2002). Phosphorylation of huntingtin by Ak t (Humbert et al., 2002) and another cell survival kinase, S G K (Rangone et al., 2004), promoted neuronal survival against the toxic effects of mutant huntingtin. 1.6.2 Transcriptional regulation The discovery that wild-type huntingtin can sequester R E S T / N R S F , and therefore enhance transcription of B D N F is just one example supporting the hypothesis that huntingtin plays a role in transcriptional regulation. Much of the evidence supporting a transcriptional regulatory role for huntingtin emerges from examination of huntingtin's interacting proteins. Huntingtin interacts with the transcriptional activator S p l (specificity protein 1) (Dunah et al., 2002;Li et al., 2002), and the C R E B ( c A M P -response element binding protein) binding protein (Steffan et al., 2000;Nucifora, Jr. et al., 2001). S p l binding sites are present in most promoters of the glutamate receptor subunits (Myers et al., 1999), thought to play an important role in the pathogenesis of H D and C R E B knock-out mice exhibit neuronal degeneration (Mantamadiotis et al., 2002), displaying the importance of both of these transcription factors in neuronal function. Other research revealed huntingtin interacts with CA150 , a transcriptional activator (Holbert et al., 2001), the transcription factor TAFII-130 (Dunah et al., 2002), p53 (Steffan et al., 2000) and the transcriptional co-repressor mSin3A (Steffan et al., 2000). In addition to data from huntingtin interacting proteins, microarray analysis of transcripts from mouse models of H D revealed altered transcription compared to wild-type controls (Luthi-Carter et al., 2000;Chan et al., 2002), demonstrating mutant huntingtin can affect transcription. 1.6.3 Endocytosis and cellular transport Clathrin-mediated endocytosis is an essential mechanism through which cells can regulate membrane composition, including the number of receptors, ion channels and transporters on the cell surface (Buckley et al., 2000;McPherson et al., 2001). In neurons, clathrin mediated endocytosis facilitates the rapid retrieval of synaptic vesicles (Sun et al., 2002) as well as regulating the surface expression of excitatory glutamate receptors (Sheng and K i m , 2002). Huntingtin has recently been shown to play a role in receptor-7 mediated endocytosis through its interaction with huntingtin-interacting protein 1 (Hip l ) . H i p l not only binds huntingtin (Kalchman et al., 1997;Gervais et al., 2002), but also the clathrin heavy chain (Metzler et al., 2001;Mishra et al., 2001;Legendre-Guillemin et al., 2002), and adaptor protein 2 (Ap2) which drives the assembly of the clathrin lattices and clathrin-coated pits (Metzler et al., 2001;Mishra et al., 2001;Waelter et al., 2001;Legendre-Guillemin et al., 2002). Mice with targeted disruption of H i p l (-/-) display neurological defects, and impaired trafficking of the A M P A glutamate receptor (Metzler et al., 2003) suggesting H i p l and huntingtin play a role in receptor trafficking. Huntingtin also interacts with Pascinl (Modregger et al., 2002), S H 3 G L 3 (Sittler et al., 1998), and H i p l 4 (Singaraja et al., 2002), three other proteins involved in endocytosis. Vesicles, including endosomes created by receptor-mediated endocytosis, can undergo axonal retrograde transport powered by the motor protein dynein. Dynein forms a complex with p l 5 0 G l u e d and other proteins to enable movement of vesicles along the microtubule (Gross, 2003). Huntingtin's interacting partner huntingtin-associated protein 1 (Hapl) (L i et al., 1996) binds to p l 5 0 G l u e d (Engelender et al., 1997) and both huntingtin and H a p l demonstrate anterograde and retrograde transport compatible with a role in axonal transport on vesicular membranes (Block-Galarza et al., 1997). In addition, reduction of endogenous huntingtin levels in Drosophila resulted in disrupted axonal transport, suggesting huntingtin is necessary for appropriate transport along axons (Gunawardena et al., 2003). 1.7 Huntingtin proteolysis The discovery of N-terminal huntingtin fragments in patient brains indicated a role for protease cleavage of huntingtin in the disease (DiFigl ia et al., 1997). A possible class of proteases involved in the generation of these N-terminal fragments had been described a year earlier when it was discovered that huntingtin is a substrate for apopain, now referred to as caspase-3 (Goldberg et al., 1996). Caspases are cysteine aspartic-acid proteases and are key participants in apoptosis, believed to be the method by which neurons die in H D . Caspase-3 is an important effector of apoptosis and cleaves huntingtin at Asp513 and Asp552 (Wellington et al., 2000). Subsequent research led to the discovery that in vitro, huntingtin can also serve as a substrate for caspase-6 8 (Wellington et al., 2000) and caspase-2 (Hermel et a l , 2004) at Asp586 and Asp552 respectively. Fragments corresponding to caspase-generated fragments are present in patient brains ( K i m et al., 2001;Wellington et al., 2002), and their appearance precedes neuronal loss both in patient and FID mouse model brain (Wellington et al., 2002). Caspases exist in two forms, both inactive zymogens, and as activated proteases, capable of cleaving their substrates and triggering apoptosis. Activated caspase-2, 6 and 7 have been detected in H D patient as well as H D transgenic mouse brain, but not control human or mouse brain (Hermel et al., 2004). Finally, treatment of a mouse model with a broad spectrum caspase inhibitor led to decreased neuropathology and mortality in a mouse model of H D (Ona et al., 1999), suggesting that caspase inhibition is a promising therapeutic target. Calpains are a family of calcium-dependent cysteine proteases that can be triggered by necrotic and apoptotic stimuli, especially those that lead to increased C a + 2 levels in the cell (Khorchid and Dcura, 2002). Recent evidence has supported a role for disrupted C a + 2 signaling in H D , including the discovery of dysfunctional mitochondrial calcium buffering in both patient lymphoblasts and H D mouse model brain (Sawa et al., 1999;Panov et al., 2002), and that the activation of the intracellular calcium channel type 1 inositol (l,4,5)-triphosphate receptor (InsP3Rl) is sensitized in the presence of mutant huntingtin (Tang et al., 2003). In addition, mutant huntingtin sensitizes the N M D A receptor, a receptor which controls C a + 2 influx into the neuron, to activation both in vitro (Zeron et al., 2001) and in vivo (Zeron et al., 2002;Zeron et al., 2004). A n y of these events would lead to increased intracellular C a + 2 levels and the potential activation of calpains. Huntingtin can be cleaved by calpains at two sites between amino acids 437-540 and fragments corresponding to those generated by in vitro recombinant calpain proteolysis are present in patient brain ( K i m et al., 2001). Mutation of the calpain cleavage sites to prevent calpain proteolysis results in reduced toxicity of huntingtin in an in vitro cell culture model (Gafni et al., 2004) similar to the decreased toxicity observed when caspase cleavage sites in huntingtin are mutated (Wellington et al., 2000). Levels of both inactive and active calpains are increased in patient compared to control brain (Gafni and Ellerby, 2002). 9 >40 36-39 <36 Huntingtin proteolysis 100 200 300 400 500 600 3144 aa Caspase C3/7-513, 552 C2 - 552 C6 - 586 Calpain 3-5 sites aa 430-550 Aspartyl protease aa 104-114 Shortstop mouse aa 117 (chapter 4) Figure 1.1: Huntingtin proteolysis. Illustration of N-terminal fragments produced by proteolytic cleavage of full-length huntingtin. The fragment expressed by the shortstop mouse is shown in red (discussed further is Chapter 4). The triangle symbolizes the polyglutamine repeat expansion with red indicating a pathogenic repeat and green a normal repeat length. Huntingtin aggregates are found both in patient and H D mouse model brain (discussed further in Section 1.8). A n antibody screen o f aggregated huntingtin inclusions revealed the presence of two previously unknown N-terminal fragments of huntingtin named cpA (104-110 amino acids in length) and cpB (146-214 amino acids in length) (Lunkes et al., 2002). Production of cpA and cpB in a cell culture system could be prevented by the addition of pepstatin, an inhibitor of aspartic endopeptidases, implicating an unknowii aspartyl protease in the cleavage event which generates these fragments (Lunkes et al., 2002). 10 t C a 2 + ^•pP^^^" Caspasi Caspase activation/1 Proteolysis Neuronal dysfunction -Q and death t Nuc lear htt Al tered gene express ion Al tered interactions Activation of toxic pathways CAG Mutant (>35) Normal (<35) Cleavage of Huntingtin J i functional wild-type htt CAG Mutant Normal \BW MMM Caspase (aa 513, 552, 586) mm Calpain (aa 436-550) | Aspartyl protease (aa 104 - 114) t toxic N-terminal f ragments from mutant htt Figure 1.2: The toxic fragment hypothesis. Proteolysis of htt leads to the generation of the toxic N-terminal htt fragments. These fragments activate toxic pathways and cause altered gene expression and altered interactions with htt interacting partners. This leads to neuronal dysfunction, increased susceptibility to excitotoxicity and increased calcium influx. These events result in increased activation of proteases, subsequent generation of N-terminal toxic fragments and an amplification cycle which ultimately results in the commitment of the neuron to programmed cell death. Figure courtesy of Cheryl Wellington. The proteolysis o f huntingtin by caspases, calpains and aspartyl proteases generates N-terminal fragments containing the polyglutamine stretch (Fig. 1.1). These data combined with the observation that N-terminal mutant huntingtin fragments are more toxic than full-length mutant huntingtin in vitro (Hackam et al., 1998;Martindale et al., 1998) led to the development of the "toxic fragment hypothesis" (Wellington and Hayden, 1997). This hypothesis states that cleavage of mutant huntingtin by proteases in H D patient neurons generates toxic N-terminal huntingtin fragments which are inefficiently degraded due to the expanded polyglutamine tract, and can therefore cause disruptions in transport, protein-protein interactions, and gene expression. These disruptions lead to increased neuronal stress, and greater sensitivity to excitotoxicity which further increases intracellular calcium levels leading to increased activation of 11 proteases and an amplification of the toxic fragment cycle until the neuron is ultimately committed to death (Fig. 1.2). 1.8 Huntingtin aggregation Huntingtin protein aggregates and/or inclusions are characteristic of H D and are present in both human patients (DiFiglia et al., 1997;Gutekunst et al., 1999) and in H D mouse models (Davies et al., 1997;Hodgson et al., 1999;Schilling et al., 1999;Wheeler et al., 2000;Slow et al., 2003). Protein aggregation is also a feature of many of the other polyglutamine disorders including S C A - 1 (Skinner et al., 1997), S C A - 3 (Paulson et al., 1997), S C A - 7 (Holmberg et al., 1998), S B M A (L i et al., 1998) and D R P L A (Becher et al., 1998), indicating a common biomarker of polyglutamine expansion despite different background protein context. Research has demonstrated ubiquitination of a subset of these inclusions in H D patients (Gutekunst et al., 1999;Sieradzan et al., 1999). In addition, various studies of inclusions indicate the ability to recruit chaperones (Jana et al., 2000), and transcription factors (Boutell et al., 1999;Suhr et al., 2001), but evidence from H D mouse models suggests this recruitment may not underlie the pathogenesis of the disorder (Yu et al., 2002;Hansson et al., 2003). Investigation into the process of huntingtin aggregation using polyglutamine peptides indicated that polyglutamine stretches are capable of supporting spontaneous, self-nucleated aggregation. Interestingly, there is a dramatic increase in the spontaneous formation of aggregates with 37 polyglutamines or higher, corresponding to the polyglutamine length separating the normal population from H D patients (Chen et al., 2001). Research with these polyglutamine containing peptides indicates that a critical concentration of aggregate precursor must be reached before the polyglutamine forms into a P-sheet structure (Chen et al., 2002). This research has contributed to the hypothesis that formation of aggregates and inclusions is a linear process, beginning with the formation of P-sheet structures, and continuing with the formation of protofibrils, fibrils, aggregation foci and finally visible aggregates (Bates, 2003). Since the initial discovery of huntingtin inclusions, there has been continual controversy over what role huntingtin aggregates play in pathogenesis of the disease. The debate is divided over whether aggregates and their precursors are in fact the toxic, 12 underlying mechanism in the neuronal loss and dysfunction of the disease, or simply benign byproducts of polyglutamine expansion. In a short fragment mouse model of H D , the R6/2 mouse, huntingtin aggregates form before the onset of neuropathology (Davies et al., 1997) supporting a causative role for aggregates in the resulting pathology. Expression of anti-aggregation peptides in a Drosophila model of H D , and treatment of the R6/2 mouse model of H D with Congo red, an azo-dye which inhibits oligomerization, both decreased inclusions and delayed lethality and neurodegeneration in the models (Kazantsev et al., 2002;Sanchez et al., 2003), indicating inhibition of aggregates could inhibit the disease. However, the appearance of huntingtin inclusions long after the onset of behavioural abnormalities as an indicator of neuronal dysfunction in full-length mouse models of H D argues against a toxic role of aggregates (Hodgson et al., 1999;Menalled et al., 2002;Menalled et al., 2003;Slow et al., 2003) and studies in human patients show little overlap between those regions most affected in the disease and those with the largest inclusion burden (Gutekunst et al., 1999). In a S C A - 1 transgenic mouse model where the self-aggregation domain of the ataxin-1 transgene was deleted, preventing the formation of aggregates, the mice still manifested ataxia and Purkinje cell degeneration, demonstrating aggregation is not necessary for neuronal dysfunction and death in this related polyglutamine disorder (Klement et al., 1998). In addition, breeding of the R6/2 truncated mouse model with tissue transglutaminase knock-out mice partially rescues the brain and body weight loss and delays mortality but is accompanied by increased huntingtin neuronal inclusions (Mastroberardino et al., 2002) further supporting a role for aggregates as a benign byproduct. A recent study demonstrated that huntingtin aggregates sequester a negative regulator of autophagy named m T O R (Ravikumar et al., 2004). This sequestration leads to increased clearance of toxic mutant huntingtin through the autophagic pathway, suggesting aggregates may in fact perform a neuroprotective role in H D through this pathway (Ravikumar et al., 2004). 1.9 Mouse models of Huntington disease 1.9.1 Cell culture and invertebrate organism models of HD The discovery of the gene and underlying mutation responsible for H D in 1993 led to the establishment of a variety of models of the disorder and use of these models to 13 both investigate the underlying mechanisms, and test potential therapeutics for this devastating disease. The simplest of these models involve cell culture systems where huntingtin constructs are stably transfected into a variety of cells. These models include inducible-huntingtin exon-1 PC12 cells (Steffan et al., 2001;Apostol et al., 2003), striatal cells expressing inducible fragments of huntingtin (Sipione et al., 2002), and immortalized striatal neurons from a mouse model with 118 C A G repeats (Trettel et al., 2000). Cel l culture models allow the dissection of basic intracellular pathways involved in the disease pathogenesis, studies of protein localization etc. in a simple, controlled system. Invertebrates including Drosophila melanogaster and Caenorhabditis elegans have also been utilized as model organisms in H D research. DrosophUa models expressing N-terminal fragments of mutant huntingtin in the photoreceptor neurons (Jackson et al., 1998;Steffan et al., 2001;Steffan et al., 2004) display a quantitative neurodegenerative phenotype. Similarly, expression of an N-terminal huntingtin fragment in the touch receptor ( P L M ) neurons (Parker et al., 2001) or in the glutamatergic A S H sensory neurons (Faber et al., 2002) of C. elegans results in neuronal dysfunction and toxicity. These models exhibit degeneration of a subset of neurons in an invertebrate organism. In addition, these models have short life-spans and the advantage of genomic and genetic tools including deletion strains to map out potential modifiers of the resulting phenotypes and give insight into the pathogenesis of H D and potential therapeutic targets. Both cell culture models and invertebrate organism models, although useful for basic research, assay development and in some cases therapeutic trials, are not capable of accurately recapitulating the human disorder, due to the relative simplicity of the systems in which they are set. H D affects a complex organ, the brain, necessitating the use of a model system which more closely replicates the physiology and neuroanatomy of humans. For this reason, considerable efforts have been undertaken to accurately recapitulate H D in mice. A number of mouse models of H D have been established (Table 1.2) since the cloning of the gene in 1993. These models differ in background strain, promoter, protein length, expression level of mutant huntingtin and polyglutamine 14 size as evidenced by Table 1.2. H D mouse models expressing truncated fragments of huntingtin and expressing full-length huntingtin have both been described. 1.9.2 Truncated mouse models Truncated mouse models of H D expressing fragments of huntingtin ranging from 67 amino acids in the R6/2 (Mangiarini et al., 1996;Davies et al., 1997;Carter et al., 1999;Lione et al., 1999;Luesse et al., 2001;Meade et al., 2002) and conditional exon 1 model (Yamamoto et al., 2000) to 1000 amino acids in the N S E model (Laforet et al., 2001) (Table 1.2) have been established. The promoters which drive the expression of the fragments of huntingtin in these mouse models are either not endogenous (NSE model, N171, conditional exon 1 model) or represent only a portion of the huntingtin endogenous promoter (R6/2). Phenotypically, the truncated mouse models of H D tend to demonstrate early onset of both behavioural (rotarod) and neuropathological (brain weight and striatal volume) phenotypes compared to full-length models. Huntingtin aggregates or inclusions develop in each of the H D mouse models, with an earlier onset of inclusion formation demonstrated in the truncated models along with a more widespread tissue distribution of inclusions (Table 1.2). Due to the presence of early behavioural and neuropathological phenotypes in the truncated mouse models and an early death phenotype, the R6/2 model and the N171 (Schilling et al., 1999;Schilling et al., 2001) models have been widely used for therapeutic trials as summarized in Beal and Ferrante in 2004 (Beal and Ferrante, 2004). The R6/2 model has also been studied to determine mechanisms which may be involved in H D pathogenesis including transcriptional changes (Luthi-Carter et al., 2000;Luthi-Carter et al., 2002a;Luthi-Carter et al., 2002b), altered neurotransmitter levels (Cha et al., 1998;Cha et al., 1999), altered dopamine signaling (Levine et al., 1999;van Dellen et al., 2000;Hickey et al., 2002) and electrophysiological changes (Klapstein et al., 2001;Cepeda et al., 2003). 15 Table 1.2: Summary of the design, behavioural and neuropathological findings in HD mouse models (adapted from a table by A.Yamamoto). Behavioural Phenotype Neurological Phenotype Truncated HD mouse Models Bates CBA/B6 1 kb human exon 1 (67aa)/ R6/2 144-190Q, R6/1 115Q not reported Hen CBA/B6 Tet-off exon 1 (67aa)/ 94Q >1X Ross/ Borchelt C3H/B6 prion 171 aa/ 82Q.44Q,18Q 1/1 OX to 1/5X Aronin/ Difiglia SJL/B6 rat NSE 3 kb (1000 aa)/100Q, 46Q, 18Q <1X Full-Length Mouse Models MacDonald 129/CD1 Hdh mouse endo./ 111Q, 92Q, 50Q 1/2-1X Myers FVB/B6 Hdh mouse endo./ 80Q, 72Q 1/2 X • Zeitlin/ Chesselet B6 Hdh mouse endo./ 140Q, 94Q, 71Q 1/2 X Detloff 129/B6 Hdh mouse endo./ 150Q 1/2 X Tagle FVB CMV full-length cDNA/ 89Q, 48Q, 16Q 1-11X Hayden FVB 25 kb human human genomic (in YAC) / 72Q, 46Q, 18Q 1/2- 1X Rotarod Activity 5 weeks (R6/2) hypoactivity (R6/2) clasping, tremors, learning deficit R6/2-10-13 weeks, R6/1 8-12 months 2.5 months hypoactivity late onset tremor/ clasping none observed 3 months hypoactivity clasping 30 weeks 3-6 months both hyper and hypoactivity clasping, irregular gait none observed not reported not measured gait changes none observed none reported not reported early onset aggressive none observed none reported hyperactivity first, hypoactivity gait changes none observed 15-40 weeks Not measured gait changes anxiety none observed 13-16 months hyperactivity , late hypoactivity circling, weight loss 30 weeks Not reported hyperactivity circling none observed 8 weeks/ 8 weeks shrunken neurons, dendrites; TUNEL, toluidine blue 6 weeks/ throughout brain not reported/ 18 weeks not reported 6 months/ throughout brain 4.5 month/ 4.5 month shrunken neurons 3.5 months/ throughout brain not reported/ 14 months none observed 3-11 months/ striatum, cortex none observed toluidine blue staining 10 months/ striatum none observed none observed not reported not reported/ 18-26 months none observed 4-6 months / striatum, olfactory, cerebellum none observed none observed 51 weeks/ striatum not observed TUNEL staining, dendritic spine loss 12 weeks/ striatum, cerebellum not reported toluidine blue staining 12 months striatum 16 The presence of early phenotypes in the R6/2 and N171 truncated H D mouse models allowed their use in a number of therapeutic trials and studies. However, these mouse models do not accurately recapitulate the protein context of the disease, since human H D patients express full-length mutant huntingtin while the R6/2 and N171 models express fragments which account for only 2% and 5%, respectively, of the full length protein. The phenotypes in these models are very similar to phenotypes observed in a transgenic mouse with 146 C A G s 'knocked-in' to the hypoxanthine phosphoribosyltransferase gene (Hprt), a gene which does not normally contain a C A G repeat tract (Ordway et al., 1997;Tallaksen-Greene et al., 2003). The presence of a phenotype in the Hprt C A G knock-in mouse suggests that expanded polyglutamines can cause toxicity and phenotypes unrelated to the specific mechanisms involved in H D . The presence of similar phenotypes in the Hprt C A G knock-in, the R6/2 and the N171 mice indicate that the phenotypes exhibited in the truncated huntingtin models may be due to non-specific polyglutamine toxicity. It is probable therefore that the results from therapeutic trials using these models wi l l reveal compounds which are efficacious against polyglutamine toxicity, and not specifically HD-related pathogenesis. Furthermore, study of truncated mouse models wi l l not reveal the mechanisms specific to H D pathogenesis. Indeed, the different cell specificity of the polyglutamine diseases (Table 1.1) despite the identical underlying mutation demonstrates the importance of factors other than the expanded polyglutamine tract in the specific pathogenesis of these disorders. 1.9.3 Full- length mouse models In recognition of the importance of protein context for the accurate recapitulation of H D , a number of full-length mouse models were established (Table 1.2). The majority of these models were created by the "knock-in" of an expanded C A G tract into the endogenous mouse H D gene. Two transgenic full-length models were established including the C M V - d r i v e n c D N A model in the Tagle lab (Reddy et al., 1998;Reddy et al., 1999a) and the yeast artificial chromosome ( Y A C ) mice in the Hayden lab (Hodgson et al., 1996;Hodgson et al., 1999) which wi l l be discussed further in Section 1.9.4. Four separate knock-in models were established in the MacDonald (Wheeler et al., 2000;Wheeler et al., 2002), Myers (Shelbourne et al., 1999), Zeitlin/Chesselet 17 (Menalled et al., 2002;Menalled et al., 2003) and Detloff (Lin et al., 2001) labs and these models most accurately recapitulate the underlying genetic mutation in H D , by expansion of a C A G tract in the endogenous mouse gene. However, the knock-in models do not manifest the motor deficits or the neuropathological phenotypes of striatal volume and brain weight loss exhibited by human patients. These models have been useful in answering questions about C A G repeat instability (Shelbourne et al., 1999;Wheeler et al., 1999;Kennedy and Shelbourne, 2000;Wheeler et al., 2003), and the importance of huntingtin for neuronal survival (White et al., 1997). The knock-in models may be beneficial for the examination of early phenotypic changes which occur long before neurodegeneration. However, it is impossible to assess the effects of a therapeutic intervention on neuronal degeneration or dysfunction using the knock-in animals, since these models do not manifest behavioural or neurodegenerative phenotypes in the lifetime of the mouse. A possible exception is the newly established Zeitlin/Chesselet 140Q knock-in mice which demonstrates both hypoactivity and striatal volume decreases (Menalled et al., 2002;Menalled et al., 2003). A full-length c D N A mouse model expressed under the C M V promoter was first described in 1998 and exhibited both neuronal dysfunction and an early death phenotype (Reddy et al., 1999a;Reddy et al., 1999b;Guidetti et al., 2001). However, these mice highly overexpress the transgene at up to 1 I X endogenous levels under a non-endogenous, C M V promoter, raising concerns about appropriate protein expression for the accurate recapitulation of the disease. 1.9.4 Y A C mouse model The yeast artificial chromosome ( Y A C ) mouse model expresses the full-length human huntingtin protein with 24 kb of upstream human promoter sequence and 18 (control), 46 or 72 C A G s (mutant). The human huntingtin expressed from these transgenes can rescue the embryonic lethality of the mouse huntingtin knock-out, indicating appropriate developmental regulation of the transgene during mouse development (Hodgson et al., 1996). Western blots revealed identical tissue distribution of transgenic huntingtin protein compared to endogenous mouse protein (Hodgson et al., 1999). Finally, the Y A C mice express transgenic protein at less than endogenous mouse 18 protein levels, removing the concern that any resulting phenotypes were due to a highly overexpressed protein. The presence of the full-length huntingtin protein expressed under 24 kb of endogenous promoter, with the identical tissue specificity and developmental expression as the mouse endogenous protein at less than endogenous mouse levels demonstrate that the Y A C mouse model accurately recapitulates the underlying genetic defect of H D . Examination of the Y A C transgenic model revealed the onset of a hyperactivity phenotype in both a lower (2511) and higher (2498) expressing Y A C 7 2 ( Y A C mouse with 72 C A G repeats) line, impairments in long term potentiation (LTP), and increased resting neuronal C a + 2 levels, indicating neuronal dysfunction in this model. Toluidine blue staining and electron microscopy demonstrated the presence of degenerating neurons in the highest expressing Y A C 7 2 at 12 months of age, and the presence of huntingtin inclusions by E M 4 8 staining. Together, these results reveal a full-length H D mouse model expressed under an appropriate promoter which demonstrates both a neuronal dysfunction and degenerative phenotype. The Y A C mouse model has helped elucidate a number of pathways and mechanisms involved in the pathogenesis of H D as summarized in Table 1.3. These pathways include wild-type huntingtin's ability to upregulate B D N F (Zuccato et al., 2001;Zuccato et al., 2003) and importance for cell survival (Leavitt et al., 2001;Zhang et al., 2003); the presence of mitochondrial dysfunction (Panov et al., 2002), caspase activation (Wellington et al., 2002;Hermel et al., 2004), and increased susceptibility to excitotoxicity (Cepeda et al., 2001;Zeron et al., 2002;Li et al., 2003b) in the disease condition. While the Y A C mice have been beneficial for investigating the underlying mechanisms involved in H D pathogenesis, the relatively late onset of neuronal dysfunction (7 months) and degenerative phenotypes (12 months) in the Y A C 7 2 , the lack of quantitatively measured phenotypes, and the presence of significant inter-animal variability were concerns for attempting to use these mice in therapeutic trials. In an effort to accelerate and establish quantifiable phenotypes, and reduce inter-animal variability, with the knowledge that increasing C A G size both increases age of onset 19 Table 1.3: Results from studies using HD YAC mice to investigate different pathways involved in the pathogenesis of HD. A * denotes a result in the YAC mice that recapitulated data from human patients. Huntingtin is symbolized by htt. Mouse Line Age Publication Results Relevance YAC18(212) YAC72 (2511) 9 months (Zuccato et al., 2001): Science BDNF levels increased in YAC 18, BDNF levels decreased in YAC72 htt effects on BDNF levels may underlie HD pathogenesis YAC 18 (212) YAC72 (2511) 9 months (Zuccato et al., 2003); Nature Genetics NRSE-controlled genes upregulated in YAC18, downregulated in YAC72 htt controls BDNF levels through interaction with REST/NRSF, a repressor of NRSE genes * YAC72 (2511) 12 months (Zala et al., 2004); Exp. Neurol. lentiviral CNTF (trophic factor) reduced number of degenerating neurons in YAC72 trophic factors are potential therapeutics YAC 18 (29) YAC46 (668) YAC72 (2511) > 4 months (Leavitt etal., 2001); Am. J . Hum. Genet. increasing endogenous levels of mouse htt rescues the testicular degeneration of YAC72 on a htt KO background wild-type htt is protective against the toxic effects of the mutant protein YAC18(212) 4-8 months (Zhang et al., 2003); J . Neurochem. overexpression of wild-type htt reduces the injury volume in a stroke model wild-type htt is protective against ischemic injury YAC72 (2511) 12-24 months (Cepeda et al., 2001); J Neurosci Res. increased responsiveness to NMDA in YAC72 with increased C a + 2 concentrations increased susceptibility to excitotoxicity may underlie HD pathogenesis YAC72(2511) primary neurons 6,10 months (Zeron et al., 2002); Neuron increased susceptibility to excitotoxicity of YAC72 striatal neurons in vivo and in culture mutant htt affects susceptibility to excitotoxicity of MSNs from birth YAC46 (668) YAC72 (2511) neuronal cultures (Zeron et al., 2004); Mol. Cell. Neurosci. increased intracellular C a ^ levels and mitochondrial depolarization upon NMDA treatment in YAC72 and YAC46 striatal neurons links increased susceptibility to excitotoxicity with apoptosis through mitochondrial depolarization, increased C a + 2 YAC 18 (29) YAC72 (2511) YAC72 (44) 4 months (Panov et al., 2002); Nat. Neurosci. mitochondria from YAC72 depolarize at a lower C a + 2 level and have lower C a + 2 retention than mitochondria from YAC18 implicates mitochondrial dysfunction in HD pathogenesis * YAC72 (44) 2, 4, 6, 8 months (Wellington et al., 2002); J . Neuroscience caspase cleavage of htt occurs before the onset of neurodegeneration in YAC72 caspase cleavage of htt is early event in HD * YAC72(2511) 12 months (Hermel et al., 2004); Cell Death Differ. caspase 2, 6 and 7 are activated in YAC72, but not caspase 3, 8, 9 specificity of caspase activation in HD * YAC72 (2511) 14 months (Ariano et al., 2002); J . Neurosci Res. D1, D2 and D3 dopamine receptor decrease in YAC72, increase in D5 striatal dopamine pathway is disrupted and may underlie HD pathogenesis YAC72 (2511) 9 months (Peel et al., 2001), Hum Mol Genet double-stranded RNA-dependent protein kinase is activated in YAC72 PKR activation may play a role in HD * 20 (1993;Brinkman et al., 1997) and decreases variability (Langbehn et al., 2004), several Y A C mouse lines with 128 C A G repeats were created. 1.10 Objectives The objective of the first part of this thesis was to characterize the Y A C 128 mouse model. We hypothesized that the Y A C 128 would demonstrate an earlier onset and less variability in H D related phenotypes compared to the Y A C 7 2 . Our objectives were to determine 1) how well it recapitulated the human disorder; 2) quantitative phenotypes that could be used in therapeutic trials; 3) the variability of these phenotypes to determine the number of mice necessary for therapeutic trials; 4) the natural history of phenotypic changes in the model. During the establishment of additional full-length Y A C mice with 128 C A G repeats, a mouse expressing a truncated N-terminal fragment of huntingtin, termed shortstop, was serendipitously generated. We hypothesized based on in vitro results with huntingtin fragments (Hackam et al., 1998;Martindale et al., 1998) that a mouse expressing a short fragment of huntingtin should present with a more severe phenotype than a mouse expressing full-length huntingtin (YAC128) . The shortstop mouse expresses a truncated huntingtin fragment from the identical promoter and upstream 24 kb of regulatory region as the full-length Y A C 1 2 8 mouse, allowing for the first time the examination of the toxic fragment hypothesis in vivo and the comparison of a truncated construct with a full-length construct in vivo. The first objective of this study was to 1) characterize the shortstop truncated transgene and determine its integration site in the mouse genome. The shortstop and full-length Y A C 128 were than examined in parallel to determine 1) huntingtin inclusion formation in a short fragment (shortstop) versus a full-length ( Y A C 128) huntingtin mouse and 2) the effect of expression of a truncated huntingtin fragment (shortstop) on the neuronal degeneration and dysfunction phenotypes discovered in the full-length Y A C 128 mouse. Q T L mapping using mouse models can be used to reveal genetic modifiers of age of onset for the purpose of discovering pathways that are involved in H D pathogenesis and potentially targeting those pathways for therapeutics. This method requires the transgene to be congenic on at least two different background strains of mice, one 21 susceptible and one resistant to the effects of the transgene. The Y A C 1 2 8 mice ( F V B / N background strain) were backcrossed onto the C57B1/6 (B6) strain with the hypothesis that the B6 strain would be resistant to the effects of the Y A C . The objectives of this study were 1) to determine i f the Y A C 128 phenotypes were robust and penetrant on multiple strains; 2) to investigate other phenotypes which may recapitulate the human disease; 3) determine i f the B6 strain is resistant to the effects of the Y A C transgene. 22 Chapter 2: Materials and Methods 2.1 YAC mutagenesis and generation of mice Y A C mutagenesis was performed as described previously (Hodgson et al., 1999) using a construct containing 128 C A G repeats. Y A C D N A was prepared for microinjection into F V B / N pronuclei, and founder pups were screened (Hodgson et al., 1996). Mice were maintained on the F V B / N (Charles River, Wilmington, M A ) background strain and are congenic on this strain. Mice for the B6 study (Chapter 5) were generated through backcrossing of the Y A C 128-line 53 for five generations (N5) onto the C57B1/6 (B6) strain (The Jackson Laboratory, Bar Harbor, M A ) . Mice were genotyped by a P C R procedure described previously (Hodgson et al., 1996). M i c e were housed, tested and tissues were harvested according to the University of British Columbia animal protocol A00-0254. 2.2 Copy number, RNA and protein analysis Southern blotting to assess copy number was performed as described previously using 12 \xg D N A (Hodgson et al., 1996) and the human specific probe cD70-2 (Hodgson et a l , 1999). 2.2.1 Protein analysis Protein lysates were prepared from whole mouse brain or dissected cortex, striatum, hippocampus and cerebellum in a procedure described previously (Hodgson et al., 1996) with a caspase inhibitor, 10 p M Z V A D (Calbiochem., San Diego, C A ) added to the lysis buffer. Protein concentration was determined by BioRad D C Protein Assay (BioRad, Hercules, C A ) . These lysates were separated on 7.5% polyacrylamide gels and blotted on P V D F membranes. Blots were probed with anti-actin (Chemicon, Temecula, C A ) , 1C2 (Chemicon, Temecula, C A ) and monoclonal antibody FED650. Quantification was done using BioRad Quantity One imaging software (BioRad). HD650 was produced against H D peptides 650-663 ( V L R D E A T E P G D Q E N ) and reacts specifically to human huntingtin. The H D peptides ( V L R D E A T E P G D N Q E N ) coupled to K L H carrier protein was used as an immunogen to inject Balb/C mice. The mouse was injected with 100 jug of the pept ide-KLH protein in Freund's complete adjuvant subcutaneously, and then followed by two additional injections of 100 ng of the 24 peptides-KLH protein in Freund's incomplete adjuvant at 14 day intervals. Three days before cell fusion, the mouse received an intravenous injection of 100 peg of the peptides via the tail vein. Splenocytes were fused with NS-1 myeloma cell, and hybridomas were selected and cloned in a procedure described previously (Davis et al., 1982). 2.2.2 RNA analysis Total R N A was extracted from mouse cortex with RNeasy Protect M i n i K i t (Qiagen, Mississauga, ON) . First-strand c D N A was prepared from ljug of total R N A using Superscript First-Strand Synthesis System for R T - P C R (Invitrogen, Burlington, O N ) in a final volume of 20 [il. A fraction (1/100) of the R T reactions was used as template in real-time P C R reactions. Real-time P C R was performed using the A B I GeneAmp 5700 Sequence Detection System instrument and S Y B R Green Two-step R T -P C R (Applied Biosystems, Foster City, C A ) using intron-spanning human specific primers. A dissociation curve confirmed the absence of nonspecific amplification. Serially diluted c D N A samples were used for standard curve calibration. A l l samples were run in quadruplicate. Expression levels were normalized to beta-actin m R N A levels. Primary data analysis was performed using system software from Applied Biosystems. 2.3 Morphological analysis Mice were terminally anesthetized by intraperitoneal injection of 2.5% avertin and perfused with 3% paraformaldehyde/0.15% glutaraldehyde in P B S . The brains were left in the skulls for 24 hours in 3% paraformaldehyde at 4°C, then removed and stored in P B S . Brains which did not perfuse well (softer than others) were removed from the groups at this point. Coronal sections of 25 pm and 50 pm thickness were cut throughout the striatum using a vibratome. Transgenic and wi ld type mice were matched based on age and sex (i.e. equal number of males and females in each group) and littermates were used whenever possible. 2.4 Quantitative analysis A l l quantitative analyses were performed blind with respect to genotype. Coronal sections (25 pm) spaced 200 pm apart throughout the striatum were stained with NeuN (Chemicon) antibody at 1:100 dilution or D A R P P - 3 2 antibody (Chemicon) at 1:500 25 dilution. Biotinylated secondary antibodies (Vector, Burlington, ON) , mouse or rabbit at 1:200 were used prior to signal amplification with an A B C Elite kit (Vector) and detection with diaminobenzidine ( D A B , Pierce). The perimeter of the striatum was traced in each of the serial sections using a 2 .5X objective and Stereoinvestigator software (Microbrightfield, Williston, Vermont). Subsequently, counts of neuronal profiles within 50 /xm X 50 /jm counting frames spaced evenly throughout the striatum (striatal grid size was 450 jum X 450 /im) was obtained using a 20X objective. Serial reconstruction of the striatum by the Stereoinvestigator software allowed estimation of total neuronal profiles and volume. Cortical volume was estimated in the region with the largest percentage of striatal tissue (centered on the landmarks of the corpus callosum and the anterior commissure crossing) totaling 6 serial sections. A l l layers and regions of cortex present in the section were outlined as a whole and volume was estimated using Neuroexplorer software (Microbrightfield, Williston, Vermont). Cross-sectional area of striatal neuronal profiles was determined by outlining the perimeter of all clearly defined neurons within 50 \im X 50 jU,m counting frames spaced evenly throughout the striatum (450 jiim X 450 pm grids). Neuronal profiles were outlined using a 100X objective in anatomically matched coronal sections. Note: Tissue from wild-type and Y A C 1 2 8 at the same time point (e.g. 9 months) were treated identically; however, there was some experimental variability between time points (e.g. variability between tissue from 6 and 9 month time points), making volume comparisons between time points invalid. 2.5 Assessment of aggregates 2.5.1 Light microscopy Twenty-five pm brain sections throughout the striatum were stained for the presence of aggregates. Sections were immuno-stained as described previously (Gutekunst et al., 1999) using polyclonal E M 4 8 antibody at 1:1000 and D A B as the chromogen (Pierce). 2.5.2 Electron microscopy For ultrastructural analysis, we used pre-embedding immunogold labeling of E M 4 8 . Sections were rinsed in P B S , processed according to manufacturer's instructions 26 using E M 4 8 antibodies at 1:500. Ultrasmall colloidal gold conjugated secondary antibody (Aurion, Wageningen, Netherlands) was used to bind the primary antibody. Following a post-fixation with 2.5% glutaraldehyde, gold particles in sections were intensified using R-gent S E - E M silver enhancement kit (Aurion, Wageningen, Netherlands). Sections were further fixed with 0.5% osmium tetroxide in 0 . 1 M P B for 15 minutes and processed for electron microscopy as described elsewhere (Gutekunst et al., 1998). Selected sections were then placed in 0.5% osmium tetroxide in 0 .1M phosphate buffer for 30 min. Sections were rinsed in P B , dehydrated in 25-100% ethanol followed by propylene oxide, infiltrated and flat embedded in Epon between sheets of Aclar and cured at 60°C for 2-3 days. 2.5.3 Aggregation foci Brains perfused in the method described in Section 2.3 with 4% paraformaldehyde were embedded and sectioned by Neuroscience Associates using their proprietary Mult iBrain™ embedding method. In short summary, sections were freeze cut from gelatin embedded blocks at 40 pm. The protocol for polyglutamine recruitment was adapted from a published immunohistochemical method (Berghorn et al., 1994) except the peroxidase was detected using nickel-enhanced diaminobenzidine (Akiyama et al., 1999). Free-floating sections were incubated for 18 hours at room temperature in biotinylated polyglutamine peptide, bQ28 (100-250 nM) or bPEGQ30 (1-25 nM), diluted in P B S T x . Biot in was detected using the 'Eli te ' A B C kit (Vector Laboratories, Burlingame C A ) and D A B (Pierce) as the chromogen. 2.6 Behavioural assessment M i c e were singly housed in microisolator cages under reverse lighting (lights off at 11:00 am, lights on at 11:00 pm) with the exception of the accelerating rotarod cohort in Chapter 4 and the fixed speed rotarod cohort in Chapter 3 as noted in Section 2.6.1. A l l mice in a testing group were cage changed on the same day and no testing was performed until 2 days after a cage change. M i c e were semi-randomly number coded (first and last half of testing group had an equal number of males/females, transgenic/wild-type). A l l behavioural testing was executed during the mouse night 27 cycle, when the mice are normally active, with testing carried out in a behavioural testing suite under red light. The same observer carried out all of the tests and was blind to the genotype of the individual mice throughout the course of the testing. 2.6.1 Rotarod analysis. Accelerating Chapter 3, 5: We used an accelerating rotarod protocol where the rotarod (San Diego Instruments, San Diego, C A ) accelerated from 0 to 45 rpm over a period of 120 seconds. Mice were trained for 3 days with 2 trials per day on an accelerating rotarod. Following this training, the mice were tested for 3 consecutive trials in one day, with 1.5 hours rest between trials. The rotarod was wiped clean with ethanol between each test subject. Chapter 4: Mice were singly housed in microisolator cages. A l l behavioural testing was executed in the light in a behavioral testing suite. Mice were tested on a rotarod (Ugo-Basile, Norfolk, England) which accelerated from 0-40 rpm over 300s. M i c e were initially trained on the accelerating rotarod for 3 days with 3 trials per day. Subsequent testing occurred over 1 day with 1.5 hour rest in between tests. Mice were tested at 12 months of age and were naive (i.e. had never been tested before). Fixed speed Chapter 3: Mice were housed in microisolator cages with siblings. A l l behavioural testing was executed in the light in a behavioural testing suite. Mice were tested on a fixed speed rotarod (Ugo-Basile, Norfolk, England) at 12, 24, 34 and 40 rpm and were initially trained at 24 rpm for 3 days with 3 trials per day. Subsequent testing occurred over a 3 day period, testing each speed once a day with 1.5 hour rest in between tests. Mice were tested every month from 3 to 12 months of age. 2.6.2 Open-field analysis. Mice were assessed using an open-field activity monitor (Med Associates Inc., St. Albans, V T ) for a period of 10 minutes. Testing began at least one hour after the beginning of the mouse night cycle. The testing chamber was wiped clean with ethanol between each test subject. Ambulatory count is defined as the number of beam breaks while the mouse is ambulating, while ambulatory episodes are the number of times the 28 mouse begins ambulating (from a resting position). Measurements were calculated by accompanying software (Med Associates). M i c e who circled for the entirety of the 10 minute interval were removed from the analysis (n=6 wi ld type, n=2 Y A C 1 2 8 , Chapter 3). 2.6.3 Locomotor activity Locomotor activity was measured for 24 hours using a Cage Rack System (San Diego Instruments) with a uniformly spaced 8 x 4 photobeam grid. The cages were 28 x 17 x 12 cm and the mice provided with food and water. Locomotor activity was calculated from the total number of beam breaks over 24 hour testing period. 2.6.4 Pre-pulse inhibition and habituation Acoustic startle, PPI and habituation were measured using the SR Labs startle response chamber (San Diego Instruments). The chosen paradigm was adapted from Carter et al. (Carter et al., 1999) and assessed startle amplitude, PPI and habituation using an acoustic stimulus of 120 dB, a single prepulse interval (100 msec) and 4 different prepulse intensities [2, 4, 8 and 16 dB above background noise (70dB)]. A mouse was placed in the startle chamber and allowed to acclimatize for 5 minutes to background noise alone. The mouse was then presented with 66 startle trials, each trial consisting of one of three conditions 1) a 30 msec, 120 dB stimulus presented alone; 2) a 30 msec, 120 dB stimulus preceded 100 msec by prepulses (30msec) that were 2, 4, 8 and 16 dB above background noise; 3) no stimulus which was used to measure baseline movement in the chamber. These 6 trial types were each repeated 11 times in a pseudorandom order such that each trial type was presented once within a block of 11 trials. Analysis for the PPI and startle magnitude was based on the mean of the 11 trials, although measurements which were 2 standard deviations from the mean were removed from the analysis. Habituation was measured by comparing the first and eleventh response to startle alone. 2.7 Statistics A l l statistics were carried out using an unpaired Student's T-test, two-way A N O V A with repeated measures or one-way A N O V A with Tukey's multiple comparison post-test. P-values, S E M , means and standard deviations were calculated using Graphpad 29 Prism version 3.0 or Microsoft® Excel 2002. SPSS 11.5 was used to calculate F and P values for behavioural measures. Correlation calculations with R and p-values were calculated by Pearson correlation using Graphpad Prism version 3.0. Power analysis numbers were calculated using the website http://www.health.ucalgary.ca/~rollin/stats/ssize/n2.html 2.8 PCR Characterization of YAC Shortstop Truncation Sites Primers were designed using Primer 3 (Rozen and Slaletsky, 1998) and synthesized by Invitrogen. Primer sequences are as follows: 9229R: 5' T T G T A G T T C T G T C A G G C T T T G C T T C 3'; 9234R: 5' T C T A C T T G T A G T T C T G T C A G G C T T T G C 3'; 927IF: 5' C G C T C T C A A T G A T T T G T A G A A C A C G 3'; 9343F: 5' T G C A A C C T C A T T G G C A T T T A C A G 3'; 9617R: 5' T T C C C T T T T G A C T T C A C T T C T G A C C 3'; 9680R: 5' C A G C A C C C C A C A A G T T T A G A A A T C 3'; M476R: 5' T T C A T C T T T G C T G G A A A C A G T G C 3'; S P L J E : 5' C G A A T C G T A A C C G T T C G T A C G A G A A 3 ' ; SPL_I : 5' T C G T A C G A G A A T C G C T G T C C T C T C C 3'; R Y A _ E : 5' G T C C G G C G T A G A G G A T C A A T T C 3'; R Y A _ I : 5' T G C A A G T C T G G G A A G T G A A T G G 3 ' . P C R s were performed used 1U Taq Polymerase (Invitrogen, Burlington, ON) or Advantage2 GC-genomic Polymerase (Clontech, Mississauga, ON) . P C R reactions and products of digestion of genomic D N A were purified using a Qiaquick P C R purification kit ( Q I A G E N Sciences, Mississauga, ON) . Ligations were performed using 10U of T4 D N A ligase (Roche, Grenzacherstrasse, Switzerland) at 4°C overnight. D N A sequencing was performed on an A B I 3100 using B i g Dye dye terminators (Applied Biosystems, Framingham, M A ) . For inverse P C R , 1 pg of shortstop genomic D N A was digested with Msp I or A l u I (New England Biolabs, Beverly, M A ) and purified. The digested D N A was then re-ligated in a dilute reaction (16 pi purified digested D N A in a total volume of 80 pi) to favour circularization of molecules. For the first round of P C R , primers 9234R and 927 IF were used to amplify 1 pi of the ligation reaction using Taq Polymerase. For the nested round of P C R , 2 pi of the primary reaction product was used for amplification with primers 9229R and 9343F. After agarose gel electrophoresis of the products, a « 30 750bp band from the Msp I digested D N A (750bp and 300bp) was purified and sequenced with primer 9229R, and identified as correctly spanning the junction between H D intron 2 and mouse genomic D N A . For splinkerette P C R (Devon et al., 1995), a variation of inverse P C R , approximately 5 pg of genomic D N A from a Y A C transgenic mouse were digested with A f l I, B a m H I, B g l II, Be l I, Spe I or X b a I (New England Biolabs), purified and ligated to a duplexed splinkerette with compatible sticky ends using a molar ratio of 1:4 genomic D N A to splinkerette. The ligation reactions were pooled and l p l of the pool was P C R amplified with primers Sp l_E and R Y A _ E using Advantage2 GC-genomic Polymerase. A second round of P C R was performed, amplifying l p l of the primary P C R reaction with primers Spl_I and R Y A _ I . After agarose gel electrophoresis, four bands (size range 200-450 bp) were purified and sequenced, all of which were identified as correctly spanning the junction between the right Y A C arm and human genomic D N A upstream of the H D gene. 2.9 Bioinformatics on Shortstop The genomic location and gene context of derived D N A sequences were identified using the human and mouse Ensembl Genome Browsers (http://www:ensembl .org) and by using blastn with default parameters at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST). Poly(A) addition signals were predicted by searching for " A A T A A A " motifs using the program P A T M A T D B within the E M B O S S suite of bioinformatics programs (http://bioinfo.pbi.nrc.ca:8090/EMBOSS/). A search for functional elements in the shortstop 3 ' U T R was performed by searching the P R O S I T E database of protein families and domains (Sigrist et al., 2002) with the H D intron 2 sequence, and by comparing the mouse and human H D intron 2 sequences with a dot-plot using PipMaker (Schwartz et al., 2000). 2.10 CAG Repeat Sizing P C R of the C A G repeat was performed as previously described (Hodgson et al., 1996) using a 6 - F A M labeled 5' primer. The products were cleaned using a QIAquick Gel Extraction K i t ( Q I A G E N Sciences, Mississauga, ON) and then applied to an A B I 31 3100 Genetic Analyzer (Amersham Biosciences, San Francisco, C A ) . The products were visualized using GeneScan Analysis 3.7.1 software (Applied Biosystems, Framingham, M A ) . 32 Chapter 3: Selective Striatal Neuronal Loss in a YAC 128 Mouse Model of Huntington Disease The work in this chapter has been published as: 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 , Hayden M R . Selective striatal neuronal loss in a Y A C 128 mouse model of Huntington disease. Hum M o l Genet. 2003 Jul l;12(13):1555-67. and presented as: Slow E, van Raamsdonk J, Rogers D , Bissada N , Oh R, Simpson E , Leavitt B , Hayden M R (2002) Huntingtin Inclusions Occur Following the Onset of Brain and Behaviour Changes in the Y A C 128 Mouse Model of H D . (August 8-11, 2002: Cambridge) Hereditary Disease Foundation General Meeting. Poster and Oral presentation Slow E, van 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 1 2 8 Huntington disease mouse model. (Apri l 26-28, 2002: Chicago) Huntington Disease Society of America- Coalition for the Cure. Oral presentation. 33 Preface I designed all of the experiments and analyses presented within the chapter and performed all with the exception of that noted below. Jeremy Van Raamsdonk helped with the striatal volume measurements and performed the striatal neuronal count and striatal neuronal surface area measurements. Rona Graham provided assistance with the Southern blot and performed the Western blot. Y u Deng performed the quantitative R T -P C R . Rosemary Oh performed the Y A C mutagenesis and identified the initial founders. Nagat Bissada assisted with the colony management and performed the mouse perfusions. Sazzad Hossain performed the twenty-four hour home cage activity measurement. Y u Zhou Yang created the F£D650 antibody and Dr. X . J . L i kindly provided the E M 4 8 antibody. Sarah Coleman and Dr. Claire-Anne Gutekunst performed the electron microscopy work. Dr. Elizabeth Simpson provided advice on behavioural analysis while Dr. Blair Leavitt provided advice on neuropathological analysis. 34 3.1 Introduction The discovery of the gene responsible for Huntington disease (HD) in 1993 facilitated the development of several genetic mouse models of this autosomal dominant, neurodegenerative disease (1993)(Table 1.2). Transgenic mouse models that expressed (in most cases) a truncated, N-terminal fragment of huntingtin under the control of a variety of promoters were the first models described (Mangiarini et al., 1996;Schilling et al., 1999;Laforet et al., 2001). The replication of the underlying genetic defect, a C A G expansion, by inserting an expanded repeat into the murine huntingtin gene, led to the creation of "knock-in" models (Shelbourne et al., 1999;Wheeler et al., 2000;Lin et al., 2001;Menalled et al., 2002). Although the knock-in models accurately replicate the underlying genetic defect of H D , they do not present with robust motor deficits or demonstrate the brain atrophy and neuronal loss that characterize the human disease (Shelbourne et al., 1999;Wheeler et al., 2000;Lin et al., 2001;Menalled et al., 2002). In contrast, the truncated, N-terminal mice exhibit brain atrophy, which is not specific to those regions affected in H D , and rapid onset of motor deficits (Mangiarini et al., 1996;Schilling et al., 1999). While a rapid onset phenotype is beneficial for study, these truncated mice lack the full-length huntingtin protein and therefore imperfectly replicate the protein context of the human condition. We previously created a yeast artificial chromosome ( Y A C ) mouse model of H D (Hodgson et al., 1996;Hodgson et al., 1999). Our goal was to establish a mouse model that expressed a full-length form of huntingtin under the control of the endogenous huntingtin promoter and regulatory elements. The Y A C fulfilled both of these requirements, spanning the entire genomic region of the human H D gene, including promoter, intronic, upstream and downstream regulatory elements. The full-length, human huntingtin protein is expressed in a developmental and tissue-specific manner identical to the endogenous mouse protein (Hodgson et al., 1996;Hodgson et al., 1999). We originally created Y A C mice with 46 and 72 C A G repeats (mutant huntingtin mice) and Y A C mice with 18 repeats in the H D gene (control mice) (Hodgson et al., 1999). These mice have helped elucidate different pathways involved in the pathogenesis 35 of H D including increased susceptibility to excitotoxic cell death of neurons with mutant huntingtin (Cepeda et al., 2001;Zeron et al., 2001;Zeron et al., 2002), the discovery of decreased B D N F production in H D (Zuccato et al., 2001;Zuccato et al., 2003), the presence of mitochondrial dysfunction (Panov et al., 2002), and the anti-apoptotic role of wild-type huntingtin (Leavitt et al., 2001). However, the hyperactivity and neuronal degeneration in the Y A C 7 2 mice manifested late in the lifetime of the mouse (7 and 12 months, respectively) and the initial assessment of the HD-related phenotypes in these mice used predominantly qualitative measures (Hodgson et al., 1999). In addition, the presence of significant inter-animal variability in HD-related changes in the mice was a concern for assessing interventions which altered the natural history of the illness. Assessment of therapeutic interventions using Y A C 7 2 mice would require the use of large numbers of animals to determine a significant effect of an intervention at a significant cost. In an effort to create a Y A C mouse with both an accelerated and quantifiable phenotype, and with the knowledge that increasing C A G repeat length leads to an earlier age of onset (Brinkman et al., 1997;Langbehn et al., 2004) and decreases variability (Langbehn et al., 2004) we created several Y A C mouse lines with 128 C A G repeats ( Y A C 128), and rigorously characterized one of these lines. Our analysis of the Y A C 1 2 8 mice reveals a hyperkinetic phenotype first manifest at 3 months of age, followed by a progressive motor deficit on the rotarod present at 6 months with eventual progression to hypokinesis by 12 months of age. These behavioural changes are followed by striatal atrophy clearly evident by 9 months of age, cortical atrophy at 12 months and a progressive loss of striatal neurons accompanied by a decrease in striatal cell surface area. The rotarod deficit in the Y A C 1 2 8 mice is highly correlated with neuronal loss. The accurate recapitulation of the human condition in the Y A C 128 mouse model, coupled with low inter-animal variability, results in an H D mouse model that is now highly suited for the assessment of different interventions on the disease phenotype. 36 3.2 Results 3.2.1 Establishment of YAC128 mice A well-characterized Y A C (353G6) spanning the entire H D gene including the promoter region was used to create the Y A C 128 mice on an F V B background strain (Hodgson et al., 1999). Homologous recombination was used to incorporate 128 C A G repeats obtained from P C R amplified, juvenile-onset H D patient D N A into the Y A C in a previously described strategy (Duff et al., 1994). A l l founders were extensively screened by P C R and Southern blot as previously described (Hodgson et al., 1996). Two founders integrated the complete Y A C and were used to establish 2 lines of Y A C mice (lines 53 and 55) with 128 C A G repeats (Fig 3.1 A ) . Southern blots from these mice revealed that line 53 integrated more copies of the transgene than line 55 or line 2511, a previously described Y A C 7 2 mouse (Hodgson et al., 1999) (Fig. 3. IB) . R N A analysis of the human (18/35 CAG) Figure 3.1: Characterization of YAC128 transgenic mouse: DNA. A, Agarose gel showing CAG repeat expansion. PCR using human specific primers amplified the expanded CAG repeat from genomic DNA. The 18, 46 and 72 repeats were amplified from previously described YAC mice (Hodgson et al., 1996;Hodgson et al., 1999), while the 128 was amplified from a YAC128 mouse (line 53). The control (+ve) is from a yeast transfer vector with 128 CAGs. Wild-type (WT) mouse is a negative control and human DNA (18 and 35 CAGs) is another positive control. The marker is a 100 bp ladder. B. Southern blot for assessment of copy number. Genomic DNA digested with EcoRl was probed with a human specific probe (CD70-2). Mouse DNA is the negative control, unaffected human is the positive control, previously described YAC72 (line 2511) and 2 lines of YAC128 (53, 55) were assessed for copy number. huntingtin transcript demonstrated that line 53 had twice the R N A levels of line 55 and three times the R N A levels of line 2511 (Fig.3.2B). Protein expression detected with HD650 (Fig. 3.2A), a human huntingtin specific antibody, correlated exactly with R N A levels (R 2=1.0, Pearson correlation) (Fig 3.2C). Line 53 had the highest level of 37 1.25 Figure 3.2: Characterization of YAC128 transgenic mouse: RNA and protein. A. Western blot showing protein expression of YAC128. WT, lines 2511, 53, 55 and human control were probed with the human specific huntingtin antibody HD650. Blots were stripped and reprobed with anti-actin antibody to demonstrate equal loading. B. Real-time quantitative RT-PCR showing human huntingtin mRNA expression levels of YAC128. RNA was isolated from mouse frontal cortex. All PCR values are expressed as relative mRNA levels normalized to beta-actin mRNA levels. Error bars represent standard deviation from mean of n=3 animals per group. C. Comparison of relative levels of human RNA and protein expression. Protein levels were calculated with NIH image densitometry software and normalized to actin. Pearson correlation revealed a R2=1.0. huntingtin protein expression by densitometric analysis at approximately 75% of endogenous levels (data not shown). We further characterized line 53 due to the higher level of huntingtin protein expression. 3.2.2 B r a i n weight decrease i n YAC128, line 53 Cohorts of line 53 mice (hereafter referred to as Y A C 128) and wild-type littermates at 6, 9 and 12 months were sacrificed and perfused and brain weight was measured. No difference in brain weight was detected in 6-month old Y A C 128s (WT= 0.421 ± 0.016 g, Y A C 1 2 8 = 0.433 ± 0.02 g, n=5) (Fig. 3.3A). However, by 9 months of age Y A C 1 2 8 mice demonstrated a 5% decrease in mean brain weight compared to wi ld-type littermates (WT= 0.414 ± 0.016 g, Y A C 1 2 8 = 0.393 ± 0.01 g, p<0.05, n=7), progressing to a 10% decrease in one year old Y A C 1 2 8 s (WT= 0.411 ± 0.009 g, Y A C 1 2 8 = 0.373 ± 0.022 g, p<0.01, n=7) (Fig. 3.3A). A N O V A analysis further revealed the effect of genotype (F!, 3 6=6.169, p=0.018), age (F 2 , 3 6 =l 1-706, p=0.0001) and the interaction of age and genotype (F2,36=5.002, p=0.012) on brain weight. The difference in brain weight did not appear to be due to generalized atrophy, but rather a region specific effect. For example, no significant difference was detected between Y A C 1 2 8 s and wild-type littermates in the weight of the cerebellum, a region not usually involved in H D pathology (Harper et al., 2002), at any of the ages examined (Fig. 3.3B). 38 Figure 3.3: Brain weight is selectively decreased in YAC128 mice. A. Perfused brain weight (including cerebellum) was compared between YAC128, line 53 and wild-type littermates at 6 (n=5), 9 (n=7) and 12 (n=7) months of age. Brain weight in 9 month YAC128s was significantly decreased (p<0.05, *) and in 12 month old YAC128s (p<0.01, **) by Student's T-test. Mean brain weight ± standard deviation of the groups is shown. B. Cerebellum weight is not decreased in YAC128s. The cerebella were removed and weight was measured. There was no significant difference at any time point. 3.2.3 Striatal and cortical volume is decreased in YAC128 mice To determine whether the brain regions that are most affected in FID patients, the striatum and cortex (Hayden, 1981;Harper et al., 2002), were also affected in Y A C 1 2 8 mice, striatal and cortical volume estimates of mice were calculated using stereological software. There was no detectable difference in striatal volume at 6 months of age in Y A C 128s compared to wild-type littermates (WT= 14.3 ± 2.39 mm 3 , Y A C 128= 14.29 ± 1.25 mm 3 , n=5) (Fig. 3.4A). However, by 9 months of age, a 15% decrease in striatal volume was evident in Y A C 1 2 8 mice (WT= 13.76 ± 0.5 mm 3 , Y A C 1 2 8 = 11.81 ± 0.36 mm 3 , p<0.001, n=7) (Fig. 3.4A), which was also seen in twelve-month old Y A C 1 2 8 mice (WT=11.27 ± 0 . 6 8 mm 3 , Y A C 1 2 8 = 9.88 ± 0 . 3 3 mm 3 , p<0.01, n=7) (Fig. 3.4A). Cortex volume was estimated from the region surrounding the crossing of the corpus callosum to the crossing of the anterior commissure (i.e. region of brain encompassing the greatest volume of striatum). In contrast to the striatum, there was no significant difference in cortical volume at 9 months of age (WT= 14.43 ± 0.79 mm 3 , Y A C 1 2 8 = 13.57 ± 0.41 mm 3 , n=5) but a 7% decrease at 12 months of age (WT= 12.83 ± 0.50 mm 3 , Y A C 1 2 8 = 39 11.88 ± 0.43 mm 3 , p<0.05, n=5) (Fig. 3.4B). Figure 3.4: Striatal and cortical volume are decreased in YAC128 mice. Perfused brains were cut coronally into 25 urn sections throughout the striatum. Every eighth section was immuno-stained with NeuN and Stereoinvestigator software was used to trace and calculate volume. YAC128s showed no difference in striatal volume at 6 months (n=5), a significant decrease at 9 months (n=7, p<0.001, ***) and 12 months of age (n=6, p<0.01, **) by Student's T-test. B. Cortical volume was not significantly different at 9 months of age (n=5) but decreased at 12 months of age (n=5, p<0.05, *). Mean volume ± standard deviation for each group is shown. 3.2.4 Neuronal loss and dysfunction i n Y A C 1 2 8 mice Striatal cell loss is a defining neuropathological characteristic of H D (Vonsattel et al., 1985). Stereological software was used to estimate the striatal neuron number in our mouse cohorts. Sections were immuno-stained with NeuN, an antibody specific for a neuronal nuclear protein, which is present in most neuronal cell types (Mullen et al., 1992). Counting of neurons, along with all other neuropathological and behavioural procedures, was performed completely blind with respect to genotype. A t 9 months of age, Y A C 1 2 8 mice exhibited a decrease of 9% in striatal neuron count, although this trend did not reach significance (p=0.1, n=3) (Table 3.1). To further examine neuronal loss at 9 months, the number of medium spiny neurons (MSNs) , was assessed by immuno-staining for D A R P P - 3 2 , a protein specific to M S N s (Ouimet et al., 1998). M S N s are the neurons that are most affected in H D (Vonsattel et al., 1985) and are the major neuronal cell type of the striatum. Y A C 1 2 8 mice exhibited a decrease of 8% in M S N count, although this trend did not reach significance (p=0.1, n=7) (data not shown). Neuronal loss was progressive and by 12 months of age, a 15% decrease in striatal neuronal count was detected in the Y A C 128 mice compared to control littermates (p<0.01, n=7) (Table 3.1). To ensure the validity of this finding, another group of Y A C 1 2 8 and control mice at 12 months of age underwent analysis. Similar to the 40 previous results, the Y A C 128 mice in this group also exhibited an 18% decrease in striatal neuron count compared to controls (p=0.01, n=5) (Table 3.1). There was no significant difference in neuronal density for the 9 (WT= 135500 ± 1611 neurons/mm3), Y A C 1 2 8 = 140700 ± 3834, p=0.28) and 12 month old (WT= 138900 ± 3927, Y A C 1 2 8 = 142500 ± 2088, p=0.43) mouse cohorts. The lack of change in neuronal density combined with the loss in striatal volume of the Y A C 128 mice provides further evidence for neuronal loss in these mice. Table 3.1: Striatal neuron number in YAC128 mice. Stereological software was used to assess the number of NeuN stained striatal neurons in serial sections of YAC128s vs. controls. YAC128s exhibit a decrease in number of neurons at 12 months of age in two different groups, but not at 9 months of age. Age (months) W T Y A C 128 N % Decrease Significance 9 1507000 ± 68530 1365000 ± 33580 3 9 NS,p=0.16 12 (i) 1724000 ± 50270 1504000 ± 23640 7 15 p=0.004 12 (ii) 1682000 ± 7 1 8 7 0 1375000 ± 65360 5 18 p=0.01 The cross-sectional area of the remaining striatal neurons was measured to determine i f the neurons were decreasing in size, a phenotype reported in other H D mouse models (Levine et al., 1999;Ferrante et al., 2000;Laforet et al., 2001). The mean area of striatal neurons randomly selected by stereological software was decreased by 18% in 12-month old Y A C 1 2 8 s compared to controls (WT= 65.71± 4.5 (im2, YAC128=54.37± 3.7 pm2, p<0.01, n=8 animals) but not in 9 month old Y A C 128s (WT= 57.01± 7.74/xm2, Y A C 1 2 8 = 60.62± 4.76 fim2, n=7 animals) (Fig. 3.5). Figure 3.5: Striatal neuron surface area is decreased in YAC128 mice. Stereoinvestigator software randomly chose 50(xm X 50|xm frames within the striatum of a coronal section immuno-stained with NeuN. The surface area of the neurons within the frames was calculated by the software (>70 neurons per animal). There is no difference in mean striatal neuron surface area in 9 month old YAC 128s (n=7 animals) but a significant decrease in surface area of striatal neurons in 12 month old YAC128s (n=6 animals, p<0.01, **) by Student's T-test. Mean striatal surface ± standard deviation for each group is shown. 41 3.2.5 Behavioural assessment Y A C 128 mice were studied at 3 month intervals from 3 to 12 months of age. Using an accelerating rotarod to examine fall latency, no difference in rotarod performance was detected between Y A C 128 and wild-type mice at 3 months (Fig. 3.6). However, by 6 months of age, a sharp decrease in rotarod performance was evident in Y A C 128 mice (p<0.01) which was further decreased at 9 and 12 months of age (p<0.05, p<0.01, respectively) (Fig. 3.6). A N O V A analysis further revealed the effect of genotype (Fi,28=6.77, p=0.015) and the interaction between genotype and age (F3,84=4.662, p=0.005) indicating a significant difference in rotarod performance between wild-type and Y A C 128s with increasing age. A N O V A also reveals the highly significant effect of age (F3!84=24.427, p<0.001) on rotarod performance for all mice. Figure 3.6: Rotarod deficit in YAC128 mice. YAC 128s (n=16) and wild-type control (n=14) littermates were tested in 3 trials on an accelerating rotarod in 3 month intervals. At 3 months of age there was no difference between the 2 groups. At 6 months of age YAC 128 rotarod performance had decreased (p<0.01, **). This deficit was present at 9 (p<0.05, *) and 12 months of age (p<0.01, **) although at these time points, wild-type mouse performance was also decreasing. ANOVA analysis revealed the significant interaction of genotype and time (F3i 84=4.662, p=0.005). Mean time spent on rotarod for each group is plotted and error bars represent SEM. Mouse activity was measured using an open-field apparatus for a period of 10 minutes at 3, 6 , 9 and 12 months of age. A N O V A analysis showed a highly significant interaction between genotype and age in distance traveled ^3,66=6.808, p<0.001), ambulatory counts (F 3 6 6=5.822, p=0.001), ambulatory episodes (F3,66=6.698, p=0.001), time spent engaged in ambulatory movements (F3 i 66=6.251, p=0.001), and time spent resting (F3,66=2.409, p=0.029), revealing the significant difference in activity levels of wild-type and Y A C 128 mice overtime. At 3 months of age, the Y A C 128 mice exhibit a hyperkinetic phenotype compared to wild-type mice, illustrated by a significant elevation in distance traveled and ambulatory measurements and time spent in ambulatory movements (Fig. 3.7A-D). Interestingly, the Y A C 1 2 8 s begin to manifest a hypokinetic 42 A 4000 -3500 -E 3000 --7- 2500 c 2000 -a 1500 • )5 1000-500 • 0 • Distance Traveled |-*-WT -YAC128 •p<0.05 "p<0.01 Age (months) B 2500 2000 I 1500 o U 1000 - 500 0 Ambulatory Counts j-*-WT -YAC12S V0.05 •*p<0.01 -4 __Age (months) 180 -160 -j 140 \ 8 1 2 0 • •a 100-.2 80 1 & 60 -40 1 20 i Ambulatory Episode's -WT -YAC 128 'p<0.01 A g e (months) Time Resting 20i 0 rWT -YAC128 «p<0.05 Age (months) Age (months) Figure 3.7: YAC128s initially exhibit hyper- followed by hypokinetic behaviour. YAC128 (n=14) and control (n=12) (WT) littermates were tested in an open-field activity box over a period of 10 minutes. Five activity parameters were measured and the mean and standard error for the groups is shown. These measures include distance traveled (A), ambulatory counts (B), ambulatory episodes (C), time spent ambulating (D) and time spent resting (E). Locomotor activity was measured in the home cage environment over 24 hours (n=8 YAC128 and WT) (F) in 12 month old YAC128 and control. The mean measurement for each group is plotted and error bars represent SEM. Significant differences in group means by T-test are represented by * and corresponding P values. phenotype at 6 months compared to wild-type littermates and this hypokinetic phenotype is progressive with age, becoming significant at 12 months of age (Fig. 3.7 A - E ) . To ensure the 10 minute open field test was accurately measuring the activity of the mice, spontaneous locomotor activity was measured over a 24 hour period in the home cage environment. Twelve-month old Y A C 128 exhibited decreased activity compared to wild-type littermates (p<0.01, n=8, F ig . 3.7F), reproducing the results obtained in the 10 minute analyses. Y A C 1 2 8 mice are heavier than their littermates at all ages examined (3, 6, 9 and 12 months). Since these mice overexpress full-length huntingtin, one possibility is the increased weight is due to the effects of full length huntingtin on survival of cells as discussed in Section 1.6.1 (Dragatsis et al., 2000;Leavitt et al., 2001). Y A C 1 8 mice which overexpress full-length huntingtin with 18 C A G repeats are also heavier than wi ld-type littermates, supporting this hypothesis (Leavitt and Hayden, in preparation). 3.2.6 Rotarod performance correlates with severity of neuronal loss Five of the 12 month old Y A C 1 2 8 mice analyzed for neuronal loss (Table 3.1) underwent rotarod assessment. Rotarod performance in this group of Y A C 128 mice was measured using a fixed speed rotarod, a testing paradigm shown to produce results that are highly correlative with results obtained using an accelerating rotarod in another 43 mouse model of H D (Luesse et al., 2001). Y A C 1 2 8 (n=9) mice demonstrated decreased performance compared to wild-type controls at 40 rpm (p<0.05) at 6 months of age, at 34 and 40 rpm (p<0.05, p=0.01) at 9 months and at 34 rpm (p<0.001) at 12 months of age. Twelve month old mice from this study were analyzed for striatal volume, brain weight and striatal neuron count and, as shown in a separate analysis (Fig. 3.3, 4, Table 3.1), Y A C 1 2 8 mice exhibited reduced striatal volume, brain weight and neuronal loss. Five Y A C 128 mice were analyzed behaviourally at 6, 9 and 12 months and also neuropathologically at 12 months. A highly significant correlation is observed between rotarod performance at 12 months of age and striatal neuronal count (p<0.01, R =0.9214) (Fig. 3.8D). A 70 -j Six-month (40 rpm) B 7 0 ~ Nine-month (34 rpin) C 45 -40 • S 3 5 • " 3^0 • g25-3 2 0 = 15 • t£ 10 Nine-month (40 rpm) 60 • § 3 0 --20 • R2=0.87I9 y / p=0.02 . / ' * 60 .-.50-X 4 0 I 30 • a r 20-R2=0.9108 • S p-0.01 y S . R3=0.764l * p-0.05 y S * * t£io • ' |«YAC128| £ 10 - ' | • YAC1281 5 • . | • YAC128| 1.1 1.2 1.3 1.4 1.5 1.6 Striatal Neuronal Count (X106) 1.1 1.2 1.3 1.4 1.5 1.6 Striatal Neuronal Count (XlO6) 0 -1.1 1.2 1.3 1.4 1.5 1.6 Striatal Neuronal Count (XlO6) ID 45 40 3 - 3 5 "^30 8* a 20 ± 15 £io 5 0 Twelve month (34rpm) • YAC128 1 1.1 1.2 1.3 1.4 1.5 Striatal Neuronal Count (X 10s) 1 "5 40 ~ 3 5 " 3 0 g 25 u. 10 5 0 Twelve month (34 rpm) • YAC128 0.4 0.45 Brain Weight (g) 45 40 . 35 • 30 ' 25 20 15 i l i 11 Twelve month (34rpm) R2=0.442 p=0.22 • YAC128 13 15 17 . Striatal Volume (mm') Figure 3.8: Impairment in rotarod activity is correlated with striatal neuronal loss. Rotarod performance at a fixed speed (34rpm or 40rpm) was measured in 6, 9 and 12 month old YAC128 mice (n=5) and correlated with neuropathological phenotypes using Pearson's correlation. Neuronal count at 12 months of age is significantly correlated with rotarod performance in 6-month old Y A C 128 mice at 40rpm (A, R2=0.8719, p=0.02), 9-month old YAC128 mice at 34 (B, R2=0.9108, p=0.01) and 40 rpm (C, R2=0.7641, p=0.05) and 12-month old YAC128 mice at 34 rpm (D, p<0.01, R2=0.9214). Rotarod performance in 12-month old YAC128 mice is weakly correlated with brain weight (E, p<0.05, R2=0.808) and striatal volume (F, p=0.22, R2=0.442) at 12 months of age. Individual YAC128 mice are represented by diamond shapes. M i c e with the greatest degrees of neuronal loss are associated with worsening performance on the rotarod. Clearly additional mice need to be analyzed to increase the power of this finding. Brain weight is also weakly correlated with rotarod performance (p<0.05, R 2=0.808) (Fig. 3.8E) and there is a trend towards a correlation between striatal 44 volume and rotarod performance (p=0.22, R 2=0.442) (Fig. 3.8F). Interestingly, striatal neuronal loss is the most highly correlated of the three neuropathological measurements with rotarod performance. Because the mice are behaviour tested over a period of months and only sacrificed for neuropathological measurements at 12 months, there is no neuropathological data for these mice at 6 or 9 months. However, the correlation between early rotarod performance and 12 month neuropathology can be examined. Interestingly, striatal neuronal count at 12 months of age was significantly correlated with rotarod performance at 6 months of age at 40rpm (R 2=0.8719, p=0.02) (Fig.3.8A) and at 9 months of age at 34 rpm (Fig. 3.8B, R 2=0.9108, p=0.01) and 40 rpm (R 2=0.7641, p=0.05) (Fig 3.8C), indicating that early rotarod performance is predictive of the degree of ensuing neuronal loss. 3.2.7 Presence of aggregates Brain sections from Y A C 128s were immuno-stained with E M 4 8 , an antibody that recognizes N-terminal huntingtin and is highly specific for aggregates (Gutekunst et al., 1999). We observed increased nuclear huntingtin staining in 12 month old Y A C 1 2 8 s using brightfield microscopy but no nuclear huntingtin inclusions (n=5) (Fig. 3.9A). Huntingtin inclusions are defined as aggregates that are visible at the light microscope level. However, by electron microscopy many small clusters of 3 to 6 immunogold particles (micro-aggregates) are visible throughout the nucleoplasm (Fig. 3.9D). E M 4 8 positive inclusions were present in striatal cells of all 18 month old Y A C 128s examined (Fig. 3.9B, C) . B y electron microscopy, these inclusions appeared as large clusters of immunogold particles (Fig. 3.9E, F). Wild-type animals did not exhibit nuclear huntingtin staining or inclusions at either 12 or 18 months (data not shown). 45 Figure 3.9: EM48 immunoreactivity in the striatum of 12 (A and D) and 18 (B, C, E and F) month old YAC128 mice. At 12 months EM48 DAB reaction product is present in many nuclei (A). EM48 immunoreactivity is seen as diffuse staining as well as small EM48 positive puncta throughout the nucleoplasm. By electron microscopy many small clusters of 3 to 6 immunogold particles forming micro-aggregates are visible throughout the nucleoplasm (D, arrows). By 18 months, in addition to the diffuse staining and micro-aggregates, EM48 DAB reaction product is also seen in inclusions (aggregates visible at the light microscope level), visible as dark EM48 positive puncta by light level microscopy (B and C, arrowheads). By electron microscopy inclusions consist of large clusters of immunogold particles (F, arrows). Scale bars: A-C: 10 microns; D-E: 1 micron; F: 270 nm 3.2.8 Low variability in the phenotypes of YAC128 mice Lowering inter-animal variability decreases the number of animals necessary to determine a significant effect due to an experimental therapeutic and is likely to yield less false negatives. As an indicator of variability, we looked at the number of Y A C 128 mice with measurements that had overlapping results with the wild-type control group. The decrease in striatal and cortical volume phenotype and the decrease in striatal neuron size phenotype showed no overlap between individual wild-type and Y A C 128 measurements (Table 3.2). 46 Table 3.2: Overlap and power analysis for quantitative phenotypes in individual YAC128 mice. The number of YAC 128 mice with measurements that overlap the measurements in the wild-type group is represented in fractions. Power analysis determines the number of YAC128 animals necessary to detect a significant (p<0.05) difference in treated versus untreated animals if you predict an 80% chance of discerning a 33%, 50% or 66% rescue of the various quantitative phenotypes. Phenotype Age (months) # overlap 33% rescue Power 50% rescue 66% rescue Striatal volume 9 0 8 4 2 Striatal neuron size 12 0 19 8 5 Brain weight 12 1/3 27 12 7 Striatal neuron count 12 1/8 30 13 7 Cortical volume 12 0 35 15 9 Rotarod 6 1/2 99 43 25 By contrast, individual brain weights showed 33% overlap and the behavioural phenotype demonstrated 50% overlap between wild-type and single Y A C 128 mice. Using the data acquired in the characterization of the Y A C 128s, we performed a power analysis to estimate the number of animals we would need to detect a 33%, 50% or 66% rescue at a significance of p<0.05 with 80% power (Table 3.2). As variability increases, the number of animals necessary to determine a significant effect on that phenotype also rises. A small number of animals are required in a therapeutic trial where the goal is to determine a 50% rescue with the neuropathological phenotypes as endpoints, ranging from 4 animals for striatal volume to 13 animals for striatal neuronal loss. For a less effective rescue of 33%, only 8 animals would be required to determine a difference using striatal volume as an endpoint. 47 3.3 Discussion The Y A C 1 2 8 mouse represents a new animal model for H D , demonstrating motor deficit, a biphasic activity profile and striatal atrophy associated with significant neuronal loss. The HD-related phenotypes exhibited in the Y A C 128 mouse model accurately recapitulate the changes observed in the human disease. Brain weight which is decreased in H D patients, (Harper et al., 2002) and progressive neuronal (Hayden, 1981;Harper et al., 2002) and volumetric loss in the striatum (Rosas et al., 2001) is accurately mirrored in the Y A C 128 mice. A decrease in cortical volume, which occurs after the onset of striatal loss, also replicates the pathology and order of progression of the disease in human patients (Vonsattel et al., 1985). Motor deficits are a hallmark of onset of H D in patients (Hayden, 1981;Harper et al., 2002) and typically present before the onset of neurodegeneration (Vonsattel et al., 1985). This natural history is replicated in the Y A C 128 model, with the onset of a motor deficit on the rotarod occurring before the onset of neuronal loss (Fig. 3.10). Finally, the initial period of increased movement, evidenced by chorea and clumsiness, which generally progresses into a more rigid state as the disease progresses (Hayden, 1981), parallels the biphasic activity profile of the Y A C 128 mice, composed of initial hyperactivity, followed by hypokinesis. Other mouse models of H D have demonstrated varying degrees of striatal atrophy (Mangiarini et al., 1996;Laforet et al., 2001;Menalled et al., 2002) non-specific brain weight loss (Mangiarini et al., 1996) and motor deficit (Carter et al., 1999;Schilling et al., 1999;Laforet et al., 2001) exhibited in human patients. However, there are no reproducible reports of quantitative striatal neuronal loss, a hallmark of the human condition (Harper et al., 2002). Although the R6 truncated mouse models of H D have been reported to exhibit T U N E L staining in the striatum (Keene et al., 2002) and abnormal striatal neurons by toluidine blue staining (Mastroberardino et al., 2002), suggestive of apoptotic processes, quantitative loss of striatal neurons has not been reported. This present study revealed a significant decrease in the number of striatal neurons in the Y A C 1 2 8 mouse model of H D . Qualitative observations of striatal neuronal degeneration, including nuclear shrinking, swelling of mitochondria and other 48 features consistent with apoptosis, has been reported before in the Y A C 7 2 model (Hodgson et a l , 1999). Human Age of onset I Clinical diagnosis motor signs, chorea Inclusions present Mood disturbances GrO (duration years) Time course (age months) Striatal neuronal loss I t I t I hyperactivity Rotarod deficit Striatal volume decrease YAC128 Striatal neuronal loss cortical volume decrease hypoactivity Inclusions present Figure 3.10: Comparison of the natural history of HD in the YAC128 mouse model and in human patients. The time course of phenotypic changes is similar in both the YAC 128 mouse model (in months) and human patients (in years) with the onset of behavioural abnormalities before the loss of striatal volume and neuronal loss. Inclusions are present late in the YAC128 mouse model and in late stage human patient brain, but more analysis is required to determine when inclusions first manifest in human patients. A major question raised by this study is why is neuronal loss present in the Y A C mice, but less evident in other models of H D ? Neuronal loss is not specifically accounted for by the appropriate developmental and cell specific regulation o f full-length huntingtin in the Y A C model, as the mice with targeted insertion of expanded polyglutamine tracts into the endogenous mouse gene have similar regulation of the H D gene, but do not show the same degree o f neuronal loss (Shelbourne et al., 1999;Lin et al., 2001;Menalled et a l , 2002). The difference in striatal neuronal loss between the Y A C mice and other knock-in animals also raises the possibility that human huntingtin with an expanded C A G tract is more toxic than the mouse gene with C A G expansion. The unique finding of neuronal loss in the Y A C mouse models compared to other H D mouse models may also be due (in part) to the differences in strains o f the various models. A n increasing body of evidence indicates a role for excitotoxic cell death in the 49 striatal specific neuron loss observed in H D (Cepeda et al., 2001;Zeron et al., 2001;Zeron et al., 2002;Zeron et al., 2004). Excitotoxic lesions of rodent striatum caused by injections of kainic acid generate a neuropathological and behavioural phenotype that mimics the human condition (Coyle and Schwarcz, 1976;McGeer and McGeer, 1976). The background strain of the Y A C mice is F V B / N , a strain that exhibits a high degree of neuronal loss when exposed to excitotoxic stress after injection of kainic acid or quinolinic acid (Schauwecker and Steward, 1997;Schauwecker, 2002;Schauwecker, 2003). In contrast, injection of excitotoxins into C57BL/6 , the background strain common to most H D mouse models, results in a much lower degree of cell death relative to other mouse stains, including F V B , especially at low doses of kainic and quinolinic acid (Schauwecker and Steward, 1997;Schauwecker, 2002;Schauwecker, 2003). Therefore, strain differences could account for differences in susceptibility to neurotoxicity of polyglutamine expansion. The striatal neuronal loss observed in Y A C 128 mice is linearly correlated with the motor deficit assayed by rotarod. This intriguing result provides the first link in an animal model of H D between behaviour and neuronal loss, suggesting a structural basis for the behavioural manifestations in the Y A C 128 mice. Interestingly, rotarod performance at 6 and 9 months of age is correlated with neuronal loss at 12 months of age. The degree of early motor dysfunction may therefore be an indicator of the severity of the extent of dysfunction of neurons present in the striatum; neurons that w i l l eventually degenerate as the animal ages. The emergence of rotarod deficits therefore, represents a time when neuronal dysfunction already has phenotypic effects, demonstrating the importance of assessing the effect of therapeutic interventions before and after this time point. The strong correlation between striatal neuronal loss and rotarod deficit indicates that neuronal loss and dysfunction are at least major contributors to the behavioural abnormalities in the Y A C 1 2 8 micev. The characterization of the natural history in the Y A C 1 2 8 mouse wi l l make the Y A C 128 line an ideal model for defining the temporal relationship of other observed changes with the loss of striatal neurons. Nuclear huntingtin inclusions visible under light microscopy are a common neuropathological marker in both human patients (DiFigl ia et al., 1997;Gutekunst et al., 1999) and H D mouse models (Davies et al., 50 1997;Hodgson et al., 1999;Schilling et al., 1999;Wheeler et al., 2000). The role of huntingtin inclusions in the pathogenesis of H D is controversial. The appearance of huntingtin inclusions prior to the development of a neurologic phenotype in some transgenic models of H D , indicated that the formation of these inclusions was potentially causative in the disease (Davies et al., 1997). However, in vitro experiments with mutant huntingtin demonstrated a distinct dissociation between the presence of huntingtin inclusions and huntingtin related cell death (Saudou et al., 1998;Kim et al., 1999). Studies in human patients showed little overlap between those cells exhibiting nuclear inclusions and the cells that undergo neurodegeneration in H D (Gutekunst et al., 1999) further supporting the lack of correlation between inclusions and neuronal loss. Huntingtin inclusions are present in 18 month old Y A C 128 mice, but not at 12 months of age, a time point when both behavioural and neuropathological changes, including neuronal loss are present, demonstrating that inclusions are not involved in the initiation of neuronal loss. This finding corresponds with results previously observed in other mouse models of H D (Hodgson et al., 1999;Menalled et al., 2002;Menalled et al., 2003), demonstrating neuronal dysfunction before the onset of huntingtin inclusions. However, inclusion formation is believed to be the end stage of a process beginning with huntingtin translocation to the nucleus and continuing with the formation of micro-aggregates (Davies et al., 1997;Wheeler et al., 2002) visible using electron microscopy. Nuclear translocation and huntingtin micro-aggregates are present in 12 month old animals, leaving the question as to the potential role of nuclear translocation and oligomerization of huntingtin as an initiating stimulus for H D earlier in the disease process. The Y A C mice wi l l be useful for the assessment of therapeutic interventions, as this model accurately recapitulates the human disease and displays phenotypes that can be measured quantitatively with low inter-animal variability. Furthermore, the Y A C 1 2 8 mice exhibit progressive, quantitative phenotypes that parallel the human condition. The significance of low inter-animal variability in the use of a mouse model for therapeutic interventions is illustrated by the power analysis estimations. The phenotype with the lowest variability, striatal volume, requires only 4 animals to determine a 50% rescue at 9 months of age. Even the neuropathological phenotypes with greater variability (e.g. neuron count) require at most, 13 animals to discern a 50% rescue. For a less robust 51 therapeutic effect (33%), only 8 animals are needed for assessment of striatal volume. This result demonstrates the potential usefulness of the Y A C 1 2 8 mice in therapeutic trials and the crucial importance of phenotypic predictability in a model used in experimental therapeutics. The precise natural history of changes in the Y A C 128 mouse model of H D allows for further investigation of the temporal sequence and inter-relationships of other H D -related changes in the pathogenesis of the disease. The role of proteolytic cleavage of mutant huntingtin in the development of the disease is a question of particular interest. Increasing evidence implicates proteolytic cleavage of huntingtin by caspases (Wellington et al., 2002), and/or calpains ( K i m et al., 2001;Gafni and Ellerby, 2002;Gafni et al., 2004), and/or other unknown proteases (Lunkes et al., 2002) in the pathogenesis of H D . The expression of a full-length form of mutant huntingtin makes the Y A C mouse model uniquely suited to test the temporal sequence of proteolysis events in vivo and discern which of the proteases are crucial for the pathogenesis of the disease. In addition, the Y A C 128 model is ideal for assessing the efficacy of inhibition of these proteases on the disease development, either through the use of compounds, or through genetic manipulation. The Y A C 128 mouse model accurately recapitulates the striatal neuronal loss that characterizes the human disease, which allows the Y A C 1 2 8 mice to be useful in assessing experimental therapeutics that provide protection against neuronal loss. The defined natural history in Y A C 128 mice permits the accurate calculation of time points for commencement of therapeutic interventions, and endpoints for the assessment of the efficacy of those interventions. Using the data reported in this chapter, we can now design experimental therapeutic trials with the Y A C 128 mice ensuring an adequate number of animals to properly assess the effect of neuroprotective strategies on the pathogenesis of H D . 52 Chapter 4: A YAC Mouse Expressing a Truncated Fragment of Huntingtin (shortstop) Displays Widespread Neuronal Inclusions without Neuronal Dysfunction or Degeneration The work presented in this chapter is in preparation as: Slow EJ, Graham R K , Devon R S , L u G , Deng Y , Pearson J, Va id K , Bissada N , Leavitt B R , Hayden M R . A Y A C mouse expressing a truncated fragment of huntingtin (shortstop) displays widespread neuronal inclusions without neuronal dysfunction, behavioural changes or degeneration, (in preparation). published in abstract form as: Graham R K , Slow EJ, Devon R S , Va id K , Bissada N , Leavitt B R , Hayden M R (2003) Full-length huntingtin protein is required for striatal specificity of aggregates in Y A C mouse models of Huntington's disease. Program No. 130.10. 2003 Abstract Viewer/Itinerary Planner. Washington. D C : Society for Neuroscience. A n d presented as: Slow E., Graham R K , Devon R S , Va id K , Bissada N , Leavitt B R , Hayden M R (2003) A Shortstop Y A C Mouse Exhibits Accelerated Accumulation and Loses Regional Specificity of Htt Inclusions. (May 4-9, 2003: II Ciocco, Italy) Gordon Conference for C A G Triplet Repeat Disorders. Poster presentation. Slow E, Graham R K , V a i d K , Devon R S , Hayden M R (2003). Full-length huntingtin protein is required for striatal specificity of aggregates in Y A C mouse models of Huntington disease. (May 2-4, 2003: Milan) Molecular Mechanisms of Neurodegeneration. Oral presentation. 53 Preface I designed all of the experiments and analyses presented in this chapter with support from Rona Graham and performed all with the exception of that noted below. Rona initially discovered the shortstop mouse in her investigation of other lines of full-length Y A C 128 mice. Rona also performed the Western blot for comparison of the level of transgenic protein and for brain distribution. Rebecca Devon discovered the insertion site of the shortstop transgene with assistance from Kuljeet Va id and performed the bioinformatics analysis on the shortstop mouse. Jacquie Pearson assisted with the rotarod analysis. Nagat Bissada assisted with the colony management and Ge L u performed the mouse perfusions. Dr. X . J . L i kindly provided the E M 4 8 antibody. Dr. A lex Osmand performed the aggregation foci study. 54 4.1 Introduction Huntingtin, the protein product encoded by the gene mutated in H D is cleaved by a number of different proteases including caspases (Goldberg et al., 1996;Wellington et al., 2000;Wellington et al., 2002), calpains ( K i m et al., 2001;Gafni and Ellerby, 2002;Gafni et al., 2004) and aspartyl proteases (Lunkes et al., 2002) resulting in the production of small, N-terminal fragments carrying the expanded polyglutamine repeat. Cel l culture experiments have demonstrated that decreasing the size of the N-terminal huntingtin fragment increases the toxicity of the fragments in vitro (Hackam et al., 1998;Lunkes and Mandel, 1998;Martindale et al., 1998;Hackam et al., 1999), leading to the toxic fragment hypothesis. The toxic fragment hypothesis postulates that N-terminal fragments of huntingtin with expanded polyglutamine repeats are capable of initiating and propagating a cycle of events which eventually leads to neuronal death. Evidence from mouse models seems to support the toxic fragment hypothesis since mouse models of H D expressing truncated huntingtin (Mangiarini et al., 1996;Schilling et al., 1999) tend to manifest a more severe phenotype than full-length mouse models (Wellington et al., 1997;Hodgson et al., 1999;Shelbourne et al., 1999;Wheeler et al., 2000;Lin et al., 2001). However, it has been difficult to directly compare the truncated mouse models with the full-length models since transgene promoters, background mouse strains and transgenic protein levels vary widely between the different models (Table 1.2), making it impossible to properly control all of the variables besides protein length that may affect the resulting phenotype. One relationship that does seem to be maintained both in vitro and in vivo is the relationship between huntingtin protein length and the formation of huntingtin inclusions. Cel l culture models demonstrated that smaller fragments of huntingtin formed inclusions more quickly in vitro and are more toxic (Hackam et al., 1998). Mouse models of H D expressing short fragments of huntingtin (Davies et al., 1997;Meade et al., 2002) form huntingtin inclusions earlier in the lifespan of the mouse than full-length H D mouse models (Hodgson et al., 1999;Wheeler et al., 2000;Menalled et al., 2002;Menalled et al., 2003;Slow et al., 2003). The role these inclusions or their aggregate precursors play in the pathogenesis is still a matter of debate with some evidence supporting a role for aggregates in toxicity (Davies et al., 1997;Chen et al., 2002) and other evidence 55 supporting a role for aggregates as a non-pathogenic bio-marker (Saudou et al., 1998;Gutekunst et al., 1999). During the development of other full-length Y A C mouse models with 128 C A G repeats, a mouse expressing a short fragment of huntingtin from a premature stop, and hence named "shortstop", was serendipitously created. The shortstop mouse expresses exons 1 and 2 of the H D gene, producing a protein similar in size to a huntingtin fragment created by aspartyl proteolysis of full-length huntingtin (Lunkes et al., 2002). The shortstop mouse possesses the identical 24 kb of upstream regulatory region as the full-length Y A C mouse model with an identical polyglutamine repeat size and level of transgenic protein expression on the identical F V B background strain. This allows for the first time the ability to characterize both a full-length and truncated H D mouse model in parallel to determine the effects of a truncated fragment on the neurodegenerative and behavioural HD-related phenotypes exhibited by the full-length Y A C 1 2 8 . Huntingtin inclusion analysis in the shortstop and the full-length Y A C 1 2 8 mice revealed more widespread huntingtin inclusion formation both in percentage of neurons and in neuronal tissue types affected in the shortstop mouse, compared to the full-length Y A C 1 2 8 . In addition, inclusions form earlier in the shortstop mouse than in the full-length Y A C 128 mouse. Surprisingly, the shortstop mouse does not demonstrate the neuronal dysfunction determined by rotarod deficit or neuronal degeneration determined by brain weight, striatal volume and striatal neuronal count decreases observed in the full-length Y A C 128 mouse model. The lack of a neurodegenerative or dysfunctional HD-related phenotype in a mouse exhibiting widespread huntingtin inclusion formation further supports a role for inclusions as harmless byproducts of polyglutamine expansion. The lack of an HD-related phenotype in the shortstop mouse expressing a truncated fragment of huntingtin does not refute the toxic fragment hypothesis, but rather supports the importance of the specificity of the N-terminal fragment for initiation of the toxic fragment cycle which leads to neuronal degeneration and dysfunction in vivo. 56 4.2 Results 4.2.1 Discovery of the shortstop mouse During generation of additional full-length Y A C mice with 128 C A G repeats, it was noted that one of the generated lines was missing the left Y A C arm ( L Y A ) by P C R screening (data not shown). The Y A C mice are genotyped by P C R using three sets of primers, one which amplifies the C A G stretch to insure the tract is not expanding or Shortstop Shortstop B -endo. , s kDa htt -fhtt-9343F/9617R 9343F I 9680R 9343F / m476R Ab: Bkpl Ab: 1C2 RYA 24,392bp exon 1 mouse chromosome 4 shortstop Y A C full-length Y A C Msel 9229R 9234R 9617R i m476R exon 3 9680R Figure 4.1: Protein discovery and shortstop localization in mouse chromosome 4. A. Western blot showing protein expression of truncated (fhtt) shortstop fragment probed with the huntingtin specific N-terminal antibody BKP1 in four separate shortstop mice. The blot was stripped and reprobed with the expanded polyglutamine-specific antibody 1C2 which recognized the same band. B. Agarose gel demonstrating integration of the shortstop transgene into mouse chromosome 4. PCR on genomic mouse DNA from shortstop (SS), YAC128 (53) and wild-type mice (WT) using primers within intron 2 of human huntingtin before the SS breakpoint (9343F/9617R) and after the shortstop breakpoint (9343F/9680R) reveal the presence of part of intron 2 in the SS mouse. PCR with primers from human intron 2 (9343F) and mouse chromosome 4 (m476R) reveal a band in SS, indicating integration into mouse chromosome 4. C. Schematic describing the genomic organization of the shortstop truncated transgene including primer positions for PCR in B. 5 7 contracting, while the other two sets amplify the Y A C arms (left and right) to insure the whole Y A C including the full-length huntingtin gene is present. The loss of the L Y A which lies 3' to the huntingtin gene, and the presence of an expanded C A G tract in this line of mice indicated that the Y A C had truncated 3'of the C A G tract. Western blots of brain lysates from the truncated mouse line revealed an approximately 75 kDa band immunostaining with B K P 1 , an antibody specific to the N-terminus of huntingtin (Fig. 4.1 A ) . Stripping the blots and reprobing with 1C2, an antibody specific to expanded polyglutamine, demonstrated an identical band at 75 kDa (Fig. 4.1 A ) . The Western results indicated that the truncated mouse line was expressing a short fragment of huntingtin from a premature stop and was therefore given the name "shortstop". 4.2.2 Characterization of the shortstop 3' YAC truncation site and insertion site To define the precise 3' truncation site of the Y A C in shortstop mice, the region was initially narrowed down by designing a series of P C R primers to amplify short (200-300 bp) regions of the human H D gene from exons 1 to 10, and testing for amplification in genomic D N A from a shortstop mouse compared with a mouse transgenic for the full length Y A C , line 53, hereafter referred to as Y A C 128. In this way the truncation site was quickly narrowed down to lying within the 12251 bp that comprises intron 2. In the same way, a second series of primer pairs was then used to narrow down the region to a 408 bp region between bases 9366 and 9773 of intron 2. The precise truncation site was identified by performing inverse P C R using primers (9229R, 9234R, 927IF, 9343F) within the known positive region immediately 5' of base 9366. A 750 bp product obtained after two rounds of nested P C R was sequenced and was found to comprise H D intron 2 until base 9628, followed by 383 bp of mouse chromosome 4. Additional evidence for the truncation of the Y A C at base 9628 of H D intron 2 was provided by testing shortstop genomic D N A for P C R amplification with primer 9343F paired with either primer 9617R (predicted to be positive) or primer 9680R (predicted to be negative) (Fig. 4. IB) . The region of mouse chromosome 4 identified in the 750 bp inverse P C R product corresponds to the insertion site of the shortstop Y A C in mouse genomic D N A . This was confirmed by P C R amplification and sequencing of a 476 bp fragment spanning the 58 junction between H D intron 2 and mouse chromosome 4 from shortstop genomic D N A only (primers 9343F and m476R; see Fig . 4.1C). According to the inverse P C R product, the insertion site of the Y A C occurred at base 63804 of the B A C clone RP23-123E1. This B A C has been mapped to band B3 of mouse chromosome 4, near marker D4Mit88. This region of the mouse genome is gene-poor, and there are no genes mapped within this B A C . According to Ensembl Mouse Genome Assembly N C B I Bu i ld 30 (updated 1 s t July 2003), the nearest centromeric gene, which is highly similar to S-adenosylmethionine decarboxylase 2 (Amd2), is located 566 kb proximal to this B A C , and the nearest known telomeric gene, structural maintenance of chromosomes 2-like 1 (Smc211) is located 627 kb distal to the B A C . Four novel predicted genes lie between the end of the B A C and Smc211; the closest is located 299 kb distal to the B A C . We can be confident then, that the insertion of the shortstop transgene does not directly disrupt the expression of another gene. It is unclear precisely where the transcript, would be terminated, however there are five potential poly(A) addition signals within the portion of intron 2 present in the shortstop Y A C at positions 709, 1924, 3766, 8783 and 8830. There is no evidence for any functional elements present within the shortstop 3 ' U T R sequence, as judged by scanning of the P R O S I T E motif database with the intron 2 shortstop sequence, and searching for sequence elements conserved between human and mouse. The first codon within H D intron 2 is a ' T A A ' stop codon, therefore there are no additional amino acids in the shortstop protein that are not present in the normal H D protein, removing the possibility for dominant negative effects. The predicted protein sequence translated from the shortstop Y A C is identical to that translated from H D exons 1 and 2 and should express a protein from amino acid 1-117 in the human sequence. The shortstop protein is therefore running above the predicted size of ~ 24 kDa (Fig. 4.1 A ) . This phenomenon has been reported previously in the R6/2 short fragment model of H D and is consistent with aberrant migration due to the polyglutamine stretch (Mangiarini et al., 1996). 59 4.2.3 Shortstop and YAC128 mice have identical promoter, regulatory elements and CAG sizes Although the approximate content of the Y A C 353G6 used to make H D transgenic mice has been defined previously (Hodgson et al., 1996), the exact extent of the Y A C was not known. Splinkerette P C R (Devon et al., 1995) was therefore used to identify the precise boundary of the Y A C at the 5' end by P C R "walking" inwards from the right Y A C arm. Two rounds of nested P C R resulted in products that were identified as spanning the junction between the right Y A C arm and human genomic sequence upstream of the H D gene. The Y A C was found to contain 24392 bp of sequence upstream of exon 1 of the H D gene. There are no additional genes within this 24kb; in fact the nearest upstream gene, G protein coupled receptor kinase 2-like ( G P R K 2 L ) lies 34kb upstream of the H D gene (Ensembl Human Genome Browser, release 18.34.1, 4 t h November 2003). It is highly likely therefore that all or most of the promoter and cis-regulatory elements required to drive correct expression of the H D gene are present in the Y A C mice including both the full-length ( Y A C 128) and shortstop mice: To insure both the full-length Y A C 128 mouse and the short-stop mouse had similar polyglutamine tract lengths, the C A G tract was P C R labeled with 6-Hex, run on an A B I and analysed with genescan. Identical peaks were observed for both shortstop and Y A C 128 at the expected size, indicating both lines have identical C A G repeat lengths (Fig. 4.2A). 4.2.4 Shortstop and YAC128 mice exhibit an identical pattern of transgenic protein expression The presence of the identical 24 kb of upstream sequence in both the Y A C 128 and shortstop mouse transgene suggests that all of the promoter and regulatory regions are present in both mouse lines. To verify the transgenes were expressed similarly throughout the brain, Western blots of different brain regions from the two lines of mice were probed with 1C2, an antibody specific to expanded polyglutamine. Expression analysis revealed similar transgenic protein distribution in Y A C 128 and shortstop brain regions, including the striatum, cortex, hippocampus and cerebellum (Fig. 4.2B). Different lines of transgenic mice often express different quantities of transgenic protein 60 and it is important to determine these relative values before making comparisons between lines. Towards this goal, protein lysates from the cortices of n=3 Y A C 1 2 8 and n=3 shortstop mice were separated on a gel and probed with 1C2 (Fig. 4.2C). Densitometric analysis revealed the shortstop mice expressed approximately 1.46X the amount of transgenic protein compared to the full-length Y A C 128 mice (Fig. 4.2D). Figure 4.2: Comparison of shortstop with YAC128, line 53. A. Genescan results from PCR using 6-FAM labeled human specific primers which flank the CAG tract demonstrate identical CAG repeat length in shortstop and YAC128. B. Western blots demonstrating similar protein expression in shortstop and YAC 128 mice across 4 different brain regions. The antibody is 1C2, specific to expanded polyglutamine. C. Western blot showing similar levels of transgenic protein expression in shortstop and YAC128 cortex (n=3) using 1C2 antibody. D. Densitometry analysis of blot from C reveals shortstop expresses 1.5X protein of YAC128, line 53. Identical C A G size combined with identical tissue distribution and similar level of transgenic protein expression in the full-length Y A C 128 and shortstop mice allows for the first time the comparison between a suitably matched and controlled full-length and truncated huntingtin mouse model. 61 4.2.5 Inclusion formation in shortstop mice Previous reports have demonstrated greater distribution o f huntingtin inclusions in the brain in short fragment mouse models compared to full-length H D mouse models (L i et al., 2000;Li et al., 2001). Using the suitably controlled Y A C 1 2 8 and shortstop mouse, the question o f the effects of expression of full-length huntingtin versus truncated huntingtin on inclusion formation in vivo can be addressed. To investigate inclusion distribution in the shortstop and full-length Y A C 128 mice, brain sections from 18 months old animals were immuno-stained with E M 4 8 , an antibody specific for huntingtin aggregates and inclusions (Gutekunst et al., 1999). Neuronal inclusions were present in the striatum (Fig. 4.3 A ) , the brain region most affected in H D patients, of the full-length Y A C 128 mouse in approximately 30% of neurons. In contrast, the inclusions in the shortstop mouse were larger (Fig. 4.3E) and present in over 95% of striatal neurons. Cortical inclusions were also noted in the Y A C 128 mouse in <10% of neurons. Cortical neuronal inclusions were also present in the shortstop mouse but were larger (Fig. 4.3F) and were present in over 90% of neurons. # * m< # « * • — WF .. • • I l Figure 4.3: Huntingtin inclusions in shortstop and YAC128 mice at 18 months. Inclusions are present in the striatum (A) and cortex (B) of YAC128 mice, but not in the dentate gyrus (C) or CA layers (D) of the hippocampus. Inclusions are present in most cells in the striatum (E), cortex (F), and dentate gyrus (G) and CA layers (H) of the hippocampus in the shortstop mouse. Scale bars indicate 10/im for A-H. The hippocampus, a brain region less frequently affected in H D brains, revealed no neuronal inclusions in the full-length Y A C 128 mouse in either the dentate gyrus or C A regions (Fig. 4.3C, D). In contrast, the dentate gyrus of the shortstop mouse exhibited 62 large inclusions in approximately 100% of neurons, and smaller inclusions in the C A region of the hippocampus, again in most of the neurons (Fig. 4.3G, H). These results demonstrate that expression of a short fragment of huntingtin (shortstop) in vivo results in the accumulation of larger and more widespread inclusions throughout different neuronal populations than expression of the full-length huntingtin protein ( Y A C 128). To determine whether the accumulation of inclusions in the shortstop mouse is accelerated compared to the full-length Y A C 1 2 8 mouse, brain sections from 3, 6, 9, 12 and 18 month old shortstop and Y A C 1 2 8 were immuno-stained with E M 4 8 and scored for the presence of inclusions as well as the localization of huntingtin in the nucleus (Table 4.1 and 4.2). The shortstop mice demonstrated nuclear huntingtin staining at 3 months of age in all regions analysed, while the Y A C 128 mice exhibited nuclear huntingtin staining selectively in the striatum at the same age (Table 4.1). Dark nuclear huntingtin staining is present in most of the brain, regions (excluding layer V of the cortex) examined in the shortstop mice at 6 months of age, while a comparable level of nuclear huntingtin staining is not detected in Y A C 128 mice until 12 months of age. Table 4.1: Nuclear localization of htt is accelerated in the shortstop mice and exhibits regional differences compared to the full-length YAC128 mouse. Sections from YAC 128 and shortstop mice at 3, 6,9, 12 and 18 months of age were immuno-stained with EM48 to determine localization of htt to the nucleus. Regions were scored as - no labeling, + light labeling, ++ dark labeling. 3 6 9 12 18 Striatum SS + ++ ++ ++ ++ YAC128 + + ++ ++ ++ Cerebral Cortex SS ++ ++ ++ ++ ++ - layers II,III YAC 128 - ++ ++ ++ ++ - layer IV SS + ++ ++ ++ ++ YAC 128 - + + + + - layer V SS + + + + ++ YAC 128 - - - - -- layer VI SS + ++ ++ ++ ++ YAC 128 - + + + + Pyriform Cortex SS + ++ ++ ++ ++ YAC 128 - ++ ++ ++ ++ Hippocampus SS ++ ++ ++ ++ ++ -DG YAC 128 - + + ++ ++ - CA layers SS ++ ++ ++ ++ ++ YAC128 - + + ++ ++ Inclusions were first present in cortical layers II-IV at 6 months of age in the shortstop mice, and in the striatum, hippocampus and all cortical layers except V at 9 months of age (Table 4.2). In contrast, Y A C 128 mice exhibit a very low percentage of 63 inclusions in the striatum at 12 months of age (1%), and inclusions in the striatum and cortex, but not the hippocampus at 18 months of age (Table 4.2). We had previously reported inclusions in the striatum of Y A C 128 mice at 18 months of age, but further analysis revealed smaller inclusions or punctuate micro-aggregates in a very low percentage (<1%) of striatal neurons. These are similar to micro-aggregates, displayed in other mouse models (Menalled et al., 2002;Menalled et al., 2003) and defined as numerous, small, punctuate aggregates. The comparison of the time course of shortstop and YAC128 inclusion formation reveals a much earlier onset of inclusion formation in the shortstop mouse, accompanied by a more widespread tissue distribution. Table 4.2: Huntingtin inclusion formation is accelerated and more widespread in the shortstop mice compared to the full-length YAC128 mouse. Sections from YAC128 and shortstop mice at 3, 6, 9, 12 and 18 months of age were immuno-stained with EM48 for huntingtin inclusions. Regions were scored as -no cells with inclusions, + few cells present nuclear inclusions (1-5%), ++ scattered cells present nuclear inclusions (<50%), +++ numerous cells present nuclear inclusions (50-100%). Area Mouse Age (months) 3 6 9 12 18 Striatum SS - - + +++ +++ YAC128 - - . + ++ Cerebral Cortex SS - + ++ +++ +++ - layers 11,111 YAC128 - - - - + - layer IV SS - ++ +++ +++ +++ YAC 128 - - - - ++ - layer V SS - - - + ++ YAC 128 - - - - -- layer VI SS - - ++ +++ +++ YAC 128 - - - - -Pyriform Cortex SS - - + +++ +++ YAC 128 - - - - + Hippocampus SS - - +++ +++ +++ -DG YAC 128 - - - - -- CA layers SS YAC 128 - ++ +++ +++ 4.2.6 Aggregation foci in shortstop and YAC128 mice A possible argument is that visible aggregates or inclusions are present in the shortstop mouse, but some of the toxic precursors are absent. To examine this hypothesis, tissue from both the YAC128 and shortstop were examined for aggregation foci, a precursor of huntingtin inclusions. Aggregation foci are intracellular structures which demonstrate polyglutamine recruitment activity, and have been identified in HD patient cortex (Osmand et al., 2002). These aggregation foci are visualized and defined by their ability to recruit biotinylated polyglutamine peptides and occur preceding 64 development of huntingtin inclusions in the linear process of huntingtin aggregation (Bates, 2003). Aggregation foci are present in both Y A C 128 and shortstop mouse brain (Fig. 4.4) at 12 months of age. The aggregation foci are at a greater density in the cortex (Fig. 4.4F) dentate gyrus (Fig. 4.4G) and hippocampus (Fig. 4.4H) in the shortstop mouse compared to the same tissues in the Y A C 128, replicating the greater density o f inclusions in the shortstop versus Y A C 128 mice. However, aggregation foci are present in the hippocampus and dentate gyrus o f the Y A C 128 mice, a tissue which does not manifest inclusions at any time point examined in the Y A C 128 mice. Surprisingly, aggregation foci are barely present in the striatum of either the shortstop (Fig. 4.4E) or Y A C 128 (Fig 4.4A), despite huntingtin inclusion staining in nearly 100% of striatal neurons in the shortstop and 30% of striatal neurons in the Y A C 128s at 18 months. Similar to the observation o f inclusion formation, the shortstop mouse displays a greater density o f aggregation foci compared to the full-length Y A C 128 mouse. Interestingly, the brain regions which manifest aggregation foci do not completely parallel those tissues which manifest inclusions (e.g. striatum). This finding indicates that some tissues may be prone to either manifest aggregation foci or inclusions, and suggests that aggregation foci are not necessarily upstream o f huntingtin inclusion formation. A i Figure 4.4: Aggregation foci in shortstop and YAC128 mice. At 12 months of age YAC128 dentate gyrus (C), hippocampus (D) and cortex (B) demonstrate less labeling of aggregation foci compared to shortstop dentate gyrus (G), hippocampus (H) and cortex (F) of shortstop mice. Surprisingly, very few aggregation foci are present in the striatum of the YAC128 (A) or shortstop (E) mice. Aggregation foci are indicated with black arrows. 65 4.2.7 Shortstop does not exhibit a rotarod deficit Previous studies of the full-length Y A C 128 mouse demonstrated a severe, progressive rotarod deficit (Slow et al., 2003) present at 6, 9 and 12 months of age. We tested the shortstop mice at 12 months of age with Y A C 128 and wild-type mice to determine i f the shortstop mice had a similar deficit compared to the Y A C 128 mice. The Y A C 128 mice performed worse than wild-type littermates in an accelerating rotarod protocol (p<0.001) (Fig. 4.5A). Surprisingly, the shortstop mice exhibited no deficit on the rotarod compared to wild-type mice and therefore performed significantly better than the full-length Y A C 1 2 8 (p<0.001), ( A N O V A p= 0.0002, n=10 wild-type, n=8 Y A C 1 2 8 , n=4 shortstop) (Fig. 4.5A). Although the number of shortstop mice in the study was low, these results replicated previous rotarod results that demonstrated the shortstop mice performing similarly to wild-type mice at 12 months of age, while the Y A C 1 2 8 were significantly worse than wild-type (p<0.01) ( A N O V A p=0.01, n=7 wild-type, n=12 Y A C 128, n=5 shortstop). 4.2.8 Shortstop does not exhibit neuronal degeneration Brain weight and striatal volume loss accompanied by striatal neuronal loss are manifest in the full-length Y A C 1 2 8 mice by 12 months of age, recapitulating the neurodegeneration present in H D patients. Cohorts of Y A C 128, wild-type littermates and shortstop mice at one year of age were perfused and brain weight was measured. Y A C 128 mouse brains weighed 8% less than wild-type littermates (p<0.01) while shortstop brain weight was not significantly decreased compared to wild-type mouse brains ( A N O V A p=0.006, n=7 wild-type, n=12 Y A C 1 2 8 , n=5 shortstop) (Fig. 4.5B). The brains were sectioned, immunostained and striatal volume and neuronal count measured. A 15% decrease in Y A C 1 2 8 striatal volume compared to wild-type controls replicated previously reported results (p<0.05). Surprisingly, striatal volume in the shortstop mice was not different compared to wild-type controls ( A N O V A p=0.02, n=5 wild-type, n=5 Y A C 1 2 8 , n=5 shortstop, F ig . 4.5C). Striatal neuronal count was significantly decreased in Y A C 1 2 8 compared to both wild-type (p<0.05) and to shortstop mice (p<0.05) (Fig. 4.5D). Shortstop mice again showed no difference in striatal neuronal count compared to wild-type mice ( A N O V A p=0.01, n=5 wild-type, n=5 66 Y A C 1 2 8 , n=5 shortstop). These results indicate that shortstop mice at 12 months of age do not manifest the HD-related phenotypes, including brain weight and striatal volume decreases with striatal neuronal loss that manifest in the full-length Y A C 1 2 8 mice at the same age. A 300-i >, 200-o £ CO _J " r o 100-u_ o-14 E E 12 E o 10 P < 0 0 0 1 p<0.001 n=10 n=8 WT YAC128 SS Genotype WT YAC128 SS Genotype B ^ 0.4-OS CD i> 0.3 0.2 p<0.05 T T • n=5 n=5 D ID o X 1.6-, 1 .4H i 1 - 2 a> z 1.0 0.8 p<0.01 n=7 WT YAC128 SS Genotype p<0.05 p<0.05 n=5 WT YAC128 Genotype SS Figure 4.5: The shortstop mouse does not manifest the neuronal dysfunction or degeneration of the full-length YAC128 model at 12 months of age. A. The shortstop mice do not exhibit a deficit on an accelerating rotarod, while the YAC128 mice demonstrate clear dysfunction compared to wild-type (WT) littermates (p<0.001). The shortstop mice do not exhibit significant brain weight decrease (B), striatal volume decrease (C) or striatal neuronal count decrease (D) when compared to wild-type mice, while the full-length Y A C 128 mice demonstrate significant decreases. Mean measurement ± standard deviation of each group is shown in B, C, D and ± S E M in A. All data was analysed by a one-way A N O V A and p-values between groups were calculated using Tukey's multiple comparison test. To ensure that these phenotypes are not present in older shortstop mice, an 18 month cohort was examined. Shortstop mice did not demonstrate significant brain weight or striatal volume decrease compared to wild-type mice, while full-length Y A C 128 exhibited a significant decrease in brain weight (p<0.05, A N O V A p=0.03, n=8 wild-type, n=10 Y A C 1 2 8 , n=7 shortstop) and striatal volume (p<0.01, A N O V A p=0.003, n=7 wild-type, n=8 Y A C 1 2 8 , n=6 shortstop) compared to wild-type littermates, 67 suggesting that even at older ages, the shortstop mice are resistant to the HD related neurodegenerative phenotypes present in the full-length YAC128 (Fig. 4.6). A S 0.4-.£ 0.3 2 GO 0.2 *p<0.05 n=8 n=8 WT YAC128 Genotype SS B 1 5 «g 14 E —-13 <u E = 12 o > 11 10 *p<0.01 **p<0.01 WT YAC128 SS Genotype Figure 4.6 Shortstop mice do not exhibit decreases in brain weight or striatal volume at 18 months of age. The shortstop mice reveal no significant brain weight decrease (A), or striatal volume decrease (B) at 18 months of age when compared to wild-type mice, while the full-length YAC 128 mice demonstrate significant decreases. Mean measurement ± standard deviation of each group is shown in A. All data was analysed by a one-way ANOVAand p-values between groups were calculated using Tukey's multiple comparison test. 68 4.3 Discussion Huntingtin protein aggregates and/or inclusions are a hallmark of Huntington disease and are present in both human patients (DiFigl ia et al., 1997;Gutekunst et al., 1999) and in H D mouse models (Davies et al., 1997;Hodgson et al., 1999;Schilling et al., 1999;Wheeler et al., 2000;Slow et al., 2003). Since the initial discovery of huntingtin inclusions, there has been continual controversy over what role huntingtin aggregates play in the pathogenesis of the disease. The debate is divided over whether aggregates and their precursors are in fact the toxic, underlying mechanism in the neuronal loss and dysfunction of the disease, or simply benign byproducts of polyglutamine expansion. The initial support for the toxicity of aggregates model resulted from cell culture assays using fragments of huntingtin. These studies demonstrated that those huntingtin fragments which were most toxic to cells also most readily formed inclusions, (Cooper et al., 1998;Hackam et al., 1998) indicating a correlation between huntingtin toxicity and huntingtin inclusion formation. In the R6/2 short fragment mouse model of H D , huntingtin aggregates form before the onset of neuropathology (Davies et al., 1997) supporting a causative role for aggregates in the resulting pathology. Experimental therapeutics lent further support to the toxicity of huntingtin aggregates hypothesis illustrated by a study of anti-aggregation peptides in a Drosophila model of H D which both decreased inclusions and delayed lethality and neurodegeneration (Kazantsev et al., 2002). Treatment of the R6/2 mouse model with trehalose (Tanaka et al., 2004), Congo red (Sanchez et al., 2003), creatine (Ferrante et al., 2000) and Coenzyme Q coupled with remacemide (Ferrante et al., 2002) both decreased aggregates in the brains of treated mice and delayed neurodegeneration and/or premature death. The hypothesis that aggregates may be a non-toxic byproduct of polyglutamine expansion was first advanced following subsequent cell culture work. One study revealed that cells transfected with mutant huntingtin could demonstrate both increased cell survival without affecting huntingtin inclusions, or decreased huntingtin inclusions with no effect on cellular toxicity depending on the type of caspase inhibitor the cells were treated with ( K i m et al., 1999). Inhibition of inclusion formation in another cell culture model of H D increased toxicity, while blocking of huntingtin-related apoptosis in this same model resulted in greater percentage of cells with inclusions (Saudou et al., 69 1998), again illustrating the dissociation of aggregate formation and toxicity. Full-length mouse models of FfD exhibit the onset of behavioural abnormalities as an indicator of neuronal dysfunction before the formation of huntingtin inclusions (Hodgson et al., 1999;Menalled et al., 2002;Menalled et al., 2003;Slow et al., 2003) and studies in human patients show little overlap between those regions most affected in the disease and those with the largest inclusion burden (Gutekunst et al., 1999). In a S C A 1 knock-in mouse model which accurately recapitulated the human disorder, the Purkinje neurons which were most susceptible to neurodegeneration were also the last to form huntingtin aggregates, indicating that the neurons which were least capable of forming aggregates were most susceptible to degeneration (Watase et al., 2002). To investigate the role of aggregation in S C A 1 , a mouse model was established with an ataxin-1 transgene which had the self-association domain deleted and therefore could not self-aggregate. This S C A 1 mouse model demonstrated ataxia and neuronal loss without formation of neuronal aggregates, indicating aggregate formation was not relevant for disease pathogenesis in this related polyglutamine disorder (Klement et al., 1998). A recent study has demonstrated that aggregates sequester mTOR, a negative regulator of autophagy. This sequestration promotes autophagy of the toxic mutant protein, indicating that huntingtin aggregates may actually play a role in neuroprotection through this mechanism (Ravikumar et al., 2004). Experimental therapeutics with H D mouse models has also provided support for the hypothesis that aggregates are a benign by-product. Treating the R6/2 model with the H D A C inhibitors S A H A (Hockly et al., 2003) and sodium butyrate (Ferrante et al., 2003) or the glutamate antagonist riluzole (Schiefer et al., 2002) resulted in decreased neuropathology with no accompanying decrease in huntingtin inclusion burden. Treatment of the R6/2 mice with cystamine revealed significant and massive decreases in huntingtin inclusions with only mild effects on neurodegenerative and early death phenotypes (Dedeoglu et al., 2002). Finally and most strikingly, crossing the R6/2 mice with tissue transglutaminase knock-out mice partially rescued the brain and body weight loss and early mortality phenotype but was accompanied by a significant increase in huntingtin neuronal inclusions (Mastroberardino et al., 2002), in contradiction of the hypothesis that aggregates are toxic. 70 In the shortstop mouse, huntingtin inclusions appear at an earlier timepoint, have a more widespread tissue distribution and are larger than those inclusions revealed in the full-length Y A C 128 mouse. Furthermore, the shortstop mouse demonstrates greater density of aggregation foci, another component of the aggregation pathway, compared to full-length Y A C 128. While the shortstop mice reveal widespread huntingtin inclusion burden, they do not manifest a behavioural, HD-related phenotype assessed on the rotarod or decreases in brain weight, striatal volume and striatal neuronal count at 12 or 18 months of age. The shortstop mouse illustrates in vivo, the lack of correlation between inclusion formation or inclusion precursor (aggregation foci) formation and HD-related behavioural and neuropathological phenotypes observed in the full-length Y A C 128 model, supporting the hypothesis that huntingtin aggregates and/or inclusions are benign. The full-length Y A C 1 2 8 model manifests huntingtin inclusions at a timepoint after initial behavioural and gross neuropathological deficits including loss of striatal volume and brain weight (Slow et al., 2003). The formation of visible huntingtin inclusions have been hypothesized to be the last stage of a complex, linear process beginning with a critical concentration of aggregate precursor and continuing through the formation of P-sheets, protofibrils, fibrils, aggregation foci and finally the production of a visible aggregate (inclusions) (Bates, 2003). A n y of these intermediates from protofibril through visible aggregate is hypothesized to be capable of causing cellular dysfunction which leads to eventual disease presentation (Bates, 2003). The toxicity of aggregate precursors has been offered as an explanation for those mouse models which manifest inclusions after pathology and behavioural changes, including the Y A C 1 2 8 mouse model (Slow et al., 2003), i.e. the inclusion precursors are causing the dysfunction not the late-forming inclusions. However, i f the formation of aggregates is a linear process, with visible aggregates as the last stage in this process, then the shortstop mouse should at one time express each of the "toxic" aggregation precursors, yet it does not present with a neurodegenerative phenotype. The full-length Y A C 1 2 8 mice clearly manifest an HD-related phenotype, therefore i f the aggregate toxicity hypothesis is correct, the aggregates must be different between the Y A C 128 and shortstop mice. E M 4 8 and aggregation foci staining indicate 71 that at least these two steps in the aggregation formation pathway are present and similar in both the full-length and shortstop Y A C mice. The presence of aggregates without neurodegeneration in the shortstop mouse and similar aggregates with neurodegeneration in a well-matched full-length Y A C 128 model support the argument that aggregates and inclusions, although part of the disease sequelae, can exist completely separate from H D -related neuronal dysfunction and degeneration. The study of the shortstop mouse in parallel with a full-length Y A C 128 mouse expressed under the same promoter and matched for C A G repeat length and protein expression level allows for the first time, the comparison of a short fragment of huntingtin with the full-length protein in vivo. Surprisingly, the short fragment huntingtin mouse does not manifest the behavioural deficits as a marker of neuronal dysfunction or the neurodegeneration displayed in the full-length Y A C 1 2 8 model. One possible explanation is that the integration site of the shortstop mouse transgene is affecting a gene involved in the pathogenesis of the disorder, but mapping of the insertion site revealed the shortstop transgene integrated into a gene-free region, ruling out this possibility. The absence of neuronal dysfunction or degeneration in the shortstop mouse and the presence in the full-length Y A C 128 mouse does not contradict the toxic fragment hypothesis. The toxic fragment hypothesis proposes that toxic, N-terminal fragments of mutant huntingtin are inefficiently degraded due to the expanded polyglutamine tract, and subsequently cause alterations in transport and/or endocytosis and/or transcription. These alterations lead to neuronal stress which makes the neuron more susceptible to excitotoxicity and C a + 2 uptake, leading to increased proteolysis and production of toxic N-terminal fragments and a steady amplification of this cycle until the neuron is ultimately committed to apoptosis (Fig. 1.2) (Wellington and Hayden, 1997). The lack of a neurodegenerative or dysfunctional phenotype in the shortstop mouse which expresses an N-terminal fragment of huntingtin, similar, although not identical, in size to a fragment generated from aspartyl proteolysis of huntingtin (Fig. 1.1), indicates that there may be specificity to the size of the N-terminal fragment which can initiate the toxic fragment cycle. The lack of a neuronal degeneration or dysfunction phenotype in the shortstop mouse, a mouse expressing a truncated N-terminal fragment of huntingtin, indicates that 72 not all fragments of huntingtin are capable of initiating the toxic fragment cycle and therefore causing an H D related phenotype in vivo. Initial work in cell culture models transfected with fragments of mutant huntingtin indicated that the length of the huntingtin fragment inversely correlated with the toxicity of the fragments (Hackam et al., 1998;Martindale et al., 1998). However, Y u et al in 2003 described an experiment where different lengths of huntingtin with the same polyglutamine stretch were transfected into H E K 2 9 3 cells and the cells were assayed for viability and caspase activation as a marker of apoptosis. A fragment of 208 amino acids in length produced the greatest caspase activation (apoptosis) and toxicity, significantly greater than that caused by an exon 1 fragment or fragments of greater length (Yu et al., 2003). The idea that different fragments of a disease-causing protein can be differentially toxic is well-recognized in the Alzheimer disease field where the AP1.42 fragment is more toxic than the Ab*1-40fragment, despite only a 2 amino acid difference (Iijima et al., 2004). Caspases cleave huntingtin, with sites for caspase-3 cleavage at amino acid 513 and 552 and caspase-6 cleavage at amino acid 586 (Wellington et al., 2000;Wellington et al., 2002;Hermel et al., 2004). Graham and Hayden (unpublished data) developed Y A C transgenic mice with amino acid substitutions which prevent cleavage of huntingtin by caspase-3 (caspase-3 resistant) or caspase-6 (caspase-6 resistant). Preventing caspase-6 cleavage protected the mice from neurodegeneration, while preventing caspase-3 cleavage provided no protection from neurodegeneration, indicating that the caspase-6 cleavage fragment of 586 amino acids is capable of causing toxicity in vivo, while the smaller caspase-3 fragments of 513 and 552 amino acids are not capable of causing toxicity (unpublished data). The lack of neuronal degeneration or dysfunction in the shortstop mouse expressing the first 117 amino acids of huntingtin with an expanded polyglutamine repeat indicates that in vivo (Fig. 1.1), this fragment is not capable of initiating the cycle of events which leads to neuronal dysfunction and death. The 117 amino acid shortstop fragment corresponds to a fragment which would be generated by aspartyl protease cleavage of huntingtin (Lunkes et al., 2002). The results of the shortstop study therefore indicate that aspartyl proteolysis is not a primary, initiating event in H D pathogenesis and is therefore not a promising therapeutic target. However, it must be noted that the 73 shortstop and aspartyl proteolysis fragments do differ by 3 amino acids (Figure 1.2), and toxicity of the specific aspartyl proteolysis fragment cannot be entirely ruled out due to the slight difference in size between this fragment and the shortstop fragment. It remains to be determined whether the shortstop fragment is capable of amplifying the toxic fragment cycle once it is initiated by the appropriate N-terminal fragment of huntingtin. The inability of the N-terminal fragment expressed by the shortstop mouse to initiate the toxic fragment cycle which ultimately leads to neuronal degeneration and dysfunction in the full-length Y A C 1 2 8 mouse indicates that there is an important role for the full-length mutant protein or at least for a longer fragment than that present in the shortstop mouse in the pathogenesis of the disorder. The necessity of the correct protein context for the appropriate recapitulation of the disease phenotype was first suggested following the comparison of the nine neurodegenerative disorders which result from polyglutamine expansion (Table 1.1). While the underlying genetic mutation, a C A G repeat expansion, is identical in these disorders, the site of pathology is different for each disorder with H D characterized by loss of striatal and cortical neurons, S B M A by motor neurons in the brainstem and spinal cord, and the spinocerebellar ataxias and D R P L A predominantly showing neuronal loss in the cerebellum (Evert et al., 2000) (Table 1.1). The differences in pathology are thought to primarily occur due to the protein context of the mutation i.e. the particular properties of the protein in which the polyglutamine tract resides and demonstrate the importance of the protein context for correct recapitulation of the disorders. Recent evidence from other polyglutamine disorders has clearly demonstrated the importance of protein context for the development of a neurodegenerative phenotype from a polyglutamine expanded protein. Evidence from two studies on S C A 1 discovered that phosphorylation of ataxin-1, the protein expressed by the gene mutated in S C A 1 , is necessary for neurodegeneration (Chen et al., 2003;Emamian et al., 2003). S B M A is caused by a C A G expansion in the androgen receptor gene located on the X-chromosome and recent research has demonstrated the necessity of testosterone for the development of neurodegeneration (Katsuno et al., 2002;Katsuno et al., 2003). The necessity of phosphorylation of ataxin-1 for neurodegeneration in S C A - 1 (Chen et al., 2003;Emamian et al., 2003) and testosterone 74 for neurodegeneration in S B M A (Katsuno et al., 2002;Katsuno et al., 2003) reveals the importance of protein domains other than the polyQ stretch for neurodegeneration. The lack of neuronal dysfunction or degeneration in the shortstop mouse indicates that other portions of huntingtin, not present in the 117 amino acid shortstop transgenic protein are required for the initiation of the toxic cycle and the ensuing degeneration. These domains may be responsible for protein-protein interactions or protein modifications which play a role in the normal functioning of huntingtin and are altered in the disease, causing increased susceptibility of the neuron to apoptotic death. Huntingtin, like ataxin-1 is phosphorylated by Akt (Humbert et al., 2002;Rangone et al., 2004) on serine 421, a residue not present in the shortstop mouse, and it remains to be seen what effect phosphorylation or other protein modifications of the mutant protein have on the development of a neurodegenerative phenotype. Huntingtin interacts with many different proteins in the cell, suggesting huntingtin may play a role in endocytosis and cellular trafficking, cellular survival and transcription as described in Section 1.6 (Harjes and Wanker, 2003). The shortstop protein does not interact with H i p l , H a p l or H i p l 4 by yeast two hybrid (Haigh and Hayden, unpublished data) and likely does not interact with many of full-length huntingtin's interacting partners. This data reveals that the shortstop fragment cannot cause alterations in endocytosis and/or transcription and/or transport through altered interactions with huntingtin's interacting proteins, alterations which may ultimately lead to neuronal stress and 'prime' the neuron for degeneration. The importance of other portions or domains of huntingtin in HD-related neurodegeneration was recently demonstrated in a study of susceptibility to excitotoxicity, a mechanism which has long been thought to underlie the selective neuronal loss in H D (Cepeda et al., 2001;Zeron et al., 2001;Zeron et al., 2002;Zeron et al., 2004). Zeron et al. in 2001 found that cells transfected with full-length mutant huntingtin were much more susceptible to NMDA-media ted excitotoxicity than cells transfected with a truncated 548 amino acid N -terminal fragment of huntingtin, indicating the necessity for full-length huntingtin (or a fragment longer than the N-terminal fragment) in increased susceptibility to excitotoxicity (Zeron et al., 2001). Other mouse models expressing truncated fragments of huntingtin have been described in the literature. The R6/2 mouse model (Mangiarini et al., 1996) expresses 75 exon 1 (67 amino acids) of human huntingtin under 1 kb of upstream promoter with polyglutamine sizes ranging from 145 to over 200. The N171 mice express a huntingtin fragment of 171 amino acids under the prion promoter with 82 polyglutamines (Schilling et al., 1999;Schilling et al., 2001). Both of these mouse models exhibit a rotarod deficit and an early death phenotype, in contrast to what is observed in the shortstop mice. While the exact reason behind the differences in the phenotypes of these huntingtin fragment mouse models remains unknown, the many differences between the three models may provide an explanation. The transgenic huntingtin fragment in the shortstop mouse is expressed under 24 kb of upstream endogenous promoter and regulatory sequences compared to only 1 kb of endogenous promoter in the R6/2 and the non-endogenous prion promoter in the N171 mouse, indicating that transgenic protein expression is most appropriate in the shortstop mouse. It is hypothesized that the phenotypes in the R6/2 and N171 mice may be due to improper regulation and expression of the transgene which leads to non-specific polyglutamine toxicity, since the phenotypes in these models are similar to phenotypes observed in a mouse model with expanded C A G s knocked into the Hprt gene, a gene without an endogenous C A G tract (Ordway et al., 1997;Tallaksen-Greene et al., 2003). The appropriate expression of the shortstop mouse may therefore prevent the initiation of mechanisms which lead to non-specific polyglutamine toxicity that is unrelated to H D pathogenesis. It is l ikely that the large polyQ repeat in the R6/2 mouse of 145 to over 200 and the potential overexpression of the transgene further contribute to polyglutamine toxicity in this model. In addition, the background strain of the three models is different with the R6 lines on a mixed B 6 / C B A strain, the N171 on a C H 3 / B 6 strain, and the shortstop on an F V B strain and non-specific polyglutamine toxicity may not be penetrant on the F V B strain. 76 Chapter 5: The YAC128 Exhibits Deficits in PPI, Cognition and Neuronal Degeneration on the B6 Strain The data in this chapter is unpublished. 77 Preface I designed all of the experiments and analyses presented within the chapter and performed all with the exception of that noted below. Rebecca Devon backcrossed the Y A C 128 onto the B6 strain. Sazzad Hossain performed the home cage activity and pre-pulse inhibition analysis. Nagat Bissada assisted with the colony management and performed the mouse perfusions. Dr. X J . L i kindly provided the E M 4 8 antibody. Dr. Elizabeth Simpson provided advice and behavioural expertise. 78 5.1 Introduction Huntington disease (HD) results from the expansion of a C A G repeat in the gene encoding the protein huntingtin, resulting in an expanded polyglutamine tract in the first exon of huntingtin (1993). Most individuals have polyglutamine tracts of 10-35 while H D results from a polyglutamine stretch of over 36 (Brinkman et al., 1997;Langbehn et al., 2004). The presence of an expanded tract over 41 C A G s in length is fully penetrant i.e. the patient wi l l experience the onset of H D related symptoms in their lifetime. The age when a patient experiences the first signs of H D , termed onset, correlates inversely with the polyglutamine repeat length i.e. the longer the polyglutamine repeat length, the earlier the onset (1993;Brinkman et al., 1997;Langbehn et al., 2004). Approximately 70% of the variability in age of onset is accounted for by the length of the polyglutamine tract (Rosenblatt et al., 2001), leaving 30% of the variability in age of onset due to modifying genes and/or environmental factors. Different strategies have been employed to discover genes that modify the age of onset in H D , for the purpose of discovering pathways that are involved in H D pathogenesis and potentially targeting those pathways for therapeutics. Studies of age of onset modifiers in H D patients have indicated potential roles for the GluR6 kainate receptor locus (Rubinsztein et al., 1997;MacDonald et al., 1999), a ubiquitin carboxy-hydrolase (Naze et al., 2002), apolipoprotein E (Panas et al., 1999) and the length of the C A G repeat in the "normal" allele (Djousse et al., 2003). More recently, a genome scan for modifiers using H D patient sibpairs found strong evidence for modifiers on human chromosome 4 and 6 (L i et al., 2003a). A n alternate method for discovering genetic modifiers of age of onset in H D is to perform quantitative trait loci (QTL) mapping in mouse models of H D . Q T L mapping relies on having at least two background strains of mice, one susceptible to the phenotype and one resistant. The resistant and susceptible strains are bred together and the resulting progeny of the F2 generation analysed both by phenotype and genotype by Q T L mapping. The "resistant" mice in the F2 generation should share the identical genomic region of the parental resistant strain where the modifier gene resides. This method has been utilized to identify potential modifiers of tumourigenesis (Dragani, 2003), 79 atherosclerosis (Smith et al., 2003), insulin resistance (Kido et al., 2000), and modifiers of the neurodegenerative disorder A L S (Kunst et al., 2000). One prerequisite of Q T L mapping studies with mouse models is the availability of background strains which are both susceptible and resistant to the resulting phenotypes. The Y A C 128 mouse model of H D is con genie on the F V B / N strain and exhibits both behavioural and neuropathological phenotypes on this strain, indicating the F V B / N strain is susceptible to the effects of the Y A C transgene. The Y A C 128 mice were backcrossed onto the C57B1/6 (hereafter referred to as B6) strain of mice, in anticipation that this strain may be resistant to the effects of the transgene. The B6 mouse strain has demonstrated resistance to a wide variety of brain insults including the knock-out of caspase-3, which causes embryonic lethality in other strains of mice due to inadequate apoptosis of neurons during development (Leonard et al., 2002), resistance to cone photoreceptor neuronal degeneration caused by the targeted disruption of the rhodopsin gene (Humphries et al., 2001) and resistance to the onset of an A L S phenotype caused by a SOD1 mutation (Kunst et al., 2000). In addition, an increasing body of evidence indicates a role for excitotoxic cell death in the striatal specific neuron loss observed in H D (Cepeda et al., 2001;Zeron et a l , 2001;Zeron et al., 2002;Zeron et al., 2004). Injection of kainic or quinolinic acid, excitotoxins that cause a high degree of neuronal loss in F V B / N mice, results in a much lower degree of cell death in B6 mice, indicating this strain is more resistant to excitotoxic cell death (Schauwecker and Steward, 1997;Schauwecker, 2002;Schauwecker, 2003) and therefore may be resistant to the H D phenotype. To determine whether the B6 strain would be resistant to the HD-related phenotypes exhibited by the Y A C 128 mouse model, and therefore a good candidate for modifier mapping, the Y A C 128 mice were backcrossed for five generations onto the B6 background (N5). Analysis revealed that the neuropathological endpoints of brain weight and striatal volume decrease were present at 12 and 18 months of age in the B 6 - Y A C 1 2 8 mice. However, these phenotypes were less severe in the B 6 - Y A C 1 2 8 s compared to the F V B - Y A C 1 2 8 s at the same age, indicating that the neuropathological phenotypes in the B 6 - Y A C 1 2 8 mice are delayed. Both F V B - Y A C 1 2 8 s and B 6 - Y A C 1 2 8 s demonstrate 80 impairment in pre-pulse inhibition and habituation, further revealing the accurate recapitulation of the human condition in this model. 5.2 Results 5.2.1 The Y A C 1 2 8 mice on a B6 background exhibit selective brain weight loss. The highest expressing Y A C 1 2 8 mouse, line 53, was backcrossed onto the B6 background for five generations (N5), at which point the B 6 - Y A C 1 2 8 were approximately 97% congenic on the B6 background. Cohorts of B 6 - Y A C 1 2 8 and wi ld-type littermates at N5 were sacrificed, perfused and brain weight was measured at 12 and 18 months of age, timepoints when F V B - Y A C 1 2 8 display significant decreases in brain weight (Slow et al., 2003). A t 12 months of age, B 6 - Y A C 1 2 8 mice demonstrate a 6% decrease in mean brain weight compared to wild-type littermates (WT= 0.3894 ± 0.02 g, B 6 - Y A C 1 2 8 = 0.3646 ± 0 . 0 1 g, p<0.05), progressing to a 10% decrease in 18 month old B 6 - Y A C 1 2 8 s (WT= 0.3951 ± 0.02 g, Y A C 1 2 8 = 0.3603 ± 0.01 g, p<0.01) (Fig. 5.1A). 0.3 12 18 Age (months) B 0.07 -i 3 0.06 -0.05 -O) 1 0.04 " E 0.03 -"aj A 0.02 -<u <u O 0.01 -• WT • YAC 128 n=8| n=5 12 18 Age (months) Figure 5.1: Brain weight is selectively decreased in B6-YAC128 mice. A. Perfused brain weight (including cerebellum) was compared between B6-YAC128, line 53 and wild-type littermates at 12 (n=8 WT, n=6 B6-YAC128) and 18 (n=5 WT, n=7 YAC128) months of age. Brain weight was significantly decreased in 12 month B6-YAC128 (p<0.05, *) and in 18 month old B6-YAC128s (p<0.01, **) by Student's T-test. Mean brain weight ± standard deviation of the groups is shown. B. Cerebellum weight is not decreased in B6-YAC128s. The cerebella were removed and weight was measured. There was no significant difference at any time point. Similar to what was demonstrated in F V B - Y A C 1 2 8 mice, the difference in brain weight did not appear to be due to generalized atrophy, but rather a region specific effect. No significant difference was detected between Y A C 1 2 8 s and wild-type littermates in the weight of the cerebellum, a region not usually involved in FfD pathology (Harper et al., 2002), at 12 or 18 months of age (Fig. 5.1B). 82 5.2.2 Striatal volume and neuronal number are decreased in B6-YAC128. The striatum is the brain region most affected in H D patients. F V B - Y A C 1 2 8 mice exhibit decreases in striatal volume beginning at 9 months of age. To determine i f this brain region is affected in B 6 - Y A C 1 2 8 , striatal volume estimates of the 12 and 18 month cohorts of B 6 - Y A C 1 2 8 mice were calculated using stereological software. A t 12 months of age, an 8% decrease in striatal volume was present in B 6 - Y A C 1 2 8 mice (WT= 12.95 ± 0.82 mm 3 , B 6 - Y A C 1 2 8 = 11.87 ± 0.42 mm 3 , p<0.05) (Fig. 5.2). The decrease in striatal volume was progressive, with 18 month old B 6 - Y A C 1 2 8 mice exhibiting a 15% decrease compared to wild-type controls (WT= 13.24 ± 0.92 mm 3 , B6 -YAC128= 11.63 ± 0.53 mm 3 , p<0.01) (Fig.5.2). Figure 5.2: Striatal volume is decreased in B6-YAC128 mice. Perfused brains were cut coronally into 25 \im sections throughout the striatum. Every eighth section was immuno-stained and Stereoinvestigator software was used to trace and calculate volume. B6-YAC128s showed significant decrease in striatal volume at 12 months (n=8 WT, n=6 B6-YAC128, p<0.05 *) and 18 months of age (n=5 WT, n=7 B6-YAC128, p<0.01, **) by Student's T-test. Mean volume ± standard deviation for each group is shown. 12 18 Age (months) Striatal neuronal count is significantly decreased in F V B - Y A C 1 2 8 mice beginning at 12 months of age, accurately recapitulating the defining neuropathological characteristic of H D (Vonsattel et al., 1985). Stereological software was used to estimate the striatal neuron number in the 12 month old B 6 - Y A C 1 2 8 . Sections were immuno-stained with NeuN, an antibody specific for a neuronal nuclear protein, which is present in most neuronal cell types (Mullen et al., 1992). Table 5.1: Neuronal decrease in B6-YAC128 mice at 12 months of age. Stereological software was used to assess the number of NeuN stained striatal neurons in serial sections of B6-YAC128 vs. B6 wild-type littermates. B6-YAC128 demonstrated a decrease in number of neurons at 12 months of age which did not reach significance. Age (months) B 6 - W T B 6 - Y A C 1 2 8 Percentage Significance N=7 N=6 Decrease 12 1640000 ± 61660 1513000 ± 5 8 4 8 0 8 NS,p=0.17 83 A t 12 months of age, B 6 - Y A C 1 2 8 mice exhibited a decrease of 8% in striatal neuron count compared to wild-type littermates although this trend did not reach significance (p=0.1, n=7 W T , n=6 B 6 - Y A C 1 2 8 ) (Table 5.1). 5.2.3 Rotarod and home cage activity i n B6-YAC128 compared to FVB-YAC128. The F V B - Y A C 1 2 8 exhibit a rotarod deficit beginning at 6 months of age (Slow et al., 2003). The B 6 - Y A C 1 2 8 mice were analysed on an accelerating rotarod at 12 months of age to determine i f they manifest a similar deficit. Because mouse behaviour testing can be affected by many uncontrollable factors, including time of year, status of animal facility etc., it is difficult to draw conclusions between studies conducted at different times, therefore an F V B - Y A C 1 2 8 cohort with wild-type littermates at 12 months of age were included for comparison. A s demonstrated previously, the F V B - Y A C 1 2 8 exhibited a significant deficit in rotarod performance compared to wild-type littermates (n=10 W T , n=14 F V B - Y A C 1 2 8 , p<0.05, Fig . 5.3). Surprisingly, B 6 - Y A C 1 2 8 mice exhibit no rotarod deficit at 12 months of age compared to wild-type littermates (n=18 W T , n=17 B 6 - Y A C 1 2 8 ) . While it seems that the B 6 - Y A C 1 2 8 mice are resistant to the rotarod deficit, it may be that the rotarod accelerated to the maximum speed (45 rpm) in too short a time frame (2 minutes) to reveal a more subtle effect in the B 6 - Y A C 1 2 8 mice. Other studies have shown that robust rotarod differences disappear when the time to maximum acceleration is reduced (Hossain et al., 2004) Figure 5.3 Rotarod deficit is dependent on strain in YAC128 mice. FVB-YAC128 (n=14) and wild-type FVB (n=10) littermates along with B6-YAC128 (n=17) and wild-type B6 (n=18) littermates were tested in 3 trials on an accelerating rotarod at 12 months of age. The rotarod accelerated from 0-45rpm in 120 seconds. The FVB-YAC128 demonstrated a deficit at this timepoint (p<0.05, *) while the B6-YAC128 exhibited no significant difference in rotarod performance using this protocol. Mean time spent on rotarod for each group is plotted and error bars represent SEM. Spontaneous locomotor activity was measured in both the B 6 - Y A C 1 2 8 and F V B -Y A C 1 2 8 in the home cage environment over 24 hours. The F V B - Y A C 1 2 8 males 84 exhibited reduced activity, compared to control littermates (n=4), which did not reach significance. The F V B - Y A C 1 2 8 females were significantly hypoactive compared to controls (n=4, p<0.01)(Fig. 5.4 A , B ) . In contrast, both the B 6 - Y A C 1 2 8 males (n=4, p<0.01) and B 6 - Y A C 1 2 8 females (n=4, p<0.01) were hypoactive compared to control littermates (Fig. 5.4 C,D) . This study indicates that the hypoactivity phenotype is more penetrant on the B6 strain compared to the F V B strain since both male and female B6-Y A C 1 2 8 demonstrate significant hypoactivity, although there is a clear trend in the F V B males. FVB Males c 3000 -I VI 2500 -a 2000 -m am 1500 -» m 1000 -ra t> 1- 500 -B6 Males - **p<0.01 • WT»YAC128 FVB Females *p<0.01 B6 Females **p<0.01 Figure 5.4: YAC128 mice exhibit hypoactivity at 12 months of age. A, B. Home cage activity was assessed in FVB-YAC128 males (n=4) (A) and females (B) (n=4) with control FVB (WT) males (n=4) and females (n=4) over 24 hours. C, D. Home cage activity was assessed in B6-YAC128 males (n=4) (C) and females (D) (n=4) with control B6 (WT) males (n=4) and females (n=4) over 24 hours. The mean measurement for each group is plotted and error bars represent SEM. Significant differences in group means by T-test are represented by * and corresponding P values. 5.2.4 YAC128 on B6 and FVB exhibit similar deficits in pre-pulse inhibition and habituation Pre-pulse inhibition (PPI) is the phenomenon where a weak pre-pulse stimulus attenuates the response to a subsequent startling stimulus. Patients with H D demonstrate impaired PPI of both the acoustic and tactile startle response (Swerdlow et al., 85 1995;Munoz et al., 2003), indicating a disruption of sensorimotor gating in the striatum. Both a mouse model of H D (Carter et al., 1999) and an acute rat model of H D have shown deficits in PPI (Kodsi and Swerdlow, 1997) and it was of interest to determine whether the Y A C 128 mice also exhibit impaired PPI. The acoustic startle response is the measure of the motor reflex response to a loud noise (startle), and this reflex is reduced (inhibited) when a low level acoustic pulse is presented before the startle. F V B - Y A C 1 2 8 and B 6 - Y A C 1 2 8 were examined with control littermates at 12 months of age using the acoustic startle response to examine PPI. The magnitude of the startle response at 120 decibels (db) was significantly reduced in the F V B - Y A C 1 2 8 mice compared to control littermates (n=8, p<0.01, Fig . 4.5A). The B 6 - Y A C 1 2 8 mice exhibited a similar magnitude of response to the startle stimulus when compared to control littermates (n=8, F ig . 4.5B). ^ 700 •g 600 .-i 500 c % 400 S 300 r. 200 CO W 100 0 FVB-YAC 128 **p<0.01 • Wild-type • YAC128 B 700 v •a 600 | 500 gMoOH | 300-I r 200 ro 35100 B6-YAC128 • Wild-type • YAC 128 Figure 5.5: Startle magnitude in YAC128 on an FVB and B6 background. The magnitude of response to a startle stimulus of 120 db was measured in FVB-YAC128 compared to control FVB littermates (n=8 mice/group) and B6-YAC128 compared to control B6 littermates (n=8 mice/group). The mean measurement for each group is plotted and error bars represent standard deviation. Significant differences in group means by T-test are represented by * and corresponding P values. The amount of PPI was assessed as the percentage reduction of the baseline startle response and is obtained using four different levels of pre-pulse including 2, 4, 8 and 16 db pre-pulse above background. The F V B - Y A C 1 2 8 mice exhibited significant impairment of PPI with a pre-pulse of 4, 8 and 16 db (n=8, p<0.05, F ig . 5.6A) while the B 6 - Y A C 1 2 8 mice exhibited impairment with a pre-pulse of 16 db (n=8, p<0.05, Fig . 5.6B). This result demonstrates that Y A C 128 mice on both strain backgrounds accurately recapitulate the PPI impairment demonstrated in human H D patients 86 (Swerdlow et al., 1995;Munoz et al., 2003). Furthermore, this phenotype is present in both strains of mice, indicating the phenotype is robust on multiple background strains. A 100 • 80 -§ 3 60 -£ 40 -ss 20 -o • FVB-Prepulse Inhibition *p<( rfi 0.05 *p<0.05 *p<0.0Sl ff l • WT • YA3128 PP2 PP4 PP8 . PP16 Prepulse intensity PP2 PP4 PP8 PP16 Prepulse Intensity C 900 <„ 800 % 600 g> 500 S 400 g 300 200 100 0 FVB-Habituation to • WT • YAC128 *p<0.05 1 Block Figure 5.6: Impairments in pre-pulse inhibition and habituation in YAC128 on the B6 and FVB background. Pre-pulse inhibition of the startle response to a 120 db pulse was measured using a pre-pulse of 2 db (PP2), 4 db (PP4), 8 db (PP8) and 18 db (PP16) above background noise level in FVB-YAC128 (A) and B6-YAC128 (B). The magnitude of response to the 120 db startle is significantly reduced during the course of the testing in the FVB-WT (C) and B6-WT (D) demonstrating the property of habituation. FVB-YAC128 (C) and B6-YAC128 do not demonstrate habituation. The mean measurement for each group is plotted (n=8 mice/group) and error bars represent SEM. Significant differences in group means by T-test are represented by * and corresponding P values. The PPI data can be further analyzed to determine i f the Y A C 1 2 8 mice demonstrate deficits in habituation. Habituation is the decline of a response due to repeated exposure to the stimulus and is a measure of cognitive impairment. Cognitive impairment is one of the cardinal features of H D and includes memory and judgment decline, disorientation and intellectual impairment (Harper et al., 2002). Habituation as a marker of cognition can be measured in the Y A C 128 mice using the data obtained from the PPI experiment. The mice are exposed to the startle stimulus 11 times in succession over the course of the test and display a lower magnitude of startle response at late startle stimuli compared to early startle stimuli due to habituation. The F V B and B6 wild-type mice exhibit a significantly decreased magnitude of response to the last startle stimuli compared to the first (n=8, p<0.05, Fig . 5.6C, D) indicating habituation to the startle 87 stimulus. In contrast, the F V B - Y A C 1 2 8 and B 6 - Y A C 1 2 8 show no significant difference between the magnitude of the response to the final startle stimuli and the first startle stimuli (n=8, Fig . 5.6C, D) , indicating an impairment in habituation, and revealing the first indication that the Y A C 1 2 8 mice have deficits in cognitive function, similar to human H D patients. It should be noted that the difference in the magnitude of the startle response between the F V B - Y A C 1 2 8 and F V B wild-type mice makes habituation more difficult to assess in the F V B - Y A C 1 2 8 , i.e. do the F V B - Y A C 1 2 8 fail to habituate or do they fail to respond to the startle stimuli at a magnitude where habituation can be assessed? 5.2.5 Inclusions are similar in the B6-YAC128 and F V B - Y A C 128 mice. Both H D patients (DiFigl ia et al., 1997;Gutekunst et al., 1999) and mouse models (Davies et al., 1997;Wheeler et al., 2000), including the Y A C 1 2 8 mouse model (Slow et al., 2003) demonstrate intranuclear, neuronal aggregates or inclusions of huntingtin. A truncated huntingtin mouse model, the R6/1 model, demonstrates differential time-course of huntingtin inclusion formation depending on the background strain of the mice (Manley et al., 2003). To determine whether the Y A C 1 2 8 demonstrated differential development of huntingtin inclusions based on strain background, sections from both F V B - Y A C 1 2 8 s and B 6 - Y A C 1 2 8 s at 12 and 18 months were immunostained with E M 4 8 , an antibody targeted to the N-terminus of huntingtin and highly specific for aggregates (Gutekunst et al., 1999). Immunostaining revealed inclusions in both F V B - Y A C 1 2 8 and B 6 - Y A C 1 2 8 striatum at 18 months of age (Fig. 5.7B, D) . A t 12 months of age a low percentage of cells in the striatum (<1%) exhibited tiny huntingtin aggregates in both the F V B - Y A C 1 2 8 s and B 6 - Y A C 1 2 8 s (Fig. 5.7A, C) . These results indicate that the time-course of inclusion formation and the morphology of the inclusions in the Y A C 1 2 8 on the B6 and F V B backgrounds was similar, indicating that potential genetic modifiers in the two strains do not differentially affect the formation of inclusions. 88 Figure 5.7: B6-YAC128 exhibit huntingtin inclusions. Striatal neurons from FVB-YAC128 (B) and B6-YAC128 (D) exhibit clear huntingtin inclusions at 18 months of age by EM48 staining (long arrows). At 12 months of age, a low percentage of striatal neurons (<1%) from FVB-YAC128 (A) and B6-YAC128 (C) demonstrate small huntingtin inclusions (short arrows). Scale bar is shown in B and is 10 pm. 5.2.6 La te r onset of neurodegeneration i n B 6 - Y A C 1 2 8 compared to F V B - Y A C 1 2 8 The neurodegenerative phenotypes of brain weight, striatal volume and striatal neuron count present in the F V B - Y A C 1 2 8 are penetrant on the B6 background. However, by comparing the B 6 - Y A C 1 2 8 data with previous data collected from the study of the F V B - Y A C 1 2 8 mice (Slow et al., 2003), an interesting result emerges. Table 5.2 summarizes the results from the F V B - Y A C 1 2 8 mice at 12 months demonstrated previously (Slow et al., 2003), with the results of the present study of the B 6 - Y A C 1 2 8 at 12 months. For every neuropathological endpoint examined, F V B - Y A C 1 2 8 demonstrated a lower % decrease compared to B 6 - Y A C 1 2 8 at the same age. For example, striatal volume is decreased by 16% in 12-month old F V B - Y A C 1 2 8 mice, while the B 6 - Y A C 1 2 8 mice exhibit an 8% decrease in striatal volume at the same time point. The most striking difference between the two backgrounds is illustrated by the difference in striatal neuronal counts. In the F V B - Y A C 1 2 8 , the neuronal counts are significantly decreased at 12 months of age, while in the B 6 - Y A C 1 2 8 , the neuronal counts are decreased by 8% but have not reached significance. The F V B - Y A C 1 2 8 89 exhibited a 9% decrease at 9 months of age which did not reach significance, similar to the result seen in the B6-YAC128 mice at 12 months of age. These results reveal that although the B6-YAC128 mice manifest the neuropathological phenotypes present in the FVB-YAC128, the phenotypes may be delayed in onset in the B6-YAC128 mice, indicating resistance of the B6 strain to the effects of the transgene. Table 5.2: Summary of the neuropathological and behavioural phenotypes exhibited in the FVB-YAC128 and B6-YAC128 mice at 12 months of age. The mean measurement for each group is shown with standard deviation for brain measurements and SEM for behavioural measurements. Student's T-test was used to determine significance. *Male and female data was pooled for home cage activity. Phenotype Strain WT YAC128 -9% P-value Brain Weight (g) FVB 0.411± 0.01 N=7 0.373± 0.02 N=7 p<0.01 B6 0.3894 ± 0.02 N=8 0.3646 ±0.01 N=6 -6.4% ' p<0.05 Striatal Volume (mm3) F V B 11.81 ±0.36 N=7 9.88 ± 0.33 N=7 -16% p<001 B6 12.95 ± 0.82 N=8 11.87 +0.44 N=6 ..- 8.4% p<0.05 Striatal Neuronal Count FVB 1724000± 123127 N=6 1504000 ± 66870 N=8 -13% p<0.01 \ 136 1640000+ 103141 N=7 1513000± 143238 N=6 NSp=().l Inclusions FVB Not present Microaggregates 12 m, inclusions 18m - -B6 Not present Microaggregates 12 m, inclusions 18m Rotarod FVB 50.70 ± 7.974 N=10 27.75 ± 4.592 N=14 -45% p=0.()l B6 58.05 ± 3.622 N=18 57.96 ± 4.827 N=17 -.02'r N.S p=0.99 Home Cage Activity* FVB 3300 ± 958.0 (n=8) 1880 ±1017(n=8) -43% p<0.05 B6 3842± 1198(n=8) 2429.5 ± 884 (n=8) -37% p<0.05 Startle Response -PPI FVB - Deficits at pp4, pp8, ppl6 - p<0.05 B6 - Deficits at ppl6 : p<0.05 Startle Response -Habituation FVB Significant habituation No habituation - -B6 Significant habituation No habituation - V Behaviourally, the B6-YAC128 mice seem resistant to the rotarod deficit but susceptible to the hypoactivity phenotype, the PPI deficit and the habituation impairment demonstrated in the FVB-YAC128. This finding indicates that there are potentially different pathways which lead to rotarod impairments and both hypoactivity and startle response deficits in the Y A C 128 model; furthermore the B6 strain demonstrates resistance to the pathway which leads to rotarod deficits. It will be interesting to examine 90 the behavioural impairments in the F V B and B6 Y A C 1 2 8 mice overtime to discern i f the B 6 - Y A C 1 2 8 manifest behavioural abnormalities later in the natural history of disease in this strain than the F V B - Y A C 1 2 8 as would be hypothesized from the neuropathological findings. Despite the apparent delay in the manifestation of neurodegeneration in the B6-Y A C 1 2 8 mice compared to the F V B - Y A C 1 2 8 mice, the formation of huntingtin inclusions is identical in the B 6 - Y A C 1 2 8 and F V B - Y A C 1 2 8 . This result indicates that inclusion formation is not correlated with neurodegeneration in the Y A C 1 2 8 mouse model. 91 5.3 Discussion Seventy percent of the variability in age of onset of symptoms in FJD patients can be accounted for by the C A G repeat length (Rosenblatt et al., 2001), leaving thirty percent of age of onset variability due to other environmental or genetic factors. One method to determine genetic factors involved in age of onset variability is the use of Q T L mapping of H D mouse models on multiple inbred strains, with one strain susceptible to the HD-related phenotypes and one resistant. The Y A C mouse model with 128 C A G repeats exhibits both behavioural and neuropathological phenotypes, including selective striatal neuronal loss on an F V B strain background, indicating the F V B strain is susceptible to the effects of the transgene. In the quest to reveal a strain that was resistant to the effects of the Y A C 128 transgene, the F V B - Y A C 1 2 8 was backcrossed for five generations onto the B6 strain. The results of the characterization of the B 6 - Y A C 1 2 8 indicate that the HD-related phenotypes exhibited by the F V B - Y A C 1 2 8 are penetrant on the B6 background. If the neuropathological phenotypes were truly related to the pathogenic effects of the H D Y A C transgene, they should be penetrant on multiple backgrounds, since a polyglutamine expansion of over 41 repeats is fully penetrant in genetically heterogeneous human populations. However, it appears that the neuropathological phenotypes of brain weight, striatal volume and striatal neuronal count decreases are less severe at the same 12 month timepoint in the B 6 - Y A C 1 2 8 compared to the F V B - Y A C 1 2 8 . The striatal neuronal count decrease phenotype is particularly interesting since the magnitude of decrease and the accompanying significance level in the 12 month-old B 6 - Y A C 1 2 8 mice is identical to that observed in the 9 month old F V B - Y A C 1 2 8 mice, indicating that the B 6 - Y A C 1 2 8 exhibit a delay of onset of this particular phenotype compared to the F V B - Y A C 1 2 8 . This result is in agreement with the original hypothesis that the B6 strain would be resistant to the effects of the transgene. This hypothesis was based on considerable evidence from experiments with various excitotoxins demonstrating that the F V B strain exhibits significant cell death when exposed to the excitotoxins kainic acid or quinolinic acid, while the B6 strain exhibits a very low percentage of cell death and in fact seems resistant to neuronal death caused by excitotoxicity (Schauwecker and Steward, 92 1997;Schauwecker, 2002;Schauwecker, 2003). Increased susceptibility to excitotoxicity has long been thought to underlie the cell specificity of H D , with the initial observation that injection of excitotoxins into rat striatum targeted the same cells as those susceptible in H D (Coyle and Schwarcz, 1976;McGeer and McGeer, 1976). More recent evidence has demonstrated increased susceptibility of striatal neurons to the excitotoxin N M D A in striatal neuronal cultures established from Y A C 1 2 8 PO pups (Zeron et al., 2002;Zeron et al., 2004), suggesting that mutant huntingtin's effects on increasing susceptibility of striatal neurons to excitotoxicity is present at birth in the organism. The delay in onset of the neuropathological phenotype in the Y A C 128 mouse on a B6 background compared to an F V B background further supports a role for excitotoxicity in the pathogenesis of the disorder. The discovery that age of onset in the B 6 - Y A C 1 2 8 is delayed may allow for the use of the B6 strain as the 'resistant' strain for modifier mapping using Q T L analysis. Clearly the neuronal count decrease phenotype seems delayed in the B 6 - Y A C 1 2 8 mice compared to the F V B - Y A C 1 2 8 mice and it may be possible to use this as a phenotypic outcome measure in Q T L mapping at 12 months of age, when the F V B - Y A C 1 2 8 demonstrate significant loss while the B 6 - Y A C 1 2 8 only exhibit a trend. However this phenotype is one of the more variable of the neuropathological phenotypes, necessitating a larger number of animals to detect a significant difference compared to some of the other phenotypes (Table 3.2), which wi l l in turn require even greater numbers of animals for Q T L mapping. The magnitude of decrease in the striatal volume of the B 6 - Y A C 1 2 8 is one half that of the F V B - Y A C 1 2 8 at 12 months of age (Table 5.2). This result indicates that the age of onset for this phenotype in the B 6 - Y A C 1 2 8 may also be delayed. Significant striatal volume decreases are first evident at nine months of age in the F V B -Y A C 1 2 8 and the data from this study would suggest that the striatal volume may not be significantly decreased in the B 6 - Y A C 1 2 8 at this timepoint. The striatal volume decrease is a robust phenotype requiring few animals to note a significant difference (Table 3.2) and would therefore require significantly less animals for Q T L mapping. A detailed study of the B 6 - Y A C 1 2 8 at 9 months of age is required before pursuing modifier mapping using this phenotype, but this is a promising endpoint. Finally, the B 6 - Y A C 1 2 8 mice exhibit no significant rotarod difference using a protocol of acceleration from 0 to 93 45rpm in 120s, while the F V B - Y A C 1 2 8 mice demonstrate a clear deficit, indicating that the B 6 - Y A C 1 2 8 are resistant to this behavioural phenotype, revealing another promising endpoint to assess for QTL-mapping. While the results show that the B 6 - Y A C 1 2 8 exhibit a delayed age of onset for the neuropathological phenotypes, it is clear that both the neuropathological and neurodegenerative phenotypes (exhibited by the behavioural deficits) are still penetrant on the B6 strain background. The susceptibility of the B6 strain to the effects of the Y A C 1 2 8 transgene has benefits for the future use of the Y A C 1 2 8 mice. The F V B strain was originally chosen as the background for the Y A C 1 2 8 due to the ability to immediately create transgenic mice on a fully congenic background, eliminating the need to backcross for multiple generations to congenic status before assessing the phenotype. While the F V B mice display excellent fertility and fecundity (Silver, 1995), and are clearly susceptible to excitotoxic cell death (Schauwecker and Steward, 1997;Schauwecker, 2002;Schauwecker, 2003) and the effects of the Y A C transgene (Hodgson et al., 1999;Slow et al., 2003), there are some disadvantages to using this strain of mice. The F V B strain carries a retinal degeneration mutation due to interruption of the Pdeb gene (Gimenez and Montoliu, 2001). The presence of retinal degeneration in the F V B strain does not affect the significance of the HD-related phenotypes present in the F V B - Y A C 1 2 8 since the control animals are always F V B littermates, and therefore also possess the retinal degeneration mutation. However, the retinal degeneration mutation is a disadvantage for further behavioural characterization of the Y A C 128 mice on the F V B background. H D patients present with cognitive dysfunction in learning and memory (Hayden, 1981;Harper et al., 2002) and it is important to determine i f cognitive impairment is accurately recapitulated in the Y A C 128 mouse, and i f so, determine how this phenotype can be utilized as an endpoint for therapeutic trials. Many of the behavioural tests which can determine cognitive deficits in mice rely on visual cues and are inappropriate to use with a visually impaired strain. In addition, analysis of the F V B strain in parallel with other mouse strain backgrounds indicates that, relative to other mouse strains (including the B6), the F V B show impaired learning (Crawley, 2000), making it difficult to discern learning and memory deficits in transgenic mice on this background strain. In contrast, the B6 strain perform well on learning and memory tasks 94 and are not visually impaired, making the B 6 - Y A C 1 2 8 an important tool for studying cognitive impairments in the Y A C 128 mice. Another potential use for the B 6 - Y A C 1 2 8 is in intercrossing experiments to test whether certain knock-outs or transgenic mice can have an impact on the phenotypes of neuronal dysfunction and neuronal degeneration. Intercrossing of multiple transgenics have been employed to tease out various pathways believed to be involved in disease pathogenesis, and are a more direct, targeted way to discern modifiers of phenotype and provide information concerning potential therapeutic targets. This strategy has been employed in H D research to test the importance of transglutaminase (Mastroberardino et al., 2002) and heat shock proteins (Hansson et al., 2003) in a mouse model, and histone deacetylases (Steffan et al., 2001) and SUMOyla t ion in a Drosophila model (Steffan et al., 2004). One disadvantage of the F V B - Y A C 1 2 8 for intercross experiments is that few transgenic mouse models of interest are congenic on the F V B background strain. This necessitates the backcrossing of the transgenic mice of interest to the F V B strain background for at least 5 generations (approximately one year time investment) in order to prevent the possibility of strain effects in the final analysis. Many transgenic mice of interest are maintained on a B6 background and can therefore be directly crossed to the B 6 - Y A C 1 2 8 mice and the phenotype of the progeny assessed. It is of the utmost importance when modeling a human disease to ensure that the model accurately recapitulates the human disorder. The Y A C 128 model has previously demonstrated the accurate recapitulation of the neuropathological changes of brain weight, striatal volume and striatal neuronal count decreases demonstrated in human H D patients along with motor deficits and changes in activity profile over time that accurately models the natural history of the human disorder. In the present study, the Y A C 1 2 8 model further displays the accurate recapitulation of the human disease due to the presence of pre-pulse inhibition impairments in both the F V B - Y A C 1 2 8 and the B 6 -Y A C 1 2 8 . Impairments in pre-pulse inhibition have been described in two separate studies of patients with H D (Swerdlow et al., 1995;Munoz et al., 2003). PPI impairments indicate a deficit in sensorimotor gating or an impairment of the process by which inhibitory neural pathways filter multiple stimuli and allow attention to be focused on one stimulus, a critical function of the striatal G A B A e r g i c pathways (Swerdlow et al., 1995). 95 The existence of PPI deficits in the Y A C 1 2 8 mice both recapitulates the human disorder, while also offering further evidence of the presence of neuronal dysfunction in the Y A C 128 mice. It wi l l be interesting to determine when deficits in PPI first occur in the natural history of disease in the Y A C 128 model and whether PPI could be utilized as a therapeutic endpoint against which to test the efficacy of therapeutics both on neuronal dysfunction and the rescue of sensorimotor gating deficits. Further analysis of the PPI data from the B 6 - Y A C 1 2 8 and F V B - Y A C 1 2 8 revealed the presence of impairments in habituation in the Y A C 1 2 8 on both background strains, a finding which was more striking in the B 6 - Y A C 1 2 8 mice. This is the first indication that the Y A C 1 2 8 mice demonstrate impaired cognitive function, and again demonstrate the accurate recapitulation of the disease as human H D patients present with learning and memory impairments (Harper et al., 2002). Further investigations into cognitive impairment in the Y A C 1 2 8 mouse model wi l l benefit from the use of the B 6 - Y A C 1 2 8 as outlined in the previous paragraph. 96 Chapter 6: Discussion and Future Directions 6.1 The YAC128 mouse model accurately recapitulates Huntington disease Animal models of human genetic diseases are important for both revealing how the underlying defect results in the disease and determining potential efficacy of therapeutics for patient treatment, since there are ethical and practical limitations to the data which can be obtained from human patients. The most valuable animal models of genetic disease recapitulate both the underlying genetic defect and the resulting disease phenotype, thereby allowing exploration of the primary molecular changes which result in the disease presentation and the targeting of these changes for therapeutic strategies. The H D Y A C mouse model encodes a transgene spanning the entire genomic region of the human H D gene, including promoter, intronic, upstream and downstream regulatory elements and expresses the full-length human protein, accurately recapitulating the genomic and protein context of human patients. Human huntingtin is 95% similar to mouse huntingtin and the ability of the human protein to substitute for the mouse protein was demonstrated by the rescue of the embryonic lethality of the huntingtin knock-out mouse by expression of Y A C transgenic human huntingtin (Hodgson et al., 1996). In addition, human huntingtin expressed under 24 kb of regulatory sequence in the Y A C mouse models exhibits identical tissue distribution as the mouse endogenous protein (Hodgson et al., 1999). Together, these results demonstrate that the human huntingtin protein is suitably similar to the mouse protein to allow phenotypic analysis of an H D mouse model which expresses the human mutant protein. The appropriate tissue and developmental expression of the full-length huntingtin protein by an endogenous promoter at less than endogenous mouse levels demonstrates that the H D Y A C mouse model accurately recapitulates the underlying genetic defect of H D . One of the main objectives of this thesis was to determine i f the Y A C 1 2 8 mouse model accurately recapitulated the phenotype of the human disease. Motor deficits evidenced by mi ld chorea tend to be one of the first identifiable, clinical manifestations in human patients. Motor deficits are clearly present in the Y A C 128 mice at 6 months of age using the rotarod apparatus, a tool for assaying deficits in motor function in rodent models which has been widely used for analysis of other H D mouse models (Carter et al., 1999;Schilling et al., 1999;Lin et al., 2001). Impairment in habituation is present in 12 month old Y A C 128 mice, for the first time indicating the presence of cognitive 98 dysfunction in the Y A C mouse model, a common characteristic of the human disorder. Pre-pulse inhibition is impaired in H D patients (Swerdlow et al., 1995;Munoz et al., 2003) and in the Y A C 128 mice. Finally, the initial period of increased movement, evidenced by chorea and clumsiness, which generally progresses into a more rigid state as the disease progresses (Hayden, 1981), parallels the biphasic activity profile of the Y A C 1 2 8 mice, composed of initial hyperactivity, followed by hypokinesis. The striatum is the brain region most affected in H D patients, demonstrated by striatal volume (Rosas et al., 2001) and striatal neuron loss (Vonsattel et al., 1985). These defining characteristics are remarkably replicated in the Y A C 128 model beginning at nine months of age with striatal volume decrease and progressing to 12 months of age with significant quantitative decrease in striatal neuron number, the first report of quantitative striatal neuron decreases in an H D mouse model. Cortical volume in Y A C 128 mice is also significantly decreased at 12 months of age, replicating the cortical thinning that is observed in H D patients (Rosas et al., 2002). Brain atrophy revealed by decreases in brain weight is evident in both human patients (Harper et al., 2002) and in 9-month old Y A C 128 mice. Finally neuronal intranuclear inclusions or aggregates of huntingtin are present late in the natural history of disease in the Y A C 128 model, similar to inclusions of huntingtin present in human tissue (DiFigl ia et al., 1997;Gutekunst et al., 1999). Besides recapitulating the genotype and phenotype of the human disease, the Y A C 128 mouse model recapitulates the natural history of changes in the disease. Motor deficits are a hallmark of onset of H D in patients (Hayden, 1981;Harper et al., 2002) and typically present before the onset of neuronal loss (Vonsattel et al., 1985). This natural history is replicated in the Y A C 128 model, with the onset of a motor deficit on the rotarod occurring before the onset of quantifiable neuronal loss (Fig. 6.1). Finally, the effects of mutant huntingtin from the Y A C 128 transgene are penetrant on multiple background strains, replicating the observation that a polyglutamine expansion above a certain size is fully penetrant in genetically heterogeneous human populations (Brinkman et al., 1997;Langbehn et al., 2004). 99 NEUROPATHOLOGY Cleavage of full-length htt and nuclear localization in striatum Age (months) I J O t t Slight hyperactivity BEHAVIOUR Rotarod deficit Brain weight decrease M S N s YAC128 MSNs Striatal volume decrease Striatal neuronal loss and shrinkage, cortical volume decrease 12 I Hypoactivity, PPI and cognitive deficits Inclusions present I 18 Figure 6.1: Natural history of HD in the YAC 128 mouse model. Neuropathological and behavioural changes during the lifetime of the YAC128 mouse. Shrinkage of striatal neurons is indicated by pictures of wild-type medium spiny neurons (WT MSNs) and YAC 128 MSNs. 6.2 The Y A C 1 2 8 mouse model is highly suited for use in therapeutic trials One o f the most important uses for animal models o f human disease is pre-clinical testing and development of therapeutics, a pressing need in H D research since there are currently no direct treatments to offer patients with this devastating disorder. A key objective o f this thesis was both to determine whether the Y A C 1 2 8 would be suitable for therapeutic trials and demonstrate that suitability. For a model to be suitable for therapeutic trials, it must accurately recapitulate the genotype and phenotype of the disorder in order to assure that the therapeutic strategies are both targeting appropriate pathways and the effects of the therapeutic can be assessed on clinically relevant phenotypes. A s outlined in the previous section, the presence of full-length huntingtin expressed under an endogenous promoter at endogenous levels in the Y A C 128 recapitulates the protein context of the human disease, allowing the appropriate targeting of the mechanisms that lead to pathogenesis. The Y A C 128 also recapitulates both the neuronal dysfunction and neuronal degeneration o f the human 100 disease; therefore, the efficacy of therapeutics can be assessed on clinically relevant phenotypes. In addition to accurate recapitulation of the disorder, it is also important that the phenotypic expression in the model display low variability, so that significant effects on these phenotypes can be assessed with low animal numbers. Power analysis on the data collected in the initial characterization of the Y A C 128 mice demonstrated that only 4 animals would be necessary to determine a significant 50% rescue of striatal volume decrease, the phenotype with the lowest variability and even the neuropathological phenotypes with greater variability (e.g. neuron count) require at most, 13 animals to discern a 50% rescue, demonstrating that low numbers of animals are required to reveal significant effects in a trial using the Y A C 1 2 8 model. Robust and reproducible phenotypes are also key characteristics for use of an animal model in therapeutic trials. The observation that significant striatal volume loss and brain weight reduction and significant hypoactivity and PPI impairments manifest on both F V B and B6 background strains indicates these disease-related phenotypes are robust and penetrant on multiple strains. The motor deficit of the Y A C 128 mice on the rotarod was first observed in the initial characterization of the Y A C 1 2 8 mice, and then subsequently recapitulated in groups of Y A C 128 mice for the shortstop (Chapter 4) and B6 (Chapter 5) studies, demonstrating the reproducibility of this neuronal dysfunction phenotype. Furthermore, phenotypes related to neuronal degeneration including brain weight, striatal volume and striatal neuronal count decreases were recapitulated in F V B -Y A C 1 2 8 mice in both 12 and 18 month old animals for the shortstop study (Chapter 4) and also recapitulated on a different background strain in the B6 study, demonstrating the reproducibility and reliability of these neurodegenerative phenotypes. The Y A C 128 mice demonstrate robust, reliable and reproducible phenotypes with low inter-animal variability which accurately recapitulates the symptoms of the human disorder, indicating the suitability of the Y A C 1 2 8 for use in therapeutic trials. Although the scope of this thesis did not encompass the use of the Y A C 1 2 8 in a standard therapeutic trial with a compound, the analysis of the shortstop mouse in parallel with the Y A C 1 2 8 mouse was similar in design to a therapeutic trial. In this study, the shortstop mouse can be considered the "therapeutic" group, i.e. what is the effect of a short-101 fragment of huntingtin on disease pathogenesis? The conclusive result that the shortstop is resistant to the neuronal dysfunction and degeneration of the full-length Y A C 1 2 8 demonstrates the applicability of the Y A C 128 for therapeutic trials. 6.2.1 Significance of Y A C 1 2 8 model for the H D field The availability of a mouse model of H D which accurately recapitulates the genotype and phenotype of the human disorder is necessary for both investigation of pathways which lead to pathogenesis and testing the efficacy of therapeutic targeting of those and other pathways in order to discover a cure or better treatments for this insidious disease. The mouse models which have previously been established in the H D field either expressed truncated forms of huntingtin from a variety of promoters, or expressed endogenous full-length mouse huntingtin with an expanded C A G repeat (Table 1.2). The truncated mouse models exhibit striatal atrophy and onset of motor deficits (Mangiarini et al., 1996;Schilling et al., 1999;Yamamoto et al., 2000), but do not exhibit quantitative loss of striatal neurons, nor do they accurately recapitulate the genomic and protein context of the human disorder i.e. do not express full-length mutant huntingtin. Data from the shortstop study suggests that full-length or a longer fragment of huntingtin than is present in the shortstop or R6/2 model is necessary for the specific recapitulation of H D in mice. This finding indicates that the phenotypes observed in the short fragment models including the R6/2 model do not necessarily result from the same mechanisms that leads to H D in patients, but may instead be due to improper regulation and expression of the transgene leading to non-specific polyglutamine toxicity. Knock-in of 146 C A G s into the mouse hypoxanthine phosphoribosyltransferase gene (Hprt), a gene which does not normally contain a C A G tract, causes many of the same phenotypes observed in the R6/2 and N171-82Q mice (Ordway et al., 1997;Tallaksen-Greene et al., 2003), suggesting that expanded polyglutamines can cause toxicity and phenotypes unrelated to the specific mechanisms involved in H D pathogenesis. Study of truncated mouse models w i l l therefore not reveal the mechanisms specific to H D pathogenesis and testing of therapeutic compounds in truncated mouse models may only reveal compounds which are efficacious against polyglutamine toxicity and not specifically H D . 102 The knock-in models accurately replicate the underlying genetic defect of H D , but do not manifest the clinical phenotypes of the human disorder including neuronal degeneration demonstrated by striatal volume atrophy accompanied with striatal neuronal loss or neuronal dysfunction demonstrated by motor deficits on the rotarod (Shelbourne et al., 1999;Wheeler et al., 2000;Lin et al., 200T;Menalled et al., 2002). This is a surprising finding, since expression of the full-length human protein in the Y A C 1 2 8 model exhibits such a striking recapitulation of the human disorder. This result indicates that human huntingtin with an expanded polyglutamine tract is more toxic than the corresponding mouse protein with polyglutamine expansion. The lack of a neurodegenerative or behavioural phenotype in the knock-in models makes it impossible to use these mice for therapeutic trials since there are no clinically relevant phenotypes with which to test the efficacy of therapeutic compounds. The work of Hodgson et al. in 1996 and 1999, demonstrated that the H D Y A C mouse expresses full-length human huntingtin in a tissue-specific and developmental-specific manner identical to the mouse endogenous huntingtin at less than endogenous mouse levels. These observations indicate that the Y A C mice correctly recapitulate the genotype and protein context of the human disorder. The work in this thesis presents data which demonstrates that the Y A C 128 model accurately recapitulates both the neuronal dysfunction and neuronal degeneration characteristic of the human disorder. The importance of this finding is two-fold - first mechanisms which underlie the dysfunction and degeneration of the human disease can be studied in an appropriate mouse model of the disease and second the Y A C 1 2 8 mice can be utilized for therapeutic trials. The timeline of changes in the Y A C 128 (Fig. 6.1) wi l l be especially useful for distinguishing which molecular changes occur before the onset of neuronal dysfunction and/or degeneration, indicating primary events which should be targeted for therapeutics, and which events occur after dysfunction/degeneration and are therefore downstream of the mechanisms which actually lead to the pathogenesis of the disease. The ability to distinguish primary events in H D pathogenesis from those downstream of dysfunction/degeneration is not possible in any other mouse model for the aforementioned reasons, making the Y A C 1 2 8 the enabling reagent in the development of a cure for H D . 103 This thesis demonstrates the YAC128 manifests robust, reliable and reproducible neuronal dysfunction and degeneration phenotypes which accurately recapitulate the human disorder with low variability, making the YAC128 ideally suited for use in therapeutic trials. These trials may consist of genetic modification by breeding the YAC128 mice to different transgenic mice of interest, or chemical modification through the delivery of various therapeutic compounds. The importance of the Y A C 128 mouse to the HD research community has been demonstrated by the request from Huntington disease funding and research organizations to import the Y A C 128 into the Jackson Laboratories for easier distribution to the research community, and to the company Psychogenics for more detailed analysis of the model for use in large-scale therapeutic trials. Collaborations with HD researchers from around the world have already been developed to further investigate the mechanisms which underlie the disorder and to begin therapeutic trials using the YAC128s (Table 6.1). Table 6.1: YAC128 collaborations. Collaborations established to investigate YAC128 for mechanisms which underlie HD, and begin treatment. Principal Investigator Institution Purpose of Y A C 128 Research Preliminary Findings (unpublished) Ilya Bezprozvanny University of Texas The role of the InsP3Rl channel in HD YAC 128 primary neurons show increased susceptibility to glutamate excitotoxicity Alex Osmand University of Tennessee Aggregation foci in HD Aggregation foci in shortstop and YAC 128 Lisa Ellerby The Buck Institute for Age Research Calpain activation In progress Lynn Raymond University of British Columbia NMDA excitotoxicity YAC 128 primary neurons display increased susceptibility to NMDA excitotoxicity Jang Ho Cha Massachusetts General Hospital Neurotransmitter receptor levels Decreased AMPA receptor binding in YAC 128 brain Nigel Bamford University of Washington Glutamate release In progress Jenny Morton University of Cambridge Cognitive dysfunction In progress De-Maw Chuang NIH Lithium and sodium valproate treatment In progress Christopher Hough NIH Intracellular calcium levels In progress Psychogenics - Large-scale therapeutic trials In progress The Jackson Laboratories - Distribution to academic researchers In progress 104 6.3 Huntingtin inclusions (aggregates) and their precursors are not toxic Aggregates or huntingtin inclusions are present in both human patient brain (DiFigl ia et al., 1997;Gutekunst et al., 1999) and in mouse models of H D (Davies et al., 1997;Hodgson et al., 1999;Schilling et al., 1999;Wheeler et al., 2000;Slow et al., 2003). Hypotheses over the role these huntingtin inclusions and their precursors play in H D range from a benign byproduct of polyglutamine expansion (Saudou et al., 1998;Gutekunst et al., 1999), to a toxic, underlying mechanism in the disease pathogenesis (Davies et al., 1997;Chen et al., 2002). Initial support for the hypothesis that huntingtin aggregates/inclusions are pathogenic came from a study of the R6/2 truncated mouse model of H D which demonstrated the presence of inclusions before the onset of motor deficits or brain and striatal volume decreases (Davies et al., 1997). Results from this thesis revealed neuronal huntingtin inclusions in the Y A C 128 mouse model at 18 months of age, months after the onset of motor impairment (6 months) and neuronal degeneration manifest as brain and striatal volume decreases (9 months), indicating that inclusions were not a primary, initiating pathogenic mechanism involved in neuronal dysfunction or degeneration in the Y A C 1 2 8 mouse model. Even though subsequent analysis of the Y A C 1 2 8 mouse striatum revealed tiny micro-aggregates present at 12 months of age, the onset of these micro-aggregates was at a later time point in the natural history of disease in the Y A C 128 model than both the onset of neuronal dysfunction and degeneration. The observation that huntingtin inclusions occur after the onset of neuronal dysfunction was also noted in the Y A C 7 2 (Hodgson et al., 1999) and in a full-length knock-in model of H D (Menalled et al., 2002;Menalled et al., 2003). Additional evidence supporting the dissociation of neuronal dysfunction and degeneration from huntingtin inclusion formation was further revealed in this thesis during the study of the Y A C 1 2 8 on the B 6 background. Despite the fact that the B 6 - Y A C 1 2 8 display delayed onset of neuronal degeneration compared to the F V B - Y A C 1 2 8 , the time course and severity of huntingtin inclusion formation is unaltered in B 6 - Y A C 1 2 8 . 105 The dissociation of inclusion formation from neuronal degeneration and dysfunction in the Y A C 128 model is in agreement with data from cell culture models (Saudou et al., 1998;Kim et al., 1999) which demonstrate inclusion formation without toxicity and toxicity without inclusion formation and also with data from therapeutic trials in mouse models of H D that reveal 1) significant improvement of neuronal degeneration without effects on inclusion formation (Schiefer et al., 2002;Ferrante et al., 2003;Hockly et al., 2003), 2) significant and marked decrease in inclusion formation with only slight improvement in neuronal degeneration (Dedeoglu et al., 2002) and most strikingly 3) significant improvement in neuronal degeneration with significant increases in inclusion formation (Mastroberardino et al., 2002). Expression of an ataxin-1 transgene which had the self-association domain deleted and therefore could not self-aggregate caused ataxia and neuronal loss in a S C A - 1 mouse model without forming neuronal aggregates, suggesting aggregate formation was not relevant for disease pathogenesis in another polyglutamine disorder (Klement et al., 1998). These results support a role for huntingtin aggregates as a non-toxic by-product. Recent research demonstrated that huntingtin aggregates promote clearance of mutant huntingtin by the process of autophagy through sequestration of mTOR, indicating that aggregates may actually have neuroprotective functions (Ravikumar et al., 2004). However, it has been proposed that visible huntingtin aggregates are simply the last component of a linear pathway of aggregate formation composed of P-sheets, protofibrils, fibrils and aggregation foci, and any of these aggregate precursors may cause toxicity in H D (Bates, 2003). It is argued that the afore-mentioned results only examine visible huntingtin aggregates and do not analyze aggregate precursors and therefore do not fully address the issue of whether aggregates and their precursors are toxic. Data from the shortstop model in this thesis directly supports the theory that both visible aggregates/inclusions and their aggregate precursors are not toxic. The shortstop mouse develops widespread huntingtin inclusions and aggregation foci but does not develop neuronal dysfunction in the form of a rotarod deficit or neuronal degeneration in the form of brain weight, striatal volume and striatal neuronal count decreases. Inclusions immuno-stained with E M 4 8 and aggregation foci which demonstrate recruitment of biotin-labeled peptide are both present in the shortstop model and in the 106 full-length Y A C 128 model, a model that manifests both neuronal degeneration and dysfunction. These observations indicate that it is not a difference in inclusions which results in the differences between the shortstop and Y A C 1 2 8 phenotypes. Finally, data from this thesis reveals aggregation foci and inclusions in the shortstop mouse, the last two constituents of the pathway of aggregate formation. If aggregate formation is a linear pathway, the shortstop mouse must at one time manifest each of the aggregation precursors, yet it does not manifest neuronal degeneration or dysfunction, indicating huntingtin inclusions and their precursors can exist completely separate from disease pathology in vivo and are therefore unlikely to underlie disease pathogenesis. 6.3.1 Significance of aggregate findings for the H D field Huntingtin aggregates exist in cell (Apostol et al., 2003), Drosophila (Kazantsev et al., 2002), C. elegans (Parker et al., 2001) and mouse (Davies et al., 1997;Hodgson et al., 1999;Schilling et al., 1999;Wheeler et al., 2000;Slow et al., 2003) models of H D and are easy to assay and quantify. The hypothesis that huntingtin aggregates/inclusions and their precursors are the toxic species underlying pathogenesis in H D have led to the search for inhibitors of aggregates in screens of therapeutic compounds. A cell culture model (Apostol et al., 2003), an automated filter retardation assay (Heiser et al., 2000;Heiser et al., 2002) and a brain slice model using R6/2 tissue (Smith et al., 2001;Smith and Bates, 2004) have been established for the primary purpose of screening compounds which can inhibit aggregation. Reduction in the number and/or size of huntingtin aggregates in therapeutic trials with Drosophila (Kazantsev et al., 2002) and mouse (Ona et al., 1999;Ferrante et al., 2000;Ferrante et al., 2002;Klivenyi et al., 2003;Tanaka et al., 2004) models of H D is considered an indication of therapeutic efficacy. The data from this thesis indicate that reduction in aggregate/inclusion number may not be a suitable endpoint to examine in therapeutic trials since the shortstop mouse demonstrates widespread aggregate accumulation without pathology. The data in this thesis also indicate that primary screening for compounds which selectively inhibit aggregates may not be beneficial in clinical trials since aggregates or their precursors are not toxic in the shortstop mouse. It is possible that these primary screens for aggregate 107 inhibitors w i l l reveal efficacious therapeutics which simultaneously target pathways involved in the pathogenesis of the disease and aggregate formation. However, it seems likely that therapeutics which can delay or halt neuronal degeneration and dysfunction in H D patients w i l l not be revealed in primary screens for aggregate inhibitors, since the data in this thesis would suggest that aggregate formation and neurodegeneration are unrelated. Furthermore, many "positive hits" using a primary screen for inhibition of aggregates wi l l l ikely have no effect on neuronal dysfunction or degeneration and therefore require further screening to be eliminated. The data in this thesis indicate it is crucial to invest time and capital in the development of other primary screens which target pathways directly related to the pathogenesis of the disease. 6.4 The shortstop mouse suggests there is specificity of the proteolytic fragment for the toxic fragment hypothesis. The toxic fragment hypothesis proposes that cleavage of huntingtin by proteases generates toxic N-terminal huntingtin fragments which are inefficiently degraded due to the expanded polyglutamine tract, and can therefore cause disruptions in transport, protein-protein interactions, and gene expression. This leads to increased neuronal stress, and greater sensitivity to excitotoxicity which increases intracellular calcium levels leading to increased activation of proteases and an amplification of the toxic fragment cycle until the neuron is ultimately committed to death (Fig. 1.2). The shortstop mouse expresses a 117 amino acid N-terminal fragment of huntingtin with an expanded polyglutamine repeat which does not result in neuronal degeneration or dysfunction. The results from the shortstop study seem to argue against the toxic fragment hypothesis but instead, actually indicate specificity of the toxic fragment which initiates this pathway. Caspases (Goldberg et al., 1996;Wellington et al., 2000;Wellington et al., 2002), calpains (Gafni and Ellerby, 2002;Kim et al., 2001;Gafni et al., 2004) and aspartyl proteases (Lunkes et al., 2002) cleave huntingtin to generate N-terminal huntingtin fragments of different sizes. The shortstop fragment most closely resembles the cpA fragment generated by aspartyl protease cleavage (Lunkes et al., 2002). The lack of a neurodegenerative or dysfunctional phenotype in the shortstop model indicates that this fragment is not capable of initiating the events which lead to increased neuronal stress, 108 excitotoxicity and C a + influx which eventually result in activation of proteases, amplification of the toxic cycle and neuronal death. Results from the shortstop model indicate that only specific N-terminal fragments of huntingtin can initiate this toxic pathway. The idea that different fragments of a disease-causing protein can be differentially toxic is well-recognized in the Alzheimer disease field where the APi_ 4 2 fragment is more toxic than the AP 1.40 fragment, despite only a 2 amino acid difference (Iijima et al., 2004). Evidence from caspase-resistant Y A C mice indicates that inhibiting caspase-6 cleavage of huntingtin is neuroprotective while inhibiting caspase-3 cleavage is not (Graham and Hayden, unpublished data), indicating caspase-6 fragments, but not caspase-3 fragments of huntingtin are capable of initiating the toxic fragment cascade. Results from the shortstop study are significant for therapeutic strategies in the H D field. The results from this study modify the toxic fragment hypothesis to indicate that there is a specific, initiating N-terminal fragment of polyglutamine-expanded huntingtin which pushes the neurons into a toxic fragment cycle that eventually leads to neuronal death. If the production of the initiating N-terminal fragment can be eliminated by the inhibition of specific proteases, then the cycle of neuronal dysfunction and death in H D can be halted. The results from the shortstop mouse indicate that inhibition of aspartyl proteases may not be efficacious since the shortstop fragment is similar in size to a fragment generated by aspartyl proteolysis and is incapable of initiating the cycle which leads to neuronal death. Other research on caspase proteolysis (Graham and Hayden, in preparation) suggests inhibition of caspase-6 may stop the initiating event in the toxic fragment pathway and therefore stop the resulting dysfunction and degeneration in H D patients. 6.5 Future directions 1. What therapeutics are efficacious in the YAC128 model? This thesis has demonstrated the suitability of the Y A C 128 mouse model for use in therapeutic trials and the next logical step is the use of this model to test therapeutic compounds. There is a limitless list of therapeutic compounds and strategies to test in the Y A C 128 model, but examples of some therapeutics with great potential are: 109 i . Li thium - Lithium has been demonstrated to have effects on many of the pathways believed to be involved in H D pathogenesis including B D N F (Hashimoto et al., 2002b), N M D A receptors (Nonaka et al., 1998;Hashimoto et al., 2002a), and A K T (Chalecka-Franaszek and Chuang, 1999). Pre-treatment with lithium reduced injury in an acute rat model of H D (Wei et al., 2001) and lithium is already approved for clinical use, significantly reducing the time it would take to begin clinical trials should it prove efficacious. i i . Caspase inhibitors- Activated caspases are present in H D patient brain (Hermel et al., 2004) and caspase-cleaved huntingtin is present in pre-symptomatic H D brain (Wellington et al., 2002), suggesting caspase cleavage of huntingtin is an initiating event in H D pathogenesis. Recent evidence from H D Y A C transgenic mouse models which are resistant to caspase cleavage of huntingtin show that blocking caspase-6 cleavage of huntingtin can prevent the resulting pathology (Graham and Hayden, in preparation) indicating inhibition of caspase-6 may be efficacious. i i i . Downregulation of mutant huntingtin through s i R N A techniques- A . Yamamoto in 2000 demonstrated using a Tet-off exon-1 model of H D that turning off the mutant protein could both prevent and reverse some of the neurodegenerative effects (Yamamoto et al., 2000). Small interfering R N A (s iRNA) is a powerful technique for the silencing of specific genes, and holds particular promise for silencing dominantly acting disease genes. Mi l l e r et al. in 2003 demonstrated allele-specific silencing of the mutant S C A 3 gene by targeting the s i R N A to a S N P which was linked to the mutant allele (Miller et al., 2003). While no linked SNPs have yet been discovered for the mutant huntingtin allele, 110 this technique holds great promise as it can directly target the primary disease causing mechanism, mutant huntingtin. Does the YAC128 model recapitulate other characteristics of the human disease? The data in this thesis demonstrates the Y A C 128 mouse accurately recapitulates many of the clinical manifestations and neuropathological characteristics of the human disorder. It w i l l be important to continue to investigate whether the Y A C 1 2 8 model reproduces other characteristics of human H D to determine how well the Y A C 128 model recapitulates all aspects of the human disease. The B6 study (Chapter 5) indicated that the Y A C 128 manifests a cognitive deficit in habituation. Mouse behavioural analysis of learning and memory tested in a time course similar to that described in the initial characterization of the Y A C 128 mouse (every 3 months of age) w i l l be useful in determining when the Y A C 1 2 8 mice manifest cognitive deficits. Similarly, the Y A C 128 mice can undergo behavioural tests for psychiatric disturbances, including depression, to determine i f the model recapitulates this aspect of the human disorder. It wi l l also be beneficial to determine when PPI impairments first manifest in the Y A C 128 model, and whether other behavioural testing apparati can be used to quantify the motor deficits in this model. Impairments or deficits in any of these behavioural tests can then be used as endpoints for therapeutic trials. Enkephalin is a marker for those striatal efferent neurons which project to the external pallidum and enkephalin expression is decreased in human and mouse model brain (Ferrante et al., 1986;Menalled et al., 2000). It w i l l be interesting to determine i f this phenotype is replicated in the Y A C 1 2 8 mouse model. The cortex is also affected in H D and although data from this thesis demonstrates decreases in cortical volume in the Y A C 128 model, it w i l l be important to investigate which layers and regions of the cortex are selectively affected and determine i f neuronal loss is present in the cortex of Y A C 128 mice. Finally, H D patients demonstrate neuronal loss in the striatum in a dorsal-ventral fashion, and it w i l l be important to determine i f there is specificity of neuronal loss within the 111 striatum in the Y A C 128 mice, both to indicate potential mechanisms and potentially refine the measurement of neuronal count decrease. 3. What is the time course of other molecular changes in the YAC128 model? The accurate recapitulation of the human disease and the detailed time course of changes in the Y A C 128 mouse allow for the investigation into the mechanisms which underlie the pathogenesis of the disorder. Collaborations have been established to investigate the role of neurotransmitters, glutamate levels, the InsP3Rl channel, calpains, intracellular calcium levels, and transglutaminase levels in the pathogenesis of disease in the mice. Investigation using the Y A C 1 2 8 mice can also help determine the role and time course of phosphorylation (Humbert et al., 2002) and caspase activation (Hermel et al., 2004) as well as other post-translational modification and proteolysis events in the disease progression. Striatal neuron surface area is decreased in the Y A C 1 2 8 at 12 months of age, indicating the neurons may be dysfunctional, however, this technique only examines the cell body and not the projections and processes which are truly important for proper functioning of the neuron. Studies in other mouse models of H D (Klapstein et al., 2001;Laforet et al., 2001) demonstrate decreases in dendritic spine density and abnormal dendritic morphology. It w i l l be important to determine i f and when these changes occur in the Y A C 128 mouse model for potential use as an endpoint in therapeutic trials. 4. What is the phenotype of the YAC128 model on different mouse background strains? It is important to continue the characterization of the Y A C 1 2 8 model on different background strains, both for the potential future use in modifier mapping studies and also to be able to work with and supply the Y A C 128 mouse on a number of different strain backgrounds. The B 6 - Y A C 1 2 8 strain should be investigated for striatal volume decreases at 9 months of age to determine i f the age of onset for this phenotype is indeed delayed for this phenotype and can potentially be used as an endpoint in modifier mapping. The B 6 - Y A C 1 2 8 mice can also be beneficial for many of the afore-mentioned behavioural studies. Finally, it is useful to investigate the Y A C 128 mice on a third mouse strain, which 112 may also be susceptible to the effects of the transgene but does not carry a retinal degeneration mutation. One possibility is the 129/Sv strain. This strain is well-characterized and also demonstrates susceptibility to excitotoxicity, similar to the F V B strain (Schauwecker and Steward, 1997). 5. What is the effect of protein dosage on the YAC 128 phenotypes? During the generation of the Y A C 1 2 8 mouse line 53 (used in this thesis) two other full-length Y A C 1 2 8 lines were established, line 54 and 55, each with different and lower levels of transgenic protein expression compared to line 53. It is important to characterize these lines both to rule out any suggestion of insertion site effects on the resulting phenotypes in the characterized Y A C 1 2 8 , line 53 and also to examine dosage. Initial results from studies of homozygous H D patients (those with 2 copies of mutant huntingtin) indicated that two copies of the mutant protein did not seem to affect age of onset (1993), while subsequent research indicated that progression of the illness was more severe in homozygous patients (Squitieri et al., 2003), indicating dosage affects progression, i f not age of onset. The different levels of mutant protein expression in the different full-length Y A C 128 lines wi l l allow investigation into the effects of dosage on the phenotypes described in the Y A C 1 2 8 characterization, and therefore the effects of dosage on the pathogenesis of the disorder. 6. What is the mechanism which underlies the difference in phenotype of the shortstop versus the YAC128 model? Although there are many possible mechanisms which underlie this difference, one hypothesis is that increased susceptibility to excitotoxicity in the Y A C 128 mice leads to neuronal degeneration and dysfunction and this susceptibility is absent in the shortstop mouse. Zeron et al. in 2001 found that cells transfected with full-length mutant huntingtin were more susceptible to NMDA-media ted excitotoxicity than cells transfected with a truncated N-terminal fragment of huntingtin, indicating the necessity for full-length huntingtin in increased susceptibility to excitotoxicity (Zeron et al., 2001). The Y A C 7 2 mice demonstrated increased susceptibility to excitotoxic death caused by injection of quinolinic acid compared to wild-type controls, and primary striatal neurons established from Y A C 7 2 mice were more 113 susceptible to N M D A treatment compared to neurons from wild-type mice (Zeron et al., 2002). The R6/2 mouse, a short fragment model of H D similar in transgenic protein size to the shortstop mouse was resistant to quinolinic acid injury compared to wild-type controls (Hansson et al., 1999). Differential susceptibility to excitotoxicity may be one of the mechanisms underlying the difference in disease manifestation in the shortstop and Y A C 1 2 8 mice. Examination of the susceptibility of both the shortstop and the Y A C 128 to quinolinic acid injection and to N M D A treatment of primary neuronal cultures wi l l help answer determine i f excitotoxicity underlies this difference. 6.6 Conclusions The primary objective of this thesis was to characterize a Y A C mouse model of H D in order to determine i f it accurately recapitulated the human disease and i f it was suitable for therapeutic trials. The data in this thesis demonstrated that both of these goals were met. These mice have now been distributed to a number of collaborators in order to characterize other aspects of the phenotype, investigate mechanisms which underlie the pathogenesis of the disease and begin using this model in therapeutic trials. Importation of the model to the Jackson laboratories w i l l facilitate the distribution of this model to other researchers. The availability of the Y A C 1 2 8 on two different background strains should also help facilitate the characterization and use of this model. A second objective was to determine i f a mouse expressing a short fragment of huntingtin would manifest the neuronal degeneration and dysfunction of the full-length Y A C 1 2 8 . The data in this thesis demonstrates the importance of the specificity of the toxic fragment to the initiation of events in the toxic fragment hypothesis which eventually lead to neuronal dysfunction and death. Furthermore, data from the shortstop mouse indicates that aggregates and their precursors are not toxic, indicating that primary screens for inhibitors of aggregates and using aggregates as endpoints for therapeutic trials is ill-advised. Hopefully the work presented in this thesis wi l l serve as a foundation for many other research avenues which together can have a positive and resounding impact on the H D field. I hope that the data I have produced in this thesis w i l l help guide research 114 efforts and therapeutic trials using these mice towards an effective cure or better treatment for H D patients. M y greatest hope for this thesis is that in 10 years time there wi l l no longer be a need for therapeutic trials, or investigation into H D mouse models due to the establishment and delivery of a viable cure to H D patients, making research into H D motivated by intellectual curiosity instead of vital necessity. 115 C h a p t e r 7: References Reference List 1. (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell 72: 971-983. 2. Akiyama H, Mori H, Saido T, Kondo H, Ikeda K, McGeer PL (1999) Occurrence of the diffuse amyloid beta-protein (Abeta) deposits with numerous Abeta-containing glial cells in the cerebral cortex of patients with Alzheimer's disease. Glia 25: 324-331. 3. Albin RL, Reiner A, Anderson KD, Penney JB, Young AB (1990) Striatal and nigral neuron subpopulations in rigid Huntington's disease: implications for the functional anatomy of chorea and rigidity-akinesia. Ann Neurol 27: 357-365. 4. Ambrose CM, Duyao MP, Barnes G, Bates GP, Lin CS, Srinidhi J, Baxendale S, Hummerich H, Lehrach H, Altherr M , . (1994) Structure and expression of the Huntington's disease gene: evidence against simple inactivation due to an expanded CAG repeat. Somat Cell Mol Genet 20: 27-38. 5. Andrade MA, Bork P (1995) HEAT repeats in the Huntington's disease protein. Nat Genet 11: 115-116. 6. Andrew SE, Goldberg YP, Kremer B, Telenius H, Theilmann J, Adam S, Starr E, Squitieri F, Lin B, Kalchman MA,. (1993) The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease. Nat Genet 4: 398-403. 7. Apostol BL, Kazantsev A, Raffioni S, Hies K, Pallos J, Bodai L, Slepko N, Bear JE, Gertler FB, Hersch S, Housman DE, Marsh JL, Thompson LM (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: 5950-5955. 8. Ariano MA, Aronin N, DiFiglia M, Tagle DA, Sibley DR, Leavitt BR, Hayden MR, Levine MS (2002) Striatal neurochemical changes in transgenic models of Huntington's disease. J Neurosci Res 68: 716-729. 9. Bates G (2003) Huntingtin aggregation and toxicity in Huntington's disease. Lancet 361:1642-1644. 10. Beal MF, Ferrante RJ (2004) Experimental therapeutics in transgenic mouse models of Huntington's disease. Nat Rev Neurosci 5: 373-384. 11. Becher MW, Kotzuk JA, Sharp AH, Davies SW, Bates GP, Price DL, Ross CA (1998) Intranuclear neuronal inclusions in Huntington's disease and dentatorubral and pallidoluysian atrophy: correlation between the density of inclusions and IT15 CAG triplet repeat length. Neurobiol Dis 4: 387-397. 117 12. Berghorn KA, Bonnett JH, Hoffman GE (1994) cFos immunoreactivity is enhanced with biotin amplification. J Histochem Cytochem 42: 1635-1642. 13. Block-Galarza J, Chase KO, Sapp E, Vaughn KT, Vallee RB, DiFiglia M, Aronin N (1997) Fast transport and retrograde movement of huntingtin and HAP 1 in axons. Neuroreport 8: 2247-2251. 14. Boutell JM, Thomas P, Neal JW, Weston VJ, Duce J, Harper PS, Jones AL (1999) Aberrant interactions of transcriptional repressor proteins with the Huntington's disease gene product, huntingtin. Hum Mol Genet 8: 1647-1655. 15. Brinkman RR, Mezei MM, Theilmann J, Almqvist E, Hayden MR (1997) The likelihood of being affected with Huntington disease by a particular age, for a specific CAG size. Am J Hum Genet 60:1202-1210. 16. Buckley KM, Melikian HE, Provoda CJ, Waring MT (2000) Regulation of neuronal function by protein trafficking: a role for the endosomal pathway. J Physiol 525 Pt 1: 11-19. 17. Carter RJ, Lione LA, Humby T, Mangiarini L, Mahal A, Bates GP, Dunnett SB, Morton AJ (1999) Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation. J Neurosci 19: 3248-3257. 18. Cepeda C, Ariano MA, Calvert CR, Flores-Hernandez J, Chandler SH, Leavitt BR, Hayden MR, Levine MS (2001) NMDA receptor function in mouse models of Huntington disease. J Neurosci Res 66: 525-539. 19. Cepeda C, Hurst RS, Calvert CR, Hernandez-Echeagaray E, Nguyen OK, Jocoy E, Christian LJ, Ariano MA, Levine MS (2003) Transient and progressive electrophysiological alterations in the corticostriatal pathway in a mouse model of Huntington's disease. J Neurosci 23: 961-969. 20. Cha JH, Frey AS, Alsdorf SA, Kerner JA, Kosinski CM, Mangiarini L, Penney JB, Jr., Davies SW, Bates GP, Young AB (1999) Altered neurotransmitter receptor expression in transgenic mouse models of Huntington's disease. Philos Trans R Soc Lond B Biol Sci 354: 981-989. 21. Cha JH, Kosinski CM, Kerner JA, Alsdorf SA, Mangiarini L, Davies SW, Penney JB, Bates GP, Young AB (1998) Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human huntington disease gene. Proc Natl Acad Sci U S A 95: 6480-6485. 22. Chalecka-Franaszek E, Chuang DM (1999) Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc Natl Acad Sci U S A 96: 8745-8750. 23. Chan EY, Luthi-Carter R, Strand A, Solano SM, Hanson SA, DeJohn MM, Kooperberg C, Chase KO, DiFiglia M, Young AB, Leavitt BR, Cha JH, Aronin N, Hayden MR, Olson JM (2002) Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease. Hum Mol Genet 11: 1939-1951. 118 24. Chen HK, Fernandez-Funez P, Acevedo SF, Lam YC, Kaytor MD, Fernandez MH, Aitken A, Skoulakis EM, Orr HT, Botas J, Zoghbi HY (2003) Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1. Cell 113: 457-468. 25. Chen S, Berthelier V, Yang W, Wetzel R (2001) Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity. J Mol Biol 311: 173-182. 26. Chen S, Ferrone FA, Wetzel R (2002) Huntington's disease age-of-onset linked to polyglutamine aggregation nucleation. Proc Natl Acad Sci U S A 99:11884-11889. 27. Cooper JK, Schilling G, Peters MF, Herring WJ, Sharp AH, Kaminsky Z, Masone J, Khan FA, Delanoy M, Borchelt DR, Dawson VL, Dawson TM, Ross CA (1998) Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture. Hum Mol Genet 7: 783-790. 28. Coyle JT, Schwarcz R (1976) Lesion of striatal neurones with kainic acid provides a model for Huntington's chorea. Nature 263: 244-246. 29. Crawley JN (2000) What's Wrong With My Mouse?: Behavioural Phenotyping of Transgenic and Knockout Mice. New York: John Wiley & Sons, Inc. 30. Davenport CB, Muncey EB (1916) Huntingtin's chorea in relation to heredity and insanity. American Journal of Insanity 73:195-222. 31. David G, Abbas N, Stevanin G, Durr A, Yvert G, Cancel G, Weber C, Imbert G, Saudou F, Antoniou E, Drabkin H, Gemmill R, Giunti P, Benomar A, Wood N, Ruberg M, Agid Y, Mandel JL, Brice A (1997) Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nat Genet 17: 65-70. 32. Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA, Scherzinger E, Wanker EE, Mangiarini L, Bates GP (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90: 537-548. 33. Davis JM, Pennington JE, Kubler AM, Conscience JF (1982) A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J Immunol Methods 50:161-171. 34. Dedeoglu A, Kubilus JK, Jeitner TM, Matson SA, Bogdanov M, Kowall NW, Matson WR, Cooper AJ, Ratan RR, Beal MF, Hersch SM, Ferrante RJ (2002) Therapeutic effects of cystamine in a murine model of Huntington's disease. J Neurosci 22: 8942-8950. 35. Devon RS, Porteous DJ, Brookes AJ (1995) Splinkerettes—improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res 23:1644-1645. 36. DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277:1990-1993. 119 37. Djousse L, Knowlton B, Hayden M, Almqvist EW, Brinkman R, Ross C, Margolis R, Rosenblatt A, Durr A, Dode C, Morrison PJ, Novelletto A, Frontali M, Trent RJ, McCusker E, Gomez-Tortosa E, Mayo D, Jones R, Zanko A, Nance M, Abramson R, Suchowersky O, Paulsen J, Harrison M, Yang Q, Cupples LA, Gusella JF, MacDonald ME, Myers RH (2003) Interaction of normal and expanded CAG repeat sizes influences age at onset of Huntington disease. Am J Med Genet 119A: 279-282. 38. Dragani TA (2003) 10 years of mouse cancer modifier loci: human relevance. Cancer Res 63: 3011-3018. 39. Dragatsis I, Levine MS, Zeitlin S (2000) Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice. Nat Genet 26: 300-306. 40. Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, Mouradian MM, Young AB, Tanese N, Krainc D (2002) Spl and TAFII130 transcriptional activity disrupted in early Huntington's disease. Science 296: 2238-2243. 41. Duyao M, Ambrose C, Myers R, Novelletto A, Persichetti F, Frontali M, Folstein S, Ross C, Franz M, Abbott M , . (1993) Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat Genet 4: 387-392. 42. Duyao MP, Auerbach AB, Ryan A, Persichetti F, Barnes GT, McNeil SM, Ge P, Vonsattel JP, Gusella JF, Joyner AL, . (1995) Inactivation of the mouse Huntington's disease gene homolog Hdh. Science 269: 407-410. 43. Emamian ES, Kaytor MD, Duvick LA, Zu T, Tousey SK, Zoghbi HY, Clark HB, Orr HT (2003) Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice. Neuron 38: 375-387. 44. Engelender S, Sharp AH, Colomer V, Tokito MK, Lanahan A, Worley P, Holzbaur EL, Ross CA (1997) Huntingtin-associated protein 1 (HAP1) interacts with the pl50Glued subunit of dynactin. Hum Mol Genet 6: 2205-2212. 45. Evert BO, Wullner U, Klockgether T (2000) Cell death in polyglutamine diseases. Cell Tissue Res 301:189-204. 46. Faber PW, Voisine C, King DC, Bates EA, Hart AC (2002) Glutamine/proline-rich PQE-1 proteins protect Caenorhabditis elegans neurons from huntingtin polyglutamine neurotoxicity. Proc Natl Acad Sci U S A 99: 17131-17136. 47. Ferrante RJ, Andreassen OA, Dedeoglu A, Ferrante KL, Jenkins BG, Hersch SM, Beal MF (2002) Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington's disease. J Neurosci 22: 1592-1599. 48. Ferrante RJ, Andreassen OA, Jenkins BG, Dedeoglu A, Kuemmerle S, Kubilus JK, Kaddurah-Daouk R, Hersch SM, Beal MF (2000) Neuroprotective effects of creatine in a transgenic mouse model of Huntington's disease. J Neurosci 20: 4389-4397. 120 49. Ferrante RJ, Kowall NW, Richardson EP, Jr., Bird ED, Martin JB (1986) Topography of enkephalin, substance P and acetylcholinesterase staining in Huntington's disease striatum. Neurosci Lett 71: 283-288. 50. Ferrante RJ, Kubilus JK, Lee J, Ryu H, Beesen A, Zucker B, Smith K, Kowall NW, Ratan RR, Luthi-Carter R, Hersch SM (2003) Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J Neurosci 23: 9418-9427. 51. Gafni J, Ellerby LM (2002) Calpain activation in Huntington's disease. J Neurosci 22:4842-4849. 52. Gafni J, Hermel E, Young JE, Wellington CL, Hayden MR, Ellerby LM (2004) Inhibition of Calpain Cleavage of Huntingtin Reduces Toxicity: Accumulation of Calpain/Caspase Fragments in the Nucleus. J Biol Chem 279: 20211-20220. 53. Gervais FG, Singaraja R, Xanthoudakis S, Gutekunst CA, Leavitt BR, Metzler M, Hackam AS, Tam J, Vaillancourt JP, Houtzager V, Rasper DM, Roy S, Hayden MR, Nicholson DW (2002) Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Nat Cell Biol 4: 95-105. 54. Gimenez E, Montoliu L (2001) A simple polymerase chain reaction assay for genotyping the retinal degeneration mutation (Pdeb(rdl)) in FVB/N-derived transgenic mice. Lab Anim 35: 153-156. 55. Goldberg YP, Nicholson DW, Rasper DM, Kalchman MA, Koide HB, Graham RK, Bromm M, Kazemi-Esfarjani P, Thornberry NA, Vaillancourt JP, Hayden MR (1996) Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract. Nat Genet 13: 442-449. 56. Gross SP (2003) Dynactin: coordinating motors with opposite inclinations. Curr Biol 13: R320-R322. 57. Guidetti P, Charles V, Chen EY, Reddy PH, Kordower JH, Whetsell WO, Jr., Schwarcz R, Tagle DA (2001) Early degenerative changes in transgenic mice expressing mutant huntingtin involve dendritic abnormalities but no impairment of mitochondrial energy production. Exp Neurol 169: 340-350. 58. Gunawardena S, Her LS, Brusch RG, Laymon RA, Niesman IR, Gordesky-Gold B, Sintasath L, Bonini NM, Goldstein LS (2003) Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron 40: 25-40. 59. Gutekunst CA, Li SH, Yi H, Ferrante RJ, Li XJ, Hersch SM (1998) The cellular and subcellular localization of huntingtin-associated protein 1 (HAP1): comparison with huntingtin in rat and human. J Neurosci 18: 7674-7686. 60. Gutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, Rye D, Ferrante RJ, Hersch SM, Li XJ (1999) Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology. J Neurosci 19: 2522-2534. 121 61. Hackam AS, Hodgson JG, Singaraja R, Zhang T, Gan L, Gutekunst CA, Hersch SM, Hayden MR (1999) Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin. Philos Trans R Soc Lond B Biol Sci 354: 1047-1055. 62. Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, Kalchman M, Hayden MR (1998) The influence of huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol 141:1097-1105. 63. Hansson O, Nylandsted J, Castilho RF, Leist M, Jaattela M, 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: 47-57. 64. Hansson O, Petersen A, Leist M, Nicotera P, Castilho RF, Brundin P (1999) Transgenic mice expressing a Huntington's disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity. Proc Natl Acad Sci U S A 96: 8727-8732. 65. Harjes P, Wanker EE (2003) The hunt for huntingtin function: interaction partners tell many different stories. Trends Biochem Sci 28: 425-433. 66. Harper P, Bates GP, Jones L (2002) Huntington's disease. New York: Oxford University Press. 67. Hashimoto R, Hough C, Nakazawa T, Yamamoto T, Chuang DM (2002a) Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation. J Neurochem 80: 589-597. 68. Hashimoto R, Takei N, Shimazu K, Christ L, Lu B, Chuang DM (2002b) Lithium induces brain-derived neurotrophic factor and activates TrkB in rodent cortical neurons: an essential step for neuroprotection against glutamate excitotoxicity. Neuropharmacology 43:1173-1179. 69. Hayden MR (1981) Huntington's Chorea. New York: Springer. 70. Heiser V, Engemann S, Brocker W, Dunkel I, Boeddrich A, Waelter S, Nordhoff E, Lurz R, Schugardt N, Rautenberg S, Herhaus C, Barnickel G, Bottcher H, Lehrach H, Wanker EE (2002) Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Huntington's disease by using an automated filter retardation assay. Proc Natl Acad Sci U S A 99 Suppl 4:16400-16406. 71. Heiser V, Scherzinger E, Boeddrich A, Nordhoff E, Lurz R, Schugardt N, Lehrach H, Wanker EE (2000) Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy. Proc Natl Acad Sci U S A 97: 6739-6744. 72. Hermel E, Garni J, Propp SS, Leavitt BR, Wellington CL, Young JE, Hackam AS, Logvinova AV, Peel AL, Chen SF, Hook V, Singaraja R, Krajewski S, Goldsmith PC, Ellerby HM, Hayden MR, Bredesen DE, Ellerby LM (2004) Specific caspase 122 interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease. Cell Death Differ. 73. Hickey MA, Reynolds GP, Morton AJ (2002) The role of dopamine in motor symptoms in the R6/2 transgenic mouse model of Huntington's disease. J Neurochem 81: 46-59. 74. Hockly E, Richon VM, Woodman B, Smith DL, Zhou X, Rosa E, Sathasivam K, Ghazi-Noori S, Mahal A, Lowden PA, Steffan JS, Marsh JL, Thompson LM, Lewis CM, Marks PA, Bates GP (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: 2041-2046. 75. Hodgson JG, Agopyan N, Gutekunst CA, Leavitt BR, LePiane F, Singaraja R, Smith DJ, Bissada N, McCutcheon K, Nasir J, Jamot L, Li XJ, Stevens ME, Rosemond E, Roder JC, Phillips AG, Rubin EM, Hersch SM, Hayden MR (1999) A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23:181-192. 76. Hodgson JG, Smith DJ, McCutcheon K, Koide HB, Nishiyama K, Dinulos MB, Stevens ME, Bissada N, Nasir J, Kanazawa I, Disteche CM, Rubin EM, Hayden MR (1996) Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype. Hum Mol Genet 5: 1875-1885. 77. Holbert S, Denghien I, Kiechle T, Rosenblatt A, Wellington C, Hayden MR, Margolis RL, Ross CA, Dausset J, Ferrante RJ, 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 98:1811-1816. 78. Holmberg M, Duyckaerts C, Durr A, Cancel G, Gourfinkel-An I, Damier P, Faucheux B, Trottier Y, Hirsch EC, Agid Y, Brice A (1998) Spinocerebellar ataxia type 7 (SCA7): a neurodegenerative disorder with neuronal intranuclear inclusions. Hum Mol Genet 7: 913-918. 79. Hossain SM, Wong BK, Simpson EM (2004) The dark phase improves genetic discrimination for some high throughput mouse behavioral phenotyping. Genes Brain Behav 3:167-177. 80. Humbert S, Bryson EA, Cordelieres FP, Connors NC, Datta SR, Finkbeiner S, Greenberg ME, Saudou F (2002) The IGF-l/Akt pathway is neuroprotective in Huntington's disease and involves Huntingtin phosphorylation by Akt. Dev Cell 2: 831-837. 81. Humphries MM, Kiang S, McNally N, Donovan MA, Sieving PA, Bush RA, Machida S, Cotter T, Hobson A, Farrar J, Humphries P, Kenna P (2001) Comparative structural and functional analysis of photoreceptor neurons of Rlio-/-mice reveal increased survival on C57BL/6J in comparison to 129Sv genetic background. Vis Neurosci 18: 437-443. 123 82. Huntington G (2003) On chorea. George Huntington, M.D. J Neuropsychiatry Clin Neurosci 15: 109-112. 83. Huq AM, Hackam A, Graham RK, Wellington CL, Hayden MR (1998) Molecular Pathogenesis of Huntington's Disease: Biochemical Studies of Huntingtin. In: Genetic Instabilities and Hereditary Neurological Diseases pp 325-354. Academic Press. 84. Iijima K, Liu HP, Chiang AS, Hearn SA, Konsolaki M, Zhong Y (2004) Dissecting the pathological effects of human Abeta40 and Abeta42 in Drosophila: a potential model for Alzheimer's disease. Proc Natl Acad Sci U S A 101: 6623-6628. 85. Imbert G, Saudou F, Yvert G, Devys D, Trottier Y, Garnier JM, Weber C, Mandel JL, Cancel G, Abbas N, Durr A, Didierjean O, Stevanin G, Agid Y, Brice A (1996) Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats. Nat Genet 14: 285-291. 86. Jackson GR, Salecker I, Dong X, Yao X, Arnheim N, Faber PW, MacDonald ME, Zipursky SL (1998) Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons. Neuron 21: 633-642. 87. Jana NR, Tanaka M, Wang G, Nukina N (2000) Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity. Hum Mol Genet 9: 2009-2018. 88. Kalchman MA, Koide HB, McCutcheon K, Graham RK, Nichol K, Nishiyama K, Kazemi-Esfarjani P, Lynn FC, Wellington C, Metzler M, Goldberg YP, Kanazawa I, Gietz RD, Hayden MR (1997) HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat Genet 16: 44-53. 89. Katsuno M, Adachi H, Doyu M, Minamiyama M, Sang C, Kobayashi Y, Inukai A, Sobue G (2003) Leuprorelin rescues polyglutamine-dependent phenotypes in a transgenic mouse model of spinal and bulbar muscular atrophy. Nat Med 9: 768-773. 90. Katsuno M, Adachi H, Kume A, Li M, Nakagomi Y, Niwa H, Sang C, Kobayashi Y, Doyu M, Sobue G (2002) Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy. Neuron 35: 843-854. 91. Kawaguchi Y, Okamoto T, Taniwaki M, Aizawa M, Inoue M, Katayama S, Kawakami H, Nakamura S, Nishimura M, Akiguchi I,. (1994) CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nat Genet 8: 221-228. 92. Kazantsev A, Walker HA, Slepko N, Bear JE, Preisinger E, Steffan JS, Zhu YZ, Gertler FB, Housman DE, Marsh JL, Thompson LM (2002) A bivalent Huntingtin binding peptide suppresses polyglutamine aggregation and pathogenesis in Drosophila. Nat Genet 30: 367-376. 124 93. Keene CD, Rodrigues CM, Eich T, Chhabra MS, Steer CJ, Low WC (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: 10671-10676. 94. Kennedy L, Shelbourne PF (2000) Dramatic mutation instability in HD mouse striatum: does polyglutamine load contribute to cell-specific vulnerability in Huntington's disease? Hum Mol Genet 9: 2539-2544. 95. Khorchid A, Ikura M (2002) How calpain is activated by calcium. Nat Struct Biol 9: 239-241. 96. Kido Y, Philippe N, Schaffer AA, Accili D (2000) Genetic modifiers of the insulin resistance phenotype in mice. Diabetes 49: 589-596. 97. Kim M, Lee HS, Laforet G, Mclntyre C, Martin EJ, Chang P, Kim TW, Williams M, Reddy PH, Tagle D, Boyce FM, Won L, Heller A, Aronin N, DiFiglia M (1999) Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition. J Neurosci 19: 964-973. 98. Kim YJ, Yi Y, Sapp E, Wang Y, Cuiffo B, Kegel KB, Qin ZH, Aronin N, DiFiglia M (2001) Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis. Proc Natl Acad Sci USA 98: 12784-12789. 99. Klapstein GJ, Fisher RS, Zanjani H, Cepeda C, Jokel ES, Chesselet MF, Levine MS (2001) Electrophysiological and morphological changes in striatal spiny neurons in R6/2 Huntington's disease transgenic mice. J Neurophysiol 86: 2667-2677. 100. Klement IA, Skinner PJ, Kaytor MD, Yi H, Hersch SM, Clark HB, Zoghbi HY, Orr HT (1998) Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell 95: 41-53. 101. Klivenyi P, Ferrante RJ, Gardian G, Browne S, Chabrier PE, Beal MF (2003) Increased survival and neuroprotective effects of BN82451 in a transgenic mouse model of Huntington's disease. J Neurochem 86: 267-272. 102. Kodsi MH, Swerdlow NR (1997) Mitochondrial toxin 3-nitropropionic acid produces startle reflex abnormalities and striatal damage in rats that model some features of Huntington's disease. Neurosci Lett 231: 103-107. 103. Koide R, Ikeuchi T, Onodera O, Tanaka H, Igarashi S, Endo K, Takahashi H, Kondo R, Ishikawa A, Hayashi T, . (1994) Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA). Nat Genet 6: 9-13. 104. Kremer B, Goldberg P, Andrew SE, Theilmann J, Telenius H, Zeisler J, Squitieri F, Lin B, Bassett A, Almqvist E , . (1994) A worldwide study of the Huntington's disease mutation. The sensitivity and specificity of measuring CAG repeats. N Engl J Med 330:1401-1406. 125 105. Kunst CB, Messer L, Gordon J, Haines J, Patterson D (2000) Genetic mapping of a mouse modifier gene that can prevent ALS onset. Genomics 70:181-189. 106. La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352: 77-79. 107. Laforet GA, Sapp E, Chase K, Mclntyre C, Boyce FM, Campbell M, Cadigan BA, Warzecki L, Tagle DA, Reddy PH, Cepeda C, Calvert CR, Jokel ES, Klapstein GJ, Ariano MA, Levine MS, DiFiglia M, 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: 9112-9123. 108. Langbehn DR, Brinkman RR, Falush D, Paulsen JS, Hayden MR (2004) A new model for prediction of the age of onset and penetrance for Huntington's disease based on CAG length. Clin Genet 65: 267-277. 109. Leavitt BR, Guttman JA, Hodgson JG, Kimel GH, Singaraja R, Vogl AW, Hayden MR (2001) Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo. Am J Hum Genet 68: 313-324. 110. Leavitt BR, Wellington CL, Hayden MR (1999) Recent insights into the molecular pathogenesis of Huntington disease. Semin Neurol 19: 385-395. 111. Legendre-Guillemin V, Metzler M, Charbonneau M, Gan L, Chopra V, Philie J, Hayden MR, McPherson PS (2002) HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin. Identification of a novel interaction with clathrin light chain. J Biol Chem 277:19897-19904. 112. Leonard JR, Klocke BJ, D'Sa C, Flavell RA, Roth KA (2002) Strain-dependent neurodevelopmental abnormalities in caspase-3-deficient mice. J Neuropathol Exp Neurol 61: 673-677. 113. Levine MS, Klapstein GJ, Koppel A, Gruen E, Cepeda C, Vargas ME, Jokel ES, Carpenter EM, Zanjani H, Hurst RS, Efstratiadis A, Zeitlin S, Chesselet MF (1999) Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington's disease. J Neurosci Res 58: 515-532. 114. Li H, Li SH, Johnston H, Shelbourne PF, Li XJ (2000) Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat Genet 25: 385-389. 115. Li H, Li SH, Yu ZX, Shelbourne P, Li XJ (2001) Huntingtin aggregate-associated axonal degeneration is an early pathological event in Huntington's disease mice. J Neurosci 21: 8473-8481. 116. Li JL, Hayden MR, Almqvist EW, Brinkman RR, Durr A, Dode C, Morrison PJ, Suchowersky O, Ross CA, Margolis RL, Rosenblatt A, Gomez-Tortosa E, Cabrero DM, Novelletto A, Frontali M, Nance M, Trent RJ, McCusker E, Jones R, Paulsen JS, Harrison M, Zanko A, Abramson RK, Russ AL, Knowlton B, Djousse L, Mysore JS, Tariot S, Gusella MF, Wheeler VC, Atwood LD, Cupples LA, Saint-Hilaire M, 126 Cha JH, Hersch SM, Koroshetz WJ, Gusella JF, MacDonald ME, Myers RH (2003a) A genome scan for modifiers of age at onset in Huntington disease: The HD MAPS study. Am J Hum Genet 73: 682-687. 117. Li L, Fan M, Icton CD, Chen N, Leavitt BR, Hayden MR, Murphy TH, Raymond LA (2003b) Role of NR2B-type NMDA receptors in selective neurodegeneration in Huntington disease. Neurobiol Aging 24: 1113-1121. 118. Li M, Miwa S, Kobayashi Y, Merry DE, Yamamoto M, Tanaka F, Doyu M, Hashizume Y, Fischbeck KH, Sobue G (1998) Nuclear inclusions of the androgen receptor protein in spinal and bulbar muscular atrophy. Ann Neurol 44: 249-254. 119. Li SH, Cheng AL, Zhou H, Lam S, Rao M, Li H, Li XJ (2002) Interaction of Huntington disease protein with transcriptional activator Spl. Mol Cell Biol 22: 1277-1287. 120. Li XJ, Sharp AH, Li SH, Dawson TM, Snyder SH, Ross CA (1996) Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase. Proc Natl Acad Sci USA 93: 4839-4844. 121. Lin CH, Tallaksen-Greene S, Chien WM, Cearley JA, Jackson WS, Crouse AB, Ren S, Li XJ, Albin RL, Detloff PJ (2001) Neurological abnormalities in a knock-in mouse model of Huntington's disease. Hum Mol Genet 10:137-144. 122. Lione LA, Carter RJ, Hunt MJ, Bates GP, Morton AJ, Dunnett SB (1999) Selective discrimination learning impairments in mice expressing the human Huntington's disease mutation. J Neurosci 19: 10428-10437. 123. Luesse HG, Schiefer J, Spruenken A, Puis C, Block F, Kosinski CM (2001) Evaluation of R6/2 HD transgenic mice for therapeutic studies in Huntington's disease: behavioral testing and impact of diabetes mellitus. Behav Brain Res 126: 185-195. 124. Lunkes A, Lindenberg KS, Ben Haiem L, Weber C, Devys D, Landwehrmeyer GB, Mandel JL, Trottier Y (2002) Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol Cell 10: 259-269. 125. Lunkes A, Mandel JL (1998) A cellular model that recapitulates major pathogenic steps of Huntington's disease. Hum Mol Genet 7:1355-1361. 126. Luthi-Carter R, Hanson SA, Strand AD, Bergstrom DA, Chun W, Peters NL, Woods AM, Chan EY, Kooperberg C, Krainc D, Young AB, Tapscott SJ, Olson JM (2002a) Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain. Hum Mol Genet 11:1911-1926. 127. Luthi-Carter R, Strand A, Peters NL, Solano SM, Hollingsworth ZR, Menon AS, Frey AS, Spektor BS, Penney EB, Schilling G, Ross CA, Borchelt DR, Tapscott SJ, Young AB, Cha JH, Olson JM (2000) Decreased expression of striatal signaling genes in a mouse model of Huntington's disease. Hum Mol Genet 9:1259-1271. 127 128. Luthi-Carter R, Strand AD, Hanson SA, Kooperberg C, Schilling G, La Spada AR, Merry DE, Young AB, Ross CA, Borchelt DR, Olson JM (2002b) Polyglutamine and transcription: gene expression changes shared by DRPLA and Huntington's disease mouse models reveal context-independent effects. Hum Mol Genet 11:1927-1937. 129. MacDonald ME, Vonsattel JP, Shrinidhi J, Couropmitree NN, Cupples LA, Bird ED, Gusella JF, Myers RH (1999) Evidence for the GluR6 gene associated with younger onset age of Huntington's disease. Neurology 53:1330-1332. 130. Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hetherington C, Lawton M, Trottier Y, Lehrach H, Davies SW, Bates GP (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493-506. 131. Manley, K., Bolivar, V. J., Wolfgang, W. J., Armour, K, McManus A., Miller, T. W., and Messer, A. Effect of genetic background on the Huntington disease R6/1 transgene: genomic, histological, and behavioral analyses. Abstract Viewer/Itinerary Planner. 2003. Ref Type: Abstract 132. Mantamadiotis T, Lemberger T, Bleckmann SC, Kern H, Kretz O, Martin VA, Tronche F, Kellendonk C, Gau D, Kapfhammer J, Otto C, Schmid W, Schutz G (2002) Disruption of CREB function in brain leads to neurodegeneration. Nat Genet 31: 47-54. 133. Martindale D, Hackam A, Wieczorek A, Ellerby L, Wellington C, McCutcheon K, Singaraja R, Kazemi-Esfarjani P, Devon R, Kim SU, Bredesen DE, Tufaro F, Hayden MR (1998) Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates. Nat Genet 18:150-154. 134. Mastroberardino PG, Iannicola C, Nardacci R, Bernassola F, De L, V, Melino G, Moreno S, Pavone F, Oliverio S, Fesus L, Piacentini M (2002) 'Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease. Cell Death Differ 9: 873-880. 135. McGeer EG, McGeer PL (1976) Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acids. Nature 263: 517-519. 136. McPherson PS, Kay BK, Hussain NK (2001) Signaling on the endocytic pathway. Traffic 2: 375-384. 137. Meade CA, Deng YP, Fusco FR, Del Mar N, Hersch S, Goldowitz D, Reiner A (2002) Cellular localization and development of neuronal intranuclear inclusions in striatal and cortical neurons in R6/2 transgenic mice. J Comp Neurol 449: 241-269. 138. Menalled L, Zanjani H, MacKenzie L, Koppel A, Carpenter E, Zeitlin S, Chesselet MF (2000) Decrease in striatal enkephalin mRNA in mouse models of Huntington's disease. Exp Neurol 162: 328-342. 128 139. Menalled LB, Sison JD, Dragatsis I, Zeitlin S, Chesselet MF (2003) Time course of early motor and neuropathological anomalies in a knock-in mouse model of Huntington's disease with 140 CAG repeats. J Comp Neurol 465: 11-26. 140. Menalled LB, Sison JD, Wu Y, Olivieri M, Li XJ, Li H, Zeitlin S, Chesselet MF (2002) Early motor dysfunction and striosomal distribution of huntingtin microaggregates in Huntington's disease knock-in mice. J Neurosci 22: 8266-8276. 141. Metzler M, Legendre-Guillemin V, Gan L, Chopra V, Kwok A, McPherson PS, Hayden MR (2001) HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2. J Biol Chem 276: 39271-39276. 142. Metzler M, Li B, Gan L, Georgiou J, Gutekunst CA, Wang Y, Torre E, Devon RS, Oh R, Legendre-Guillemin V, Rich M, Alvarez C, Gertsenstein M, McPherson PS, Nagy A, Wang YT, Roder JC, Raymond LA, Hayden MR (2003) Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking. EMBO J 22: 3254-3266. 143. Miller VM, Xia H, Marrs GL, Gouvion CM, Lee G, Davidson BL, Paulson HL (2003) Allele-specific silencing of dominant disease genes. Proc Natl Acad Sci U S A 100: 7195-7200. 144. Mishra SK, Agostinelli NR, Brett TJ, Mizukami I, Ross TS, Traub LM (2001) Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins. J Biol Chem 276: 46230-46236. 145. Mizuno K, Carnahan J, Nawa H (1994) Brain-derived neurotrophic factor promotes differentiation of striatal GABAergic neurons. Dev Biol 165: 243-256. 146. Modregger J, DiProspero NA, Charles V, Tagle DA, Plomann M (2002) PACSIN 1 interacts with huntingtin and is absent from synaptic varicosities in presymptomatic Huntington's disease brains. Hum Mol Genet 11: 2547-2558. 147. Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116: 201-211. 148. Munoz E, Cervera A, Valls-Sole J (2003) Neurophysiological study of facial chorea in patients with Huntington's disease. Clin Neurophysiol 114:1246-1252. 149. Myers SJ, Dingledine R, Borges K (1999) Genetic regulation of glutamate receptor ion channels. Annu Rev Pharmacol Toxicol 39: 221-241. 150. Nasir J, Floresco SB, O'Kusky JR, Diewert VM, Richman JM, Zeisler J, Borowski A, Marth JD, Phillips AG, Hayden MR (1995) Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 81: 811-823. 151. Naze P, Vuillaume I, Destee A, Pasquier F, Sablonniere B (2002) Mutation analysis and association studies of the ubiquitin carboxy-terminal hydrolase LI gene in Huntington's disease. Neurosci Lett 328:1-4. 129 152. Nonaka S, Hough CJ, Chuang DM (1998) Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium influx. Proc Natl Acad Sci U S A 95: 2642-2647. 153. Nucifora FC, Jr., Sasaki M, Peters MF, Huang H, Cooper JK, Yamada M, Takahashi H, Tsuji S, Troncoso J, Dawson VL, Dawson TM, Ross CA (2001) Interference by huntingtin and atrophia-1 with cbp-mediated transcription leading to cellular toxicity. Science 291: 2423-2428. 154. O'Kusky JR, Nasir J, Cicchetti F, Parent A, Hayden MR (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: 468-479. 155. Ona VO, Li M, Vonsattel JP, Andrews LJ, Khan SQ, Chung WM, Frey AS, Menon AS, Li XJ, Stieg PE, Yuan J, Penney JB, Young AB, Cha JH, Friedlander RM (1999) Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease. Nature 399: 263-267. 156. Ordway JM, Tallaksen-Greene S, Gutekunst CA, Bernstein EM, Cearley JA, Wiener HW, Dure LS, Lindsey R, Hersch SM, Jope RS, Albin RL, Detloff PJ (1997) Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell 91: 753-763. 157. Orr HT, Chung MY, Banfl S, Kwiatkowski TJ, Jr., Servadio A, Beaudet AL, McCall AE, Duvick LA, Ranum LP, Zoghbi HY (1993) Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat Genet 4: 221-226. 158. Osmand, A. P., Berthelier, V., and Wetzel, R. Identification of aggregation foci, intracellular neuronal structures in the neocortex in Huntington's disease capable of recruiting polyglutamine. Soc.Neurosci.Abst 293[6]. 2002. Ref Type: Abstract 159. Ouimet CC, Langley-Gullion KC, Greengard P (1998) Quantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen. Brain Res 808: 8-12. 160. Panas M, Avramopoulos D, Karadima G, Petersen MB, Vassilopoulos D (1999) Apolipoprotein E and presenilin-1 genotypes in Huntington's disease. J Neurol 246: 574-577. 161. Panov AV, Gutekunst CA, Leavitt BR, Hayden MR, Burke JR, Strittmatter WJ, Greenamyre JT (2002) Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines. Nat Neurosci 5: 731-736. 162. Parker JA, Connolly JB, Wellington C, Hayden M, Dausset J, Neri C (2001) Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. Proc Natl Acad Sci U S A 98:13318-13323. 130 163. Paulson HL, Perez MK, Trottier Y, Trojanowski JQ, Subramony SH, Das SS, Vig P, Mandel JL, Fischbeck KH, Pittman RN (1997) Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. Neuron 19: 333-344. 164. Peel AL, Rao RV, Cottrell BA, Hayden MR, Ellerby LM, Bredesen DE (2001) Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue. Hum Mol Genet 10:1531-1538. 165. Pulst SM, Nechiporuk A, Nechiporuk T, Gispert S, Chen XN, Lopes-Cendes I, Pearlman S, Starkman S, Orozco-Diaz G, Lunkes A, Dejong P, Rouleau GA, Auburger G, Korenberg JR, Figueroa C, Sahba S (1996) Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nat Genet 14: 269-276. 166. Rangone H, Poizat G, Troncoso J, Ross CA, MacDonald ME, Saudou F, Humbert S (2004) The serum- and glucocorticoid-induced kinase SGK inhibits mutant huntingtin-induced toxicity by phosphorylating serine 421 of huntingtin. Eur J Neurosci 19: 273-279. 167. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein DC (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36: 585-595. 168. Reddy PH, Charles V, Williams M, Miller G, Whetsell WO, Jr., Tagle DA (1999a) Transgenic mice expressing mutated full-length HD cDNA: a paradigm for locomotor changes and selective neuronal loss in Huntington's disease. Philos Trans R Soc Lond B Biol Sci 354:1035-1045. 169. Reddy PH, Williams M, Charles V, Garrett L, Pike-Buchanan L, Whetsell WO, Jr., Miller G, Tagle DA (1998) Behavioural abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA. Nat Genet 20:198-202. 170. Reddy PH, Williams M, Tagle DA (1999b) Recent advances in understanding the pathogenesis of Huntington's disease. Trends Neurosci 22: 248-255. 171. Rigamonti D, Bauer JH, De Fraja C, Conti L, Sipione S, Sciorati C, Clementi E, Hackam A, Hayden MR, Li Y, Cooper JK, Ross CA, Govoni S, Vincenz C, Cattaneo E (2000) Wild-type huntingtin protects from apoptosis upstream of caspase-3. J Neurosci 20: 3705-3713. 172. Rosas HD, Goodman J, Chen YI, Jenkins BG, Kennedy DN, Makris N, Patti M, Seidman LJ, Beal MF, Koroshetz WJ (2001) Striatal volume loss in HD as measured by MRI and the influence of CAG repeat. Neurology 57:1025-1028. 173. Rosas HD, Liu AK, Hersch S, Glessner M, Ferrante RJ, Salat DH, van der KA, Jenkins BG, Dale AM, Fischl B (2002) Regional and progressive thinning of the cortical ribbon in Huntington's disease. Neurology 58: 695-701. 131 174. Rosenblatt A, Brinkman RR, Liang KY, Almqvist EW, Margolis RL, Huang CY, Sherr M, Franz ML, Abbott MH, Hayden MR, Ross CA (2001) Familial influence on age of onset among siblings with Huntington disease. Am J Med Genet 105: 399-403. 175. Rozen, S. and Slaletsky, H. J. Primer3. 1998. Ref Type: Computer Program 176. Rubinsztein DC, Leggo J, Chiano M, Dodge A, Norbury G, Rosser E, 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: 3872-3876. 177. Sanchez I, Mahlke C, Yuan J (2003) Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature 421: 373-379. 178. Sanpei K, Takano H, Igarashi S, Sato T, Oyake M, Sasaki H, Wakisaka A, Tashiro K, Ishida Y, Ikeuchi T, Koide R, Saito M, Sato A, Tanaka T, Hanyu S, Takiyama Y, Nishizawa M, Shimizu N, Nomura Y, Segawa M, Iwabuchi K, Eguchi I, Tanaka H, Takahashi H, Tsuji S (1996) Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT. Nat Genet 14: 277-284. 179. Saudou F, Finkbeiner S, Devys D, Greenberg ME (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95: 55-66. 180. Sawa A, Wiegand GW, Cooper J, Margolis RL, Sharp AH, Lawler JF, Jr., Greenamyre JT, Snyder SH, Ross CA (1999) Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization. Nat Med 5:1194-1198. 181. Schauwecker PE (2002) Modulation of cell death by mouse genotype: differential vulnerability to excitatory amino acid-induced lesions. Exp Neurol 178: 219-235. 182. Schauwecker PE (2003) Genetic basis of kainate-induced excitotoxicity in mice: phenotypic modulation of seizure-induced cell death. Epilepsy Res 55: 201-210. 183. Schauwecker PE, Steward O (1997) Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc Natl Acad Sci U S A 94: 4103-4108. 184. Schiefer J, Landwehrmeyer GB, Luesse HG, Sprunken A, Puis C, Milkereit A, Milkereit E, Kosinski CM (2002) Riluzole prolongs survival time and alters nuclear inclusion formation in a transgenic mouse model of Huntington's disease. Mov Disord 17: 748-757. 185. Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, Copeland NG, Price DL, Ross CA, Borchelt DR (1999) Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet 8: 397-407. 132 186. Schilling G, Jinnah HA, Gonzales V, Coonfield ML, Kim Y, Wood JD, Price DL, Li XJ, Jenkins N, Copeland N, Moran T, Ross CA, Borchelt DR (2001) Distinct behavioral and neuropathological abnormalities in transgenic mouse models of HD and DRPLA. Neurobiol Dis 8: 405-418. 187. Schwartz S, Zhang Z, Frazer KA, Smit A, Riemer C, Bouck J, Gibbs R, Hardison R, Miller W (2000) PipMaker--a web server for aligning two genomic DNA sequences. Genome Res 10: 577-586. 188. Shelbourne PF, Killeen N, Hevner RF, Johnston HM, Tecott L, Lewandoski M, Ennis M, Ramirez L, Li Z, Iannicola C, Littman DR, Myers RM (1999) A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice. Hum Mol Genet 8: 763-774. 189. Sheng M, Kim MJ (2002) Postsynaptic signaling and plasticity mechanisms. Science 298: 776-780. 190. Sieradzan KA, Mechan AO, Jones L, Wanker EE, Nukina N, Mann DM (1999) Huntington's disease intranuclear inclusions contain truncated, ubiquitinated huntingtin protein. Exp Neurol 156: 92-99. 191. Silver LM (1995) Mouse Genetics: Concepts and Applications. New York: Oxford University Press, Inc. 192. Singaraja RR, Hadano S, Metzler M, Givan S, Wellington CL, Warby S, Yanai A, Gutekunst CA, Leavitt BR, Yi H, Fichter K, Gan L, McCutcheon K, Chopra V, Michel J, Hersch SM, Ikeda JE, Hayden MR (2002) HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis. Hum Mol Genet 11: 2815-2828. 193. Sipione S, Rigamonti D, Valenza M, Zuccato C, Conti L, Pritchard J, Kooperberg C, Olson JM, Cattaneo E (2002) Early transcriptional profiles in huntingtin-inducible striatal cells by microarray analyses. Hum Mol Genet 11:1953-1965. 194. Sittler A, Walter S, Wedemeyer N, Hasenbank R, Scherzinger E, Eickhoff H, Bates GP, Lehrach H, Wanker EE (1998) SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polygln-containing protein aggregates. Mol Cell 2: 427-436. 195. Skinner PJ, Koshy BT, Cummings CJ, Klement IA, Helin K, Servadio A, Zoghbi HY, Orr HT (1997) Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures. Nature 389: 971-974. 196. Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, Deng Y, Oh R, Bissada N, Hossain SM, Yang YZ, Li XJ, Simpson EM, Gutekunst CA, Leavitt BR, Hayden MR (2003) Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. Hum Mol Genet 12:1555-1567. 197. Smith DL, Bates GP (2004) Monitoring aggregate formation in organotypic slice cultures from transgenic mice. Methods Mol Biol 277: 161-172. 133 198. Smith DL, Portier R, Woodman B, Hockly E, Mahal A, Klunk WE, Li XJ, Wanker E, Murray KD, Bates GP (2001) Inhibition of polyglutamine aggregation in R6/2 HD brain slices-complex dose-response profiles. Neurobiol Dis 8:1017-1026. 199. Smith JD, James D, Dansky HM, Wittkowski KM, Moore KJ, Breslow JL (2003) In silico quantitative trait locus map for atherosclerosis susceptibility in apolipoprotein E-deficient mice. Arterioscler Thromb Vase Biol 23:117-122. 200. Snell RG, MacMillan JC, Cheadle JP, Fenton I, Lazarou LP, Davies P, MacDonald ME, Gusella JF, Harper PS, Shaw DJ (1993) Relationship between trinucleotide repeat expansion and phenotypic variation in Huntington's disease. Nat Genet 4: 393-397. 201. Squitieri F, Gellera C, Cannella M, Mariotti C, Cislaghi G, Rubinsztein DC, Almqvist EW, Turner D, Bachoud-Levi AC, Simpson SA, Delatycki M, Maglione V, Hayden MR, Donato SD (2003) Homozygosity for CAG mutation in Huntington disease is associated with a more severe clinical course. Brain 126: 946-955. 202. Steffan JS, Agrawal N, Pallos J, Rockabrand E, Trotman LC, Slepko N, Illes K, Lukacsovich T, Zhu YZ, Cattaneo E, Pandolfi PP, Thompson LM, Marsh JL (2004) SUMO modification of Huntingtin and Huntington's disease pathology. Science 304: 100-104. 203. Steffan JS, Bodai L, Pallos J, Poelman M, McCampbell A, Apostol BL, Kazantsev A, Schmidt E, Zhu YZ, Greenwald M, Kurokawa R, Housman DE, Jackson GR, Marsh JL, Thompson LM (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413: 739-743. 204. Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, Wanker EE, Bates GP, Housman DE, Thompson LM (2000) The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc Natl Acad Sci U S A 97: 6763-6768. 205. Suhr ST, Senut MC, Whitelegge JP, Faull KF, Cuizon DB, Gage FH (2001) Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression. J Cell Biol 153: 283-294. 206. Sun JY, Wu XS, Wu LG (2002) Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse. Nature 417: 555-559. 207. Swerdlow NR, Paulsen J, Braff DL, Butters N, Geyer MA, Swenson MR (1995) Impaired prepulse inhibition of acoustic and tactile startle response in patients with Huntington's disease. J Neurol Neurosurg Psychiatry 58:192-200. 208. Takano H, Gusella JF (2002) The predominantly HEAT-like motif structure of huntingtin and its association and coincident nuclear entry with dorsal, an NF-kB/Rel/dorsal family transcription factor. BMC Neurosci 3: 15. 209. Tallaksen-Greene SJ, Ordway JM, Crouse AB, Jackson WS, Detloff PJ, Albin RL (2003) Hprt(CAG)146 mice: age of onset of behavioral abnormalities, time course of neuronal intranuclear inclusion accumulation, neurotransmitter marker alterations, 134 mitochondrial function markers, and susceptibility to l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine. J Comp Neurol 465: 205-219. 210. Tanaka M, Machida Y, Niu S, Ikeda T, Jana NR, Doi H, Kurosawa M, Nekooki M, Nukina N (2004) Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat Med 10:148-154. 211. Tang TS, Tu H, Chan EY, Maximov A, Wang Z, Wellington CL, Hayden MR, Bezprozvanny I (2003) Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(l,4,5) triphosphate receptor type 1. Neuron 39: 227-239. 212. Trettel F, Rigamonti D, Hilditch-Maguire P, Wheeler VC, Sharp AH, Persichetti F, Cattaneo E, MacDonald ME (2000) Dominant phenotypes produced by the HD mutation in STHdh(Qlll) striatal cells. Hum Mol Genet 9: 2799-2809. 213. Tsuji S (2004) Spinocerebellar ataxia type 17: latest member of polyglutamine disease group highlights unanswered questions. Arch Neurol 61: 183-184. 214. van Dellen A, Welch J, Dixon RM, Cordery P, York D, Styles P, Blakemore C, Hannan AJ (2000) N-Acetylaspartate and DARPP-32 levels decrease in the corpus striatum of Huntington's disease mice. Neuroreport 11: 3751-3757. 215. Ventimiglia R, Mather PE, Jones BE, Lindsay RM (1995) The neurotrophins BDNF, NT-3 and NT-4/5 promote survival and morphological and biochemical differentiation of striatal neurons in vitro. Eur J Neurosci 7: 213-222. 216. Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP, Jr. (1985) Neuropathological classification of Huntington's disease. J Neuropathol Exp Neurol 44: 559-577. 217. Waelter S, Scherzinger E, Hasenbank R, Nordhoff E, Lurz R, Goehler H, Gauss C, Sathasivam K, Bates GP, Lehrach H, Wanker EE (2001) The huntingtin interacting protein HIP1 is a clathrin and alpha-adaptin-binding protein involved in receptor-mediated endocytosis. Hum Mol Genet 10:1807-1817. 218. Watase K, Weeber EJ, Xu B, Antalffy B, Yuva-Paylor L, Hashimoto K, Kano M, Atkinson R, Sun Y, Armstrong DL, Sweatt JD, Orr HT, Paylor R, Zoghbi HY (2002) A long CAG repeat in the mouse Seal locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration. Neuron 34: 905-919. 219. Wei H, Qin ZH, Senatorov VV, Wei W, Wang Y, Qian Y, Chuang DM (2001) Lithium suppresses excitotoxicity-induced striatal lesions in a rat model of Huntington's disease. Neuroscience 106: 603-612. 220. Wellington CL, Brinkman RR, O'Kusky JR, Hayden MR (1997) Toward understanding the molecular pathology of Huntington's disease. Brain Pathol 7: 979-1002. 135 221. Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, Graham RK, Loubser O, van Raamsdonk J, Singaraja R, Yang YZ, Gafni J, Bredesen D, Hersch SM, Leavitt BR, Roy S, Nicholson DW, Hayden MR (2002) Caspase cleavage of mutant huntingtin precedes neurodegeneration in Huntington's disease. J Neurosci 22: 7862-7872. 222. Wellington CL, Hayden MR (1997) Of molecular interactions, mice and mechanisms: new insights into Huntington's disease. Curr Opin Neurol 10: 291-298. 223. Wellington CL, Singaraja R, Ellerby L, Savill J, Roy S, Leavitt B, Cattaneo E, Hackam A, Sharp A, Thornberry N, Nicholson DW, Bredesen DE, Hayden MR (2000) Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J Biol Chem 275:19831-19838. 224. Wheeler VC, Auerbach W, White JK, Srinidhi J, Auerbach A, Ryan A, Duyao MP, Vrbanac V, Weaver M, Gusella JF, Joyner AL, MacDonald ME (1999) Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse. Hum Mol Genet 8: 115-122. 225. Wheeler VC, Gutekunst CA, Vrbanac V, Lebel LA, Schilling G, Hersch S, Friedlander RM, Gusella JF, Vonsattel JP, Borchelt DR, MacDonald ME (2002) Early phenotypes that presage late-onset neurodegenerative disease allow testing of modifiers in Hdh CAG knock-in mice. Hum Mol Genet 11: 633-640. 226. Wheeler VC, Lebel LA, Vrbanac V, Teed A, te RH, MacDonald ME (2003) Mismatch repair gene Msh2 modifies the timing of early disease in Hdh(Qlll) striatum. Hum Mol Genet 12: 273-281. 227. Wheeler VC, White JK, Gutekunst CA, Vrbanac V, Weaver M, Li XJ, Li SH, Yi H, Vonsattel JP, Gusella JF, Hersch S, Auerbach W, Joyner AL, MacDonald ME (2000) Long glutamine tracts cause nuclear localization of a novel form of huntingtin in medium spiny striatal neurons in HdhQ92 and HdhQlll knock-in mice. Hum Mol Genet 9: 503-513. 228. White JK, Auerbach W, Duyao MP, Vonsattel JP, Gusella JF, Joyner AL, MacDonald ME (1997) Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion. Nat Genet 17: 404-410. 229. Xia J, Lee DH, Taylor J, Vandelft M, Truant R (2003) Huntingtin contains a highly conserved nuclear export signal. Hum Mol Genet 12: 1393-1403. 230. Yamamoto A, Lucas JJ, Hen R (2000) Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease. Cell 101: 57-66. 231. Yu ZX, Li SH, Evans J, Pillarisetti A, Li H, Li XJ (2003) Mutant huntingtin causes context-dependent neurodegeneration in mice with Huntington's disease. J Neurosci 23: 2193-2202. 232. Yu ZX, Li SH, Nguyen HP, Li XJ (2002) Huntingtin inclusions do not deplete polyglutamine-containing transcription factors in HD mice. Hum Mol Genet 11: 905-914. 136 233. Zala D, Bensadoun JC, Pereira dA, Leavitt BR, Gutekunst CA, Aebischer P, Hayden MR, Deglon N (2004) Long-term lentiviral-mediated expression of ciliary neurotrophic factor in the striatum of Huntington's disease transgenic mice. Exp Neurol 185: 26-35. 234. Zeitlin S, Liu JP, Chapman DL, Papaioannou VE, Efstratiadis A (1995) Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nat Genet 11:155-163. 235. Zeron MM, Chen N, Moshaver A, Lee AT, Wellington CL, Hayden MR, Raymond LA (2001) Mutant huntingtin enhances excitotoxic cell death. Mol Cell Neurosci 17: 41-53. 236. Zeron MM, Fernandes HB, Krebs C, Shehadeh J, Wellington CL, Leavitt BR, Baimbridge KG, Hayden MR, Raymond LA (2004) Potentiation of NMDA receptor-mediated excitotoxicity linked with intrinsic apoptotic pathway in YAC transgenic mouse model of Huntington's disease. Mol Cell Neurosci 25: 469-479. 237. Zeron MM, Hansson O, Chen N, Wellington CL, Leavitt BR, Brundin P, Hayden MR, Raymond LA (2002) Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease. Neuron 33: 849-860. 238. Zhang Y, Li M, Drozda M, Chen M, Ren S, Mejia Sanchez RO, Leavitt BR, Cattaneo E, Ferrante RJ, Hayden MR, Friedlander RM (2003) Depletion of wild-type huntingtin in mouse models of neurologic diseases. J Neurochem 87:101-106. 239. Zhuchenko O, Bailey J, Bonnen P, Ashizawa T, Stockton DW,,Amos C, Dobyns WB, Subramony SH, Zoghbi HY, Lee CC (1997) Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 15: 62-69. 240. Zuccato C, Ciammola A, Rigamonti D, Leavitt BR, Goffredo D, Conti L, MacDonald ME, Friedlander RM, Silani V, Hayden MR, Timmusk T, Sipione S, Cattaneo E (2001) Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease. Science 293: 493-498. 241. Zuccato C, Tartari M, Crotti A, Goffredo D, Valenza M, Conti L, Cataudella T, Leavitt BR, Hayden MR, Timmusk T, Rigamonti D, Cattaneo E (2003) Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes. Nat Genet 35: 76-83. 137 

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