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In vivo characterization of caspase resistant huntingtin : insights into the pathogenic mechanism of.. Graham, Rona Kyrenia 2006

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IN VIVO C H A R A C T E R I Z A T I O N OF C A S P A S E RESISTANT HUNTINGTIN: INSIGHTS INTO T H E PATHOGENIC M E C H A N I S M OF H U N T I N G T O N DISEASE  by RONA KYRENIA G R A H A M B . S c , Concordia University 1987  A THESIS S U B M I T T E D IN P A R T I A L F U L L F I L L M E N T OF THE R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES  Medical Genetics  THE U N I V E R S I T Y OF BRITISH C O L U M B I A January 2006  © R o n a K y r e n i a G r a h a m 2006  Abstract Proteolytic cleavage of htt is regarded as a critical event in the pathogenesis of H D . Expression of htt fragments containing an expanded polyglutamine repeat are toxic in vitro and in vivo, and accumulation of N-terminal truncated products of htt are observed in human and mouse HD brain. Notably, the presence of htt fragments prior to clinical onset of H D suggests that htt cleavage may be a crucial, causal event in the pathogenesis of HD, rather than simply resulting from nonselective activation of proteolytic pathways in the late stage of disease. However the relationship between specific huntingtin fragments and the pathogenesis of H D is unknown. Mutagenesis of all caspase sites in mutant huntingtin prevents toxicity in cultured cells and caspase inhibitors improve survival of neurons transfected with mutant htt. Caspase resistant (CR) htt mouse models therefore would be ideal systems in which to assess whether creation of caspase generated fragments of htt underlye the pathogenesis of H D in vivo. To examine whether a specific caspase cleavage fragment of mutant huntingtin is responsible for the selective neurodegeneration observed in HD, we generated Y A C transgenic mice expressing selective mutations of the caspase cleavage sites within mutant huntingtin. We show, using sequential mutagenesis, that caspase-6 and not caspase-3, mediated cleavage of mutant htt is responsible for the HD-related behavioural phenotype and selective striatal neurodegeneration observed in the Y A C 128 model of HD. Activation of caspase-6 and nuclear translocation of htt fragments coincide with onset of motor dysfunction in the Y A C 128 model, supporting a role for a specific nuclear htt fragment in initiating neuronal dysfunction. Furthermore, caspase-6 cleavage of mutant htt influences susceptibility to excitotoxic stress highlighting caspase-6 mediated proteolysis of htt and excitotoxicity as a primary mechanism underlying motor dysfunction and neuropathology in HD. The results presented in this thesis support and further refine the toxic fragment hypothesis by identifying a specific proteolytic cleavage site in htt that is required for initiating a sequence of events which culminate in the death of selective neurons affected in HD. This evidence demonstrates that generation of a specific fragment of mutant htt in vivo represents a primary, initiating event in the pathogenesis of H D and identifies novel approaches for inhibiting cell death in neurodegenerative disorders such as HD.  Table of Contents Abstract  '.  ii  Table of Contents List of Tables List of Figures  iii '.  vii •  viii  List of Abbreviations  ix  Acknowledgements  xi  Chapter 1: Introduction  1  1.1 History of H D and clinical features  2  1.2 Neuropathology  4  1.3 Genetics  7  1.3.1 Inheritance  7  1.3.2 C A G size and Age of Onset  8  1.4 Cellular function of huntingtin  9  1.4.1 Role in development  10  1.4.2 Interacting Proteins  11  1.4.3 Role in transcription  13  1.4.4 Role in neuroprotection  14  1.4.5 Posttranslational modification of huntingtin  15  1.5 Mutant huntingtin  16  1.5.1 Gain of function/loss of function  17  1.5.1.2 Complete vs. Intermediate Dominance  18  1.6 Huntingtin proteolysis  19  1.6.1 Proteases that cleave huntingtin  21  1.6.2 Caspase cleavage of huntingtin  22  1.6.3 Caspase resistant huntingtin  24  1.6.4 Toxic fragment hypothesis  24  1.7 Huntingtin Inclusions  26  1.7.1 History and definition  26  1.7.2 Relation to toxicity  26  1.8 Apoptosis 1.8.1 Caspase activation pathways in H D 1.9 Excitotoxicity in H D  28 32 33  iii  1.9.1 Acute model of HD: In vivo Quinolinic acid mediated neurotoxicity 1.10 H D mouse models  35 36  1.10.1 Full length mouse models of H D  38  1.10.2 Truncated H D mouse models  39  1.11 Hypothesis and Objectives  41  Chapter 2: Material and Methods  44  2.1 Mutagenesis of huntingtin caspase sites  45  2.1.1 Mutagenesis constructs  45  2.1.2 Yeast growth and transformation:  46  2.1.3 Isolation of yeast genomic D N A for Y A C screening  46  2.1.4 Determination of C A G Stability  47  2.1.5 SSCP to detect mutant sequence  47  2.1.6 Restriction analysis of PCR products  47  2.1.7 Southern blot analysis for Y A C integrity  48  2.1.8 PCR analyses for Y A C integrity  48  2.2. Generation and characterization of C R Y A C transgenic mice  48  2.2.1 Generation of C R Y A C mice  49  2.2.2 Real-time quantitative RT-PCR to determine transgene expression level  49  2.2.3 Protein analysis and Western blotting  50  2.2.4 Ex vivo confirmation of caspase resistant htt  50  2.2.5 Rescue of embryonic lethality of htt deficient mice  50  2.3 Generation and characterization of caspase cleavable Y A C 128 controls 2.3.1 C A G size  ,  2.3.2 mRNA and protein transgene expression levels  51 51 51  2.4 Quantitative Analysis  52  2.5 Behaviour  52  2.5.1 Rotarod analysis  52  2.5.2 Open Field Analysis  53  2.6 Assessment of nuclear huntingtin and inclusions  53  2.7 Analysis of caspase amplification and activation  53  2.7.1 Immunohistochemistry  53  2.7.2 Protein analysis, Antibodies and Western blotting.  54  2.8 Generation of homozygotes Y A C 128 and Caspase Resistant huntingtin mice 2.8.1 Real-time quantitative PCR to determine zygosity  54 54  . 2.8.2 FISH  55  2.9 Assessment of susceptibility to Excitotoxicity  55  2.9.1 NMDAR-mediated excitotoxicity  55  iv  2.9.2 Quinolinic acid injections  56  2.10 Statistical analysis  56  Chapter 3: Levels of mutant huntingtin influence the phenotypic severity of Huntington Disease in YAC128 mouse models  57  3.1 Introduction  59  3.2 Results  61  3.2.1 Generation of H D Y A C transgenic mice  61  3.2.2 Generation and characterization of HD55 homozygote  62  3.2.3 Mutant huntingtin levels modulate onset and progression of the behavioural phenotype  64  3.2.4 The degree of neurodegeneration is influenced by levels of mutant huntingtin 67 3.2.5 Degree of nuclear translocation of huntingtin and inclusion formation increases with increasing levels of mutant huntingtin  70  3.2.6 Levels of mutant huntingtin influence NMDAR-mediated excitotoxicty  71  3.3 Discussion  73  Chapter 4: Cleavage at the caspase-6 site in huntingtin is required for motor dysfunction, neurodegeneration and excitotoxicity in Huntington Disease  77  4.1 Introduction  79  4.2 Results  81  4.2.1 Generation of Caspase Resistant Lines  81  4.2.2 Characterization of C A G size and transgene expression levels in C R and H D mice  84  4.2.3 Caspase resistant htt expressed in C R mice is resistant to caspase proteolysis86 4.2.4 Caspase resistant mutant htt retains developmental functions in vivo  88  4.2.5 Inhibition of caspase cleavage of huntingtin prevents neurodegeneration in vivo  89  4.2.6 Inhibition of caspase cleavage of mutant huntingtin protects against motor dysfunction  92  4.2.7 Altered nuclear translocation of huntingtin in the striatum of C6R mice  94  4.2.8 Caspase 6 cleavage of mutant htt triggers caspase activation pathways  96  4.2.9 Similar apoptotic pathways are observed in the Y A C 128 model and human H D brain  98  4.2.10 C6R mice are resistant to excitotoxicity  99  4.3 Discussion Chapter 5: Discussion and Future Directions 5.1 Proteolysis of huntingtin is required for neurodegeneration in H D  103 109 110  v  5.2 Specificity of the fragment  116  5.3 Model for H D  118  5.4 Future directions 5.5 Conclusion Chapter 6: Bibliography  ;  131  *  133 .  134  vi  List of Tables  Table 1.1 Huntingtin interacting proteins  13  Table 1.2 H D mouse models  38  Table 1.3 Disease progession in Y A C 128 model is influenced by mutant htt levels  69  Table 4.1. Caspase Resistant mutant huntingtin retains developmental functions in vivo 89 Table 5.1. Excitotoxicity in mouse models of H D  115  vii  L i s t of Figures  Figure 1.1 Neuropathology in H D  5  Figure 1.2 C A G repeat sizing in H D  9  Figure 1.3 Model for the pathogenesis of H D  19  Figure 1.4 Huntingtin Proteolysis  22  Figure 1. 5 The toxic fragment hypothesis in H D  25  Figure 1. 6 Absence of toxicity associated with inclusions levels in vitro and in vivo .... 28 Figure 1.7 Caspase structure and classification  30  Figure 1. 8 Order of caspases activation in Apoptosis  32  Figure 1.9 Evidence supports a role for mhtt in enhancing response of postsynaptic N M DA receptors  35  Fig. 3.1.CAG size and transgene expression levels in YAC128 mouse models  62  Fig. 3.2. Generation and characterization of HD55 homozygotes  63  Fig. 3.3 Motor dysfunction correlates with levels of mutant huntingtin in YAC128 mouse models  67  Fig. 3.4 Degree of neurodegeneration is influenced by levels of mutant huntingtin  68  Fig. 3.5 Accelerated nuclear translocation of huntingtin and inclusions formation with increasing levels of mutant huntingtin  71  Fig. 3.6 Levels of mutant huntingtin influence NMDAR-mediated excitotoxicity  73  Fig. 4.1 Y A C mutagenesis. SSCP and sequence analysis of pop-in and pop-out clones for exon 12 and exon 13 mutations  83  Fig. 4.2 Identification and assessment of Y A C integrity in C R founder lines  84  Fig. 4.3. Characterization of Caspase Resistant and control Y A C lines  86  Fig. 4.4 Caspase resistant huntingtin expressed in the transgenic mice is resistant to specific caspase cleavage  88  Fig. 4.5. Inhibition of caspase cleavage of huntingtin prevents neurodegeneration in vivo. 91 Fig.4.6. Inhibition of caspase cleavage of mutant huntingtin protects against neuronal dysfunction  94  Fig. 4.7. Altered nuclear translocation of huntingtin in C6R mice  95  Fig.4. 8 Expression pattern of apoptotic markers in striatum of wt, HD53 and C6R  97  Fig. 4.9 Altered caspase expression levels are observed in human presymptomatic H D striatum compared to control brain  98  Fig. 4.10 Caspase cleavage of mutant huntingtin influences susceptibility to excitotoxicity in vitro and in vivo  Fig. 5.1 Model for H D  102  130  viii  List of Abbreviations a-aminno-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( A M P A ) Adenosine triphosphate (ATP) Amino acid (aa) Analysis of Variance ( A N O V A ) Base pair (bp) Brain-derived neurotrophic factor (BDNF) Calcium (Ca^) cAMP-response element binding protein (CREB) Caspase Resistant (CR) C R E B binding protein (CBP) Cytomegalovirus ( C M V ) Diaminobenzidine (DAB) Dentatorubral-pallidoluysian (DRPLA) Figure (Fig) Huntingtin (htt) Huntingtin associated protein (HAP) Huntingtin interacting protein (HIP) Huntington Disease (HD) Ibotenic Acid (IA) Kainic Acid (KA) Kilo-basepair (kb) Kilo-dalton (kDa) Knockout (KO) Left Y A C arm ( L Y A ) Medium spiny neuron (MSN) Molecular Weight (MW) N-methyl-D-aspartate (NMDA) Polymerase chain reaction (PCR) Quinolinic Acid (QA) Repressor element-1 transcription factor (REST)  ix  Reverse transcription (RT) Right Y A C arm (RYA) Small interfering R N A (siRNA) Spinal and Bulbar Muscular Atrophy (SBMA) Spinocerebellar ataxia (SCA) Standard deviation (SD) Standard error of the mean (SEM) Tdt-mediated dUTP-biotin nick end labelling (TUNEL) Untranslated region (UTR) Wild type (wt) Yeast Artificial Chromosome (YAC) 1 inositol (l,4,5)-triphosphate receptor (InsP3Rl)  Acknowledgements I would like to acknowledge the many people who have supported my ambition to get my PhD. I could not have not done it without them, and while words alone cannot express my gratitude, I think they know how much it meant to me and how grateful I am. I would like to thank Dr Michael Hayden, my supervisor, for his support and encouragement not only during the Ph.D but also beforehand. Thank you Michael for your guidance, challenges and many experiences during my Ph.D. I have grown in many ways and you are responsible for a significant part of that. I would also like to thank members of the H D group at the C M M T and personal friends. In particular I would like to thank Sue Jenkins and Stephane Abran, Elizabeth Slow, Martina Metzler and Jax Shehadeh for the many wonderful and supportive interactions over the years and also great friendship. I received wonderful technical support, above all from Deborah Deng, she is an amazing scientist and provided much support and friendship. I would also like to thank Nagat Bissada, Zoe Murphy and Ge L u for their support and making it all so much fun and Helen, A l i , Simon, Jamie, Anat, Jeff and Yvonne for good advice and friendship over the years. I would like to acknowledge the support of Blair Leavitt, Lynn Raymond, Don Nicholson, Sophie Roy and in particular Cheryl Wellington, for advice and insight during my Ph.D and the members of my committee for their advice and enjoyable meetings over the years. Last, and in some ways most of all, I would like to acknowledge the loving, support and incredible encouragment I received from my family, Duncan, Maggi, Fiona and Malcolm (and their significant others Caroline and Molly). I simply could not have accomplished this without them and for that I owe them a great debt. To Keegan and Kevin, thank you for all your love and energy, and showing me that even sap on a tree can be incredibly exciting. To my daugter Kamelot, I want to thank you for letting me go after my dreams, for all your wonderful laughter, and for just being you. You have given me such tremendous, unconditional love and grounded me in ways that only you can, all of which significantly contributed to my ability to handle the demands of the Ph.D and being a mother. Finally I would like to thank Danilo, for his enthusiam, laughter and unfailing support during the most difficult part of my Ph.D.  xi  Chapter 1: Introduction  1.1 History of HD and clinical features Huntington Disease (HD), a debilitating neurological disorder, was originally categorized within the Chorea-type disorders. However, in 1840, H D was further described as Chronic Hereditary Chorea as physicians around the world noticed that the involuntary movements and mental disturbances found in H D individuals were inherited from affected parents. George Huntington, in a landmark paper published in 1872, is credited with describing H D with a particular emphasis on the hereditary nature of H D (Huntington, 2003). George Huntington came from a long line of physicians, which proved to be instrumental in his discovery of the hereditary nature of HD. He was able to draw from almost eighty years of observations made previously by his father and grandfather on patients with chorea and their families living in Long Island, New York. He believed that the distinguishing features of this disorder, namely the late onset and hereditary nature, were sufficiently different from previously described Chorea-type disorders to warrant a separate category. From 1900 to 1970 there was growing interest in H D as researchers determined the primary site of neuropathology in H D to be the striatum and in 1967, the famous American songwriter and poet Woody Guthrie, died of HD. His wife, Marjorie, was instrumental in creating the Committee to Combat H D (now called the Huntington Disease Society of America (HDSA)) to provide public health and outreach for H D families and raise funds for research into a cure for H D . The Guthrie family, now through Woody's daughter Nora, continues to play an active role in raising awareness about H D and, almost 40 years later, help to generate significant funding for H D research. The first international Symposium for H D was held on the 100 anniversary of th  George Huntington's publication (1972), and while significant progress had been made in understanding the inheritance pattern and neuropathology, the underlying genetic defect responsible for causing H D was still unknown. A landmark discovery in the search for the H D gene was a publication by Americo Negrette which described a community in Lake Maracaibo, Venezuela that contained a large number of individuals affected with H D (Negrette, A., 1955). Inspired by Dr Negrette's findings, a research team headed by Nancy Wexler went to Venezuela 2  and obtained detailed clinical notes and blood samples from affected and unaffected family members. This would prove to be pivotal in identifying the candidate region for the H D gene and later the genetic defect that causes H D . HD, an adult onset disorder, affects approximately 1 in 10,000 individuals in Canada and the United States (Hayden., 1981). The frequency of H D world-wide varies depending on founder and modifier effects. Some populations of western European origin have a very high incidence (i.e. Venezuela-Lake Maracaibo region (7/1000) (Negrette., 1955), Island of Mauritius (4.6/1000) (Hayden et al., 1981) and Tasmania (2/1000), whereas Finland, Japan and African Blacks have a very low incidence of H D (1/100,000) (Hayden and Beighton, 1977;Hayden et al., 1980). The course of the disease is progressive with death, generally due to pneumonia, malnutrition and/or heart failure, occurring -15 years after onset. The clinical features of H D include a combination of neurological and psychiatric abnormalities and progression varies from person to person, even within the same family. The psychiatric symptoms such as irritability, depression, anxiety, and aggression, represent some of the earliest signs of functional impairment (Burns et al., 1990;Cummings, 1995). Abnormal eye movements are also detected early in the course of the disease (Kremer et al., 1992;Lasker and Zee, 1997). Onset of motor dysfunction presents with incoordination and difficulties with speech, balance and walking. The classical defining symptom of H D is chorea, which refers to the characteristic peculiar movement disorder which begins subtly and progresses to a dance-like motion that with time involves the entire body. As the disease progresses, the choreic movements are replaced with dystonia, bradykinesia, and rigidity (Harper et al., 2002). Cognitive impairments include short-term memory deficits, difficulties in changing strategies and concentrating (Lawrence et al., 1998;Watkins et al., 2000). The rate of progression of cognitive decline can also vary substantially amongst H D patients (Gusella, 1991). Approximately 10% of H D cases start prior to the age of 20 and are termed juvenile H D (Hayden, 1981). The juvenile form has been described as more parkinsonian in nature with the prominent features including bradykinesia, rigidity, tremor and epilepsy (van Dijk et al., 1986).  3  1.2 Neuropathology The cardinal pathological feature of HD is a progressive dysfunction and degeneration of neurons predominantly in the striatum (Vonsattel et al., 1985), a structure within the basal ganglia comprising the caudate and putamen. The basal ganglia, which also includes the globus pallidus and amygdala, is a large collection of neurons buried deep within the cerebral hemispheres. These neurons receive much of their input via the cortex (cortico-striatal fibers); the information is then processed and passed on to the globus pallidus (striato-pallidal fibers). Output is via the thalamus to the cerebral cortex. Thus the basal ganglia acts as a type of sub-loop of the motor system by altering cortical activity. It is involved in control of complex patterns of motor activity including initiation and quality of movement and influences cognitive aspects of motor control (Albin et al., 1989a; 1989b). In normal individuals the inhibitory projection neurons of the striatum work to inhibit the excitatory signals from the motor cortex. Abnormalities of the basal ganglia result in motor dysfunction including chorea, tremors and dykinesia (Marsden, 1982; Albin et al., 1989), symptoms present in adult and juvenile onset H D (Harper., 2002; Hayden., 1981). Atrophy of the caudate and putamen is progressive in H D and commences prior to onset of chorea (Aylward et al., 1994;Aylward et al., 1996). Striatal degeneration occurs in an ordered and topographic distribution with the tail of the caudate nucleus demonstrating the most prominent degeneration and the caudal portion of the putamen more involved than the rostral (Vonsattel et al., 1985). As the disease progresses the striatal degeneration moves in a caudo-rostral and dorso-ventral/medial-lateral direction and is accompanied by enlargement of the lateral ventricles (Fig. 1.1).  4  Normal Brain  HD Grade 2  HD Grade 4  Figure 1.1 Neuropathology in HD. A system for grading HD brains was developed by Vonsattel. In total there are 5 grades. Grade 0 has no gross pathology while grade 4 has severe atrophy of the striatum. As the disease progresses the striatal degeneration moves in a caudo-rostral and dorso-ventral/medial-lateral direction. Figure adapted from Myers et al, 1998.  Vonsattel eloquently delineated the striatal atrophy observed and defined 5 grades of H D neuropathology (Vonsattel et al., 1985). Grade 0/1 HD brain have no gross striatal atrophy however, grade 1 has microscopic changes including moderate fibrillary astrocytosis and up to 50% neuronal loss. In grade 2, striatal atrophy and gliosis is observed, however, the caudate nucleus remains convex while in grade 3 the caudate  5  nucleus is flat. In the most advanced grade (4) there is severe striatal atrophy with up to 90% of striatal neurons lost and the medial surface of the nucleus is concave. Atrophy of the globus pallidus, cortex, thalamus and subthalamic nucleus may also be observed and overall brain weight is 10-20% less than age-matched controls. The selective striatal neuropathology in H D is further refined within the neurons that comprise the neuronal population of the striatum. In particular, the y-aminobutyric acid (GABA)-containing medium spiny neurons, which account for 90% of neurons within the striatum (Ferrante et al., 1985), show preferential susceptibility, while the multiple types of large interneurons remain largely unaffected (Perry et al., 1973;Beal et al., 1988;Young et al., 1988). The projection neurons of the striatum are subdivided into substance P containing neurons, which project to the medial globus pallidus and are the first portion of the direct pathway. Enkephalin containing neurons, which project to the lateral globus pallidus, represent the first portion of the indirect pathway (Albin and Gilman, 1989; Albin et al., 1989a). Activation of the direct pathway tends to increase movement while activation of the indirect pathway decreases movement. It is the enkephalin containing neurons which are affected first in HD, leading to decreased activation of the indirect pathway and increased movement (Emson et al., 1980;Richfield et al., 1995). As the medium spiny neurons in H D brain degenerate, a corresponding decrease in the neurochemicals they contain decrease. Reduced levels of G A B A , substance P and enkephalin have been found in H D patient brain (Carter, 1984;Albin et al., 1991;Augood et al., 1996). Several mechanisms have been proposed to explain the selective neuronal death characteristic of H D including excitotoxicity, oxidative stress, impaired energy metabolism and apoptosis. The excitotoxicity hypothesis suggests that overstimulation of glutamate receptors, in particular N M D A receptors, cause increased C a ^ release into the cell triggering C a  ++  overload, caspase activation, mitochondrial dysfunction and apoptotic  cell death. The mitochondrial hypothesis suggests that defects in mitochondrial metabolism cause a chronic depletion of cellular A T P and a lowering of the threshold for apoptosis. There is evidence to support both hypotheses as excitotoxic damage would cause an energy defect and contribute to free radical production and mitochondrial dysfunction, all of which would decrease the threshold for apoptosis. The most recent  6  evidence supports excitotoxicity as an underlying primary mechanism with mitochondrial dysfunction a secondary effect of excitotoxic damage. The excitotoxicity hypothesis is discussed in detail under the section 'Excitotoxicity in H D ' and in the discussion under 'Model for H D ' .  1.3 Genetics 1.3.1 Inheritance H D is inherited as an autosomal dominant disorder (Hayden, 1981). In 1983, primarily through linkage analysis of the Venezuela H D pedigree, the gene responsible for causing H D was linked to the short arm of chromosome 4 (Gusella et al., 1983). During the ensuing 10 years, analysis of transcripts within the candidate region, as determined by positional cloning, eventually led to the discovery of the H D gene and the underlying genetic mutation in 1993 by the H D consortium (1993). H D is caused by a C A G trinucleotide expansion within exon 1 of the H D gene. The H D gene spans 185kb of genomic D N A and contains 67 exons. Two major transcripts, 10.5kb and 13.7kb, have been identified which are identical except for their 3'untranslated region (UTR) (Lin et al., 1993). The resultant protein, huntingtin (htt), is ~350kDa. H D is one of nine neurodegenerative disorders that are due to a C A G repeat expansion within the respective protein. These include D R P L A (Dentatorubropallidoluysian Atrophy), S B M A (Spinobulbar Muscular Atrophy), SCA1 (Spinocerebellar Ataxia Type 1), SCA2, SCA3 (Spinocerebellar Ataxia Type 3 or Machado-Joseph Disease), SCA6, SCA7 and SCA17. The C A G trinucleotide disorders have several features in common in addition to the expanded C A G tract including: adult onset, progressive degeneration of select neuronal cell populations, and a slow rate of disease progression (10-20 years). While there is some overlap in the symptoms, the neuropathology amongst the C A G repeat disorders is remarkably dissimilar. Thus the protein context and protein-protein interactions must also play a major role in the pathogenesis of the respective disorder.  7  1.3.2 C A G size and Age of Onset Normal individuals have a C A G size range from 9 to 35 repeats within the H D gene (Kremer et al., 1994) (Fig. 1.2). Individuals with an allele containing 36-39 C A G repeats are i n the affected range, however, not all w i l l develop H D (risk 10-100%) as at this C A G size the H D gene is not fully penetrant (Langbehn et al., 2004). H D is fully penetrant with allele sizes o f >40 C A G repeats (Leavitt et al., 1999). The majority o f adult onset cases fall within 40-55 C A G repeat range (Kremer et al., 1994). The C A G size i n the juvenile form o f H D is >60 repeats, with as many as 250 observed, resulting i n an age o f onset o f 4 years. Individuals expressing alleles containing 27-35 C A G repeats, termed intermediate alleles, w i l l not be affected with H D . However, these allele sizes are prone to expansion and may result i n new mutations (Goldberg et al., 1995). U p to 10% o f H D cases have been shown to be the result o f new mutations which have all expanded from an intermediate allele size (Goldberg et al., 1995;Almqvist et al., 2001;Creighton et al., 2003). C A G repeats within the normal range (9-26), excluding intermediate alleles, have been shown to be stably inherited with less than 1% showing meiotic instability (Goldberg et al., 1995). In contrast, expanded C A G tracts are prone to instability and tend to expand from generation to generation, a phenomenon known as genetic anticipation (Duyao et al., 1993). A s the C A G repeat expands, instability occurs during gametogenesis and this event is much more apparent i n male transmissions. This has been attributed to the continual mitotic divisions during spermatogenesis. Thus individuals who inherit an expanded allele from their father (paternal transmission) generally develop symptoms at an earlier age. In H D cases that develop symptoms before the age o f 10, 90% w i l l have inherited the mutant H D allele from their father (Telenius et al., 1993). Detailed analysis o f C A G size and age o f onset i n H D patients has revealed an inverse relationship between C A G size and age o f onset (Duyao et al., 1993;Brinkman et al., 1997). The correlation is stronger as the C A G size increases and i n juvenile H D cases is the major determinant o f age o f onset. A weaker correlation is observed between the lower C A G size range and age o f onset where modifying factors have a more pronounced influence.  8  >  H D affected alleles >36 C A G repeats  Intermediate alleles 27-35 C A G repeats  HD normal alleles 9-26 C A G repeats  Figure 1.2 C A G repeat sizing in H D . Alleles  greater than 36 C A G repeats cause H D . N o r m a l allele sizes  of H D are from 9 to 26 C A G repeats. Intermediate alleles, which range from 27 to 35 repeats, do not cause H D but are prone to expansion resulting in future generations with allele sizes in the H D range.  1.4 Cellular function of huntingtin Wild type htt, a 350kDa cytoplasmic protein (DiFiglia et al., 1995), is predominantly found in brain and testes, with low levels in other peripheral tissues (Trottier et al., 1995). Throughout the brain htt is observed in the cytoplasm of cell bodies, dendrites, and in axon terminals (Sharp et al., 1995). The enhanced susceptibility of medium spiny neurons of the striatum to the mutant htt protein is not explained by the protein expression pattern of htt as there is no correlation between htt expression and disease pathology. In contrast, htt is highly expressed in regions of the brain which are relatively spared in H D such as the cerebellum, and expression is lower in the striatum, the primary area of neurodegeneration (Landwehrmeyer et al., 1995;Schilling et al., 1995). Bioinformatic analysis of the htt protein has revealed the presence of numerous motifs including H E A T repeats (Andrade and Bork, 1995), caspase recognition sites (Wellington et al., 1998), and phosphorlyation sites (Warby et al., 2005). The H E A T repeats may facilitate the many protein-protein interactions observed with htt and/or facilitate htt's role in transport (Takano and Gusella, 2002). Htt is a highly conserved protein with orthologues present in many species including rat, mouse (90% conserved), fugu (85%), zebrafish (71%) and drosphila (30%). 9  Furthermore, htt is involved in a wide range of cellular activities including embryogenesis (Nasir et al., 1995;Zeitlin et al., 1995), intracellular signalling and vesicle trafficking (DiFiglia et al., 1995;Velier et al., 1998), transcriptional regulation (Kegel et al., 2002;Zuccato et al., 2003), neuroprotection/survival (Leavitt et al., 2001;Zhang et al., 2003;Gauthier et al., 2004) and apoptosis (Rigamonti et al., 2000;Hermel et al., 2004), highlighting the importance of htt function.  1.4.1 R o l e i n development  The primary indication of htt's prominent role during embryonic development derives from studies which inactivated the murine Hdh gene and demonstrated that embryos deficient in htt die during gestation (E8.5) (Duyao et al., 1995;Nasir et al., 1995;Zeitlin et al., 1995). Interestingly, while htt is essential for embryogenesis, htt expression is downregulated during development (Schmitt et al., 1995). Furthermore, differential expression of the two htt transcripts is observed in brain and peripheral tissues (Lin et al., 1993;Schmitt et al., 1995). Northern blot analysis has revealed that the larger, 13.7kb fragment is the predominant transcript in brain, while the smaller 10.5kb transcript is highly expressed in peripheral tissues. As the two transcripts are identical except for the 3'UTR region, this suggests that the two observed m R N A species originate from a single gene and that differential polyadenylation leads to transcripts of different sizes. The relative abundance of the larger transcript in brain may provide important information regarding the mechanism underlying the selective cell death observed in HD. The longer 3'UTR region (3,319kb) on the 13.7 kb transcript may be an important regulator of htt function, localization, stability or influence on other genes as has been observed for some genes (Rastinejad et al., 1993;Jackson, 1993). In contrast to the embryonic lethality observed in vivo, a lack of htt in vitro does not kill cells nor disrupt differentiation of embryonic Hdh-/- stem cells into neurons, suggesting an essential function for htt in gastrulation (Metzler et al., 1999). In addition, wild type extra embryonic tissue can rescue the lethality of Hdh-/- embryos suggesting a defect in nutritional support (Dragatsis et al., 1998). Hemizygous htt mice show neuronal loss in the subthalmic nucleus and the globus pallidus (O'Kusky et al., 1999). In addition, gene targeted mice that express <50% htt levels show extensive mid and hind brain 10  abnormalities at El8.5 and die shortly thereafter (White et al., 1997;Auerbach et al., 2001), indicating a primary role for htt in brain development. While the htt deficient mice have provided important clues regarding htt function during development, it has not explained the function of htt in the mature brain, and in particular in the medium spiny neurons of the striatum. What critical role does htt perform in the striatum and/or conversely what gain of function is conferred by mutant htt that selective destroys these neurons in HD? One way to answer this question is to determine which proteins htt interacts with and whether altered interactions are observed in the presence of the expanded polyglutamine tract. This will provide additional clues regarding the function of htt and may highlight reasons for the selective vulnerability of medium spiny neurons to mutant htt.  1.4.2 Interacting Proteins Htt interacts with numerous proteins, involved in many distinct pathways, making it difficult to define a single role for htt (Table 1.1). Nonetheless, htt interacting proteins, the majority of which bind the N-terminal region, provide valuable information that has helped elucidate the function of htt and the mechanisms involved in the pathogenesis of HD. The predominant theme among htt's interacting partners is a role in glutamate induced intracellular signalling (Sun et al., 2001;Metzler et al., 2003), vesicle transport and endocytosis (Velier et al., 1998;Metzler et al., 2001), gene transcription (discussed separately), and apoptosis (discussed separately). Htt purifies with vesicle fractions (DiFiglia et al., 1995), associates with microtubules (Tukamoto et al., 1997), and interacts with several proteins involved in transport (Metzler et al., 2001;Singaraja et al., 2002;Gauthier et al., 2004), suggesting a role for htt in vesicle transport. HIP-1, a htt interacting protein that binds tubulin , is a critical component of clathrin-coated pits (Metzler et al., 2001). A decreased interaction of htt and HIP1 is observed in the presence of the expanded polyglutamine tract. HAP-1, a novel protein that also interacts with htt and HIP1, binds the pi50 subunit of dynactin, a motor protein involved in moving vesicles along microtubules (Li et al., 1998). In contrast to HIP1, enhanced binding of H A P 1 with mutant htt is observed. Alterations in the interactions of mutant htt with HAP1 and HIP1 may contribute to the defects in 11  axonal trafficking observed in HD, causing the dystrophic neurites present in HD brain (DiFiglia et al., 1997). Furthermore, alterations in endocytosis and recycling of vesicles at the synaptic cleft may contribute to pathogenesis through decreased binding of mutant htt with HIP 1. HIP1 has been shown to be essential for glutamate triggered clathrin-mediated endocytosis of GluRl containing A M P A receptors and HIP1 knockout mice present with a severe, progressive neurological phenotype including wasting, tremor and gait ataxia (Metzler et al., 2001;Metzler et al., 2003). This evidence suggests that htt may be involved in intracellular signalling mediated by glutamate receptors and furthermore that HIP1 may link intracellular transport machinery and degradation. Htt also interacts with PSD95, a scaffold protein which mediates N M D A receptor clustering at the membrane (Sun et al., 2001). A reduced association of PSD95 and htt is observed in the presence of the expanded polyglutamine tract and N-terminal htt fragments bind synaptic vesicles and inhibit glutamate uptake in vitro (Li et al., 2003), suggesting alterations in NMDAR-mediated signalling is involved in the pathogenesis of HD.  12  Name  Function  CAG influence  Reference  CBP  Transcription factor  enhances  Nucifora et al., 2001, Steffan et al., 2000  SP1  Transcription factor  enhances  Li et al., 2002, Dunah et al., 2002  HAP1  Trafficking, endocytosis  enhances  Li et al., 1998  CAM  Ca++ binding protein  enhances  Baoetal., 1996  IP31  Ca++ release channel  enhances  Tang etal., 2003  GAPDH  Glycolytic enzyme  enhances  Burke et al., 1996  p53  Transcription factor  enhances  Steffan et al., 2000  NFKB  Transcription factor  enhances  Takano et al., 2002, Khoshnan et al, 2004  CtBP  Transcription factor  decreases  Kegel et al., 2002  REST  Transcription suppressor  decreases  Zuccato et al., 2003  HIP1  Endocytosis, pro-apoptotic  decreases  Kalchman et al., 1997, Metzler et al., 2001  HIP14  Trafficking, palmitoylation  decreases  Singaraja et al., 2002, Huang et al., 2004  PSD95  Synaptic scaffolding  decreases  Sunet al., 2001  CA150  Transcription factor  none  Holbert etal., 2001  none  Kalchman et al., 1996  unknown  Huang etal., 1998  HIP2 TBP  Ubiquitin-congugating  enzyme  Transcription factor  Table 1.1 Huntingtin interacting proteins. Proteins which associate with huntingtin are involved in a diverse array of functions including transcription, trafficking and endyocytosis, signalling and metabolism.  1.4.3 Role in transcription  Htt contains polyglutamine and polyproline tracts which are features commonly observed in proteins involved in transcription, and alterations in gene expression are detected in HD, suggesting a role for htt as a transcription factor (Nucifora, Jr. et al., 2001;Zuccato et al., 2001;Yohrling et al., 2003). Htt interacts with a number of transcription factors including p53, C R E B binding protein (CBP), mSin3a and b (Steffan et al., 2000), TFIID (Dunah et al., 2002) and functional studies suggest that htt can repress transcription in vitro through a p53 reporter complex (Steffan et al., 2000). P53 is 13  a tumor suppressor gene that has multiple functions including a predominant role in apoptosis and as a direct regulator of the intiator caspase-6. Overexpression of p53 in cell culture has been demonstrated to cause an increase in procaspase-6 levels which is followed by an increase in caspase-6 activity and cleavage of caspase-6 specific substrates (MachLachlan et al, 2002). CBP, a transcription factor that associates with p53 and is activated in response to intracellular signaling pathways, mediates transcription of the trophic factor B D N F (Ikeda et al., 2000). The expanded polyglutamine tract in mutant htt increases the CBP/htt interaction (Zuccato et al., 2001) and C B P is observed sequestered in aggregates (Nucifora, Jr. et al., 2001), suggesting a possible mechanism for the observed transcriptional dysregulation and decreased histone acetylation observed in HD. Furthermore, decreased levels of B D N F are observed in H D which may contribute to the pathogenesis of H D (Zuccato et al., 2001). Several genes are downregulated in H D including dopamine D l and D2 receptors (Weeks et al., 1996), NR1 and NR2B subunits of N M D A receptors, substance P, and enkephalin at the mRNA and protein level (Young et al., 1988; Albin et al., 1990). Decreased protein levels may simply reflect loss of neurons that abundantly express these proteins. However, mRNA decreases (observed for enkephalin and D l and D2 dopamine receptors) support the theory that an alteration in transcriptional regulation is involved in the disease.  1.4.4 Role in neuroprotection Evidence of htt's neuroprotective role was suggested from studies of cell culture models which overexpressed wild type htt. Decreased cell death is observed in cells exposed to serum deprivation, mitochondrial toxins and increased intracellular C a  ++  in  the presence of increasing levels of wild type htt, suggesting htt modulates neuronal susceptibility to stress (Rigamonti et al., 2000;Rigamonti et al., 2001). The neuroprotective effect of htt has also been observed in vivo. Mouse models over expressing wild type htt and exposed to ischemia or quinolinic-acid (QA) induced neurotoxicity show significantly reduced neuronal death compared to normal mice  14  (Zhang et al., 2003;Leavitt et al., submitted). These observations suggest that blocking excitotoxicity is one mechanism by which excess wild type htt mediates neuroprotection. Increased apoptotic cell death is evident in embryos deficient in htt, and mice hemizygous for the H D murine gene display increased motor activity and significant neuronal loss in the subthalmic nucleus, supporting an anti-apoptotic function for htt (Nasir et al., 1995;0'Kusky et al., 1999). In addition, transgenic htt mice expressing low levels of htt (<50%) present with phenotypes reminiscent of caspase-3 knockout mice (White et al., 1997) and conditional htt knockouts demonstrate neurological deficits and neurodegeneration (Yamamoto et al., 2000), implying alterations in apoptotic pathways leading to decreased apoptosis. Degeneration of axon fibers is also observed suggesting that htt affects neuronal stability. Wild type htt mediates upregulation of B D N F (Zuccato et al., 2001), a prosurvival factor produced by cortical neurons and delivered to striatal neurons via the corticostriatal afferents, which may contribute to htt's neuroprotective effect. The beneficial activity of wild type htt in B D N F regulation is lost when htt is mutated by the C A G expansion, resulting in decreased production of cortical B D N F and insufficient trophic support for striatal neurons. The underlying mechanism of wild type htt's neuroprotective effect is currently unclear. However, wild type htt has been shown to directly interact with (initiator) caspase-2 suggesting a possible role for htt as a direct caspase inhibitor (Hermel et al., 2004). Concurrent with this is the finding that wild type htt's neuroprotective role lies upstream of caspase-3 and may take place at the level of initiator caspases such as caspase-9 (Rigamonti et al., 2001).  1.4.5 Posttranslational modification of huntingtin A number of posttranslation modification sites have been characterized within the N-terminal region of htt including phosphorylation at amino acid (aa) 421 and 434 (Humbert et al., 2002;Warby et al., 2005). Htt is phosphorylated on serine 421 by the pro-survival signalling protein kinase Akt (PKB), and in vitro evidence suggests that phosphorylation of htt by Akt is protective against the toxicity of the expanded C A G tract and abrogates its proapoptotic activity. In vivo, phosphorylation of mutant htt is reduced 15  relative to wild type htt and in the striatum of HD patients cleaved fragments of Akt are observed by western blot, suggesting proteolysis and possible inactivation of Akt may contribute to the pathogenesis of HD. Interestingly, the pattern of htt phosphorylation in normal murine brain is highest in the cerebellum, a region unaffected in H D and is least in the striatum, the primary area of neurodegeneration in HD (Warby et al., 2005). Htt is also phosphorylated by cdk5, a member of the serine/theorine cdk family, at position 434aa (Luo et al., 2005). Phosphorylation of htt at this site has been demonstrated to reduce caspase-3 cleavage of htt at 513aa and toxicity in cells expressing an N-terminal fragment of htt (l-588aa). Htt furthermore contains a palmitoylation site at 214aa and preliminary evidence suggests that a decrease in htt palmitoylation occurs in the presence of the expanded polyglutamine tract (Yanai et al., 2005 abstr). Palmitoylation of proteins regulates trafficking and function of signalling molecules and receptors (El-Husseini and Bredt, 2002). Furthermore, palmitoylation of key neuronal proteins plays a critical role in axon pathfinding and clustering of scaffolding proteins such as PSD95 (Strittmatter et al., 1995;Gauthier-Campbell et al., 2004). Alterations in posttranslation modification of htt, due to the expanded C A G tract and/or cleavage of htt, may increase the toxicity of mutant htt, causing cellular stress to striatal neurons and altering normal signalling pathways.  1.5 Mutant huntingtin The distinguishing feature of mutant htt is the expanded polyglutamine containing stretch in the N-terminal region of the protein. Elongation of the polyglutamine tract alters the configuration of the protein, modifying its solubility and interaction with other cellular proteins. Efforts to assess the intracellular localization of mutant vs. wild type htt have been hampered by inadequate antisera. However, considerable progress has been made using homozygote H D patient brain and subcellular fractionation studies of htt. Mutant htt is expressed at a similar level as wild type htt in all regions of the brains. Thus the selectivity of medium spiny neurons to full length mutant htt induced neurotoxicity cannot be explained simply by differential expression (Aronin et al., 1995).  1.6  However, in H D cortex and striatum, altered intracellular localization of htt fragments and perinuclear accumulation of htt in vesicles has been observed, suggesting altered proteolysis and trafficking as defects contributing to the pathogenesis of H D (Velier et al., 1998;Kim et al., 2001). Furthermore, in human and murine H D brain, labelling of neuronal inclusions and dystrophic neurites in the cortex and striatum is apparent (DiFiglia et al., 1997). Ubiquitin also co-distributes with mhtt in the inclusions suggesting that ubiquitin-dependent proteolysis of mutant htt is incomplete (vesRodrigues et al., 1998).  1.5.1 Gain of function/loss of function Similar to several trinucleotide repeat disorders, H D is believed to be caused by a toxic gain of function of the mutant protein as complete loss of htt results in embryonic lethality (Duyao et al., 1995;Nasir et al., 1995;Zeitlin et al., 1995) and heterozygous inactivation of htt in humans does not cause an H D phenotype (Ambrose et al., 1994) . Furthermore, nuclear localization of mutant htt fragments is observed in murine and human H D striatum, the primary area of neurodegeneration in H D patients (Van Raamsdonk et al., submitted) and intracellular inclusion formation and sequestration of transcription factors have been observed in vitro and in vivo (Kosinski et al., . 1999;Schilling et al., 1999;Suhr et al., 2001). The expanded polyglutamine region of htt directly binds to C B P and p300, transcription factors associated with acetyltransferase activity and observed sequestered in inclusions (Nucifora, Jr. et al., 2001). Decreasing intracellular levels of C B P andp300 through appropriation in inclusions may cause reduced histone acetylation and altered transcription observed in HD. Several htt interacting proteins have been shown to interact more strongly with mutant htt including HAP1 (Li et al., 1995), calmodulin (Bao et al., 1996), glyceraldehydes-3-phosphate dehydrogenase (Burke et al., 1996), and SH3GL3 (Sittler et al., 1998), which may cause a decrease in free intracellular levels of these proteins. G A P D H activity has been shown to be decreased in subcellular fractions of H D fibroblasts suggesting impairment of G A P D H glycolytic function in H D (Mazzola et al., 2001). 17  1.5.1.2 Complete vs. Intermediate Dominance More recently, the idea that loss of function of wild type htt may contribute to the pathogenesis of HD, originally proposed more than 10 years ago, has gained prominence (Nasir et al., 1995). The evidence against loss of htt function contributing to the pathogenesis in H D was derived from studies of individuals expressing deleted areas of chromosome 4 which contained the H D gene yet did not present with HD-like symptoms (Ambrose et al., 1994). Furthermore, limited studies of human H D patients suggested that H D presents with a complete dominant phenotype, with no increase in the severity of the phenotype of individuals homozygous for the mutation (Wexler et al., 1987;Myers et al., 1989;Gusella, 1991). This would also argue against loss of function contributing to pathogenesis. In contrast to the early work, a more detailed clinical assessment and review of neuropathological findings from heterozygote and homozygote patients for H D did show increased rate of disease progression in homozygotes for the mutation in humans (Squitieri et al., 2003). Studies in transgenic mice and in vitro also argue in favour of an intermediate dominant phenotype in H D (Davies et al., 1997;Reddy et al., 1998;Narain et al., 1999;Hodgson et al., 1999;Lin et al., 2001). H D knock-in mice demonstrate earlier onset of nuclear htt fragment accumulation and a more severe behavioural deficit in the homozygote (Lin et al., 2001 ;Wheeler et al., 2002). Most poly (CAG) disorders, including D R P L A , SCA2, SCA3, SCA6, and SCA18, among others, demonstrate increased severity in homozygotes (Zlotogora, 1997;Durr et al., 1999). This includes both earlier onset and more severe evolution of the disease in the individuals homozygous for the mutation. Intermediate dominance implies that the wild type allele could influence disease severity, and thus an important question in H D research is whether loss of htt function contributes to the pathogenesis of the disease (Fig. 1.3). Wild type htt is neuroprotective in a number of stress induced model systems (Rigamonti et al., 2000;Leavitt et al., 2001;Zhang et al., 2003) and mice expressing reduced levels of htt demonstrate neurological deficits, suggesting that loss of function of wild type htt may contribute to disease pathogenesis. Furthermore, wild type htt is observed sequestered in aggregates and cleavage of htt detected in H D brain tissue (Kim et al., 2001;Wellington et al., 2002)  18  which would decrease levels of neuroprotective htt. In the Y A C 128 model, levels of wild type htt have been shown to influence motor function and activity (Van Raamsdonk et al., 2005a). P a t h o g e n e s i s o f Huntington D i s e a s e Cleavage of huntingtin  Gain of function  Toxic fragment  \  Loss of function  Reduced neuroprotective huntingtin  \  Pathogenesis  F i g u r e 1.3 M o d e l f o r the pathogenesis o f HD. Increased cleavage o f htt leads to generation o f toxic mutant htt fragments and decreased levels o f neuroprotective htt, both o f which contribute to the pathogenesis o f H D . 1.6  H u n t i n g t i n proteolysis  The expression pattern of mutant htt does not explain the selective death of medium spiny neurons observed in HD. Why then are these neurons so affected by the mutant form of the protein? Does proteolysis of mutant htt affect the toxicity and/or subcellular localization of mutant htt fragments? It may be that it is not the level of full length mutant htt but rather the amount and subcellular localization of specific mutant htt fragments that cause the select death of striatal neurons in HD. Proteases are important mediators of protein function. Proteolytic cleavage of a protein has been shown to result in protein fragments with altered function compared to the intact parent molecule (Rao et al., 1996;01iver et al., 1998;Cohen et al., 2005). Several lines of evidence suggest that htt function may be mediated by proteolytic cleavage. First, numerous proteases cleave htt, including caspases (discussed separately), suggesting that htt is organized into several functional domains that are released by proteolytic cleavage, a common theme for many caspase substrates. Second, a large body of evidence supports the dual role of htt in anti-apoptotic, survival functions (discussed separately) and in cell death pro-apoptotic pathways (discussed separately).  19  A number of caspase substrates have been identified to have active roles in apoptosis and preventing caspase cleavage of the caspase substrate (i.e. by generating a caspase resistant form) has been shown to influence downstream cell death by abrogation of their inhibitory function. For example, caspase-resistant lamin protects cells against chromatin condensation and nuclear shrinkage (Rao et al., 1996) and death triggered by CD95 activation is reduced in cells expressing caspase-resistant poly (ADP-ribose) polymerase (Oliver et al., 1998). Furthermore, excessive signalling via the Notchl receptor has been shown to inhibit apoptosis which is abrogated by caspase cleavage (Cohen et al., 2005). This evidence, combined with the neuroprotective results observed with overexpression of htt and toxicity of htt fragments, provides support for the hypothesis that caspase-cleavage of htt may result in abrogation of htt's anti-apoptotic role, with htt fragments now promoting apoptosis. For htt with a normal C A G size, there would be a balance of pro and anti-apoptotic levels. However in the presence of the expanded C A G tract this may be shifted towards increased levels of N-terminal htt fragments containing the polyglutamine. This may reduce proteasomal function and protein degradation, alter interactions with protein partners and /or cause neuronal dysfunction through aberrant signalling, all of which would contribute to decreasing the threshold for apoptosis. If proteolysis of mutant htt is involved in the onset or progression of HD, then which of the many htt fragments observed promote apoptotsis and/or are some fragments actually neuroprotective vs. htt fragments simply generated as a result of protein degradation and clearance? The evidence that certain fragments of htt are protective under conditions of excitotoxic stress implicates an association between proteolytic fragments of htt, excitotoxicity and the disease (discussed in more detail in the Discussion under 'Model for H D ' , Chapter 5). In cell culture models, enhanced NMDAR-mediated excitotoxicity is observed with full length mutant htt, but not with expression of a truncated (l-548aa) fragment (Zeron et al., 2001). Furthermore, a 548aa N-terminal fragment of htt is protective under conditions of serum deprivation, a function that is lacking in a shorter, 63aa htt fragment (Rigamonti et al., 2001). These observations suggest that the toxicity of mutant htt may be mediated by specific htt fragments.  20  1.6.1 Proteases that cleave huntingtin The N-terminal region of htt is highly sensitive to proteolysis with numerous proteases, including caspases, calpains, and aspartyl endopeptidases cleaving htt (Wellington et a l , 1998;Lunkes et al., 2002;Kim et al., 2003) (Fig. 1.4). In normal brain, the released N-terminal fragments would be efficiently cleared, presumably through the ubiquitin-proteasome pathway. However, release of htt fragments containing polyglutamine expansions would generate products that are not efficiently cleared by the proteasome. Therefore, even a normal cleavage event may lead to the generation of toxic htt fragments which may themselves initiate a plethora of additional routes to neuronal cell death. Htt is cleaved by caspase-2 at 552aa, caspase-3 at 513 and 552aa, and caspase-6 at 586aa. Htt also contains two other putative caspase consensus sites at positions 530 and 589aa, that do not appear to be cleaved by any caspase in cell culture models (Wellington et al., 1998;Wellington et al., 2000). This redundancy suggests that cleavage of htt by caspases may represent a functional property of htt, with the cleavage event altering or inactivating functional domains. Further discussion of the role of caspase cleavage of htt is presented in the next section. There are numerous calpain cleavage sites in the amino terminus of htt, five of which have been mapped to aa positions 437, 451, 469, 536 and 540 (Garni and Ellerby, 2002). The contribution of calpain cleavage to the pathogenesis of H D is currently unknown. However, enhanced immunoreactivity for pand m-calpain has been observed in the caudate nucleus of H D patients, and inhibition of calpain cleavage of htt by site directed mutagenesis of htt's calpain sites reduces toxicity in vitro (Garni et al., 2004). Furthermore, calpain activation is detected in the 3-NP model of H D and this is associated with calpain-dependent cleavage of htt (Bizat et al., 2003). Selective calpain inhibition reduces the size of the striatal lesion and abolishes 3-NP induced D N A fragmentation in striatal cells. Interestingly, in rats, intra-cerebro-ventricular infusion of z V A D , a broad spectrum caspase inhibitor, significantly reduced 3NP-induced striatal degeneration, and decreased the 3NP-induced activation of calpain (Bizat et al., 2005). Further cleavage of caspase derived htt fragments by calpains has been suggested as a  21  mechanism for generating htt fragments capable of passive diffusion into the nucleus (Kim etal., 2001). The cleavage sites of aspartyl proteases are not yet defined, but they reside within a domain from aa 104-114 (CpA) and between aa 146-214 (CpB) of htt. As CpA and CpB have a predicted M W of only 30-35kDa, they could passively enter the nucleus. Characterization of htt breakdown products in nuclear inclusions of HD brain reveals the main fragment present is CpA (Lunkes et al., 2002). Aspartyl proteases, acting in concert with the proteasome to ensure the normal clearance of htt fragments, may in the presence of mutant htt, generate products with high aggregation potential.  Figure 1.4 Huntingtin Proteolysis. Graphic representation of protease cleavage sites within the N terminal region of huntingtin. Triangle represents the C A G repeat. The size of the htt fragments in the truncated H D mouse models, shortstop and R6/2. is included and discussed separately under truncated models of HD.  1.6.2 Caspase cleavage of huntingtin Despite the evidence regarding N-terminal cleavage of htt and toxicity of mutant htt fragments, it has not been demonstrated how these events lead to striatal vulnerability. What are the specific proteases and resultant mutant htt fragments responsible for initiating pathogenesis of HD in vivo"? And more importantly, will inhibiting htt proteolysis be effective in delaying the onset or progression of HD?  22  Cell type dependent cleavage of htt by proteases may explain the selective neurodegeneration observed in HD. Htt is cleaved in vitro by caspase-2, -3 and -6, resulting in N-terminal fragments containing the C A G repeat (Wellington et al., 1998;2000). Importantly, caspase cleavage of htt has been demonstrated to precede neurodegeneration in human H D brain (Kim et al., 2001;Wellington et al., 2002). Using antibodies designed to selectively detect only caspase cleavage products of htt, it has been demonstrated that htt cleavage products ending at aa 552 are particularly abundant in cortical projection neurons in a tissue sample from a presymptomatic individual, as well as in samples of an early neuropathological grade (Vonsattel grade I) where gross neurodegeneration is not yet observed. Caspase-3 cleavage of htt is also observed in human control brain, suggesting a physiological role for caspase-3 cleavage of wild type htt (Wellington et al., 2002). In addition, caspase generated fragments of htt accumulate with synaptic vesicles and inhibit glutamate uptake, possibly altering the normally efficient and rapid removal of glutamate from the synaptic cleft (Li et al., 2003). Difficulties in determining in vivo htt fragments vs. ex vivo generated fragments (due to post mortem interval etc) present in human H D tissue by western blot have lead to conflicting reports regarding htt fragments present in H D vs. control brain. Two studies have reported similar, caspase size, fragments of htt are present in H D and control tissues (Kim et al., 2001;Wellington et al., 2002), while another study observed smaller, potential calpain fragments only in H D tissue (Gafni and Ellerby, 2002). Elevated levels of N-terminal htt fragments, smaller in size than calpain-derived fragments, have also been described in H D as compared to control brain tissue. As numerous htt fragments are observed on western blot of human H D tissue, correlating the fragment to cleavage by a specific protease is complex. In addition, difficulties in obtaining sufficient samples of presymptomatic H D human brain tissue and a lack of htt neo-epitope antibodies to the various characterized fragments, preclude delineating initial cleavage events from downstream further proteolysis of htt in human H D brain. The use of full length htt animal models of HD is therefore necessary to study the primary events in disease pathogenesis so that curative therapeutic targets can be identified.  23  1.6.3 Caspase resistant huntingtin Systematic mutagenesis of all of the caspase cleavage sites in htt renders htt nontoxic in transfected 293T or hippocampal HN33 cells, even in the presence of a polyglutamine expansion and in the face of multiple stressors including tamoxifen and serum starvation (Wellington et al., 2000). These results suggest that caspase cleavage may be an important early event in the pathway by which expanded htt causes cell death, and that inhibiting htt cleavage ameliorates toxicity even when all other apoptotic pathways are left intact.  1.6.4 Toxic fragment hypothesis Several observations in vitro and in vivo support the hypothesis that mutant htt toxicity in H D occurs via formation of N-terminal fragments of htt. N-terminal htt fragments containing expanded C A G repeats have enhanced cytotoxicity in H D cell culture and fly models (Hackam et al., 1998;Cooper et al., 1998;Jana et al., 2001), and some truncated H D mouse models demonstrate behavioural deficits and neuronal inclusions (Mangiarini et al., 1996;Davies et al., 1997;Schilling et al., 1999). However, not all models of H D demonstrate the specific pathology observed in H D . Furthermore, caspase activation in an H D cell culture model has been shown to be dependent upon the protein context of the mhtt fragments (Yu et al., 2003). This may argue that specific fragments of mutant htt are required for initiation of the toxic cycle that leads to neuronal dysfunction, including behavioural and cognitive deficits, and the neuropathological abnormalities present in HD. These studies support the toxic fragment hypothesis of HD, which suggests that specific N-terminal htt fragments released by proteolytic cleavage of htt are crucial for disease onset and/or progression (Goldberg et al., 1996). Alterations of intracellular htt interactions, with selectively expressed cellular proteins, due to htt cleavage may increase susceptibility to excitotoxicity leading to further proteolysis. Amplification of the cycle would ultimately result in commitment to apoptosis and death of the neuron (Fig. 1.5).  24  brain cell  \ ^  C  a  \ /full length mutant huntingtin v  I  t Cleavage of huntingtin  ^id-type htt  ^7  Generation of toxic huntingtin fragment  Activation of toxic pathways  Neuronal r dysfunction and death  ^  J&£\  Figure 1. 5 The toxic fragment hypothesis in HD.  Proteolytic cleavage o f htt results in elevated levels o f  toxic htt fragments. Alterations o f intracellular htt interactions, with selectively expressed cellular proteins, due to htt cleavage increases susceptibility to excitotoxicity leading to further proteolysis. Amplification o f the cycle ultimately results in commitment to apoptosis and death o f the neuron.  25  1.7 Huntingtin Inclusions 1.7.1 History and definition The history of inclusions and their role in the pathogenesis of HD has been one of the most debated areas of H D research. Htt is normally a cytoplasmic protein with a diffuse pattern of staining. However, cleavage of the htt protein releases fragments containing the expanded polyglutamine repeat which accumulate and form inclusions. Relocation of htt to the nucleus and formation of inclusions are a hallmark of the human disease and are present in the brains of human patients (DiFiglia et al., 1997;Gutekunst et a l , 1999) and in H D mouse models (Li et al., 1999;Morton and Leavens, 2000;Smith et al., 2001;Slow et al., 2003). Both nuclear and cytoplasmic inclusions are observed and various sizes have been described. However, they are all membrane free with fibrillar morphology and composed of electron dense material (Huang et al., 1998;Georgalis et al., 1998). In vitro studies have determined that expanded polyglutamine tracts can spontaneously aggregate in a time, concentration, and C A G dependent fashion, possibly through formation of polar zippers (Perutz et al., 1994). The failure to detect inclusions and nuclear htt using C-terminal htt antibodies (DiFiglia et al., 1997;Becher et al., 1998;Gutekunst et al., 1999), coupled with the increase of inclusions observed in truncated models of HD, suggest N-terminal proteolytic processing of htt plays a critical role in the pathogenesis of HD. Numerous in vitro and in vivo studies use inclusion formation as a marker of toxicity and much therapeutic research is focused on screening compounds for their ability to inhibit inclusion formation. A decrease in inclusion formation is interpreted as a positive outcome and the compound is then further tested in H D mouse models.  1.7.2 Relation to toxicity Polyglutamine tracts are found in numerous proteins, many of which function as transcription factors. Increased interaction of expanded htt with transcription factors such as CBP, and recruitment into inclusions (Nucifora, Jr. et al., 2001) is hypothesised to  26  cause the transcriptional dysregulation observed in HD. Conversely, it has been demonstrated that total free intracellular levels of the proteins observed sequestered in inclusions, remains within normal levels (Yu et al., 2002) . In vitro and in vivo evidence clearly demonstrates that expression of mutant htt, in particular N-terminal products, result in inclusion formation. In addition, ubiquitin colocalizes with inclusions suggesting inadequate degradation by the ubiquitin proteasome system (DiFiglia et al., 1997;Sieradzan et al., 1999). However, the correlation between inclusions and toxicity has not been directly established, nor is it known whether it is instead the soluble form of the mutant protein that causes the toxicity, prior to overt inclusion formation. Therefore the question of whether inclusions are causative in the disease has been unknown. Furthermore, it has been shown that the presence of inclusions does not correlate with cell death in striatal cells transfected with mutant htt. Rather the evidence suggested that inclusions may reflect a cellular mechanism to protect against mutant htt induced cell death (Kim et al., 1999). Additional reports in the past year also provide compelling evidence in H D cell culture models and in vivo which supports the hypothesis that, contrary to expectation, inclusions are not pathogenic, do not correlate with neuronal cell death and may actually be neuroprotective. Using survival analysis in real time, it has been shown that there was an increased likelihood of survival in neurons with inclusions compared to those without inclusions (Arraste et al., 2004). In addition, shortstop, a truncated H D mouse model with no clinical phenotype or neurodegeneration contains numerous non-specific inclusions throughout the brain (Slow etal., 2005) (Fig. 1.6). Difficulties in assessing inclusions levels in human H D brain, due to insufficient samples available over the course of the illness, preclude a natural history characterization of inclusions in human HD. Limited studies done on human tissue reveal that inclusions are not found in the primary sites of neuropathology, and in contrast, are observed in regions less affected in H D (Kuemmerle et al 1999, Gutekunst et al 1999).  27  HD Full length mutant huntingtin Proteolytic cleavaqe  Cell death! I Caspase activation  toxic huntingtin  \f^__^ Neuronal dysfunction Transcriptional dysregulation Altered interactions  In vitro  In vivo (Shortstop)  Full length  mutant huntingtin+ Caspase inhibitor c  Neuronal survival r~~,  5x  truncated mutant huntinqtin  non-caspase \\ mediated proteolytic )) cleavage of htt  huntingtin fragment  Inclusion formation  Neuronal survival  No clinical phenotype, No neurodegeneration  huntingtin fragment inclusions present throughout the brain  Figure 1. 6 Absence of toxicity associated with inclusions levels in  vitro and in vivo. There is an increased likelihood o f survival in neurons with inclusions compared to those without inclusions ( K i m et al., 1999;Arraste et al., 2004) and shortstop, a truncated HD mouse model with no clinical phenotype or neurodegeneration, contains numerous non-specific inclusions throughout the brain (Slow et al., 2005).  1.8 Apoptosis Apoptosis, the predominant form of cell death in chronic neurological disease, is a genetically programmed form of cell death that utilizes caspases, a family of cysteine proteases that are expressed as latent zymogens. There are in total 14 caspase which are divided into initiators, effectors and caspases involved in cytokine processing, depending on the presence of the prodomain (Fig. 1.7). Caspase are activated by proteolytic processing and dimerization (Garcia-Calvo et al., 1999). Initiator caspases are involved upstream in the programmed cell death pathway and represent the initial signal that the  28  cell is undergoing stress. Executioner caspases on the other hand are responsible for cleaving structural proteins (in addition to activating other caspases) and this ultimately leads to the death of the cell. The pathways leading to caspase activation vary depending on the initial cytotoxic stimulus and include excitotoxic stress, D N A damage and C a  + +  overload among others. As many as 200 proteins have been identified as caspase substrates, many of which, similar to htt, have demonstrated anti-apoptotic roles (Rao et al., 1996;Chau et al., 2002;Cohen et al., 2005).  29  Caspase structure  Caspase subgroups  B  /  Inflammation Group I Aap-x pfc-doman (3-24 kDa)  sutounrt  Sarge 07-21 kOa)  small subunit (10-13 kDa)  caspase 1 caspase 4 caspase 5 caspase  Q i active s  13  Apoptosis Group II and III  large suburot/amail subunit hetenxUmers  Initiator caspases caspase 2 caspase 6  \  Executioner caspases  caspase 8 caspase 9  caspase 3  caspase  caspase 6  10  caspase 7  Figure 1.7 Caspase structure and classification. A ) Caspases are activated by proteolytic processing and dimerization. The size of the pro-domain ranges from 3-24kDa depending on the particular caspase. B) There are currently 3 caspase subgroups. Group I are caspases involved in inflammation and cytokine processing. Group II caspases are observed early in programmed cell death and are described as initiator caspases while Group III caspases execute the apoptotic cell death program.  Apoptosis is characterized by morphological changes including membrane blebbing, nuclear condensation, and pyknosis. Upregulation of specific markers including caspases, bax, bid, and bad, and generation of free radicals, all play a role in mediating the suicide cell death program. Two main pathways are described in apoptosis, the extrinsic, primarily death receptor-mediated pathway, and the intrinsic, mitochondrial  30  pathway (Fig. 1.8). The death receptor pathway is triggered by ligands outside the cell whereas the mitochondrial intrinsic pathway is set off from stress signals originating within the cell including D N A damage and caspase-2 and -6 activation. Caspase-2 and -6 cause mitochondrial permeabilization triggering cytochrome c release and activation of executioner caspases such as caspase-3 (Xanthoudakis et al., 1999;Lassus etal., 2002). It must be emphasized that the decision to die is not taken lightly and numerous checkpoints are seen throughout the apoptotic process to ensure the cell's full commitment to death. Mammalian inhibitors of apoptosis, including X I A P and NIAP, are effective against caspase-2 and-3 apoptotic pathways (Deveraux et al., 1999). Mammalian inhibitors for caspase-6 are yet to be described. Several peptide based approaches also selectively inhibit caspases (Garcia-Calvo et al., 1998;Nicholson, 1999). However, difficulties in the penetration of the blood brain barrier precludes the wide spread use of these in therapeutics. The ability to distinguish, using established markers, the apoptotic pathway triggered in H D and in the particular the key upstream caspase(s), is critical for the success of a target-based strategy for designing ultimate therapeutics and inhibiting select pathways directly relevant to HD.  31  Intrinsic  Extrinsic  pathway  pathway  flAft Fas Cytoplasmic membrane  o > > CO CO ER stress  TJ ro  Bax DNA damage  I  T3  a  i  o  > > CO 4—• CO •o  CO  > CO  •o  TJ a  CO  o > > CO —> CO •o  13  INS  I I  Cleavage of substrates  Apoptosis Figure 1. 8 Order of caspases activation in Apoptosis. The activation of the initiator caspases such as -2, -6, -8 and -10 are observed early post cytotoxic insult. These caspases then set off a cascade of signalling events, resulting in cleavage of caspase substrates (including downstream caspases) and mitochondrial permeablizaton. Two main pathways are described, the extrinsic, primarily death receptor-mediated pathway and the intrinsic, mitochondrial-mediated pathway. Figure adapted from Cirayo 2002. 1.8.1  Caspase activation pathways inH D  There is evidence to suggest that apoptosis plays a role in the neurodegeneration observed in HD. Increased D N A fragmentation and typical apoptotic, T U N E L positive  32  cells have been detected in human HD brain, and expression of the pro-apoptotic protein Bax is increased compared to controls. Furthermore, increased levels of cytoplasmic PARP expression, in neurons and cellular processes of activated astrocytes, is also observed and suggests a role for the D N A repair enyzyme P A R P in neurodegeneration in H D (Vis et al., 2005). With regards to caspase activation, caspase-3 cleaved fragments of htt have been detected in HD human brain (Kim et al., 2001;Wellington et al., 2002) and enhanced immunoreactivity for caspase-2 and caspase-6 is observed in the medium spiny neurons of the striatum in human and mouse HD brain (Hermel et al., 2004). Caspase-3 expression has been described in HD as weak and most likely depicting pigment-laden macrophages (Vis et al., 2005). A further study reports that degenerating astrocytes in the caudate of HD postmortem tissue were immunoreactive for caspase-3 (Hermel et al., 2004). Furthermore, experimental models of HD (including K A , Q A and 3-NP) demonstrate nuclear D N A fragmentation and enhanced expression of apoptotic related markers including caspase-1, -2, -3 and -6 (Ferrer et al., 2000; Henshall et al., 2002). What has not been established is the order of caspase activation and/or if this is a critical, primary event in the disease. Is caspase activation involved in the initial stage of HD? And i f so, which one(s) and do they generate specific fragments of mutant htt that are responsible for initiating the pathogenesis of the disease?  1.9 Excitotoxicity i n H D  Numerous studies highlight excitotoxicity as playing an important role in the pathogenesis of HD (Albin et al., 1990;DiFiglia, 1990;Beal et al., 1991;Beal, 1992; Young, 1997;Zeron et al., 2001;2002;2004). The excitotoxicity hypothesis suggests that over-stimulation of glutamate receptors, in particular N M D A receptors, cause increased C a  ++  influx into the cell triggering C a ^ overload, caspase activation,  mitochondrial dysfunction and apoptotic cell death. Medium spiny neurons of the neostriatum receive glutamatergic input from most cortical regions, and overactivation of N M D A receptors is likely to play a role in the death of these neurons. Importantly, expression of the N M D A receptor subunit NR2B, relative to other NR2 subunits, is increased in striatal MSNs (Landwehrmeyer et al., 33  1995) and results of radio-labeled ligand binding assays demonstrate decreased binding of N M D A R in presymptomatic human HD striatum, suggesting that neuronal cells which highly express these glutamate receptors are particularly vulnerable (Young et al., 1988;Albin et al., 1990). Increased levels of glutamate have been observed in H D patient brain and intrastriatal injection of quinolinic acid, an N M D A agonist, produces behavioral features in primates reminiscent of HD motor dysfunction and similar striatal lesions as observed in H D human brain (Ferrante et al., 1993; Burns et al., 1995). Furthermore, significantly increased NMDAR-mediated current and enhanced excitotoxicity following glutamate/NMDA stimulation has been demonstrated in MSNs in HD mouse models (Hodgson et al., 1999;Levine et al., 1999;Cepeda et al., 2001;Zeron et al., 2002;2004;Tang et al., 2005;Shehadeh et al., 2005). In contrast, cultured striatal neurons containing NADPH-diaphorase are relatively resistant to NMDAR-mediated toxicity as are cerebellar granule cells (Zeron et al., 2002), consistent with the pattern of sensitivity in human HD. Current density is also increased in N M D A receptor transfected nonneuronal cells co-expressing expanded huntingtin, where the effect is specific for NR1/NR2B (Chen et al., 1999). Moreover, mutant huntingtin-transfected cell lines or primary medium spiny neurons from YAC72 mice were more vulnerable to NR2B-specific induced cell death. In these paradigms, increased caspase-3 activity was observed in medium spiny neurons expressing expanded huntingtin relative to wild-type huntingtin (Zeron et al., 2002;2004). These observations strongly support a role for expanded huntingtin in augmenting an NR2B-dependent pathway that leads to Ca^-induced cellular dysfunction and ultimately neuronal cell death (Fig. 1.9). Rises in intracellular C a , as a result of excessive or enhanced activation of ++  specific C a  + +  channels such as the N M D A receptor, act as intracellular mediators of  excitotoxicity. Increased C a  + +  levels cause activation of caspases, marking the initiation  of apoptosis and programmed cell death. Caspase-mediated proteolytic cleavage of substrates, such as htt, would generate mutant htt fragments that may be toxic and act in a positive feedback loop to further increase excitotoxicity, caspase activation, and mitochondrial dysfunction. The excitotoxicity hypothesis in H D is discussed in detail in the discussion under 'Model for H D ' (Chapter 5).  34  t glutamate release (cortical efferents)  1 1 synaptic stimulation  I t N M D A R mediated current  |Ca++  Caspase activation Toxic fragments mitochondrial dysfunction  Nucleus  transcription dysregulation i proteasome inhibition  Figure 1.9 Evidence supports a role for mhtt in enhancing response of postsynaptic N M D A  receptors. The mechanism of increased N M D A R current in response to synaptic stimulation would result in increased C a influx that may chronically stress MSNs which contain mhtt and cause activation of the programmed cell death pathway. + +  1.9.1 Acute model of HD: In vivo Quinolinic acid mediated neurotoxicity Intrastriatal injection of glutamate analogs including Kainic acid (KA), Ibotenic acid (IA) and QA, in mice and primates creates lesions similar to those observed in H D patients (McGeer et al., 1978;Isacson et al., 1985;Beal et al., 1991). Q A mediated neurotoxicity most faithfully replicates H D pathology, as observed by the selective loss of G A B A and substance P containing neurons while sparing N A D P H diaphorase positive neurons (Beal et al., 1991). In contrast, lesions caused by K A and IA affect all striatal cell 35  types indiscriminately (Beal et al., 1986). Furthermore, Q A is an agonist of N M D A R (Levivier and Przedborski, 1998) and possibly a specific subset, and therefore would be expected to have the same effect as N M D A , which enhances current density and apoptotic cell death in several mouse models of H D (Levine et al., 1999;Zeron et al., 2002; 2004). In addition, Q A lesion models demonstrate accumulation of p53, N F K B and activation of caspases (Qin et al., 1999;Nakai et al., 2000), molecular changes also observed in the brains of patients with H D (Davies and Roberts, 1988;Hermel et al., 2004;Khoshnan et a l , 2004;Bae et al., 2005) 1.10 H D mouse models Studies on human H D post mortem tissue have elucidated several critical characteristics of neuropathology, including selective striatal cell loss, caspase activation, inclusions, and cleavage of htt. However, difficulties in obtaining sufficient human tissue throughout the course of the disease preclude a natural history characterization of H D in human brain. Transgenic mouse models of H D circumvent this and enable a detailed assessment of all aspects of neurodegeneration including delineation of important early primary events and not simply secondary effects due to cell loss. However, a mouse model is only useful if it recapitulates key features of the disorder observed in humans and may in fact be detrimental by highlighting inappropriate pathways not activated or relevant to the human condition. There are currently several mouse models of H D (Table 1.2). A number of techniques have been used to generate full length htt mouse models of H D including Y A C , knockin and c D N A approaches. Truncated htt mouse models have also been developed and the R6/2 model is used extensively in H D research. A common hallmark in both full length and truncated htt mouse models of H D is the presence of intranuclear inclusions, which are also observed in human H D brain and cell culture models of H D (DiFiglia et al., 1997;Cooper et al., 1998;Martindale et al., 1998;Gutekunst et al., 1999). Representative mouse models of H D have played a fundamental role in the understanding of the pathogenesis of H D and are essential reagents for assessment of therapeutic approaches. In addition to mouse models there is also a fly (drosophila melanogaster) and a C. elegans model (Jackson et al., 1998;Faber et al., 1999). The ease of genetic manipulation in these 36  organisms has enabled identification of genes that modify certain aspects of the H D phenotype (Warrick et al., 1999;Satyal et al., 2000;Kazemi-Esfarjani and Benzer, 2000).  37  Model/design R 6 line/truncated,  CAG Behavior -250  Mangiarini et al., 1996  N o evidence o f neuronal  deficiency,early death  loss  Tremors, hypokinesis,  Neuronal degeneration in  E a r l y death  striatum (caspase activation)  120  N o n e observed  N o n e observed  Slow et al., 2005  100  Hyperactivity, endstage  20%cell loss (8 months) in  Laforet et al., 2001  hypoactive  some animals  Hyperactive and circling  Neuro-degeneration in  82  M o u s e prion promoter Shortstop/truncated,  Reference  Tremors, abnormal gait, rotarod  l k b o f H D promoter  N171/truncated,  Neuropath  Schilling et al., 1999 Yu et al., 2003  25kb H D promoter 3kb N-ter htt/Rat neuron specific enolase promoter Y A C 7 2 / f u l l length  72  human htt, 25kb H D  Hodgson et al., 1999  striatum  promoter Y A C 1 2 8 / f u l l length  128  Cognitive deficits, motor  C e l l loss in the striatum  abnormalities  Decreased striatal volume  C i r c l i n g , hyperactivity, endstage  20% cell loss (striatum) in  hypoactivity  some animals  human htt, 25kb H D  Slow et al., 2003  promoter F u l l length h t t / C M V  89  promoter  Reddyetal., 1998  K n o c k i n / H d h promoter  80  aggressive  N o n e observed  Shelbourne et al., 1999  K n o c k i n / H d h promoter  94  N o behavior but N M D A  N o n e observed  Levine et al., 1999  N o n e observed  Lin etal., 2001  sensitive K n o c k i n / H d h promoter  150  Clasping, gait deficit  Table 1.2 HD mouse models. Outline o f  the construct, behaviour and neuropathological findings in H D  mouse models.  1.10.1 Full length mouse models of HD The first described full length HD model was the HD89 line under the control of the cytomegalovirus promoter (Reddy et al., 1998). A n initial hyperactive phase is followed by hypoactivity. Non-selective neuronal loss and astrogliosis is observed in some animals across several areas of the brain in the end stage of the disease. The presence of degenerating, T U N E L positive neurons and dystrophic neurites, suggest  38  activation of the apoptotic cell death pathway. A significant decrease in the number of dendritic spines is also reported. The Y A C transgenic mice were developed using the 350 kb Y A C 353G6, which contains all 67 exons of the human H D gene and its endogenous regulatory sequences (Hodgson et al., 1999). The human transgene has been shown to functionally compensate for the loss of the endogenous murine gene and rescue the embryonic lethal phenotype of htt null mice (Hodgson et a l , 1996). The Y A C transgenic animals display enhanced susceptibility to excitotoxic stress, cleavage of htt by caspases and cognitive dysfunction early in the phenotype, followed by motor deficits and selective striatal degeneration (Wellington et al., 2002; Slow et al., 2003; 2005; Van Raamsdonk et al., 2005a, 2005b; Tang et al., 2005; Shehadeh et al., 2005). Neurodegenerative changes, including toluidine blue stained degenerating striatal neurons, are also observed and demonstrate a striatal gradient of degeneration similar to that observed in humans (Hodgson et al., 1999). A less successful approach was the gene targeted knockin mice independently generated by four research groups (Levine et al., 1999;Shelbourne et al., 1999;Wheeler et al., 2000;Lin et al., 2001). The C A G size ranges from 50-150 C A G repeats and expression levels are in general higher than other full length H D models. However, the only line to develop a overt phenotype is the 150Q line which shows a progressive tendency to inactivity, clasping and rotarod deficit (Lin et al., 2001). The Hdh 94Q line does however show enhanced sensitivity to N M D A (Levine et al.,1999).  1.10.2 Truncated H D mouse models The R6/2 transgenic mice were the first described H D mouse model (Mangiarini et al., 1996). The R6/2 line expresses the exon 1 fragment of htt containing - 1 5 0 C A G repeats which is significantly larger than observed in humans. In contrast to htt expression in humans, the transgene in R6/2 is ubiquitously expressed at similar levels in all tissues. These mice appear normal at birth; however, they go on to develop symptoms by 5-8 weeks that include rotarod deficits, clasping and abnormal gait. Inclusions are an early event in the phenotype and are found through out the brain. Death occurs by 13/14 weeks of age. Neurotransmitter abnormalities, similar to what has been described in 39  humans are observed and precede onset of clinical symptoms (Cha et al., 1998). Dark degenerating neurons and dysmorphic neurites have been observed in the R6 line, however, there is no astrogliosis and evidence of neuronal loss is controversial. Using T U N E L analysis, G F A P immunostaining and electron microscopy, no apoptotic neurons have been observed in the R6 line (Yu et al., 2003). In contrast, neuropathological assessment of R6 striatum using stereology has demonstrated a significant reduction in striatal volume and neuronal counts by 2 months of age (Stack et al., 2005) and loss of orexin neurons in the lateral hypothalamus by 4 months of age (Petersen et al., 2005). Widespread atrophy in the brains of R6 mice has also been reported including significant decreases in the volume of the striatum, cortex, globus pallidus, cerebellum and hippocampus (Van Raamsdonk et al., 2005). Furthermore, the R6 line exhibits nonselective nuclear detection of mutant htt (Van Raamsdonk et al., 2005). In the R6/2, full length c D N A and knockin lines, polyglutamine aggregation is observed prior to overt behavioural deficits (Reddy et al., 1998;Morton et al., 2000;Laforet et al., 2001 ;Lin et al., 2001). In contrast, inclusions occur late in the Y A C model and present after behaviour and neuronal loss have occurred, similar to limited studies available in human H D (Gutekunst et al., 1999;Slow et al., 2003;2005). Interestingly, the frequency of striatal inclusions is not associated with reduced rotarod performance in the H D I 00 line. However, diffuse nuclear localization of htt in cortical neurons is predictive of neurological impairment (Laforet et al., 2001). The N171 truncated model expresses the first 171aa of htt with 82CAG repeats driven by the prion promoter (Schilling et al., 1999). Onset of a behavioral phenotype is at 2 months and life span varies from 6-11 months. The N171 model demonstrates a progressive neurological phenotype which includes clasping, rotarod defict and abnormal gait. Similar to other truncated models of HD, the N171 mice display extensive EM48 positive aggregates in the brain. The truncated H D mouse model, shortstop, expresses exon 1 and 2 of htt and contains 120CAG repeats (Slow et al., 2005). Interestingly, frequent and widespread htt inclusions occur early in shortstop yet these mice display no evidence of neuronal dysfunction or neurodegeneration clearly demonstrating in vivo that inclusions are not pathogenic.  40  1.11 Hypothesis and Objectives A fundamental question in H D research is how proteolytic cleavage of htt contributes to the initiation and/or progression of HD. It is well established that htt is a substrate for several proteases and caspase-mediated fragments of htt are present in H D brain prior to clinical onset of HD. Furthermore, inhibiting caspase cleavage of htt (caspase resistant htt) in an in vitro model reduces toxicity in neuronal and nonneuronal cells. These observations provide strong support for the toxic fragment hypothesis which proposes that htt cleavage is a crucial determinant of pathology in H D and furthermore suggest that caspase-resistant htt may delay or prevent the onset or progression of H D in vivo. The primary objective of this thesis was to determine if proteolysis of mutant htt is a primary event in the pathogenesis of HD. M y second objective was to determine i f a specific fragment of mutant htt causes the toxicity observed in select neuronal populations and whether inhibiting generation of this mutant htt fragment leads to protection against the toxic effects of the expanded polyglutamine tract.  Specific goal 1: To generate YAC transgenic mice expressing caspase resistant huntingtin with an expanded polyglutamine tract. In order to firmly establish the toxic fragment hypothesis and its role in the pathogenesis of HD, a detailed in vivo study of caspase resistant htt with expanded polyglutamine tracts is essential. Site directed mutagenesis of htt's caspase-3 and -6 sites was used to change the PI aspartate to an alanine. Using a homologous recombination strategy, the htt fragments containing site directed mutations of caspase sites, were targeted into the Y A C 128 used previously to generate Y A C 128 transgenic mice. Verified C R Y A C ' s were injected into F V B pronuclei to generate founder lines.  Specific goal 2: Do levels of mutant huntingtin influence the phenotypic severity in the YAC128 model?  41  We have previously demonstrated that a Y A C mouse model of HD containing the entire human huntingtin gene with 120 C A G repeats ( Y A C 128 line 53) develops agedependent motor abnormalities and selective striatal atrophy (Slow et al., 2003). These phenotypes indicate that the Y A C 128 model accurately replicates human H D in vivo, making the Y A C 128 model an ideal one with which to assess underlying hypotheses in HD. The caspase resistant Y A C mice do not all express mutant htt at a similar level to the previously described Y A C 128 line 53, therefore it was necessary to determine whether levels of mutant htt influence the phenotypic severity of H D in the intermediate and lower expressing mutant htt control Y A C 128 lines HD55 and HD54.  Specific goal 3. Is proteolysis of htt required for neurodegeneration in the Y A C transgenic animal model of H D ? To determine if caspase cleavage of mutant htt is required to observe striatal atrophy in the Y A C 128 model, a neuropathological assessment of control ( Y A C 128 lines HD53, 54 and 55) and C R lines matched for equivalent C A G size and transgene expression levels was performed. A l l lines were aged to 18 months to maximize the potential for neurodegeneration to occur. The primary outcome measure was an unbiased estimate of striatal volume.  Specific goal 4: Does selective mutation of specific caspase cleavage sites in expanded human huntingtin delay the onset or progression of behavioral changes in the Y A C transgenic animal model of H D ? Cognitive and motor deficits are a clinical hallmark of onset of HD. The Y A C 128 mice exhibit cognitive deficits and motor abnormalities which parallel those observed in human HD (Slow et al., 2003). The caspase resistant mutant htt mice were assessed for rotarod performance and open field activity to define whether generation of a specific mutant htt fragment underlies neuronal dysfunction in the Y A C 128 model. These experiments provide proof of principle that neuroprotection strategies will alleviate the clinical motor symptoms in an in vivo model of HD. Specifically, we will determine  42  whether caspase inhibitors may be useful strategies for both the neuropathological and behavioral aspects of HD.  Specific goal 5: Does inhibiting caspase-6 cleavage of mutant htt provide protection from excitotoxic neuronal death ex vivo and in vivo?. Work on the role of NMDAR-mediated excitotoxicity in HD has highlighted excitotoxicity as a fundamental aspect in the earliest stages of the pathogenesis of HD. The caspase resistant (CR) mice allow investigation of the pathways by which excitotoxicity, caspase activation, proteolysis of mutant htt and neuronal death are related.  Conclusion My overall objective is to elucidate whether proteolysis of mutant htt is a primary event in the pathogenesis of HD and furthermore to determine if a specific fragment of mutant htt initiates neuronal dysfunction and/or downsteam neurodegeneration. It is my hope that the answers obtained from these studies will highlight which caspase plays a crucial role in generating fragments of mutant htt that are a primary event in the pathogenesis of the disease and provide proof of principle for caspase inhibitors as therapeutic agents in HD and the necessary evidence required to design phase I clinical trials.  43  Chapter 2: Material and Methods  2.1 Mutagenesis of huntingtin caspase sites 2.1.1 Mutagenesis constructs Mutagenesis to create the P l aspartate to alanine mutations in htt cDNA has been previously described (Wellington et al., 2000). These cDNA constructs and the Y A C 353G6 D N A , which contains the full length genomic H D gene, were used as templates in PCR reactions designed to include relevant exon-intron sequences as follows: The exon 12 construct (pRS406-exl2) contained 1634 bp of genomic htt D N A consisting of 627 bp from intron 11, the entire exon 12 (341 bp), and 666 bp from intron 12 and was assembled using the following primers: NF1 -1F: 5 ' - G G A C G A A G C T T A C A T G C T T A C C G A C C C A A C T - 3 ' NF1-2R: 5 ' - G A G G C T G A A G A C A G A G A A A C A C T C - 3 ' NF1-2F: 5 ' - G A G T G T T T C T C T G T C T T C A G C C T C A G T G A A G G A T G - 3 ' 2RS: 5 ' - C T G T C T G A A G G G G T A A C A G C T G - 3 ' NF1-3F: 5 ' - G A T T C A G C T G T T A C C C C T T C A G - 3 ' N F 1-4R: 5 ' - G G T C T C C A G G G A A G A A C T C A - 3 ' NF1-1F and NF1-2R were used to amplify intron 11 from Y A C 353G6 D N A , NF1-2F and 2RS were used to amplify exon 12 cDNA containing the mutations from pCIneo-3949-15 quint, and NF1-3F and NF1-4R were used to amplify intron 12 from Y A C 353G6 D N A . Then, intron 11 and exon 12 were joined using NF1-1F and 2RS, and extended to intron 12 using NF1-1F and NF1-4R. The final PCR product was digested with Hindlll (underlined in NF1-1F) and subcloned into Hindlll, Smal digested pRS406 to generate pRS406-exonl2. Prior to transformation, pRS406-exonl2 was linearized with B g l l l , which is located within intron 11 at a site 452 bp upstream of the intron 11/exon 12 boundary. The exon 13 construct (pRS406-exl3) contained 1148 bp of genomic htt D N A consisting of 500 bp from intron 12, the entire exon 13 (124 bp), and 490 bp from intron 13 and was assembled using the following primers: C6AF: C6BF: C6CF: C6AR: C6BR:  5 '-GG A C G C T C G A G A C A C A G G A C A G T G G A-3' 5'-CTCACAGCCCCCCTTGACCGT-3' 5'-CATGGGTATGTGGACTACAGGTG-3' 5'-ACGGTTCAGGGGGGCTGTGAG 5'-CACCTGTAGTCCACATACCCATG-3'  45  C6CR: 5 ' - A T T T A A G G C C C A G G G A T G - 3 ' Introns 12 and 13 were amplified from Y A C 353G6 D N A using C6AF and C 6 A R and C6CF and C6CR respectively, and C6BF and C6BR were used to amplify htt c D N A containing the exon 13 mutations from pCIneo-3949-15 quint. These fragments were joined using C6AF and C6BR, and then C6AF and C6CR. The final PCR product was digested with Xhol and subcloned into XhoI-Smal-digested pRS406 to generate pRS406exonl3. Prior to transformation, pRS406- exon 13 was linearized with Hindlll, which is located within intron 12 at a site 350 bp upstream of the mutation at amino acid 586. Linear D N A fragments were gel purified and used for yeast transformation.  2.1.2 Yeast growth and transformation: Yeast were grown in liquid culture at 30°C in synthetic dropout media without lysine (lys) and tryptophan (tip) to select for the previously retrofitted Y A C 353G6. Yeast were transformed using the spheroplast method as previously described (Hodgson et al., 1999) and plated on selective media. After 4-5 days of incubation at 30°C, colonies were grown in lystrpura" selective liquid media for two nights to obtain D N A for verification. Pop-out clones were selected for by plating verified pop-in clones on lystrp" plates containing lmg/ml 5-fluoroorotic acid (5-FOA). Yeast containing the uracil gene cannot grow in the presence of 5-FOA and will excise the uracil gene by homologous recombination. Colonies were selected after 4-5 days at 30°C.  2.1.3 Isolation of yeast genomic DNA for YAC screening Total yeast genomic D N A was isolated using glass beads, which yields high quality D N A suitable for PCR and southern analysis. Pop-in or pop-out candidate clones were grown in selective media at 30°C for 46 hours, washed in H 0 and resuspended in 2  200uX of GDIS (2% Triton XlOO, 1% SDS, 100 m M NaCl, 10 m M Tris-HCl, I m M EDTA). Approximately 0.35 g of acid-washed glass beads and 200ul of PCI (25:24:1 phenol: chloroform: isoamylalcohol) were added to the cells and vortexed for 2-5 minutes. After the addition of 200u\l of water, lysates were again vortexed and centrifuged for 4 minutes at maximum speed. D N A was precipitated from the aqueous  46  layer with 100% ethanol, dissolved in 400LI1 of water and cleared of R N A with a 10 minute incubation at 37°C with RNase A . Following ethanol precipitation, D N A was resuspended in water and stored at -20°C.  2.1.4 Determination of CAG Stability The C A G size of Y A C clones was assessed by PCR amplification of the C A G region using primers HD 344 ( 5 ' - C C T T C G A G T C C C T C A A G T C C T T C - 3 ' ) and H D 482 ( 5 ' - C C G A C T C C T T C G A C T C C T C - 3 ' ) with final concentrations of 2mM M g C l , 3.5% 2  formamide, 15% glycerol, 0.2mM dNTPs, 0.5uM of each primer as previously described (Hodgson etal., 1996).  2.1.5 SSCP to detect mutant sequence SSCP was done as an initial screening step at both the pop-in and pop-out stages to detect mutant sequences within candidate Y A C clones from the exon 12 construct. Potential clones for exon 12 mutagenesis were amplified using primers 1FS (5'G A G T G T T T C T C T G T C T T C A G C C T C - 3 ' ) and 2RS (5'C T G T C T G A A G G G G T A A C A G C T G - 3 ' ) to amplify a 350 bp product. PCR products were denatured and electrophoresed on 8% native polyacrylamide gels at 4°C. Gels were fixed in 10% ethanol, 0.5% acetic acid for 15 minutes at room temperature, stained in 0.1% A g N 0 for 15 minutes and developed in 1.5% NaOH, 0.01% N a B H , 0.148% 3  4  formaldehyde until silver stained bands were evident. Wild-type and mutant controls were prepared by amplification from Y A C 18 and mutant construct pRS406-exl2, respectively.  2.1.6 Restriction analysis of PCR products Mutagenesis at the caspase-6 site (aa 586) in exon 13 resulted in the gain of a BsrFI restriction site that was used to detect the presence of mutant sequences after amplification of D N A . A 168 bp fragment was amplified from candidate exon 13 pop-in or pop-out clones using the forward primer 3FS ( 5 ' - C T C A C A G C C C C C C T T G A A C C G T 3') and the reverse primer 4RS ( 5 ' - C A C C T G T A G T C C A C A T A C C C A T G - 3 ' ) . P C R products were purified using Qiaquick (Qiagene) and digested with BsrFI (New England  47  Biolabs) at 37°C for 2 hours prior to resolution on a nondenaturing 12% polyacrylamide gel.  2.1.7 Southern blot analysis for YAC integrity Yeast D N A was digested overnight with Bglll for exon 12 clones and with Hindlll for exon 13 clones and separated on a 0.7% agarose gel for 5-6 hours at 120V. D N A was denatured and transferred to Hybond N+ nylon membrane (Amersham) overnight using standard procedures. The membranes were fixed by U V crosslinking, rinsed briefly in 2xSSC and prehybridized for 30 minutes in Rapid-hyb Buffer (Amersham) at 65°C. Probes were generated by amplifying wild-type D N A from Y A C 353G6 D N A using the primers 1FS (5'-CTAGTGTTTCTCTGTCTTCAGCCTG-3') and 2RS to generate a 352 bp product to detect exon 12 and with NF2-SF and 4RS (5'C A C C T G A G T C C A C A T A C C C A T G - 3 ' ) to generate a 344 bp product to detect exon 13. PCR products were purified using Qiaquick (Qiagene), radioactively labeled with oc- P 32  dCTP (Amersham) using the rediphme random primer labeling system (Amersham), and purified on a Sephadex G-50 N I C K column (Pharmacia). After a 1 hour hybridization, membranes were washed in 2xSSC/0.1%SDS at room temperature for 15 minutes and 0.1xSSC/0.1%SDS at 65°C for 3-4 washes each for 15 minutes prior to autoradiography. Analysis of the promoter region to ensure the presence of intact proper regulatory elements was also assessed by Southern analysis. Y A C genomic D N A was digested with EcoRI overnight and followed blotting and hybridization conditions described above using a 4. lkb probe spanning the 5' promoter region of H D as described.  2.1.8 PCR analyses for YAC integrity The maintenance of various Y A C markers spanning Y A C 353G6 was assessed by PCR assays. Primer pairs were used to identify Y A C sequences from the left and right Y A C arms ( L Y A and R Y A ) , the C A repeat in intron 1 and the A G in exon 59 of the H D gene as described.  2.2. Generation and characterization of CR YAC transgenic mice  48  2.2.1 Generation of CR YAC mice Verified C R Y A C s were purified by pulse-field electrophoresis and microinjected into F V B / N oocytes. Founder mice were identified by PCR detection of Y A C L Y A , R Y A , C A , A G , and C A G PCR in a procedure described previously (Hodgson et al., 1999) and by sequence confirmation of the mutation site. The C A G repeat was also amplified by PCR using a 6 - F A M labeled 5' primer. PCR products were cleaned using a QIAquick gel extraction Kit (Amersham). Genescan Analysis 3.7.1 software was used to visualize the products. Lines were established from each founder animal, and at least two independent lines were established for each C R Y A C construct.  2.2.2 Real-time quantitative RT-PCR to determine transgene expression level Total R N A was extracted from mouse cortex with RNeasy Protect Mini Kit (Qiagen), and treated with Amplification Grade DNasel (Invitrogen). First-strand c D N A was prepared from 1 pg of total R N A using Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). Approximately 700pg of first-strand cDNA was used as template in real-time PCR reaction in a final volume of 25 ul. Human specific huntingtin (HD) primers and mouse specific B-actin primers were designed to meet specific criteria by using Primer Express software (Perkin Elmer). The sequences of the H D primers were 5' G A A A G T C A G T C C G G G T A G A A C T T C 3 ' and 5' C A G A T A C C C G C T C C A T A G C A A 3 ' and the B-actin primers were 5' A C G G C C A G G T C A T C A C T A T T G 3' and 5' C A A G A A G G A A G G C T G G A A A A G A 3'. Real-time PCR was performed using S Y B R Green PCR Master M i x (Applied Biosystems) and the A B I PRISM 7000 Sequence Detection System. A l l samples were run in triplicate. Primary data analysis was performed using system software from Applied Biosystems. For each experimental sample, the amount of H D gene and endogenous reference (B-actin) was determined from a standard curve. The standard curve was constructed with threefold serial dilutions of pooled cDNA samples (4500pg to 170pg) and was run in triplicate during every experiment. The amount of HD mRNA level was divided by the amount of B-actin m R N A level to obtain a normalized H D gene expression value.  49  2.2.3 Protein analysis and Western blotting Protein was prepared from mouse brain lysed in homogenization buffer containing 0.3M sucrose, 20mM Tris-HCl, I m M M g C l , 0.5mM E D T A , I m M PMSF, 1 2  complete protease tablet (Roche) and 10 u M Z V A D (Calbiochem., San Diego, C A ) per 30ml volume. Protein concentration was determined by Bio Rad D C Protein Assay and lOOjig run on 7.5% or 10% low bis polyacrylamide gels. Proteins were transferred to P V D F membranes then probed with B K P 1 , an N-terminal htt antibody or HD650 a human specific htt antibody described previously (Hodgson et al., 1999) and anti-Bactin (Chemicon, Temecula, CA).  2.2.4 E x vivo confirmation of caspase resistant htt Whole mouse brain protein lysates were prepared as described above without caspase inhibitor added to the lysis buffer. Protein concentration was determined by Bio Rad D C Protein Assay. To cleave htt, 40p. g of protein was incubated in cleavage buffer alone (50nM HEPES/KOH, pH 7.0, 10% (w/v) sucrose, 2mM E D T A , 0.1% (w/v) 3-[(3cholamidopropyl) dimethylammonio]-l-propanesulfonate, 5mM DTT) or with 2nM of the respective caspase for 60mins at 37°C. After addition of SDS loading buffer the samples were boiled and loaded onto 7.5% SDS-PAGE gels and ran at 100V for one hour. Protein was transferred onto P V D F membranes (Millipore) and then probed with either htt552, BKP1 (described previously (Hodgson et a l , 1999)) or M A B 2166 (Chemicon).  2.2.5 Rescue of embryonic lethality of htt deficient mice F V B / N mice expressing the H D or caspase-resistant (CR) Y A C were crossed to mice heterozygous for the murine H D gene. Tail D N A was extracted from the offspring and screened for the R Y A , L Y A , C A G repeat and genotyped at the H D locus for the null  50  allele using primers P8 and P9 as described previously (Nasir et al., 1994; Hodgson et al., 1999). Mice containing the H D or C R Y A C and heterozygous for the murine H D gene were crossed to mice heterozygous for the murine H D gene. Progeny from these matings were screened in a similar manner and all genotypes from the heterozygous breedings entered into GraphPad Prism 4.0 for chi-square analysis.  2.3 Generation and characterization of caspase cleavable YAC128 controls A 350kb Y A C (353G6), containing 25kb of upstream regulatory sequences and the entire H D gene comprising all 67 exons and 120C A G repeat was purified by pulsefield electrophoresis and microinjected into F V B / N pronuclei. Founder mice were identified by Southern blot and detection of Y A C L Y A , R Y A , C A , A G , and C A G P C R as described previously (Slow et al., 2003). In total three lines (HD53, 54 and 55) with varying mutant transgene expression levels were established. Mice were maintained on the F V B / N (Charles River, Wilmington, M A ) background strain and are congenic on this strain. A l l mice were housed, tested and tissues harvested according to the animal protocol A00-0254 of the University of British Columbia.  2.3.1 CAG size D N A was extracted from mouse tails for PCR analysis in a procedure described previously. PCR using primer pairs encompassing the C A G repeat were used to amplify the C A G tract and products run on a 1% agarose gel with known C A G size standards. PCR of the C A G repeat was also amplified using a 6-F A M labeled 5' primer. P C R products were cleaned using a QIAquick gel extration Kit (Qiagen, Germany). Genescan Analysis 3.7.1 software was used to visualize the products.  2.3.2 mRNA and protein transgene expression levels Total R N A was extracted from mouse cortex as described in section 2.2.2. Whole mouse brain protein lysates were prepared as described previously (section 2.2.3). Protein concentration was determined by Bio-Rad D C Protein Assay (Biorad, Hercules,  51  CA) and lOOug run on 10% low-bis polyacrylamide gels. Proteins were transferred to P V D F membranes (Millipore, Bedford, M A ) then probed with B K P 1 , an N-terminal htt antibody described previously (Hodgson et al., 1999). Biorad anti-secondary antibodies were used at 1:3000 and detection done with E C L kit from Amersham (England). Protein quantification was done using Quantity 1.0 software from Bio-rad.  2.4 Quantitative Analysis A l l quantitative analysis was done blind with respect to genotype. Mice were weighed then terminally anesthetised by intraperitoneal injection of 2.5% avertin and perfused with 3% paraformaldehyde/0.15 % glutaraldehyde in PBS. The brains were stored in 3% paraformaldehyde for 24hrs at 4.C then removed and stored in PBS. Mouse brains were cut using a vibratome and coronal sections (25 pm) spaced 200pm apart throughout the striatum were stained with Neu N antibody (Chemicon, Temecula, C A ) at 1:100 dilution. Biotinylated secondary antibodies (Vector) were used at 1:200 prior to signal amplification with an A B C Elite kit (Vector) and detection with D A B (Pierce, Rockford, IL). The perimeter of the striatum for each section was traced using Stereo investigator software (Microbrightfield, Williston, V T , USA).  2.5 Behaviour Mice were group housed in microisolator cages with a normal light-dark cycle (lights on at 6:00 A M , lights off at 8:00 PM). A l l cage changing was done on the same day and no testing performed until 2 days after a cage change. Mice were randomly coded and the tester was blind to the genotypes at all times. A l l testing was done during the mouse light cycle.  2.5.1 Rotarod analysis Rotarod analysis was done on an accelerating rotarod (UGO Basile) which accelerated from 5 to 40 R P M over a period of 300s. During training mice were given 3 trials per day for 3 consecutive days. Rotarod testing scores were the average of 3 trials spaced 2 hours apart, with testing performed at 4, 6, 9 and 12 months of age. The Rotarod was wiped clean with ethanol between each test subject.  52  2.5.2 Open Field Analysis Open field analysis was conducted in the dark in an automated open-field activity apparatus (San Diego Instruments) for a period of 30mins with activity measured in 5min periods. Testing began at least lhr after the beginning of the mouse light cycle. Measurements were calculated by accompanying software, Photobeam Analysis System (PAS) (San Diego Instruments). The PAS measured the fine and ambulatory movement of the mouse. Ambulatory movement is measured as the frequency with which the mouse crosses the beams, while fine movement is defined as when the mouse enters but does not cross the beam.  2.6 Assessment of nuclear huntingtin and inclusions Perfused murine wt and transgenic brain sections of 25pm thickness were immunoassayed with the monoclonal antibody EM48 to assess for the presence of nuclear htt and inclusions as described previously (Gutekunst et al.,1999) using EM48 (polyclonal) at 1:500 and D A B as the chromogen (Vector). Htt inclusions were defined as EM48 positive puncta visible at the light microscope levels. Quantification of inclusions was determined by counting a minimum of 300 neurons across the striatum and determining the percentage of neurons which displayed EM48 positive puncta (inclusions). A similar analysis was performed for cortical inclusion levels. Photographs were taken on a light microscope (Zeiss) using 100X objective.  2.7 Analysis of caspase amplification and activation 2.7.1 Immunohistochemistry Vibratome cut coronal sections (25pm) from wt, H53 and C6R mouse brains perfused with 3% paraformaldehyde/0.15% glutaraldehyde were probed with activated caspase-6 antibody (9761, Cell Signaling) or activated caspase-2 (ab2552, Abeam) at 1:500 dilution. Biotinylated secondary antibodies (Vector) were used at 1:200 prior to signal amplification with an A B C Elite kit (Vector) and detection with D A B (Pierce). Photographs were taken on a light microscope using 100X objective.  53  2.7.2 Protein analysis, Antibodies and Western blotting Protein was prepared from mouse brain lysed in homogenization buffer containing 0.3M sucrose, 20mM Tris-HCl, l m M M g C l , 0.5mM E D T A , I m M PMSF, 1 2  complete protease tablet (Roche) and 10 u M Z V A D (Calbiochem., San Diego, CA) per 30ml volume. Protein concentration was determined by Bio-Rad D C Protein Assay and lOOpg run on 7.5% or 10% low bis polyacrylamide gels. Proteins were transferred to P V D F membranes then probed with B K P 1 , an N-terminal htt antibody or HD650 a human specific htt antibody described previously (Hodgson et al., 1999) and anti-Bactin (Chemicon, Temecula, CA). Human H D and control striatum and cortex (20pm) were prepared in a similar manner. Western blots of human tissue was probed with caspase-6 (9762, Cell Signaling) or caspase-2 (sc625, Santa Cruz) at 1:1000 and 1:250 dilutions respectively. Bio-Rad anti-secondary antibodies were used at 1:3000 and detection was done with enhanced chemiluminescence kit from Amersham or Supersignal West Pico Chemiluminescent Substrate (34080, PIERCE). Protein quantification was done using Quantity 1.0 software from Bio-Rad.  2.8 Generation of homozygotes YAC128 and Caspase Resistant huntingtin mice 2.8.1 Real-time quantitative PCR to determine zygosity Mouse genomic D N A was extracted from tail or ear notch and diluted to 0.08ng/ul. Human specific huntingtin (HD) primers and mouse specific B-actin primers with the same sequences as above were used for the real-time P C R reaction at the final concentration of lOOnM. 2.5ul of the genomic D N A was used in a final volume of 25ul. The real-time PCR was performed using the A B I PRISM Sequence Detection System and S Y B R Green PCR Master Mix. Fourfold serial dilutions of one D N A sample (3200pg to 50pg) were used to construct the standard curve. A l l samples were run in triplicate. Primary data analysis was performed using system software from A B I . The amount of H D gene and endogenous control gene (B-actin) was determined from the standard curve.  54  2.8.2 FISH Lymphocytes from mouse peripheral blood (60ul) were cultured for 3 days with Concanavalin A (Sigma, St Louis, Missouri) and Lipopolysaccharide (Sigma). Metaphase cells were obtained by treating cultures with Colcemid (Invitrogen). Chromosome spreads were fixed on microscope slides using methanol/acetic acid (Fisher, Fair Lawn, New Jersey). Biotinylated-DNA probes were generated from the H D Y A C (353G6 Y A C ) using BioPrime D N A Labeling System (Invitrogen). Before hybridization, probes and target D N A fixed on slides were denatured at 75°C. Hybridizations were performed at 37°C overnight, and subsequent washes were performed at 45°C in 50% formamide/2XSSC for 10 minutes and in 2XSSC for 10 minutes. Specific hybridization signals were detected by incubating the hybridized slides in Fluorescein Avidin and Fluorescein Anti-Avidin (Vector, Burlingame, CA) followed by counterstaining with DAPI (Molecular Probes, Eugene, Oregon). FISH images were captured and analyzed with Northern Eclipse 6.0 (Empix Imaging Inc.).  2.9 Assessment of susceptibility to Excitotoxicity 2.9.1 NMDAR-mediated excitotoxicity Pooled cultures (6-10 pups) of primary medium spiny striatal neurons were prepared from newborn pups (P0-1) of CQR, C3R, C6R and YAC128 (HD55) and W T in a procedure described previously (Zeron et al. 2002). Cultures were maintained in vitro for 9-10 days, after which they were exposed to balanced salt solution, 500uM N M D A or 500uM N M D A + 20uM MK801 for 10 minutes. Cells were rinsed and the conditioned media was replaced, and the cells maintained as usual. Twenty-four hours later, cultures were fixed (4% PFA) and assessed for apoptotic cell death using T U N E L staining (Roche -following manufacturers' instructions) and morphological criteria (small, condensed and blebbed nuclei) by propidium iodide counterstaining. Exposure of MSNs to staurosporine was for 24hrs using lOOnM, l u M and lOpM doses. For each experiment (n=3), all treatments were done blind, in triplicate, and a minimum of 1,000 cells were counted.  55  2.9.2 Quinolinic acid injections Quinolinic acid (Sigma) was dissolved into 0.1M PBS. Mice were anesthetized with isofluorane and received bilateral intrastriatal injections of 4 n M quinolinic acid. Seven days post injection, mice were terminally anesthetized as described above and analysed in a previously described procedure (Zeron et al., 2002a).  2.10 Statistical analysis A l l statistical analysis were done using unpaired, two tailed Student's Mest or one-way A N O V A (in cases of significant effect of genotype, post-hoc comparisons between genotypes were performed using the Tukey test) with the following exceptions: rotarod data were analyzed with repeated measures A N O V A and rate of disease progression analyzed with TWO-factor A N O V A . P values, S E M , means and standard deviations were calculated using Graphpad Prism version 4.0. Linear regression analysis for r and p values was calculated by Pearson correlation coefficient with Graphpad 2  Prism version 4.0.  56  Chapter 3 : Levels of mutant huntingtin influence the phenotypic severity of Huntington Disease in YAC128 mouse models  The work in this chapter has been published as:  Rona K . Graham, Elizabeth J. Slow, Y u Deng, Nagat Bissada, Ge Lu, Jacqueline Pearson, Jacqueline Shehadeh, Blair R. Leavitt, Lynn A . Raymond and Michael R. Hayden. Levels of mutant huntingtin influence the phenotypic severity of Huntington Disease in Y A C 128 mouse models. Neurobiology of Disease, in press, 2005  57  Preface I designed all of the experiments and analyses presented within this chapter and performed all with the exception of that noted below. Furthermore, while I developed the HD55 homozygote line, the HD53, HD54 and HD55 (heterozygote) lines were previously established in the laboratory. Deborah Deng helped with the FISH insitu hybridization and performed the Real time Q-PCR for human transgenic htt from mouse genomic D N A and RT-PCR for mRNA transgene levels. She also provided assistance with the 12 month striatal volume measurements in HD54. Jacqueline Pearson performed the behavioral analysis on the mice. Nagat Bissada assisted with the colony management and performed, with the help of Ge Lu, the mouse perfusions. Dr X . J . L i kindly provided the EM48 antibody. Dr. Blair Leavitt provided neuropathological advice and expertise.  58  3.1 Introduction Huntington Disease (HD) is an adult onset neurodegenerative disorder caused by a C A G expansion in the H D gene. Several neurodegenerative disorders including spinocerebellar ataxia ( S C A ) l , SCA2, Machado-Joseph Disease, dentatorubral pallidoluysian atrophy (DRPLA) and spinal bulbar muscular atrophy (SBMA) are also caused by a C A G repeat expansion in the respective genes with selective neuronal degeneration as a result of a toxic gain of function mechanism (Martin, 1999;Ross, 2002;Oda et al., 2004). There is still significant controversy as to whether mutant huntingtin (htt) demonstrates complete dominance. Early studies described H D as showing complete dominance with no increase in the severity of the phenotype of individuals homozygous for the mutation (Wexler et al., 1987;Myers et al., 1989;Gusella, 1991). This conclusion was based on the clinical evaluation of eight potential homozyogotes, as this was prior to the discovery of the H D gene and the authors could not rule out possible recombination events nor determine C A G size. As there is an inverse correlation between C A G size and age of onset (Andrew et al., 1993;Brinkman et al., 1997;Langbehn et al., 2004) this lack of information could have influenced interpretation of results. A further case report by Durr et al reported a similar age of onset in one confirmed H D patient homozygous for C A G expansion and his heterozygote brother (Durr et al., 1999). Thus clinical assessment of 9 (8 potential and 1 confirmed) homozygotes has led to the classification of H D as demonstrating complete dominance. In contrast to the early work, a more detailed clinical assessment and review of neuropathological findings from heterozygote and homozygote patients for H D did show increased rate of disease progression in the homozygote in humans but with no difference in age of onset (Squitieri et al., 2003). Studies in transgenic mice and in vitro also argue in favour of an intermediate dominant phenotype in H D (Davies et al., 1997;Reddy et al., 1998;Narain et al., 1999;Hodgson et al., 1999;Lin et al., 2001). H D knock-in mice demonstrate earlier onset of nuclear htt fragment accumulation and a more severe behavioural deficit in the homozygote (Lin et al., 2001;Wheeler et al., 2002). However,  59  there has been insufficient data to assess the impact of homozygozity for the H D mutation on the neuropathology of this disease. Most poly (CAG) disorders, including D R P L A , SCA2, SCA3, SCA6, SCA17 and SCA18 among others, demonstrate increased severity in homozygotes (Zlotogora, 1997;Durr et al., 1999). This includes both earlier onset and more severe evolution of the disease in the individuals homozygous for the mutation. Evidence from these studies and animal models of C A G repeat disorders argues for a gain of function mechanism accelerated by increased dose of the mutant protein (Bates and Davies, 1997;Burright et al., 1997;Warrick et al., 1998). H D is unique in being described as showing complete dominance and is currently the exception to what has been published for other polyglutamine expansion disorders. A n important unresolved question therefore, is whether H D demonstrates complete dominance. If this were the case, this would imply that the motor, cognitive and psychiatric symptoms are not influenced by dosage nor potentially mitigated by the normal allele (Wexler et al., 1987). Complete dominance implies a complete, saturating gain of function mechanism and argues against loss of function contributing to the disease phenotype and the effect of the other H D allele would not be expected to influence the phenotype irrespective of its being mutant or wild type. Intermediate dominance suggests that there is an underlying, cumulative toxic gain of function mechanism which is exacerbated by age and does not eliminate the possibility that there may be a concurrent contribution from altered function or activity of wild type htt. To resolve this issue requires an accurate and rigorous characterization of the H D phenotype in a controlled environment preferably with little potential for other genetic factors to influence the natural history of HD. The reason this question has not been fully addressed has been the absence of subjects and clearly quantifiable and reliable endpoints. Key reagents now available for this purpose are congenic mice carrying the H D mutation with altered levels of mutant htt and clearly defined and validated behavioural and neuropathological phenotypic endpoints. In order to address the fundamental question as to whether the H D phenotype is influenced by levels of mutant htt, we have performed a behavioural and neuropathological assessment of yeast artificial chromosome (YAC) 128 mouse lines with  60  varying levels of mutant htt. We have established Y A C 128 lines HD53, 54 and 55. HD53 is the highest expressing line. HD55 and HD54 express intermediate and lower levels of htt respectively. In addition we have used breeding strategies with HD55+/- to generate homozygous HD55+/+ mice. Behavioural and neuropathological assessment of four Y A C 128 lines provide clear evidence in vivo that the onset of the HD phenotype is exacerbated by increasing levels of mutant htt. Furthermore our data demonstrate a strong correlation between higher levels of mutant htt and a more rapid decline in behaviour and increased severity of neurodegeneration. This suggests that age of onset and disease progression in H D are directly influenced by levels of mutant htt and supports the hypothesis that H D displays an intermediate dominant phenotype with a more severe manifestation in individuals homozygous for the mutation underlying HD.  3.2 Results 3.2.1 G e n e r a t i o n o f H D YAC transgenic mice  A l l Y A C lines were generated using the well characterized 353G6 Y A C in a procedure described previously (Slow et al., 2003). Founders obtained were screened by southern blot using H D gene specific probes and by PCRs spanning the entire Y A C . One founder (HD53+/-) has been previously described (Slow et al., 2003;Van Raamsdonk et al., 2005b). Two additional founders were generated which incorporated the entire Y A C and showed altered patterns of mutant htt expression levels (HD54+/- and HD55+/-). PCR of D N A extracted from transgenic mice was performed using intragenic primers surrounding the C A G tract as described formerly (Hodgson et al., 1996). Transgene C A G sizing was done using the A B I Genescan system. Line HD54 and 55 demonstrate - 1 2 0 C A G repeat size similar to line HD53 (Fig.3.1 A). In the past, C A G sizing was done using a C A G PCR and the products run out on agarose gels (Fig. 3.IB), however, Genescan represents a more accurate estimation of the C A G size. Transgene m R N A (Fig.3.1C) and protein expression levels (Fig.3.1D) were determined using quantitative real time PCR and high resolution western blotting respectively. HD53, the highest expressing line contains approximately 75% of endogenous htt levels (Fig.3.1D). HD55 61  and HD54 contain approximately 30% and 15% of endogenous htt levels respectively (Fig.3.1D).  CAG HD53  I  I  C  'HD  5  05D  HD54  HD55  mRNA  YAC WT18 46 72 53 54 55  HD54  CAG  HD55  HD53  Protein  Fig. 3.1.CAG size and transgene expression levels in YAC128 mouse models. ( A )  Genescan results for  the expanded C A G repeat in H D 5 3 , 54 and 55. P C R o f the C A G tract was amplified using a 6 - F A M labelled 5' primer. A l l lines demonstrate identical 1 2 0 C A G repeat size. (B) Agarose gel showing P C R for C A G tract o f transgenic human htt in Y A C 18, 46, 72 lines (described previously, Hodgson et al., 1996) and H D 5 3 , 54 and 55. W T was included as a negative control. P C R conditions using human specific primers are as previously described (Hodgson et al., 1996). (C) Real-time quantitative R T - P C R showing m R N A transgene expression levels for H D 5 4 , 55 and 53. Transgenic htt levels have been normalized to actin. H D 5 3 is the highest expressing line; H D 5 5 and H D 5 4 demonstrate intermediate and low levels o f mutant htt respectively.  (D) H i g h resolution western blot o f H D 5 4 , 55 and 53 brain tissues probed with B K P 1 htt  antibody. Endogenous htt serves as loading control.  3.2.2 Generation and characterization of HD55 homozygote The previously published Y A C 128 model (HD53) is the standard against which we are assessing potential therapeutics and discovering underlying mechanisms involved in the pathogenesis of HD (Slow et al., 2003;Van Raamsdonk et al., 2005a;Tang et al., 2005). We also have established Y A C 128 lines HD54 and 55. Furthermore, using breeding strategies with HD55+/- we have developed a homozygote HD55+/+ line. Line HD55+/- contains approximately 40% the amount of mutant htt levels as HD53. Thus homozygote HD55 mice will express levels of mutant htt close to HD53 (80%).  62  A.  Q-PCR  WT  HD55+/-  HMS**  mRNA  HD55+/+  HD5J  Protein  Fig. 3.2. Generation and characterization of H D 5 5 homozygotes. (A) Real time Q-PCR for human transgenic htt from mouse genomic D N A demonstrates increased signal in HD55+/+ vs. HD55+/-. Five potential homozygotes (arrows) have been identified and are further confirmed homozygote by FISH analysis. (B) FISH analysis on murine chromosome metaphase spreads from W T , HD55+/- and HD55+/+ lymphoblasts. Human specific H D Y A C D N A (353G6) was used as a probe. W T was included as a negative control. HD55+/- lymphoblast metaphase spreads demonstrate only one signal. HD55+/+ shows two signals on metaphase chromosome spreads and in interphase nuclei. (C) Real-time quantitative RT-PCR demonstrating increased mRNA expression of human transgenic htt levels in HD55+/+ vs. HD55+/-. Y A C 128 homozygotes (HD55+/+) show transgene expression levels close to HD53. Huntingtin transgene RT-PCR values have been normalized to actin. (D) Human htt protein expression levels in brain tissue from HD55+/-, HD55+/+ and HD53. Western blots were probed with BKP1, an htt specific antibody. Endogenous htt serves as a loading control.  Generation of Y A C 128 mice with varying amounts of mutant htt have allowed us to directly address the question of whether mutant htt levels modulate age of onset and disease progression in an HD mouse model by phenotypic assessment of the different transgene expressing lines. In addition, generation of homozygotes has enabled us to more easily generate pooled striatal cultures for assessment of the endpoints including  63  susceptibility to N M D A R (N-methyl-D-aspartate receptor)-mediated excitotoxicity in the Y A C 128 medium spiny neurons (MSNs). In order to distinguish homozygote (HD55+/+) mice derived from heterozygote breedings (HD55+/-), we developed a real-time quantitative PCR (Q-PCR) method which involved Q- PCR of mouse D N A using human specific htt and B-actin primers. The expression level of the H D gene, normalized against B-actin, was determined from the standard curve. Using this method, homozygotes were easily distinguished from heterozygotes (Fig.3.2A). Blood from homozygotes detected in the Q-PCR screen were then used to establish lymphocyte cultures for fluorescent in situ hybridization (FISH) analysis. Metaphase chromosome spreads were probed with a human specific htt D N A derived from the 353G6 Y A C . Wild type (WT) mice did not show any signal by FISH analysis confirming the human specific nature of the htt probe used. In the heterozygotes, one signal is detected demonstrating that the H D Y A C transgene had inserted at only one site. In the homozygote two signals are detected (Fig.3.2B). As expected, transgene m R N A and protein levels in HD55+/+ demonstrate an approximate doubling of the mutant transgene expression level compared to HD55+/- and at levels similar to but just less than that seen in HD53 (Fig.3.2C and D).  3.2.3 Mutant huntingtin levels modulate onset and progression of the behavioural phenotype As previously demonstrated, the YAC128 mice (HD53) exhibit cognitive deficits including difficulties in changing strategies, delayed platform finding and impaired motor learning on rotarod beginning at 2 months of age (Van Raamsdonk et al., 2005b). Similar cognitive deficits have previously been shown in individuals affected with H D (HahnBarma et al., 1998;Snowden et al., 1998). In addition, the Y A C 128 mice (HD53) develop motor abnormalities which parallel those seen in the clinical course of H D (Slow et al., 2003). Specifically, the Y A C 128 mice exhibit a biphasic activity profile including hyperactivity at 2 months of age followed by the onset of a motor deficiency beginning after 3 months of age. Loss of motor function has been assessed both by a decrease in spontaneous ambulation in open field testing, as well as by decreased rotarod  64  performance, a measurement of motor co-ordination and balance. HD53 mice have motor co-ordination and balance deficits and fall off an accelerating rotating rod by 4 months of age. Furthermore, severity of rotarod dysfunction is highly correlated with striatal neuronal loss at later time points in the Y A C 128 model (Slow et al., 2003), suggesting that selective striatal neurodegeneration in Y A C 128 mice underlies their behavioural phenotype. The initial onset of rotarod deficits occur at a time when neuronal dysfunction has phenotypic effects, but prior to significant striatal cell loss. The YAC128 lines (HD53, HD55+/+, HD55+/-) and WT were assessed at 4, 6, 9 and 12 months of age for rotarod deficits. HD55+/- shows late onset of motor dysfunction, as defined by rotarod deficit, starting at 9 months of age (p<0.05, Fig. 3.3A). In contrast both HD53 and HD55+/+ demonstrate an earlier rotarod deficit clearly evident by 4 months of age (p<0.01 and p<0.01 respectively, A N O V A , Fig.3.3A). This result implies that increasing levels of mutant htt, as seen in lines HD53 and HD55+/+ (and as would be found in humans homozygous for the HD mutation), is associated with earlier onset of a motor dysfunction in the Y A C 128 mice. In order to evaluate disease progression in the Y A C 128 models, we assessed rotarod performance over time. At 4 months of age, A N O V A reveals a clear effect of mutant huntingtin levels on rotarod performance  (F 4n 2)  =6.713, p=0.0009). This effect  continued to increase with age as demonstrated by 6 months (F ,4o =6.46, p=0.001), 9 2  months (F , o =9.932, p=0.0001) and 12 months (F , o =14.66, p=0.0001) A N O V A 2 4  2 4  analysis (Fig.3.3A). Significantly, comparison of fall latency in the Y A C 128 heterozygote (HD55+/-) and homozygote (HD55+/+) mice at 6 months reveals a worsening performance on rotarod in line HD55+/+ compared to line HD55+/- (p=0.008, Fig.3.3A). Combining all data from 4 to 12 months reveals a clear effect of mutant htt levels on disease progression as defined by the slope of rotarod performance over time ( A N O V A p=0.002, HD55+/- p=0.07, HD55+/+ p=0.01 and HD53 p=0.005 compared against WT). Accelerated disease progression in HD53 compared against HD55+/- was evident, which approached statistical significance (p=0.06). Linear regression analysis of rotarod performance (12 months) and levels of mutant htt reveals a strong correlation between these outcomes (r =0.9813, p=0.009, Fig.3.3B). A similar correlation was 2  observed at 4 (r =0.9132, p=0.04) and 9 month (r =0.9580, p=0.002) time points (data 2  2  65  not shown). These data demonstrate that the level of mutant htt modulates progression of neuronal dysfunction in the Y A C 128 mouse models.We have previously demonstrated that the HD53 exhibit a biphasic activity profile with an initial hyperactive phase at 2 months which gradually progresses to a hypoactive phenotype by 6 months (Slow et al., 2003;Van Raamsdonk et al., 2005a). We therefore assessed open field activity at 12 months in the Y A C 128 mouse models to determine the influence of mutant htt expression on this phenotype. Similar to previous reports, the HD53 line demonstrated a hypokinetic phenotype at 12 months of age compared to wild type (p=0.04). The HD55+/+ revealed a similar deficit compared to wild type which approached statistical significance (p=0.06). In the HD55+/-, which contains lower levels of mutant htt, this trend is not apparent at 12 months of age (Fig.3.3C). It is clear from the above behavioral analysis in the Y A C 128 models that both age of onset and disease progression, as defined by motor deficit and hypokinetic phenotype, is modulated by levels of mutant htt.  66  A  Rotarod  1 2  Age (months)  4 months  6 months  02  0.4  06  OS  transgene protein level  1.Q  12 months  Open field activity  Rotarod/mhtt levels at 12 months  00  9 months  W T  H055W-  HOS5-./.  12 months  Fig. 3.3 Motor dysfunction correlates with levels of mutant huntingtin in Y A C 1 2 8 mouse models. (A) Assessment of rotarod performance, using an accelerating rotarod, in WT, HD55+/-, HD55+/+ and HD53 mice (n=10) demonstrate onset of motor dysfunction in HD55+/+ (p<0.01) and HD53 (p<0.01) at 4 months. The lower expressing line, HD55+/-, demonstrates onset at 9 months (p<0.05) ( A N O V A ) . Rotarod performance continues to deteriorate in HD55+/+ and HD53 at 6 (p=0.001) and 9 (p=0.0001) and 12 (p=0.000T) months of age compared against WT ( A N O V A ) . HD55+/- demonstrates motor dysfunction at 9 (p<0.05) and 12 months (p<0.01) of age ( A N O V A ) . Motor dysfunction in HD55+/- compared against HD55+/+ is significantly different at 6 months of age (p=0.008). (B) Linear regression analysis of rotarod performance at 12 months and levels of mutant htt reveal a strong correlation between these outcomes (1^=0.9813, p=0.009). (C) The hypokinetic phenotype in the Y A C 128 mouse models is influenced by levels of mutant huntingtin. In contrast to HD55+/-, HD53 mice have decreased activity, as defined by total ambulatory and fine movements, compared against W T at 12 months (p<0.05). Time on rotarod(s) and activity (beam breaks and crosses) is the mean value ±SEM. Significant differences are represented by asterisks or P value given.*p<0.05; **p<0.01; ***p<0.001.  3.2.4 The degree of neurodegeneration is influenced by levels of mutant huntingtin  In order to examine the effect of altered mutant htt levels on the selective neuropathological phenotype, we assessed striatal volume in HD53, HD55+/+, HD55+/and HD54 mice at 12 months (n=7). We have previously defined striatal volume loss as our most robust morphologic endpoint in the HD53 line (Slow et al., 2003).  67  Analysis of striatal volume at 12 months in the various Y A C 128 mouse lines revealed a clear correlation between levels of mutant htt and onset and severity of selective striatal volume loss. Our lowest expressing line, H D 5 4 , does not demonstrate striatal volume loss at 12 months ( H D 5 4 = 1 2 . 6 ± 0 . 6 7  Fig. 3.4 Degree of neurodegeneration is influenced by levels of mutant huntingtin  (A) Striatal volume at 12 months in W T , HD54, HD55+/-, HD55+/+ and HD53 mice (n=7). Perfused brains were cut coronally into 25u.m sections throughout the striatum. Every 8th section was immunostained with NeuN and Steroinvestigator software used to calculate total striatal volume across the brain. The lowest expressing line, HD54, does not demonstrate striatal atrophy at 12 months (HD54=12.6±0.67 mm vs. WT=13.0±0.66mm , p= 0.28). This is in contrast to HD55+/-, HD55+/+ and HD53 which all show significant striatal volume loss at 12 months compared to W T (HD55+/—12.1 ±0.66mm , HD55+/+ =11.8±0.55mm\ HD53=11.3±0.67 mm , A N O V A p=0.0002). Furthermore, comparison of striatal volume in HD54 line compared to HD55+/+ and HD53 is significantly different (p=0.03 and p=0.008 respectively). Striatal volume in HD55+/- vs. HD53 is also significantly different (p=0.04). (B) Linear regression analysis of striatal volume at 12 months and levels of mutant huntingtin reveal a correlation between these variables (r^O.9037, p=0.01). (C) HD54, HD55+/- and HD53 all show significant striatal volume loss at 18 months ( A N O V A p=0.0001). Furthermore there is a significant difference in striatal volume between HD54 and HD53 (HD54=12.0±0.65mm , HD53=10.9±0.40mm ,p=0.01). The difference in striatal volume between HD55+/- and HD53 approached statistical significance (HD55+/-=11.5±0.51mm , HD53=10.9±0.40mm , p=0.06). (D) Linear regression analysis revealed a strong correlation between striatal volume loss and levels of mutant huntingtin at 18 months (r=0.9898, p=0.005). Mean striatal measurements are given ±SD. Significant differences are represented by asterisks or P value given.*p<0.05; **p<0.01; ***p<0.001. 3  3  3  3  3  3  3  68  mm vs. WT=13.0±0.66mm , p= 0.28, Fig.3.4A). This is in contrast to HD55+/-, 3  3  HD55+/+ and HD53 which all show significant striatal volume loss at this time point compared to WT (ANOVA p=0.0002, Fig.3.4A) with severity corresponding with the level of mutant htt. Furthermore, comparison of striatal volume in the lower expressing HD54 line compared to HD55 +/+ and HD53 is significantly different (p=0.03 and p=0.008 respectively). Striatal volume in HD55+/- vs. HD53 is also significantly different (p=0.04) at 12 months. Linear regression analysis reveals a significant correlation between levels of mutant htt and striatal volume at 12 (r =0.9037, p=0.01 Fig. 4B) and 18 months (r =0.9898, p=0.005, Fig.3.4D). 2  In all instances, there was a clear gradient between levels of mutant htt and severity of striatal volume loss (Fig.3.4B and D). Furthermore, disease progression, as defined by striatal volume loss over time is significantly different in HD54 (0.72± 0.07) and HD55+/- (0.84±0.17) compared to HD53 (2.21± 0.35), (p=0.005 and p=0.04 respectively, Table 3.1). The results of the neuropathological assessment in the YAC 128 models reveal a clear relationship between levels of mutant htt and disease severity, marked by loss of striatal volume, and disease progression, indicated by striatal volume loss over time. This data implies that levels of mutant htt directly affect onset and rate of neurodegeneration in the YAC 128 mouse models.  Y A C line  Percent striatal  Slope of  slope  volume loss  striatal  vs. HD53  volume ;  12mo  18mo  loss/time  p  3.8  9.1  0:72  0.005  6.9  12.9  0.84  0.04  HD55+/+  9.2  ND  ND  ND  HD53  131  17.4  2.21  HD54  HD55+/-  Table 3.1 Disease progression of the neurological phenotype is influenced by levels of mutant huntingtin.  69  3.2.5 Degree of nuclear translocation of huntingtin and inclusion formation increases with increasing levels of mutant huntingtin Relocation of htt to the nucleus and formation of inclusions are a hallmark of the human disease and are present in the brains of human patients (DiFiglia et al., 1997;Gutekunst et al., 1999) and in H D mouse models (Li et al., 1999;Slow et al., 2003). In order to investigate whether levels of mutant htt influence nuclear translocation of htt and inclusion formation, brain sections from HD53, 54 and 55 were immunoassayed with the EM48 antibody, an antibody that recognizes N-terminal htt and is highly specific for inclusions (Gutekunst et al., 1999). Inclusion formation was assessed at the light microscope level. At 18 months, HD53 demonstrates nuclear localization of mutant htt by EM48 staining in the striatum (Fig.3.5G), and predominantly in cortical layers II/III, IV and V I (Fig.3.5H). HD55+/+ demonstrated a similar pattern of EM48 staining at slightly reduced levels compared to HD53 (Fig.3.5E and F). HD55+/- demonstrates nuclear localization of mutant htt in the striatum (Fig.3.5C), at lower levels than HD55+/+ and HD53, and with just detectable levels in the cortex (Fig.3.5D). In contrast, HD54 demonstrates nuclear localization of mutant htt selectively in the striatum (Fig. 3.5 A), albeit at lower levels than HD55+/- at the same time point, and not in the cortex (Fig.3.5B). Htt nuclear staining is first present in the striatum of HD53 and HD55+/+ prior to 6 months of age (data not shown). This selective nuclear localization of mutant htt is delayed in the HD55+/- and HD54 lines, occurring at 9 months and after 12 months respectively (data not shown). Analysis of inclusion levels in HD53 demonstrates that 34% of striatal neurons and 10% of cortical neurons contain htt inclusions at 18 months (Fig.3.5G and H). A slightly reduced level of inclusions are observed in HD55+/+ with 21% of striatal neurons and 7% of cortical neurons containing htt inclusions at the same time point (Fig. 5E and F). In contrast HD54 and HD55 +/- do not show neuronal inclusions at 18 months despite showing significant striatal volume loss (Fig.3.5A and C) (Striatal inclusions A N O V A pO.0001, cortical inclusions A N O V A pO.OOOl). The results of this study reveal that mutant htt has a dose-dependent effect on the time course, frequency of htt inclusions and nuclear localization of htt.  70  HD54  HD55+/-  HD55+/+  HD53  Fig. 3.5 Accelerated nuclear translocation of huntingtin and inclusions formation with increasing levels of mutant huntingtin. E M 4 8 immunostaining, assessed by light level microscope, at 18 months i n the striatum and cortex o f H D 5 4 ( A , B ) , H D 5 5 + / - ( C , D ) HD55+/+ ( E , F) and H D 5 3 ( G , H ) . A t 18 months H D 5 3 and HD55+/+ show clear diffuse nuclear htt staining i n the striatum ( E , G ) and cortex (layer V I , F , H ) w i t h ~ 3 0 % and 2 1 % o f striatal neurons containing inclusions respectively. In contrast H D 5 5 + / - shows clear diffuse nuclear htt staining i n the striatum ( C ) w i t h just detectable levels present i n the cortex (layer V I , D ) . H D 5 4 ( A , B ) demonstrates selective nuclear htt staining only i n the striatum, w i t h a reduced level vs. H D 5 5 + / - . Inclusions are not observed i n H D 5 4 and H D 5 5 + / - at 18 months. Scale bars A - H , 10u.m.  3.2.6 Levels of mutant huntingtin influence NMDAR-mediated excitotoxicty Previous studies have suggested that excitotoxicity is an important mechanism leading to neuronal death in H D ( A l b i n et al., 1990;DiFiglia, 1990;Beal et al., 1991;Beal, 1992;Young, 1997). W e have previously demonstrated that primary M S N s isolated from Y A C 7 2 striata at birth were more vulnerable to NR2B-type N M D A R - m e d i a t e d cell death and displayed increased in vivo susceptibility to quinolinic acid-mediated neurotoxicity compared to wild-type neurons (Zeron et al., 2002). W e therefore wanted to determine whether the relationship between htt levels and onset and progression o f illness, and the relationship to striatal pathology, were also reflected i n changes in excitotoxicity. W e chose to analyze neurons from HD55+/- and HD55+/+ as they were identical to each other in every respect except for levels o f mutant htt. M S N s from P0 W T , HD55+/- and HD55+/+ were cultured and analyzed at 9-10 D I V for susceptibility to N M D A R - m e d i a t e d excitotoxicity as assessed by morphological criteria and T U N E L (terminal deoxynucleotidyl transferase-mediated d U T P fluorescein nick end-label) analysis (n=3 separate litters). Each experiment was done blind, i n triplicate and a minimum o f 1000 cells were counted per treatment. The percentage o f apoptotic cells in  71  control conditions (i.e. treatment with balanced salt solution) was not significantly different for WT, HD55+/- and HD55+/+ (3.8±1.4, 5.9±3.1 and 4.8±2.4 respectively, A N O V A p=0.68) and was subtracted from percentage of apoptotic cells after treatment with N M D A . Y A C 128 MSNs from both heterozygote and homozygotes demonstrated increased apoptotic cells vs. W T upon 500uM N M D A treatment (HD55+/+ p<0.001, HD55+/p<0.01, A N O V A , Fig.3.6A). Furthermore, increased apoptotic cell death was evident in HD55+/+ than HD55+/- upon exposure to 500pm N M D A (37.2±4.4% vs. 27.9±3.5%, p<0.05, A N O V A , Fig.3.6A). To confirm that the effect we observed was due to N M D A R activation we also treated the MSNs with MK801 (20uM), a N M D A R specific antagonist, in addition to 500pM N M D A . Under these conditions, cell death was reduced to virtually baseline conditions in all cultures. The results of the ex vivo excitotoxicity study demonstrate that increasing levels of mutant htt is paralleled by an increase of susceptibility to NMDAR-mediated cell death.  72  500Ljm  [NMDA]  Control  B pi  TUNEL  I I  WT  r~\ •  HD55+/HD55-/+  500um+ MK801 500pm NMDA PI  TUNEL  WT  HD55+/-  Fig. 3.6 Levels of mutant huntingtin influence NMDAR-mediated excitotoxicity (A) M S N s from H D 5 5 + / - and H D 5 5 + / + demonstrate increased cell death upon N M D A (500jim) application vs. W T ( A N O V A , p<0.0001). Furthermore, increased apoptotic cells are evident in H D 5 5 + / + vs. H D 5 5 + / - upon exposure to N M D A (p<0.05). M S N ' s from H D 5 5 + / - and H D 5 5 + / + pretreated with M K 8 0 1 (20nm), a N M D A R specific antagonist, do not show significant cell death compared against W T . Apoptotic neurons were assessed using T U N E L staining and morphological criteria by propidium iodide (PI) counterstaining. M e a n percent T U N E L positive cells are given ± S D . Significant differences are represented by asterisks or P value given.*p<0.05; **p<0.01; ***p<0.001. (B) Representative fields o f M S N s are shown 24hr after exposure to balanced salt solution (control) or N M D A followed by T U N E L staining and PI counterstaining.  3.3 D i s c u s s i o n  We have demonstrated that onset and progression of HD is directly modulated by levels of mutant htt in different Y A C 128 mouse models. Using previously validated  73  phenotypic endpoints (Slow et al., 2003;Van Raamsdonk et al., 2005a), we demonstrate earlier age of onset and exacerbated disease progression, as defined by motor deficit and striatal volume loss, in mice expressing higher levels of mutant htt. In addition, levels of mutant htt directly influence nuclear translocation of htt and susceptibility to N M D A R mediated cell death. These data demonstrate that both onset and disease progression of H D are modulated by levels of mutant htt providing evidence for a cumulative model for H D and show that H D displays an intermediate dominant phenotype in the Y A C 128 mouse models. Intermediate dominance implies that the wild type allele could influence disease severity. The delayed age of onset in the heterozygote, clearly due to lower levels of mutant htt, may also be influenced by the presence of wild type htt. Wild type htt is neuroprotective in a number of stress induced model systems. (Rigamonti et al., 2000;Leavitt et al., 2001;Zhang et al., 2003). Furthermore, an interaction between the normal and mutant htt protein may mitigate the effect of mutant huntingtin and thus delay age of onset (Djousse et al., 2003). We have recently shown that levels of wild type htt may influence motor function and activity in the Y A C 128 mice (Van Raamsdonk et al., 2005a). Behavioural analysis of the Y A C 128 mouse models has demonstrated clearly that increasing levels of mutant htt leads to earlier onset of a behavioural phenotype. Prior studies on the Hdh knock in Q150 mice and H D c D N A mouse models also revealed earlier onset of all behavioural deficits tested in the homozygote vs. heterozygote lines (Lin et al., 2001) . This is in sharp contrast to reports comparing human H D heterozygotes and homozygote patients where no difference in age of onset, as defined by presence of chorea, has been observed (Wexler et al., 1987; Myers et al., 1989; Durr et al., 1999). The main reasons for these discrepancies may be due to the small number of individuals homozygous for the H D mutation and difficulty in defining onset. Furthermore, the variability in age of onset of heterozygote H D individuals for a given C A G size (25-52 yrs) (Brinkman et a l , 1997;Durr et al., 1999;Squitieri et al., 2000) may obscure an accelerated phenotype in the human H D homozygote. A more recent neuropathological analysis of human H D heterozygote and homozygote individuals with known C A G sizes did reveal increased rate of disease  74  progression in the H D homozygote (Squitieri et al., 2003). Neuroimaging results demonstrated more extensive and severe progressive brain atrophy in the homozygotes and analysis of HD stage (grade) and mean duration showed a shift to the left in the H D homozygotes compared to the heterozgote individuals (Squitieri et al., 2003). We have demonstrated that mutant htt influences age of onset and disease progression of both a behavioural phenotype and striatal volume loss. In order to obtain earlier endpoints, we extended our previous analysis to include nuclear htt assessment and susceptibility to N M D A R - mediated cell death studies of excitotoxicity on cultured neurons from mice in the neonatal period. A clear relationship between levels of nuclear htt and severity of the H D phenotype as defined by motor deficit and striatal volume loss is also evident. There has been controversy over the relationship between subcellular location of htt and its toxicity (Dyer and McMurray, 2001 ;Kim et al., 2001). This data is consistent with nuclear translocation of htt being an important part of the pathogenesis of this illness. Dose dependent acceleration of nuclear htt has previously been described in the HD knock in mouse model and nuclear localization in this model is also associated with enhanced neuropathology (Wheeler et al., 2002). In addition, the HdhQl 11 knock-in mice crossed to the N171-82Q line, demonstrated that the presence of nuclear mutant htt fragments accelerated early pathological changes (Wheeler et al., 2002). Timing of nuclear relocation of htt corresponds with onset of motor deficits in the Y A C 128 lines. Specifically we observe translocation of htt and onset of a motor deficit by 4 months of age in HD53 and 9 months of age in HD55+/-. This result suggests that proteolysis of htt is necessary before nuclear translocation to occur and that generation of a cleaved htt fragment may significantly contribute to the neuronal dysfunction observed in the Y A C 128 lines. Proteolytic cleavage of htt by caspases with concomitant generation of htt fragments has been well characterized in vitro (Wellington et al., 1998; Wellington et al., 2000;Kim et al., 2001). Numerous studies have highlighted excitotoxicity as playing an important role in the pathogenesis of HD (Albin et al., 1990;DiFiglia, 1990;Beal et al., 1991;Beal, 1992;Young, 1997;Zeron et al., 2002;2004). Medium spiny neurons of the neostriatum receive glutamatergic input from most cortical regions, and over activation of N M D A  75  receptors is likely to play a role in the death of these neurons. Importantly, expression of the N M D A receptor subunit NR2B relative to other NR2 subunits is increased in striatal MSNs (Landwehrmeyer et al., 1995) and results of radiolabeled ligand binding assays demonstrate decreased binding of N M D A R in presymtomatic human HD striatum suggesting that neuronal cells which express these glutamate receptors are particularly vulnerable (Young et al., 1988;Albin et al., 1990). Furthermore, significantly increased NMDAR-mediated current has been found in HD mouse models (Hodgson et al., 1999;Levine et al., 1999;Cepeda et al., 2001a;Zeron et al., 2002;2004) and enhanced excitotoxicity following glutamate stimulation have been demonstrated in MSNs from Y A C 128 (HD53) (Tang et al., 2005). The evidence for enhanced excitoxocity in the Y A C 128 lines and its clear modulation by levels of mutant htt reveals that these are indeed very early changes, occurring significantly before motor deficit or other signs of neuronal dysfunction. The relationship between increasing levels of mutant htt and enhanced vulnerability to excitotoxic stress suggests that excitotoxicity may be a cumulative underlying primary mechanism of striatal atrophy which terminates in apoptotic cell death. These observations strongly support a role for expanded htt in augmenting an NR2B-dependent pathway that leads to calcium-induced cellular dysfunction and ultimately neuronal cell death. The present study has demonstrated in vivo that age of onset and disease progression of the HD phenotype in the Y A C 128 mouse models is modulated by levels of mutant htt. As the HD gene is expressed throughout the life of the individual, this would support a model for cumulative, toxic gain of function underlying HD. This would argue that the disease initiating mechanism in HD commences very early and would indicate that early intervention in the presymptomatic stage may be necessary to prevent the cumulative gain of toxic function. This also predicts that homozygotes for HD would have a more severe phenotype, and emphasizes the need for additional clinical trials to clearly establish whether H D in humans demonstrates an intermediate dominant phenotype.  76  Chapter 4: Cleavage at the caspase-6 site in huntingtin is required for motor dysfunction, neurodegeneration and excitotoxicity in Huntington Disease  Rona K. Graham, Y u Deng, Elisabeth J. Slow, Nagat Bissada, Ge Lu, Jacqueline Pearson, Lisa Bertram, Zoe Murphy, Jacqueline Shehadeh, Simon C. Warby, Sophie Roy, Cheryl L . Wellington, Blair R. Leavitt, Lynn A . Raymond, Donald W. Nicholson and Michael R. Hayden  The work is this chapter has been presented as:  Rona K Graham, Y u Deng, Elizabeth Slow, Brendan Haigh, Nagat Bissada, Ge Lu, Rosemary Oh, Lisa Bertram, Kuljeet Vaid, Sophie Roy, Donald W Nicholson, Blair R Leavitt, Cheryl L Wellington and Michael R Hayden (2004). In vivo inhibition of caspase-6 cleavage of expanded huntingtin protects against neurodegeneration. (August 12-14, 2004: Boston). Hereditary Disease Foundation General Meeting, Poster and Oral presentation. Rona K Graham, Y u Deng, Elizabeth Slow, Brendan Haigh, Nagat Bissada, Ge Lu, Rosemary Oh, Lisa Bertram, Kuljeet Vaid, Sophie Roy, Donald W Nicholson, Blair R Leavitt, Cheryl L Wellington and Michael R Hayden (2004). In vivo inhibition of caspase-6 cleavage of expanded huntingtin protects against neurodegeneration. (November 15-21, 2004: San Diego). Neuroscience Annual Meeting, Oral presentation.  A version of this chapter has been submitted for publication: Rona K. Graham, Y u Deng, Elizabeth J. Slow, Nagat Bissada, Ge Lu, Jacqueline Pearson, Lisa Bertram, Jacqueline Shehadeh, Simon C. Warby, Sophie Roy, Cheryl L . Wellington, Blair R. Leavitt, Lynn A . Raymond, Donald W. Nicholson and Michael R. Hayden. Cleavage at the caspase-6 site in huntingtin is required for motor dysfunction, neurodegeneration and excitotoxicity in Huntington Disease. Cell. Resubmitted Jan 29 2006.  77  Preface I designed all of the experiments and analyses presented within this chapter and performed all with the exception of that noted below. Deborah Deng did the Y A C mutagenesis for each of the caspase resistant constructs. Rosemary Oh performed the Southern blot and provided assistance with the PCR on the C R Y A C constructs. The C M M T transgenic unit microinjected the purified C R Y A C constructs into F V B pronuclei. Deborah Deng performed RT-PCR for D N A and m R N A transgene levels in the H D and C R mice, and the 12 month striatal volume measurements and neuronal counts for C6R7. She also determined the lesion volume in the QA injected mice. Jacqueline Pearson performed the behavioral analysis on the mice. L i l i Wang provided technical support for the culture of primary medium spiny neurons (MSN). Jacqueline Shehadeh performed the staurosporine experiment in the M S N cultures. Nagat Bissada and Lisa Bertram assisted with the colony management and performed, with the help of Ge Lu, the mouse perfusions. Ge L u also performed the QA intrastriatal injections. Zoe Murphy helped with the caspase immunohistochemical study. Dr X . J . L i kindly provided the EM48 antibody. Dr. Blair Leavitt, Dr. Cheryl Wellington and Dr Lynn Raymond provided advice and expertise.  78  4.1 Introduction Huntington Disease (HD), a neurodegenerative disorder characterized by progressive deterioration of cognitive and motor functions, is caused by an expansion of a trinucleotide (CAG) repeat encoding glutamine in the N-terminus of huntingtin (htt). Polyglutamine expansion results in selective loss of GABAnergic medium spiny striatal neurons as well as glutamatergic cortical neurons that project to the striatum (Vonsattel et al., 1985; Albin et al., 1990). A neuropathogical hallmark in human H D and mouse models is the intracellular accumulation of N-terminal htt fragments (Gutekunst et al., 1999; K i m et al., 2001; Wellington et al., 2002), suggesting that aberrant htt proteolysis and/or dysfunctional clearance of htt fragments may underlie the neuropathology in HD. It is well established that several proteases, including caspases, calpains and aspartyl endopeptidases, cleave htt within the N-terminal region (Wellington et al., 1998; Wellington et al., 2000; Lunkes et al., 2002; Garni et al., 2004). Numerous in vitro studies have demonstrated that expanded N-terminal htt fragments have enhanced cytotoxicity (Hackam et al., 1998; Cooper et al., 1998; Jana et al., 2001) and in vitro caspase activation in H D cell culture models is dependent upon the protein context of the htt fragments (Yu et al., 2003). Furthermore, although some transgenic H D mouse models expressing truncated mutant htt (mhtt) demonstrate behavioral deficits and neuronal inclusions (Mangiarini et al., 1996; Davies et al., 1997; Schilling et al., 1999), in vivo expression of an expanded N-terminal htt fragment containing exon 1 and 2 does not result in a behavioral or neurodegenerative phenotype (Slow et al, 2005), demonstrating that N-terminal htt fragments per se do not invariably result in pathology. In addition, while several studies have shown translocation of htt to the nucleus to be associated with increased toxicity in vitro and neuropathology in vivo (DiFiglia et al., 1997; Wheeler et al., 2000; K i m et al., 2001; Wellington et al., 2002; Slow et al., 2003), the shortstop mouse exhibits widespread diffuse nuclear htt but no neurotoxicity, suggesting either that N-terminal fragments containing the mutation alone are insufficient to cause the disease or that a specific nuclear htt fragment underlies the neuronal degeneration observed in HD. In the Y A C 128 model, which expresses full length mutant htt, nuclear localization of htt fragments is earliest and to the greatest extent in the striatum, correlating with the  79  neuropathology observed in the Y A C model and the primary area of neurodegeneration in H D patients (Van Raamsdonk et al., submitted). These data argue that specific mhtt fragments are required to initiate a toxic cycle that leads to neuronal dysfunction, behavioral and cognitive deficits, and the neuropathological abnormalities present in H D . Htt is cleaved by caspase-3 and -6 in vitro (Goldberg et al., 1996; Wellington et al., 2000), and caspase-3-cleaved htt fragments are detectable in H D brain prior to the clinical onset of H D (Wellington et al., 2002; K i m et al.,2001). In addition, site-directed mutagenesis of all five known or potential caspase cleavage sites in mhtt blocks cytoxicity in vitro (Wellington et al., 2000). Several other polyglutamine containing proteins are also substrates for caspases, including atrophin-1, the androgen receptor and ataxin 3 (Wellington et al.,1998) and inhibiting caspase cleavage of the mutant forms of the androgen receptor and atrophin-1 also reduces cytoxicity in vitro (Ellerby et al., 1999a; 1999b). These observations raise the question of whether the caspase specific fragments are important in the pathogenesis of H D and inhibiting caspase cleavage of mhtt may be protective in vivo. We have previously developed an animal model for H D using the 350 kb Y A C 353G6, which contains all 67 exons of the human H D gene and its endogenous regulatory sequences (Hodgson et al., 1999). Mice harbouring Y A C 353G6 faithfully express fulllength human mhtt in a tissue-specific and developmentally appropriate manner (Hodgson et al., 1996; 1999), and display age and CAG-dependent phenotypes that mimic the human disease. Specifically, enhanced susceptibility to excitotoxic stress, cleavage and nuclear localization of htt, and cognitive dysfunction are followed by motor deficits and selective striatal degeneration (Wellington et al., 2002; Slow et al., 2003; Van Raamsdonk et al., 2005b; Tang et al., 2005; Graham et al, in press), indicating that the Y A C 128 model accurately replicates human H D in vivo and is therefore an ideal model to assess interventions on disease phenotype. To determine i f caspase cleavage of mhtt represents a primary event in the pathogenesis of H D in vivo, we generated Y A C transgenic mice expressing expanded htt containing selective mutations of the caspase-3 and caspase-6 cleavages sites. The resulting lines of caspase resistant htt (CR) mice (CQR -resistant to caspase-3 and caspase-6, C3R - resistant to caspase-3, C6R - resistant to caspase-6) were compared to  80  Y A C 128 mice expressing caspase-cleavable mhtt using previously validated endpoints previously (Slow et al., 2003; Graham et al, in press). Here we report that cleavage at the caspase-6 site in htt is required for the hallmark behavioral and neuropathological features of H D to be present. Selective elimination of the caspase-6, but not caspase-3, cleavage site in mhtt is sufficient to provide protection from neuronal dysfunction and neurodegeneration in vivo. These data argue that specific mhtt fragments are required to initiate a toxic cycle that leads to neuronal dysfunction, behavioral and cognitive deficits, and the neuropathological abnormalities present in HD. Additionally, primary striatal neurons derived from C6R mice are resistant to NMDAR-mediated excitotoxicity and staurosporine-induced cell death in vitro and quinolinic-acid (QA) induced neurotoxicity in vivo. Our results demonstrate that in vivo proteolytic cleavage of mhtt by caspase-6 is a crucial and rate limiting event in the pathogenesis of H D and suggests that preventing caspase-6 cleavage of htt may be of therapeutic interest for this disease.  4.2 Results  4.2.1 Generation of Caspase Resistant Lines The caspase sites in the human htt gene.are located in exons 12 and 13. Exon 12 encodes htt amino acid 470 to 583 and contains two caspase-3 sites at amino acid 513 (  510  DSVD  513  ) and 5 52 (  (  527  DEED  530  ) (Wellington et al., 1998). Exon 13 encodes htt amino acid 584 to 624 and  549  DLND  552  ) , as well as one silent caspase-3 site at position 530  contains the caspase-6 site at position 5 86 ( 3 site at position 5 89 (  586  DGTD  589  583  IVLD  586  ) as well as another silent caspase-  ) (Wellington et al., 2000). Therefore, specific  mutations of the PI aspartate at positions 513, 530 and 552 would be expected to generate caspase-3-resistant htt, and mutations at residues 586 and 589 would be expected to generate caspase-6-resistant htt, as the PI site is the major specificity determinant for caspase binding (Nicholson, 1999). To ensure that the silent caspase-3 sites would be unable to be cryptically activated in vivo, we elected to mutagenize their  81  P l aspartate residues also. The successful site-directed replacement of the P l aspartate with alanine in each position (caspase-3: D513A, D530A, D552A and caspase-6: D586A and D589A) was confirmed by D N A sequencing (Wellington et al., 2000). Homologous recombination in yeast was used to introduce the mutagenized exons into YAC353G6 (Fig 4.1). Maintenance of Y A C integrity following mutagenesis was confirmed by Southern blot (data not shown) and by P C R amplification of 5 regions spanning the Y A C (data not shown). These C R Y A C s are the genomic equivalent of our previously described htt c D N A C R mutants (Wellington et al., 2000). Verified C R Y A C s were purified by pulse-field electrophoresis and microinjected into F V B / N pronuclei as described previously (Hodgson et al., 1999). Founders were confirmed by a 5-point P C R screen (Fig 4.2) as well as by sequence confirmation (data not shown). Using this strategy, we generated two independent transgenic mouse lines expressing human mhtt resistant to both caspase-3 and -6, known as caspase cmint resistant (CQR) lines 24 (CQR24) and 26 (CQR26). We also developed two independent lines selectively resistant to caspase-3, known as C3R8 and C3R9, and two independent lines resistant to caspase-6, known as C6R7 and C6R13.  82  A  Popin: Exon 12 S S C P W M r *  B  M W W M  ^A *****  * ^ A* *  Popout: Exon 12 S S C P  ** * ***  WM  IILlilllllllillll IHIiiiWI! C  D  A±C  E Popin: Exon 13 BsrF1 digest F Popout: Exon 13 BsrF1 digest  F i g . 4.1 Y A C mutagenesis. S S C P a n d sequence analysis o f p o p - i n a n d p o p - o u t clones for exon 12 a n d e x o n 13 mutations. ( A and B ) S S C P analysis for pop-in ( A ) and pop-out ( B ) candidate clones for exon 12. Control S S C P reactions using known wild-type ( W ) or mutant ( M ) P C R products are designated b y arrows. Positive clones are indicated with an asterisk (*) and contain both wild-type and mutant S S C P patterns for the p o p - i n clones ( A ) and only the mutant pattern for the pop-out clones (B). ( C ) shows the presence o f both wild-type and mutant nucleotides in a representative pop-in clone, and ( D ) shows that only the mutant sequence is retained after the pop-out stage. ( E and F) P C R restriction analysis using B s r F l for p o p - i n ( E ) and pop-out (F) candidate clones for exon 13. Control digest reactions using known w i l d type ( W ) or mutant ( M ) P C R products are designated b y arrows. Positive clones are indicated with an asterisk (*) and contain both w i l d type and mutant restriction patterns for the pop-in clones ( E ) and only the mutant pattern for the pop-out clones (F). (G) shows the presence o f both w i l d type and mutant nucleotides in a representative pop-in clones, and ( H ) shows that only the mutant sequence is retained after the pop-out stage.  83  WT YAC 128  CQR  53 54 26 24  C3R C6R 8  9  7 13  —  ^TLYA ~^RYA mm  1 -•  C A repeat  AG  Fig. 4.2 Identification and assessment of Y A C integrity in Caspase Resistant ( C R ) founder lines. DNA  was extracted from mouse tails for P C R analysis o f 5 regions spanning the Y A C (described  previously, H o d g s o n et al, 1999) including C A G (result shown in F i g 1 A ) , R Y A , L Y A , C A repeat and delta G . A l l C R founders demonstrate integration o f the entire Y A C as shown by the presence o f a P C R product for each reaction in all lines.  4.2.2 Characterization of C A G size and transgene expression levels in CR and HD mice The phenotype of HD in the Y A C 128 mice is significantly influenced by levels of expression of mhtt (Graham et al, in press). Therefore it is essential to control not only for C A G size but also levels of transgene expression to obtain an accurate comparison between caspase-cleavable and C R htt. We have previously published the phenotype in the high expressing YAC128 mouse model (HD53) (Slow et al., 2003; Tang et al., 2005; Van Raamsdonk et al., 2005a,b). We have furthermore characterized two additional Y A C 128 lines, HD54 and HD55 which contain intermediate and lower levels of mhtt respectively, and shown that these lines demonstrate behavioral and neuropathological changes consistent with an H D phenotype with onset and progression of the phenotype directly influenced by levels of mhtt (Graham et al, in press). PCR-mediated C A G sizing was first used to ensure equivalent C A G sizes among caspase cleavable and C R lines (Fig. 4.3A). Lines HD53, HD54, CQR24, C3R8 and  C3R9 all containl20 C A G repeats. Lines C6R7 and C6R13 each have 133 CAGs, and CQR26 contains two C A G sizes of 120 and 83 CAGs respectively. Thus, all of the C R lines have C A G sizes at least as large as the control Y A C lines to which they were compared. Quantitative mRNA and western blotting was then used to match transgene expression in each CR murine brain tissue to the most appropriate caspase cleavable Y A C 128 line. Htt expression levels in CQR24 and CQR26 (upper allele) were found to be equivalent to HD54 (Fig.4.3B and C). The CQR26 mice display two distinct protein sizes on Western blots (Fig4.3C), which were accounted by a spontaneous contraction of the glutamine tract in one copy of the Y A C in CQR26 animals. Htt expression analysis in C3R8 and C3R9 demonstrate that both of these lines also express human mhtt at levels equivalent to HD54 (Fig.4.3B and C) whereas transgenic mhtt levels in C6R7 and C6R13 mice are similar to the previously described HD53 mice (Fig.4.3B and C). These experiments defined the control line to which each C R line should be compared as HD54 for the C Q R and C3R lines and HD53 for the C6R lines.  85  WT  HD YAC  CQR C3R  C6R  18 46 72 53 54 26 24 8 9 7 13  C3R  CQR 1J*i  mRNA  C6R  I.*-,  l u l l i l t 111 HD54  Protein J o.»  I DM  CQR24  CQR26  i.o-  I  ...  //  HDM  C3W  CJ'.J  1.0.  HMJ  I / /  C«KT  C«H13  / 'Z  l u l ::Wmm :illll HD54  COR24  CQR!«  CJRfl  C3R9  H053  CSR7  C6R13  Fig. 4.3. Characterization of Caspase Resistant and control Y A C lines. (A) Agarose gel showing PCR  for C A G tract of transgenic human htt in Y A C 18, 46, 72 and 128 ( HD53 and 54) lines ( described previously, Hodgson et al., 1996, Graham et al, in press) and CQR24, CQR26, C3R8, C3R9, C6R7 and C6R13. wt is included as negative control. Line HD53, HD54, CQR24, C3R8 and C3R9 all display a 120CAG repeat size. Line C6R7 and C6R13 have 133 C A G repeats and CQR26 contains two C A G sizes of 120 and 83 respectively. (B) Real-time quantitative RT-PCR showing mRNA transgene expression levels in C R murine brain compared against Y A C 128 lines. Transgenic htt levels have been normalized to actin. Mhtt levels in CQR24 are equivalent to HD54. CQR26 demonstrates -doubling of mhtt levels due to contribution from two alleles (left panel). Transgene levels in C3R8 and C3R9 are equivalent to HD54 (middle panel). Mhtt expression levels in C6R7 and C6R13 are similar to HD53 (right panel). (C) High resolution Western blot of CR and Y A C 128 brain tissue probed with BKP1 htt antibody (upper panel). Mhtt level of upper allele in CQR26 (120CAG) is similar to HD54.Quantification of mhtt protein levels in CR and Y A C 128 murine brain (lower panels) correlates with mRNA transgenic levels. Transgene protein levels have been normalized to endogenous htt levels.  4.2.3 Caspase resistant htt expressed in CR mice is resistant to caspase proteolysis To confirm that the transgenic htt expressed in CR mice is resistant to caspase cleavage, whole brain lysates were digested with recombinant caspases. Western blot  86  analysis shows that caspase-3 and -6 do not cleave transgenic htt in CQR24 or CQR26 lysates (Fig.4.4A). These results confirm that the mutations introduced into CQR mhtt block cleavage of transgenic htt by caspases-3 and -6. In contrast, HD54 lysates give rise to the expected htt caspase fragments (70kDa for endogenous and 98kDa for mhtt) (Fig.4.4A). Only the caspase-3 fragments of htt are observed as the htt fragments generated by caspase-6 are further digested by caspase-3. Western blotting, using the previously described neoepitope antibody to the caspase-3-derived htt 552aa fragment (Wellington et al., 2002), demonstrates that recombinant caspase-3 cleaves only endogenous murine htt (70kDa) in lysates prepared from C3R8 and C3R9 mice (Fig.4.4B). In contrast, caspase-3 treatment of lysate prepared from HD54 brain generated both the 70kDa fragment from endogenous htt and the 98kDa fragment from mhtt. Incubation of C3R lysate with caspase-6 generated the expected caspase-6 fragments from endogenous and mhtt, demonstrating that C3R mhtt is only resistant to cleavage by caspase-3 (Fig. 4.4D). Conversely treatment of lysate from HD53 with caspase-6 generated the expected caspase-6 fragments from transgenic (115kDa) and endogenous murine htt (90kDa) (Fig.4.4C). As expected, only endogenous htt is cleaved by caspase-6 in lysates from C6R7 and C6R13 mice, resulting in a 90kDa htt fragment (Fig.4.4C). Incubation of C6R lysate with recombinant caspase-3 results in caspase-3 murine and mhtt fragments, demonstrating that C6R mhtt is selectively resistant to cleavage by caspase-6 (Fig. 4.4E).  87  Fig. 4.4 Caspase resistant huntingtin expressed in the transgenic mice is resistant to specific caspase cleavage. ( A ) W h o l e brain lysates were subject to digestion with the respective caspase. Western blot analysis demonstrate that caspase-3 and-6 only cleave mhtt (98 k D a fragment) in H D 5 4 (probed with B K P 1 antibody), the 7 0 k D a band (present in H D 5 4 , C Q R 2 4 and C Q R 2 6 ) represents endogenous htt (left panel). (B) Caspase-3 cleaves mhtt only in H D 5 4 (middle panel, probed with htt552 neoepitope antibody). (C) Caspase-6 cleaves mhtt selectively in H D 5 3 , and not C 6 R 7 and C 6 R 1 3 , murine brain resulting in 115kDa fragment, 9 0 k D a band represents cleaved endogenous htt (right panel, probed with B K P 1 antibody). (D) Western blot analysis demonstrates caspase-6 cleaves mutant htt (115 k D a fragment) and endogenous (90kDa) in C 3 R transgenic mice (probed with B K P 1 antibody). (E) Conversely, caspase-3 cleaves mutant htt in C 6 R transgenic mice resulting i n 9 8 k D a fragment, 7 0 k D a band represents cleaved endogenous htt (probed with B K P 1 antibody).  4.2.4 Caspase resistant mutant htt retains developmental functions in vivo Transgenic htt expressed from a Y A C is capable of rescuing the embryonic lethal phenotype of htt-deficient animals, demonstrating that postnatal viability can be used as an indicator of preserved htt function during development (Hodgson, 1996; Van Raamsdonk et al., 2005a). To determine whether mutagenesis of htt disrupted this function, we tested the ability of CR Y A C lines to rescue the survival deficit of httdeficient mice. We observed that C R lines rescue the embryonic lethality of htt-knockout mice as efficiently as Y A C transgenic mice expressing caspase-cleavable htt demonstrating that mutagenesis of the caspase sites in mhtt do not impair htt function in vivo during development (Table 4.1). In Y A C mice expressing low levels of mhtt (HD54 and C3R9) only partial rescue is observed, consistent with previous observations that  88  developmental viability requires a threshold level of htt expression (White et al., 1997; Auerbach e t a l , 2001).  YAC  CAG  Line  K/O Strain  WT WT WT WT WT WT  18 46 72 128 128 128  29 668 2511 53 55  BI6/FVB BI6/FVB BI6/FVB FVB FVB  C3.6 C3 C6  128 128 128  CQR26 C3R9 C6R7  FVB FVB FVB  Genotype of offspring +/+ +/-/19 17 2Q 19 7 18  35 60 36 43 14  11 12 26  21 27 43  P  15 . 31 18 26 12 5  0.782 0.084 0.922 0.560 0.321 0.027  7 4 16  0.606 0.055 0.307  a  a  a L o w levels o f htt expression cause failure to rescue htt's embryonic lethality  Table 4.1. Caspase Resistant mutant huntingtin retains developmental functions in vivo F V B / N mice containing the Y A C 128 or C R Y A C and heterozygous for the murine H D gene were crossed to mice heterozygous for the murine H D gene. Progeny from these matings were screened for the presence o f the Y A C and murine H d h allele and genotype frequencies entered into G r a p h p a d Prism 4.0. for c h i square analysis. C R lines ( C Q R 2 6 , C 3 R 9 , and C 6 R 7 ) rescue the embryonic lethality o f htt-deficient mice with equal efficiency as Y A C 128 ( H D 5 3 , 54 and 55). Data for Y A C 18, 46, 72 and 128 ( H D 5 3 ) were described previously ( H o d g s o n et al., 1996, V a n Raamsdonk et al, 2005). T h e distribution o f the observed genotypes were compared to expected Mendelian distribution and p value given.  4.2.5 Inhibition of caspase cleavage of huntingtin prevents neurodegeneration in vivo We next examined neuropathology in the C R mice using validated endpoints previously defined in the YAC128 models (Slow et al., 2003; Van Raamsdonk et al., 2005b; Graham et al., in press). Significant brain atrophy and loss of striatal volume have previously been reported in HD53 mice by 9 months of age (Slow et al., 2003) and by 18 months of age in line HD54 which expresses lower levels of mutant htt (Graham et al., in press). As a first step therefore, we measured brain weights and striatal volumes in CQR and HD54 mice at 18 months of age (n=8). No reduction in brain weight is observed for either CQR24 or CQR26 line compared to wild type (wt) mice (CQR (pooled)= 0.39±0.01g, wt= 0.40±0.02g, p=0.14, Fig.4.5A). As expected, HD54 brain weights are significantly lower than wt controls at 18 months of age (HD54= 0.37±0.01g, p=0.02). Similarly, striatal volume is reduced by 9% in HD54 mice relative to wt controls (HD54= 11.9±0.65mm , wt= 13.2±0.96 mm , p=0.03, Fig.4.5A). B y contrast, CQR24 3  3  89  and CQR26 mice do not exhibit any reductions in striatal volume at 18 months compared to wt mice (CQR (pooled) = 12.6±0.74 mm , p=0.22). These observations demonstrate that caspase cleavage of htt is required for reduction of striatal volume in the presence of an expanded polyglutamine tract. Furthermore, this demonstrates that htt is the only causative caspase substrate required to initiate H D neuropathology, as preventing the cleavage of a single caspase substrate, htt, is sufficient to prevent neurodegeneration in vivo. To distinguish whether caspase-3 or caspase-6 cleavage of mhtt, or both, are critical for mediating striatal atrophy, we next evaluated brain weights and striatal volumes in both of the C3R lines relative to HD54 and wt mice (n=8). At 18 months of age, brain weight and striatal volumes in the C3R lines (pooled) are indistinguishable from HD54 (brain weight C3R= 0.37±0.02g, HD54= 0.37±0.01g, p=0.75; striatal volume C3R= 11.8±0.90 mm , HD54= 11.9±0.65 mm , p=0.84, Fig.4.5B) and significantly less 3  3  than wt controls (brain weight wt= 0.40±0.002g, p=0.002, striatal volume wt= 13.2±0.96 mm , p=0.003). These findings demonstrate that prevention of caspase-3 cleavage of 3  mhtt provides no protection from striatal atrophy in vivo. Notably, mice expressing C3R mhtt still generate the caspase-6 cleavage fragment of mhtt (Fig.4.4D). Because mutagenesis of the caspase-3 site in mhtt did not prevent striatal volume loss, we next evaluated whether selective inhibition of caspase-6 cleavage of mhtt influenced brain weight and striatal volume in vivo relative to HD53 mice. As expected (Graham et al., in press), HD53 brain weight and striatal volume are reduced compared to wt controls at 18 months of age (n=8, brain weight HD53= 0.35±0.01g, wt= 0.38±0.02g, p=0.04; striatal volume HD53= 10.9±0.40 mm , wt= 12.9±0.42 mm , pO.0001, 3  3  Fig.4.5C). B y contrast, brain weight and striatal volume from C6R7 and C6R13 lines (pooled) are indistinguishable from wt controls (n=8, brain weight C6R= 0.39±0.03g, p=0.61; striatal volume C6R= 12.3±0.83 mm , p=0.10) and striatal volume is 3  significantly increased relative to HD53 (p<0.01, A N O V A p=0.0004, Fig.4.5C). These data demonstrate that selective mutation of the caspase-6 site in expanded htt is sufficient to protect mice from the neurotoxic effects of polyglutamine expansion and neurodegeneration in vivo. 91  90  CQR-18m  1"  f | * H40-  £ B a  DI  g vi  WT  HD5*  cart  HOS*  con  C3R-18m  B ass  DB  1  D4*  f  340  ba  6  I IB  I" II-  E  II  IS  HD5*  HDM  C6R-18m 3 31  b E  D  Vr  HDSJ  CM  CM C6R-12m  1»  •  III  e  I-  HDSJ  cm  WT  HDSJ  CS"!  Fig. 4.5. Inhibition of caspase 6 cleavage of huntingtin prevents neurodegeneration in vivo. (A) Brain weight and striatal volume at 18 months in W T , H D 5 4 and C Q R mice (n=8). Perfused brains were weighed and cut coronally into 25um sections throughout the striatum. Every 8 section was immunostained with N e u N antibody and Steroinvestigator software was used to calculate total striatal volume across the brain. Brain weight and striatal volume is preserved in C Q R mice and similar to W T at 18 months (p=0.14 and p=0.22 respectively). H D 5 4 brain weight and striatal volume is significantly reduced vs. W T (p=0.02, p=0.03, respectively). ( B ) Brain weight and striatal volume in C 3 R lines (pooled) is indistinguishable from H D 5 4 (brain weight p=0.75, striatal volume p=0.84) and significantly less than W T controls at 18 months (p=0.002, p=0.003 respectively) (n=8). (C) H D 5 3 brain weight and striatal volume is significantly reduced compared to W T controls (p=0.04, p<0.0001 respectively). B y contrast, brain weight and striatal volume in C 6 R lines (pooled) are indistinguishable from W T controls (p=0.61, p=0.10 respectively) and striatal volume is significantly increased vs. H D 5 3 at 18 months of age (p<0.01, A N O V A p=0.0004) (n=8). (D) Striatal volume (left panel) and neuronal profiles (right panel) at 12 months in W T , H D 5 3 and C 6 R mice (n=8). H D 5 3 mice demonstrate significant striatal volume and neuronal loss th  91  (p<0.001 and p<0.05 respectively) at 12 months. T h e C 6 R mice show preserved striatal volume and no neuronal loss at the same time point and are significantly different from H D 5 3 (striatal volume: C 6 R vs. W T p=0.57, C 6 R vs. H D 5 3 p<0.001, A N O V A p=0.0002; neuronal counts: C 6 R vs. W T p=0.74, C 6 R vs. H D 5 3 p=0.02). M e a n measurements are g i v e n ± S D . Significant differences are represented by asterisks: *p<0.05; **p<0.01; ***p<0.001.  We then assessed striatal volume and neuronal counts in C6R mice compared to HD53 animals at 12 months of age (n=7). As described previously (Slow et al., 2003), HD53 mice demonstrate significant striatal volume and neuronal loss at 12 months (striatal volume HD53= 11.0±0.74 mm , wt= 12.6±0.84± mm , pO.OOl; neuronal counts 3  3  HD53= 12.4±0.89X10 , wt= 13.7±1.31X10 , p<0.05, Fig.4.5D). Similar to that observed 5  5  at 18 months of age, C6R mice exhibit preserved striatal volume and no neuronal loss at 12 months and are significantly different from HD53 mice (striatal volume: C6R= 12.4±0.46 mm , C6R vs. wt p=0.57, C6R vs. HD53 pO.OOl, A N O V A p=0.0002; 3  neuronal counts C6R= 13.9±1.25X10 , C6R vs. wt p=0.74, C6R vs. HD53 p=0.02, Fig 5  4.5D). Mice expressing C6R htt still generate a caspase-3 cleavage fragment of mhtt (Fig.4.4E), which clearly has no toxicity in vivo. These observations establish that selective cleavage of mhtt by caspase-6, but not caspase-3, is a critical step in the pathogenesis of HD.  4.2.6 Inhibition of caspase cleavage of mutant huntingtin protects against motor dysfunction HD53 mice develop cognitive and motor abnormalities that recapitulate the clinical features of H D (Slow et al., 2003; Van Raamsdonk et al., 2005b). Motor abnormalities consist of progressive hypoactivity and onset of a rotarod deficit before 6 months of age. Importantly, the severity of rotarod deficit correlates with striatal neuronal loss, suggesting that the selective striatal neurodegeneration in Y A C 128 mice underlie their behavioural phenotype (Slow et al., 2003). To investigate whether inhibition of caspase-6 cleavage of mhtt protects against motor dysfunction, HD53 and C6R mice (sex and weight matched, n=9) were assessed using an accelerating rotarod. As expected, HD53 mice show a significant deficit commencing at 2 months of age compared to wt (p<0.01, Fig.4.6A). By contrast, C6R mice perform as well as wt animals up to and including 10 months of age (the latest time tested), and perform significantly better than  92  HD53 mice at 2 (p<0.05), 6 (p<0.05) and 8 (p<0.05) months of age ( A N O V A pO.0001, Fig.4.6A). Open field activity was also assessed in wt, HD53 and C6R mice (sex and weight matched, n=9). The HD53 mice demonstrate a progressive hypokinetic phenotype commencing at 4 months of age compared to wt during a 30 min open field trial (p<0.01, Fig.4.6B) . We have previously reported similar findings in a 10 minute open field trial (Slow et al., 2003). The first 10 mins of open field analysis represent exploratory activity and habituation to a new environment and a longer time in the open field is more representative of overall activity levels. In contrast to the HD53 mice, the C6R mice display activity levels that were indistinguishable from wt at all time points tested. Furthermore, the C6R mice perform significantly better than HD53 mice at 6 (p<0.05), 8 (pO.Ol) and 10 (pO.Ol) months ( A N O V A pO.0001, Fig.4.6B). These results demonstrate that preventing the generation of the caspase-6 cleavage fragment of mhtt is sufficient to prevent the motor dysfunction observed in a Y A C animal model of HD.  93  Rotarod 50Ch  • w  400-I T i m e o n rotarod(s)  •  H D 5 ™>-\  •  C6  * W T vs. HD53 # C 6 R vs. HD53  300  J.  6  i  8  -2-  HD53  6 months  10  months  10  8  Open Field Activity B Acfivfffy ( b e a m c r o s s e s a n d b r e a k s )  •  w  •  HD5  •  C6  1200* W T vs. HD53 # C 6 R vs.  *** *  1000  * *  900-  i  -  '  IBM HI  300 600 400  J  800-  * C 6 R  700-  600  200  #  ##  0'  6  months  10  2  4  6 months  8  10  Fig.4.6. Inhibition of caspase-6 cleavage of mutant huntingtin protects against neuronal dysfunction. (A) Assessment of rotarod performance, using an accelerating rotarod, in WT, H D 5 3 and C6R mice (n=9). H D 5 3 mice show a significant deficit on rotarod commencing at 2 months of age compared to W T (p<0.01). C6R mice do not demonstrate a deficit compared to WT up to and including 10 months of age and are significantly different from H D 5 3 mice at 2 (p<0.05), 6 (p<0.05) and 8 (p<0.05) months of age ( A N O V A p O . 0 0 0 1 ) . (B) Open field activity in W T , H D 5 3 and C6R mice (n=9). H D 5 3 mice demonstrate a progressive hypokinetic phenotype commencing at 4 months of age (p<0.01) compared to W T during a 30 minute open field trial. C6R mice display activity levels indistinguishable to WT at all time points tested and are significantly different from H D 5 3 at 6 (p<0.05), 8 (p<0.01) and 10 (p<0.01) month of age ( A N O V A p<0.0001). Time on rotarod(s) and activity (beam breaks and crosses) is the mean value ±SEM. Significant differences are represented by asterisks or P value given.*p<0.05; **p<0.01; ***p<0.001.  4 . 2 . 7 Altered nuclear translocation of huntingtin in the striatum of C 6 R mice  Translocation of htt to the nucleus and formation of inclusions are neuropathological hallmarks of human H D (DiFiglia et al., 1997; Gutekunst et al., 1999) and HD mouse models (Li et al., 1999; Slow et al., 2003). Nuclear localization of mutant htt in the Y A C 128 model coincides with onset of motor abnormalities and occurs earliest and to the greatest extent in the striatum, the region most affected in H D (Slow et al., 94  2003;2005;Van Raamsdonk et al., submitted). To investigate whether inhibiting caspase6 cleavage of mhtt alters nuclear translocation of htt, brain sections from wt, HD53, C6R7 and C6R13 were immunostained with EM48, an antibody that recognizes N terminal htt fragments and is highly specific for inclusions (Gutekunst et al., 1999). As described previously (Slow et al, 2005), diffuse nuclear htt is observed at 3 months of age in the striatum of HD53 mice (Fig.4.7A). The level of nuclear htt increases with age and by 12 months the majority of striatal neurons demonstrate extensive EM48 staining (Fig.4.7C). In contrast, selective nuclear localization of mhtt is delayed in the C6R lines, with barely detectable levels in the striatum at 9 months of age (Fig.4.7B). Between 9 and 12 months there is a significant increase in nuclear htt in the striatum of C6R mice, suggesting that other proteases are activated and generate htt fragments that are not associated with pathology (Fig.4.7B, C). This evidence indicates that nuclear translocation of the caspase-6 fragment of mhtt represents an early neuropathological event in the Y A C 128 model of HD and supports the specificity of the htt fragment in initiating neuronal dysfunction and degeneration.  WT  HD53  C6R7  C6R13  Fig. 4.7. Altered nuclear translocation of huntingtin in C 6 R mice.  staining in the striatum of HD53 at 3 months of age show clear diffuse nuclear htt with no inclusions. The level of nuclear htt increases with age in HD53 striatum, and by 12 months the majority of striatal neurons demonstrate extensive E M 4 8 staining ((B)9mths), ( Q - 12mths)). In contrast, selective nuclear localization of mhtt is delayed in the C 6 R lines, with barely detectable levels in the striatum at 9 months of age (B). Between 9 (B) and 12 ( C ) months there is a significant increase in nuclear htt in the striatum of C 6 R mice, wt is included as negative control. Scale bars A - C = 1 0 n m . (A) E M 4 8  95  4.2.8 Caspase-6 cleavage of mutant htt triggers caspase activation pathways Accumulation of caspase-cleavage fragments of htt represent an early neuropathogical change in human H D brain and in a Y A C transgenic model of H D (Kim et al., 2001; Wellington et al., 2002). Furthermore, specific caspase amplification/activation has been identified by immunohistochemistry, including increased activation of caspase-2 and caspase-6 in human H D brain compared to control (Hermel et al., 2004). Translocation of nuclear htt is delayed in the C6R mice, suggesting that inhibiting caspase-6 cleavage of mutant htt alters caspase activation pathways active in the early stages of HD. To determine whether protection from striatal cell loss results from decreased activation of neuronal cell death pathways operative in H D , we investigated in vivo caspase activation in the striatum and cortex of wt, HD53 and C6R mice. Perfused coronal sections (25 pm) of wt, HD53 and C6R mice were stained with an antibody specific for the active form of caspase-6 at 3, 6, 9, 12 and 18 months of age (n=3). In murine wt brain, expression of active caspase-6 is observed predominantly in the nuclei of striatal neurons and in pyramidal neurons in layer 2/3 of the cortex commencing at 9 months of age with a moderate increase observed at 12 and 18 months (Fig.4.8A, B, C). In contrast, active caspase-6 is present by 3 months of age in HD53 striatum (in a similar pattern as wt at 9 months), with levels increasing with age (Fig.4.8E, F, G). Activated caspase-6 is observed at 3 months of age in mice expressing C6R mutant htt, however levels remain constant with age (Fig.4.8: C6R7 - 1 , J, K , C 6 R 1 3 - M , N , O). Enhanced immunoreactivity for activated caspase-2 has previously been observed in medium spiny neurons of the striatum from post-mortem human and mouse H D brain tissue (Hermel et al., 2004). Evaluation of activated caspase-2 expression in perfused brain sections from wt, HD53 and C6R shows that low levels of activated caspase-2 are present in wt and HD53 striatum at 3 months of age (data not shown). However, by 12 and 18 months of age, expression levels, while present in the neuronal nuclei and cytoplasm of wt striatum, is increased in the striatum of HD53 mice (Fig.4.8 D, H). In sharp contrast, activated caspase-2 is undetectable in two independent C6R htt lines, C6R 7 and C6R13, which show only light background staining in the striatum at 18 months of  96  age (Fig.4.8 L, P). A similar lack of activated caspase-2 expression is observed in mice over expressing wild type htt ( Y A C 18) (data not shown) and suggests that the neuroprotective effect of wild type htt appears to be preserved in mutant htt resistant to cleavage by caspase-6. These results demonstrate that inhibiting caspase-6 cleavage of mutant htt alters caspase cascades in vivo, and support the hypothesis that C6R htt protects against neurodegeneration by interfering with activation of neuronal cell death pathways. These findings provide additional support for the toxic fragment hypothesis and suggest that mutant htt can be rendered non-toxic via inhibition of caspase-6 cleavage. activated caspase 2  activated caspase 6 3m  18m  9m  C  B  D  W T  F  H  i  HD53  41  0 K  fl* ~  C6R7  M  N  C6R13  Fig.4. 8 Expression pattern of apoptotic markers in striatum of wt, H D 5 3 and C6R striatum. Perfused coronal sections were probed with antibodies to activated caspase-6, and activated caspase-2. (A) In wt brain, low level punctuate immunoreactivity is observed for activated caspase-6 expression predominantly in the nuclei of striatal neurons commencing at 9 months (B) with a moderate increase at 18 months of age (C). Medium sized neurons in the striatum of HD53 brain display enhanced immunoreactivity to activated caspase-6 at 3 months compared to control striatum (A,E), with levels increasing with age (F,G,). In two lines of mice expressing caspase-6 resistant mutant htt, activated caspase-6 is observed at 3 months (I,M)  97  with levels remaining constant with age (J, K , N , O ) . Enhanced immunoreactivity for activated caspase-2 is present in a diffuse pattern predominantly in the nucleus o f medium sized striatal neurons in 18 month old H D 5 3 brain (H), compared against control brain (D). O n l y background immunoreactivity for activated caspase-2 is observed in C 6 R striatum at 18 months of age ( L , P). Activated caspase-2 and -6 antibodies were verified by western blot analysis using recombinant caspases and transfected cell lysates (data not shown). Controls were performed for all immunostaining and included the use o f no primary antibody, each control showed no immunoreactivity. Scale bars A-P=10|xm  4.2.9 Similar apoptotic pathways are observed in the Y A C 128 model and human H D brain In order to determine if apoptotic pathways in human HD brain correlate with the findings in the Y A C 128 mice, we examined expression levels of pro-caspase-2 and -6 in presymptomatic human HD and control striatum by western blot. Concurrent with the findings in the Y A C 128 mice, levels of pro-caspase-6 in presymptomatic HD striatum is decreased compared to control tissue, suggesting that caspase-6 is processed and activated early in the pathogenesis of HD (Fig.4.9A). In contrast, expression levels of pro-caspase-2 are increased (Fig.4.9A) demonstrating activation of caspase-6 precedes caspase-2 up-regulation. striatum  cortex B  PMI Sin  *?  n>" <K> .XT  A S> r>  Pro-Caspase-6  Pro-Caspase-6 Pro-Caspase-2  ^  PMI 8hr>  —-  GAPDH  Pro-Caspase-2  actin  Fig. 4.9 Altered caspase expression levels are observed in human presymptomatic H D striatum compared to control brain. ( A ) Western blots o f human tissue were probed with antibodies which recognize the proform o f caspase-6 (upper panel) and caspase-2 (middle panel). H u m a n presymptomatic H D striatum demonstrate a decrease in pro-caspase-6 levels compared against control striatum (n=2). In contrast, pro-caspase-2 is increased. G A D P H demonstrates equal loading for all tissues (lower panel). (B) Western blot analysis o f human grade 3/4 H D and control cortex demonstrate a decrease in levels o f procaspase-2 and caspase-6 in cortex from H D patients compared to control tissue. A c t i n demonstrates equal loading for all tissues. T o avoid bias due to ex vivo proteolysis during post mortem interval ( P M I ) all H D tissue were compared against control tissue with a similar P M I . T h e activated form o f each caspase was not detected in human tissue by western blot.  98  No alteration in caspase expression levels were observed in striatum from grade 3/4 human HD brain compared to control tissue by western blot (data not shown). However the majority of the striatum has atrophied by this stage and the remaining tissue may not be sufficient to detect obvious changes as would be required for western blot analysis. Analysis of grade 3/4 human HD and control cortex demonstrates a decrease in the levels of pro-caspase-2 and caspase-6 in the brains of H D patients compared to control tissue, implying cleavage and activation of caspase-2 and -6 is also involved in end stage H D (Fig.4.9B).  4.2.10 C6R mice are resistant to excitotoxicity Over-activation of glutamate receptors has long been considered a fundamental underlying mechanism in the early stages of HD (Albin et al., 1992; Beal, 1992; Young, 1997; Levine et al., 1999; Zeron et al., 2002; 2004). We have previously demonstrated enhanced susceptibility to glutamate and NMDAR-mediated excitotoxity in neonatal medium spiny neurons (MSNs) from multiple Y A C lines compared to wildtype (wt) mice (Zeron et al., 2002;2004; Tang et al., 2005; Slow et al, 2005; Graham et al, in press). Conversely, MSNs overexpressing htt with a normal C A G size (18CAG) ( Y A C 18) show notably less cell death than wt cultures upon N M D A treatment (Leavitt et al, submitted), suggesting that htt mediates neuroprotection by blocking excitoxic cell death. To determine whether changes in excitotoxicity mirror the neuropathological and behavioural protection observed in the C6R mice, we assessed vulnerability to N M D A R mediated excitotoxicity in primary neonatal MSNs from wt, H D and CR neonatal pups. To be able to use pooled cultures to generate sufficient quantity of primary striatal cultures we first generated homozygous H D and C R lines that were matched for equivalent mhtt protein levels (Fig.4.10A). Furthermore we used well established techniques in our laboratory to generate primary striatal cultures that contain a high level of morphological and DARPP-32 containing medium spiny neurons (>85%) and low levels of glia (<10%) (Shehadeh et al., 2005).  99  Protection from NMDAR-mediated excitotoxic death is observed in MSNs that express CQR mhtt compared with HD55 (CQR= 2.6±1.2, HD55= 27.9±6.1, p<0.01) and wt (wt= 8.6±3.5, p<0.05, Fig.4.10B). In contrast, MSNs expressing C3R mhtt exhibit increased susceptibility to NMDAR-mediated excitotoxicity compared to wt (C3R= 26.8±1.1, wt=8.6±3.5, p<0.01) and a similar level of vulnerability as HD55 (HD55=27.9±6.1, p>0.05, Fig.4.10C). Furthermore, C6R neurons are also protected against NMDAR-mediated excitotoxicity compared with HD55 (C6R= 2.4±0.73, HD55= 37.2±7.7, p<0.001) and wt (wt=8.6±3.4, p<0.05, Fig.4.10D). To confirm that protection was due to N M D A R activation, we also treated MSNs with MK801 (20uM), a N M D A R antagonist, in addition to 500uM N M D A . Under these conditions, cell death was reduced to virtually baseline conditions in all cultures (Fig.4.10B-D). These data demonstrate that MSNs expressing CQR or C6R mhtt are no longer susceptible to NMDAR-mediated toxicity as seen in MSNs expressing caspase-cleavable (HD55) or C3R mhtt. In addition, primary MSNs expressing CQR or C6R mhtt demonstrate significantly less N M D A R mediated apoptotic death than wt neurons, suggesting that inhibiting caspase-6 cleavage of mhtt converts a neurotoxic species into a neuroprotective one. To determine if the resistance to excitotoxicity is also observed in vivo, we next examined susceptibility to QA-mediated neurotoxicity in 3 month old wt, HD55 and C6R mice. As expected, HD55 animals show enhanced lesion size compared to either C6R (HD55= 1.9±0.28mm , C6R= 0.17±0.08mm , p<0.0001) and wt mice (wt= 3  3  0.91±0.25mm , p<0.05, A N O V A pO.0001, Fig.4.10E). In contrast, C6R mice exhibit 3  reduced lesion volume even compared to wt mice (p<0.05), showing that C6R htt protects from acute excitotoxicity despite the presence of an expanded polyglutamine tract. To examine whether caspase-6 cleavage of mhtt also influences susceptibility to other, more general, apoptotic stimuli, we also assessed cell death upon exposure to staurosporine. The kinase inhibitor, staurosporine has been demonstrated to stress mitochondria through upstream inhibition of Bcl-2, production of reactive oxygen species and activation of caspases (Kruman et al, 1998). As shown previously (Shehadeh et al., 2005), HD55 MSNs are more vulnerable to staurosporine-mediated death (lOuM) compared to wt MSNs (HD55= 42.7±2.4, wt= 36.8±1.3, p<0.05), while Y A C 18 MSNs  100  demonstrate neuroprotection and reduced cell death compared to wt in this assay (YAC18= 17.4±0.9, pO.OOl, F i g A l O F ) . Notably, MSNs expressing C6R mhtt are less susceptible to staurosporine-mediated cell death than wt (C6R= 20.0±1.2, pO.OOl) and display a similar level of cell death as MSNs over expressing neuroprotective wt htt ( Y A C 18 vs. C6Rp>0.05, F i g A l O F ) ( A N O V A pO.0001). These data demonstrate that caspase-6 cleavage of mhtt releases a fragment that is essential for apoptotic-mediated cell death in a number of in vitro and in vivo paradigms. Inactivating caspase-6 proteolysis of htt prevents this function and results in neuroprotection, even in the presence of an expanded polyglutamine tract.  101  YAC  HD55  C3R  C6R  CQR  CQR  Line  55  C3R8  C6R7  CR24  CQR26  o  Protein level  o  *.  E  <B  O  ~ CO  o  E  o  Transgene Endogenous  WT  HD55 CQR NMDA  ^  WT h  HD55 CQR MK801  WT  HD55  C3R  I NMDA ...... I  WT  I  HD55 C3R MK801  I  WT  I  HD55 NMDA  C6R  WT  ***  CD O  HD55 C6R  I MK801 . •  I  I  ***  > 40 + _J LU Z =>  WT  staurosporine  Lesion volume %change vs WT  WT  HD55  C6R  0.91±0.25  1.9±0.28  0.17.1*0.08  t108%  J.81%  Y18 HD55 C6R  100nM  WT  Y18  HD55 C6R  10uM  Fig. 4.10 Caspase cleavage of mutant huntingtin influences susceptibility to excitotoxicity in vitro and in vivo. (A)YAC128 and  CR lines were matched for equivalent mhtt protein levels. Western blot of murine brain lysate from heterozygote and homozygote YAC 128 and CR mice, probed with BKP1 .(B) MSNs from CQR striata demonstrate a decrease in cell death post NMDA application vs. HD55 (p<0.01). Furthermore a decrease in apoptotic cells is evident in CQR vs. WT (p<0.05). (C) MSNs from C3R striata demonstrate an increase in susceptibility to NMDAR-mediated excitotoxicity vs. WT (p<0.01) and a similar level of cell death vs. HD55 (p>0.05) post NMDA treatment. (D) MSNs from C6R striata show protection from NMDAR-mediated excitotoxicity with a decrease in cell death vs. WT (p<0.05) and HD55 (p<0.001). Mean percent apoptotic cells are given ± SD. (E) Intrastriatal injection of QA in 3 month old mice reveal virtually no fluorojade positive cells are present in coronal sections of C6R brain. In contrast, in YAC 128 (HD55) brain, a large area of the striatum is fluorojade positive, significantly larger in size compared against the C6R lesion volume (p<0.0001) and wt p(<0.05). Lesion volume is ± SEM. (F) Staurosporine result HD55 MSNs are more vulnerable to staurosporine-mediated death (lOuM) compared to wt MSNs (p<0.05), while YAC18 MSN's demonstrate neuroprotection and reduced cell death compared to wt MSNs in this assay (p<0.001). Notably, MSNs expressing C6R mhtt are less susceptible to staurosporine-mediated cell death than wt (p<0.001) and display a similar level of cell death as MSNs over expressing neuroprotective wt htt (YAC18 vs. C6R p>0.05) (ANOVA p<0.0001). Significant differences are represented by asterisks: *p<0.05; **p<0.01; ***p<0.001.  102  4.3 Discussion The results presented in this study identify caspase cleavage of mhtt as a primary event in the pathogenesis of HD. Here we have demonstrated that specific proteolytic cleavage at the caspase-6 site in mhtt is crucial for the HD-related behavioural phenotype and selective striatal neurodegeneration observed in the Y A C 128 model of HD. Activation of caspase-6 and nuclear translocation of fragments of htt coincide with onset of motor dysfunction in the Y A C 128 model, supporting a role for a specific nuclear htt fragment in initiating neuronal dysfunction. Furthermore, caspase-6 cleavage of mhtt influences susceptibility to excitotoxic stress, highlighting caspase 6-mediated proteolysis of htt and excitotoxicity as primary mechanisms of striatal neuronal loss in HD. This evidence demonstrates that generation of a specific fragment of mhtt in vivo represents an initiating, primary event in the pathogenesis of H D . Numerous in vitro and in vivo studies have demonstrated that mhtt toxicity occurs via formation of N-terminal htt fragments containing expanded C A G repeats (Mangiarini et al., 1996; Davies et al., 1997;Hackam et al., 1998; Cooper et al., 1998; Schilling et a l , 1999;Jana et al., 2001). However, not all N-terminal fragments of mhtt demonstrate selective HD-related toxicity. HD cell culture models that selectively express the exon 1 fragment of mhtt have been the most extensively studied, yet no protease described thus far cleaves htt to release a fragment that corresponds to exon 1. In addition, most in vitro studies employ a variety of cell types including HEK293, lymphoblast and COS cells, which are not representative of the specific cell type vulnerability observed in human HD. Furthermore, nonselective expression of a pure expanded C A G tract also results in in vitro and in vivo toxicity, but this represents general polyglutamine toxicity and does not address pathways specific to HD (Ordway et al., 1997). Additionally, increasing evidence in vitro and in vivo support the specific protein context of the mhtt fragments in initiating toxicity (Yu et al., 2003; Slow et al., 2005). These observations suggest that the selective neurodegeneration observed in H D may be mediated by a specific N-terminal fragment of mhtt. Using striatal volume as a primary endpoint, we have shown that caspase fragments of mhtt are responsible for neurodegeneration in vivo. Specifically, inhibiting  103  caspase-6 and not caspase-3, cleavage of mhtt protects against striatal volume and neuronal loss. This result highlights the caspase-6 fragment of htt (586aa) as the toxic fragment, and caspase-2 (552aa) and caspase-3 (531, 552aa) htt fragments as nontoxic. The hypothesis that a specific fragment may underlie disease pathogenesis has been well characterized in Alzheimer Disease (AD). Amyloid-B precursor protein (APP), is also cleaved by proteases, leading to generation of AB42 fragments which play an essential role in initiating the neurodegeneration observed in A D (Pellegrini et al., 1999; Gervais et al., 2002). Selective down regulation of the toxic A1342 fragment, and not AB40, by therapeutic drugs such as NSAID, have been shown to ameliorate the disease phenotype in mouse models of A D showing differences in toxicity levels of fragments that have only a 2 amino acid difference (Eriksen et al., 2003). A significant finding in the C6R mice, in addition to the absence of neurodegeneration, is the lack of a behavioral phenotype, indicating that the caspase-6 fragment of mhtt is critical for the neuronal dysfunction observed in the Y A C 128 model. In the Y A C 128 model, which expresses full length mutant htt, nuclear localization of htt is earliest and to the greatest extent in the striatum, correlating with the neuropathology observed in the Y A C model and in H D patients (Van Raamsdonk et al, submitted). Translocation of fragments of htt to the nucleus coincide with onset of motor abnormalities in the Y A C 128 model, suggesting that neuronal dysfunction may be triggered by translocation of this fragment. In contrast, blocking formation and translocation of the caspase-6 fragment in the C6R mice, completely prevents the behavioral phenotype. Translocation of htt to the nucleus is associated with increased toxicity in vitro and neuropathology in vivo (DiFiglia et al., 1997; Wheeler et al., 2000; K i m et al., 2001; K i m et al., 2001; Wellington et al., 2002; Slow et al., 2003). However, the shortstop mouse exhibits widespread diffuse nuclear htt yet demonstrates no clinical phenotype (Slow et al., 2005), supporting the hypothesis that it is a specific nuclear htt fragment that underlies the neuronal degeneration observed in HD. Here we show that nuclear translocation of htt fragments does occur in the C6R mice but is delayed compared to HD53 mice. Because the caspase-6 fragment is not produced in C6R mice, the htt fragments detected in the nuclei of C6R mice at later timepoints must result from alternative proteolytic pathways, which may include calpains  104  and other caspases such as caspase-3. Calpain activation is detected in the 3-NP model of H D and this was associated with calpain-dependent cleavage of htt (Bizat et al., 2003). Furthermore, selective calpain inhibition reduced the size of the striatal lesion and abolished 3-NP induced D N A fragmentation in striatal cells. Interestingly, in rats, intracerebro-ventricular infusion of z V A D , a broad spectrum caspase inhibitor, significantly reduced 3NP-induced striatal degeneration, and decreased the 3NP-induced activation of calpain (Bizat et al., 2005). These observations support the hypothesis that there may be sequential pathways of htt proteolysis, and that further cleavage of caspase derived htt fragments by calpains may generate fragments capable of passive diffusion into the nucleus (Kim et al., 2001). However, the results of our study place cleavage of htt at the caspase-6 site as a crucial rate limiting event, as blocking htt cleavage at this site is sufficient to provide in vitro and in vivo protection from a variety of toxic insults. We have shown that activation of caspase-6 represents an early event in the Y A C 128 model and in human H D brain. Similar to the findings in this paper, a number of studies have demonstrated a critical role for caspase-6 in A D . It has previously been shown that caspase-6 and not caspase-3 is activated in an in vitro model of A D (LeBlanc et al., 1999) and increased levels of activated caspase-6 are observed in A D human brain (Guo et al., 2004). This has been substantiated with studies using a neo-epitope antibody to caspase-6 cleaved tau which preferentially binds in the neuropil threads, neuritic plaques and neurofibrillary tangles present in the A D human brain (Guo et al., 2004). Caspase-6 has been demonstrated to selectively cleave C R E B binding protein (CBP) (Rouaux et al., 2004), suggesting a function for caspase-6 in altering gene transcription in H D through cleavage of transcription factors. Interaction of CBP with mutant htt and its localization within aggregates has been suggested to explain the aberrant transcriptional regulation observed in HD. A n alternative explanation for the transcriptional dysregulation observed may be caspase-6 mediated proteolysis of CBP leading to altered transcription of p53 regulated promoters. In addition, caspase-6 is the major protease which cleaves cleaves Akt (Medina et al., 2005), a serine/theronine kinase that has demonstrated protective effects in a number of model systems (Datta et al., 1999; Noshita et al., 2002). Decreased phosphorylation of mutant htt by Akt has been described  105  in human and mouse models of H D and loss of its protective effect implicated in the pathogenesis of H D (Humbert et al., 2002;Warby et al., 2005). Similar to previously described findings (Hermel et al., 200) we found enhanced immunoreactivity for activated caspase-2 in murine H D brain vs. control. Furthermore, we show that caspase-2 is upregulated in human presymptomatic human H D striatum. Caspase-2 is also implicated in A D and has been suggested to mediate neuronal cell death induced by (3-amyloid (Troy et al., 2000). Specific downregulation of caspase-2, but not caspase-1 or -3, protects distinct neuronal populations from cell death induced by A l l Activation of caspase-2 is required for permeablization of mitochondria causing amplification of the caspase response and eventual cell death (Miyashita et al., 1998). Excitotoxicity has been implicated as a fundamental pathway in the earliest stages of the pathogenesis of H D (Albin et al., 1992; Beal, 1992; Young, 1997; Levine et al., 1999; Zeron et al., 2002;2004). Significantly increased NMDAR-mediated current density and/or C a ^ levels have been observed in numerous H D mouse models and enhanced susceptibility to glutamate and NMDAR-mediated excitotoxity is observed in neonatal MSNs from three Y A C 128 and a Y A C 7 2 mouse line compared to wt (Zeron et al., 2002;2004; Tang et al., 2005; Shehadeh et al, 2005; Graham et al, in press). Furthermore, striatal glutamate receptors show disproportional loss in H D human brain (Albin et al., 1992; Young, 1997) and excitotoxic lesions from glutamate intrastriatal injections in mice and primates closely resemble the neuropathology in H D (Schwarcz and Kohler,1983; Beal et al., 1986; Ferrante et al., 1993). In contrast, overexpression of wt htt has been shown to be neuroprotective in a number of stress-induced model systems, including NMDA-mediated excitotoxicity, Q A induced neurotoxicity and ischemia (Leavitt et al., 2001; Zhang et al., 2003; Leavitt et al, submitted). These observations suggest that blocking excitotoxicity is one mechanism by which excess htt mediates neuroprotection. While the underlying mechanism by which htt mediates neuroprotection is currently unclear, htt has been shown to directly interact with caspase-2 suggesting a possible role for htt as a direct caspase inhibitor (Hermel et al., 2004). Concurrent with this finding, it has been demonstrated that the neuroprotective role of htt lies upstream of caspase-3 and may take place at the level of initiator caspases (Rigamonti et al., 2001). The parallel observed between mice overexpressing (wild type)  106  htt ( Y A C 18) and the C6R mice supports the hypothesis that the caspase-6 cleavage site in htt modulates susceptibility to stress and that this domain is a key regulator of htt's anti vs. pro-apoptotic properties. Numerous caspase substrates, which demonstrate dual survival/anti-apoptotic vs. apoptotic properties, are mediated through proteolytic cleavage. In this study, we demonstrate that caspase-6, but not caspase-3 resistant mhtt, fully protects from excitotoxic stress, both in vitro and in vivo, supporting the hypothesis that not all mhtt fragments contribute to excitotoxic death. It has been demonstrated in vitro that enhanced NMDAR-mediated cell death is not observed with a 548aa N terminal fragment of mutant htt which is very similar in size to the caspase-3 fragment of htt with 552aa (Zeron et al., 2001). In addition, truncated mouse models including R6/2, N171 and shortstop, demonstrate resistance to QA induced excitotoxicity (Hansson et al., 1999; Jarabek., 2004;Slow et al., 2005). Thus no excitotoxicity is observed in vivo with expression of a 97, 117, 171,513 and 552aa htt fragment containing the expanded polyglutamine tract. These findings demonstrate that caspase-6 cleavage of mhtt selectively mediates susceptibility to excitotoxic stress via a specific mhtt fragment. In addition to htt, a number of caspase substrates have been identified to have active roles in apoptosis. Preventing caspase cleavage of the caspase substrate (i.e. caspase resistant) has been shown to influence downstream cell death by abrogation of their inhibitory function (Rao et al., 1999; Oliver et al., 1998; Cohen et al., 2005). For example, caspase-resistant lamin protects cells against chromatin condensation and nuclear shrinkage (Rao et al., 1999) and death triggered by CD95 activation is reduced in cells expressing caspase-resistant poly (ADP-ribose) polymerase (Oliver et al., 1998). Excessive signalling via the Notch 1 receptor has been shown to inhibit apoptosis which is abrogated by caspase cleavage (Cohen et al., 2005) and caspase-6-mediated cleavage of the transcription factor N F - K B , an important regulator of gene expression, leads to a transcriptionally inactive p65 molecule (Levkau et al.,1999). The p65 truncated fragment acts as a dominant-negative inhibitor of N F - K B , promoting apoptosis, whereas caspaseresistant p65 protects cells from apoptosis. Generation of the dominant-negative fragment of p65 during apoptosis may be an efficient pro-apoptotic feedback mechanism between caspase activation and N F - K B inactivation. Here we demonstrate that preventing  107  cleavage of a single caspase substrate block's further progression to cell death in a neurodegenerative disease in vivo. In this study, we demonstrate specificity of the mhtt fragment in generating the selective striatal neurodegeneration observed in the Y A C 128 model. Using appropriately matched controls, validated endpoints and specific forms of C R mhtt, we have shown that selective inhibition of caspase-6 cleavage of mhtt protects against excitotoxicity and neurodegeneration in vivo. Furthermore the protection observed from expression of C6R mhtt is independent of the particular apoptotic inducing signal as protection from staurosporine is also observed. While all the evidence supports the 586aa site in htt as a caspase-6 site in vitro, it cannot be definitively excluded that in vivo other proteases may cleave htt at this site. Precisely how the caspase-6 mhtt fragment leads to the selective neuronal degeneration and behavioral abnormalities seen in this Y A C model is unclear. This could either be mediated by the toxicity of this fragment itself or via activation of other pathways crucial to the pathogenesis of HD. Our in vivo and in vitro characterization of caspase-resistant mhtt provides substantial support for the hypothesis that cleavage at the caspase-6 site in mhtt represents a crucial rate limiting event in the pathogenesis of H D . Furthermore, we demonstrate a strong correlation between excitotoxicity and the disease phenotype, suggesting links between proteolysis of htt and excitotoxicity. Our study highlights the importance of developing drug targets against caspase-6 and proteins implicated in mediating excitotoxicity as potential therapeutic approaches for HD.  108  Chapter 5: Discussion and Future Directions  5.1 Proteolysis of huntingtin is required for neurodegeneration in HD The use of animal models of human disease, that accurately reproduce the symptoms and pathology observed in humans, significantly contributes to understanding the mechanisms and primary events underlying disease pathogenesis. Furthermore, animal models enable assessment of phenotype modulation due to genetic manipulation and, once targets have been identified, are critical for validation of the therapeutic efficacy of inhibiting the target in vivo. The Y A C model of H D faithfully recapitulates key features of H D observed in humans. The previously developed model for HD (using the 350 kb Y A C 353G6), contains all 67 exons of the human HD gene and its endogenous regulatory sequences (Hodgson et al., 1999). Mice harbouring Y A C 353G6 faithfully express full-length human mhtt in a tissue-specific and developmentally appropriate manner (Hodgson et al., 1999) and display age and C AG-dependent phenotypes that mimic the human disease. Specifically, enhanced susceptibility to excitotoxic stress, cleavage and nuclear translocation of htt, and cognitive dysfunction are followed by motor deficits and selective striatal degeneration (Wellington et al., 2002; Slow et al., 2003; Van Raamsdonk et al., 2005a;b; submitted; Tang et al., 2005; Shehadeh et al., 2005), indicating that the Y A C 128 model accurately replicates human H D in vivo and is therefore an ideal model to assess interventions on disease phenotype. The primary objective of this thesis was to determine if proteolysis of mutant htt is a primary event in the pathogenesis of H D and secondly to determine if a specific fragment of mutant htt initiates pathology. Expression of htt fragments containing expanded polyglutamine repeats are toxic in vitro and in vivo (Mangiarini et al., 1996; Davies et al., 1997; Hackam et al., 1998; Cooper et al., 1998; Schilling et al., 1999;Jana et al., 2001), and accumulation of N-terminal truncated products of htt are observed in human and mouse H D brain (Kim et al., 2001;Wellington et al., 2002). Notably, the presence of caspase cleaved htt fragments prior to clinical onset of H D suggests that htt cleavage may be a crucial, primary event in the pathogenesis of H D . Having an established, representative animal model of H D ( Y A C 128 line HD53) was a necessary prerequisite for the in vivo evaluation of C R mutant htt. However, as the 110  multiple lines of C R mice express a range of mutant htt levels, it was necessary to further characterize the intermediate and lower expressing Y A C 128 lines (HD55, 54). This would first determine whether levels of mutant htt influence the phenotype in the Y A C 128 model and second establish the appropriate caspase-cleavable Y A C 128 controls for the C R mutant htt study. There has been significant controversy in the literature regarding the dosage effect of mutant htt and whether increased levels of mutant htt, observed in human H D patients homozygous for the mutation, cause a corresponding increase in disease severity. The results presented in this thesis demonstrate in vivo that age of onset and disease progression of the H D phenotype in the Y A C 128 mouse models is modulated by levels of mutant htt. Furthermore, the relationship between increasing levels of mutant htt and enhanced vulnerability to excitotoxic stress, suggests that excitotoxicity may be a cumulative underlying primary mechanism of striatal atrophy which terminates in apoptotic cell death. This would argue that H D in the Y A C 128 model presents with an intermediate dominant phenotype and highlights the need for further clinical studies in humans to establish whether H D in humans also presents with an intermediate dominant phenotype rather then complete dominance as is currently thought. The natural history characterization of the Y A C 128 lines established the necessary controls (HD53 and 54), primary (striatal volume) and secondary endpoints (behaviour, susceptibility to excitotoxicity and nuclear htt accumulation) and time line (18 months of age) for the C R mutant htt characterization. The striatum is the brain region most affected in H D patients (Vonsattel et al., 1985) and this defining characteristic is recapitulated in the YAC128 models (Slow et al., 2003;Van Raamsdonk et al., 2005a). In contrast, striatal volume loss is notably absent in transgenic mice expressing CQR (all caspases sites mutated) mutant htt, demonstrating that proteolysis of htt is a critical event in the neurodegeneration observed in HD. Further investigation of mice expressing specific forms of C R mutant htt revealed that inhibiting expression of caspase-3 fragments of mutant htt did not protect against striatal volume loss, indicating that the caspase-6 fragment of mutant htt is toxic in an in vivo model of HD. Conversely, mice expressing C6R mutant htt demonstrate preserved striatal volume and neuronal number, revealing that expression of caspase-3 fragments of mutant htt are  111  not toxic in vivo . This is a surprising finding as an overwhelming body of evidence supports the role of caspase-3 in execution of the apoptotic program of cell death. However, while much information is available regarding targets of caspase-3 and its role has been extensively studied, previous information on the role of caspase-6 was limited. More recently (within the last 5 years), evidence regarding the function of caspase-6 in apoptosis and programmed cell death, has revealed the important upstream contribution of this caspase in inducing cell death under multiple paradigms including neurodegeneration (LeBlanc et al., 1999;Pellegrini et al., 1999;Guo et al., 2004;Horowitz et al., 2004), K A and/or Q A induced neurotoxicity (Narkilahti et al., 2005), ischemia (Harrison et al., 2001; Singh et al., 2002;Nu et al., 2004) and temporal lobe epilepsy (Narkilahti et al., 2005). The Y A C 128 mice exhibit cognitive deficits and motor abnormalities which parallel those observed in human H D patients. Cognitive deficits include difficulties in changing strategies, delayed platform finding and impaired motor learning on rotarod beginning at 2 months of age (Van Raamsdonk et al., 2005b). Motor abnormalities include progressive hypoactivity and onset of a motor deficit before 4 months of age (Slow et al., 2003;Van Raamsdonk et al., submitted). In contrast to the YAC128 mice, the C6R mice perform similar to wild type mice on rotarod and in the open field, supporting the critical role of the caspase-6 fragment of mutant htt in the neuronal dysfunction observed in the Y A C 128 model. This is the first time, to my knowledge, that a therapeutic strategy in H D results in complete rescue of the behavioural and neuropathological symptoms present in an H D mouse model. These experiments provide proof of principle that neuroprotection strategies will alleviate the clinical motor symptoms in an in vivo model of H D and suggest that caspase inhibitors may be useful strategies for both the neuropathological and behavioural aspects of HD. Amplification and activation of caspase-6 and caspase-2 has previously been observed in post mortem human caudate of patients with H D and in the Y A C 7 2 model (Hermel et al., 2004). However, the order of caspase activation was not established, nor was it determined whether activation of caspase-6 and caspase-2 was an early event in the disease. The work presented this thesis demonstrates that activation of caspase-6 represents an early event in the Y A C 128 model and in human H D brain. Furthermore, the  112  order of caspase activation in H D brain suggests caspase-2 is subsequent to caspase-6 involvement. Caspase-6, unlike other caspases, has been described as both an initiator (Xanthoudakis et al., 1999;Allsopp et al., 2000;Henshall et al., 2002) and executioner caspase (Srinivasula et al., 1996) and its activation observed post excitotoxic stress (Harrison et al., 2001;Krajewska et al., 2004;Chi et a., 2005;Narkilahti et al., 2005). Caspase-6 is able to cleave and activate caspase-2 (Henshall et al., 2002) supporting a role for this caspase upstream of caspase-2 and mitochondrial permeablization in H D . Caspase-2, the most evolutionarily conserved caspase, acts upstream of mitochondria by inducing Bid cleavage and Bax translocation to the mitochondria and cytochrome c release (Lassus et al., 2002; Srinivasa et al., 2002;Troy and Shelanski, 2003). Similar to the findings decribed in this thesis, a number of studies have demonstrated an upstream role for caspase-6 in A D . It has previously been shown that caspase-6 and not caspase-3 is activated in an in vitro model of A D (LeBlanc et al., 1999) and increased levels of activated caspase-6 are observed in A D human brain (Guo et al., 2004). Caspase-2 is also implicated in A D and has been suggested to mediate neuronal cell death induced by Bamyloid. Specific downregulation of caspase-2, but not caspase-1 or -3, protects distinct neuronal populations from cell death induced by AB (Troy et al., 2000). In contrast to the Y A C 128 mice, and similar to transgenic mice overexpressing wild type htt ( Y A C 18), the C6R mice exhibit low levels of activated caspase-6 and caspase -2. Overexpression of wild type htt has been shown to be neuroprotective in a number of stress-induced model systems including glutamate/NMDA-mediated excitotoxicity , Q A induced neurotoxicity and ischemia (Rigamonti et al., 2000;Leavitt et al., 2001 ;Sun et al., 2001b;Zhang et al., 2003;Leavitt et al., submitted). The parallel observed between mice overexpressing (wild type) htt (YAC18) and the C6R mice supports the hypothesis that the caspase-6 cleavage site in htt modulates susceptibility to stress and that this domain is a key regulator of htt's anti vs. pro-apoptotic properties. Numerous caspase substrates, which demonstrate dual survival/anti-apoptotic vs. apoptotic properties, are mediated through proteolytic cleavage (Levkau et al., 1999;Chau et al., 2002;Cohen et al., 2005).  113  The protection observed from neuronal dysfunction and neurodegeneration in the C6R mice is further paralleled by changes in excitotoxicity. Inhibiting caspase-6 cleavage of mutant htt results in neuroprotection against excitotoxic stress rather than neurotoxicity in vitro and in vivo. This evidence, together with the characterization of other mouse models of H D provides valuable information regarding the role of excitotoxicity in HD. Full length models of H D including Y A C 7 2 , YAC128(s) and the knockin (Hdh94), demonstrate enhanced sensitivity to excitotoxicity (Levine et al., 1999;Zeron et al., 2002;2004;Tang et al., 2005). In contrast, the truncated H D models shortstop, R6/1, R6/2 and N171 are resistant to Q A induced excitotoxic cell death (Hansson et al., 1999;MacGibbon et al., 2002;Jarabek et a l , 2004;Slow et al., 2005) (Table 5.1). Excitotoxicity resulting from overstimulation of N M D A receptors triggers C a ^ dependent proteolytic enzymes, such as caspases and calpains, which would decrease levels of full length htt and generate specific mutant htt fragments that may cause cellular dysfunction and death. The evidence that certain fragments of htt are protective under conditions of excitotoxic stress implicates an association between proteolytic fragments of htt and excitotoxicity.This may argue that the full length form of the protein is required for mediating the excitotoxicity observed and that subsequent proteolysis and release of a specific mutant htt fragment contributes to excitotoxic current increase, C a ^ influx and downstream cell death. These observations provide strong support for the hypothesis that proteolysis of htt and excitotoxicity are primary mechanism of neuronal dysfunction and neurodegeneration in HD.  114  line  CAG  method  Age/wks  Result vs. wt  phenotype  strain  Ref  R6/1  -115  QA/30nM  3  similar  presymptomatic  CBA/ C57BL6  Hansson et al., 2001  QA/30nM  8  resistant  presymptomatic  Hansson et al., 2001  QA/ 30nM  18  resistant  symptomatic  Hansson et al., 2001  QA/30nM  18  resistant  symptomatic  Hansson et al.. 1999  QA/30nM  3  partial resistance  presymptomatic  QA/30nM  6  resistant  presymptomatic  Hansson et al., 2001  KA/20mg/kg  3 and 9  resistant  pre/symptomatic  Morton et al.,2000  NMDA /50pm/slice  <8  similar  presymptomatic  Levine et ai., 1999  >8  enhanced  symptomatic  Levine et al.. 1999  QA/30nM  10  resistant  symptomatic  MacGibbon et al., 2002  16-28 32  similar  presymptomatic/ symptomatic  S5U C57BL6  Petersen et al., 2002  P0  resistant  no phenotype  FVB  Slowet al., 2005  QA/4nM  12,24,40  resistant  71  NMDA /50pm/slice  <12  similar  presymptomatic  C57BL6  Levine et al., 1999  71  NMDA /50um/,slice  >12  similar  presymptomatic  C57BL6  Levine et al., 1999  94  NMDA /50pm/slice  <12  enhanced  presymptomatic  C57BL6  Levine et al., 1999  NMDA  >12  enhanced  presymptomatic  C57BL6  Levine et al., 1999  NMDA/1<*«  P0  enhanced  presymptomatic  FVB  Zeron et al., 2002  QA/8nM  24, 40  enhanced  symptomatic  P0  enhanced  presymptomatic  FVB  Graham et al  QA/4nM  12  enhanced  symptomatic  NMDA/1<*<>  P0  enhanced  presymptomatic  FVB  Graham et al  R6/2  -150/ 200  tg46/ tg100  46/ 100  QA/ 30nM  YAC 128/ shortstop  120  NMDA/1  knockin  knockin  94  ere  CBA/ C57BL6  /50Lim/slice  YAC72  YAC128/ HD55+/+ YAC128/ HD53  72  120  120  NMDA/1  ere  Hansson et al., 2001  Table 5.1 E x c i t o t o x i c i t y in H D mouse models. F u l l length models o f H D demonstrate enhanced sensitivity to excitotoxicity. In contrast, the truncated H D models shortstop, R 6 / 1 , R6/2 and N171 are resistant to Q A - i n d u c e d excitotoxic cell death.  115  5.2 Specificity of the fragment Caspases, calpains and aspartyl endopeptidases, cleave htt within the N-terminal region (Goldberg et al., 1996; Wellington et al., 1998; Wellington et al., 2000; Lunkes et al., 2002; Gafni et al., 2004), releasing fragments of mutant htt containing expanded polyglutamine repeats. Numerous in vitro and in vivo studies have demonstrated that mhtt toxicity occurs via formation of N-terminal fragments (Mangiarini et al., 1996; Davies et al., 1997; Hackam et al., 1998; Cooper et al., 1998; Schilling et al., 1999;Jana et al., 2001). However, not all N-terminal fragments of mhtt demonstrate selective HD-related toxicity. Furthermore, characterization of the shortstop H D mouse model reveals that expression of an N-terminal fragment of mutant htt containing exon 1 and 2 does not result in neurotoxicity, demonstrating that N-terminal htt fragments per se do not invariably result in pathology (Slow et al, 2005). In addition, while several studies have shown translocation of htt to the nucleus to be associated with increased toxicity in vitro and neuropathology in vivo (DiFiglia et al., 1997; Wheeler et al., 2000; K i m et al., 2001; Wellington et al., 2002; Slow et al., 2003; Van Raamsdonk et al., submitted), the shortstop mouse exhibits widespread diffuse nuclear htt (Slow et al., 2005), suggesting either that N-terminal fragments containing the mutation alone are insufficient to cause the disease or that a specific nuclear htt fragment underlies the neuronal degeneration observed in HD. In the Y A C 128 model, which expresses full length mutant htt, nuclear localization of htt fragments is earliest and to the greatest extent in the striatum, correlating with the neuropathology observed in the Y A C model and the primary area of neurodegeneration in H D patients (Van Raamsdonk et al., submitted). Activation of caspase-6 and nuclear translocation of fragments of mutant htt coincide with onset of motor abnormalities in the Y A C 128 model. In contrast, mice expressing mutant htt resistant to cleavage by caspase-6 perform similar to wild type mice on rotarod and in the open field, and translocation of nuclear htt is delayed. These observations suggest that nuclear translocation of a specific fragment of mutant htt may be crucial for triggering neuronal dysfunction and that inhibiting caspase-6 cleavage of mutant htt alters proteolytic pathways operative in the early stages of HD.  116  This evidence supports and further refines the toxic fragment hypothesis, originally proposed almost 10 years ago (Goldberg et al., 1996), by demonstrating that generation of a specific fragment of mutant htt in vivo represents a primary, initiating event in the pathogenesis of HD. The hypothesis that a specific fragment may underlie disease pathogenesis has been well characterized in Alzheimer Disease (AD). Amyloid-13 precursor protein (APP), is also cleaved by proteases, leading to generation of AB42 fragments which play an essential role in initiating the neurodegeneration observed in A D (Pellegrini et al., 1999; Gervais et al., 2002). Selective down regulation of the toxic AB42 fragment, and not A840, by therapeutic drugs such as NSAID, have been shown to ameliorate the disease phenotype in mouse models of A D clearly showing differences in toxicity levels of fragments which have only a 2 amino acid difference (Eriksen et al., 2003) The difference in amino acid length between the caspase-3 fragment (552aa) and the caspase-6 fragment of htt (586aa) is 34aa. Bioinformatic analysis of this region for posttranslation recognition sites and/or domain structures, which may explain the increased toxicity of the caspase-6 fragment of mutant htt, highlight 566 to 569aa as a potential Casein kinase II phosphorylation site, 553-558 as a possible N-myristoylation site and 471-581aa as a serine rich region. A number of posttranslation modification sites have been characterized within the N-terminal region of htt including phosphorylation at 421 and 434aa. (Humbert et al., 2002;Warby et al., 2005) and a palmitoylation site at 214aa (Yanai et al., 2005 abstr). Posttranslational modification of proteins such as phosphorylation and palmitoylation regulates trafficking and function of signalling molecules and receptors (El Husseini and Bredt, 2002). Furthermore, palmitoylation of key neuronal proteins plays a critical role in axon pathfinding and clustering of scaffolding proteins such as PSD95 (Strittmatter et al., 1995;Gauthier-Campbell et al., 2004). A cleavage event may disrupt these domains, altering normal signalling pathways and contribute to neurodegeneration.  117  5.3 Model for H D  The characterization of transgenic mice expressing specific forms of C R mutant htt has identified upstream molecular events underlying neurodegeneration in H D and highlighted the significant involvement of caspase-proteolysis of htt and excitotoxicity in initiating pathogenesis of HD. The mechanism underlying caspase-6 activation has not been determined in this thesis. However, the findings in the C6R mice and the extensive body of literature regarding excitotoxicity, apoptosis and HD, enables creation of a potential model for HD, detailing the sequence of molecular events which may underlie the pathogenesis of this disorder (Fig. 5.3). Work on the role of NMDAR-mediated excitotoxicity in H D has shown that excitotoxicity represents an early event and strongly implicates aberrant glutamate signalling and disrupted neuronal C a ^ handling in the pathogenesis of H D (Albin et al., 1990;DiFiglia, 1990;Beal et al., 1991;Beal, 1992;Young, 1997;Zeron et al.,2002; 2004;Slow et al., 2005;Tang et al., 2005). The knockin (Hdh94) and Y A C full length models of H D demonstrate increased susceptibility to excitotoxicity (Levine et al., 1999;Zeron et al., 2002; 2004;Tang et al., 2005). In sharp contrast, the results presented in this thesis show that caspase-6, but not caspase-3 resistant mutant htt, fully protects from excitotoxic stress, both in vitro and in vivo. Truncated H D mouse models including R6, N171 and shortstop also demonstrate resistance to Q A induced excitotoxicity (Hansson et al., 1999;Jarabek et al., 2004;Slow et al., 2005) and enhanced NMDAR-mediated cell death is not observed with a 548aa N terminal fragment of mutant htt (Zeron et al., 2001). The relationship between excitotoxicity and HD-specific pathology in mouse models of the disease suggests that caspase-6 cleavage of mutant htt mediates susceptibility to excitotoxic stress via a specific mutant htt fragment.  Excitotoxicity and the calcium source specificity hypothesis  118  Over-activation of glutamate receptors has long been considered an underlying mechanism leading to neurodegeneration by increased C a  ++  influx and downstream  activation of caspases, mitochondrial dysfunction and cell death (McGeer and McGeer, 1976;Coyle and Schwarcz, 1976;Beal et al., 1991). Furthermore, Ca"* " transients derived -1  from N M D A receptor do not lead to the same effect as C a ^ influx through voltagesensitive C a the C a  ++  ++  channels (Bading et al., 1993;Lerea and McNamara, 1993), giving rise to  source specificity hypothesis (Tymianski et al., 1993;Sattler et al., 1998). As one  example, the kinetics of the N M D A receptor-mediated current flux consists of single openings, and bursts or clusters of bursts (McBain and Mayer, 1994). Additionally, specificity of C a  + +  entry can be given by distinct assemblies of C a  + +  response components  of further signalling pathways around the mouth of the channel, as exemplified by the association of neuronal nitric oxide synthase (nNos) with the N M D A receptor through PDZ proteins (Kornau et al., 1995;Brenman et al., 1996). This link to other downstream molecules such as nNos, is a well established consequence of N M D A R channel opening.  PSD95 links NMDA receptors to downstream signaling molecules  N M D A receptors also interact with PSD95 (Brenman et al., 1996), a scaffolding protein critical for proper receptor clustering on the postsynaptic membrane (Craven and Bredt, 1998). PSD95 links the N M D A receptor to other downstream signalling molecules (Sattler et al., 1999) and increased levels of PSD95 have been shown to affect N M D A receptor signalling (Sun et al., 2001). Genetic manipulations resulting in decreased levels of PSD95 reduce NMDAR-mediated cell death by a pathway that involves reduced activity of nNos (Sattler et al., 1999). Furthermore, physical disruption of the N M D A receptor/PSD95 interaction via peptides dissociates N M D A receptors from downstream neurotoxic signalling in a stroke model (Aarts et al., 2002). Htt interacts with PSD95 and this interaction is decreased in the presence of the expanded polyglutamine tract, resulting in reduced PSD95/htt complex in HD human brain (Sun et al., 2001). A n alteration in intracellular levels of PSD95 and/or its interaction with mutant htt in H D brain may contribute to altered susceptibility to excitotoxicity.  119  The truncated htt expressing H D model N171, which demonstrates protection against Q A induced neurotoxicity, appears to generate a series of compensatory mechanisms to reduce the toxicity initiated by mutant htt in the presymptomatic stage (Jarabek et al., 2004). N M D A R subunit proteins show no alteration, however, a decrease in phosphorylation of NR1 at Ser 897 is observed which would predict a decrease in N M D A receptor current. Concurrent with this, the dopamine D l receptor, responsible for protein kinase A activation and phosphorylation of NR1, and levels of nNos and PSD95like proteins, were also reduced suggesting a neuroprotective mechanism to reduce N M D A R induced activity and excitotoxicity.  p53 is upregulated in response to excitotoxic stress  It is a well established fact that accumulation of the transcription factor, p53, is a consequence of glutamate induced neurotoxicity (Xiang et al., 1998;Culmsee et al., 2001;Liang et al., 2005) and also ischemia (Morrison and Kinoshita., 1996;Napieralski et al., 1999). P53 is upregulated in response to a wide variety of insults including D N A damage (Kruman et al., 2004;Joers et al., 2004), oxidative stress (Tamagno et al., 2003;Morrison et al., 2003) and cellular C a ^ overload (Chan et al., 2002) and downregulation of p53 (ie genetic deficiency, sRNAi or chemical inhibitors) protects neurons against excitotoxicity (Morrison et al., 1996;Lakkaraju et al., 2001 ;Culmsee et al., 2001;Morrison et al., 2003). Htt with an expanded polyglutamine binds p53 and upregulates nuclear p53, as well as p53 transcriptional activity (Steffan et al., 2000;Sipione et al., 2002;Bae et al., 2005). As caspase-6 is directly regulated by p53, increasing p53 levels may result in a decrease in the threshold for apoptosis execution through activation of caspase-6 (MacLachlan et al., 2002). Increased p53 levels are observed in brains of H D patients and transgenic mice (Trettel et al., 2000;Bae et al., 2005), which correlates with the low incidence of cancer reported in H D individuals (Sorensen et al., 1999). In addition, disruption of p53 activity prevents mitochondrial membrane depolarization and cytotoxicity in H D cells and suppresses neurodegeneration in transgenic flies and  120  behavioral abnormalities in transgenic H D mice (Bae et al., 2005). Furthermore, an increase in p53 accumulation following excitotoxic insult has been well characterized (Morrison et al., 1996;Liang et al., 2005) and over expression of p53 in primary neuronal cultures elicits neuronal death (Jordan et al., 1997). Upregulation of p53 by mutant htt and excitotoxicity may modulate excitotoxic induced C a  + +  overload where increased  caspase activation is already established. Evidence for p53's role in caspase activation is derived from studies of tumor derived p53 mutants where severe defects in regulation of pro-apoptotic genes are observed (Culmsee et al., 2001). p53 signaling and production of ROS are upstream of mitochondrial damage A three step model for p53 induced cell death has been developed based on transcripts induced by p53 expression before onset of apoptosis (Polyak et al., 1997). The initial transcriptional induction of redox-related genes is followed by formation of reactive O2 species (ROS) and oxidative degradation of mitochondrial components. ROS, which are observed post viral-mediated p53 over expression (Xiang et al., 1996;Sharpless and DePinho, 2002), are strong inducers of apoptosis and cause significant injury to mitochondria through generation of oxidative stress and caspase activation (Johnson et al., 1996;Kroemer et al., 1997). A potential explanation for the selective striatal cell death observed in HD, in addition to the cell type specific expression of caspases (Hermel et al., 2004), may be a reduced ability to cope with oxidative stress. Indeed the response to ROS has been shown to vary significantly with cell type and growth conditions (Kroemer et al., 1997). Treatment of cells with bongkrekic acid, which blocks mitochondrial permeability transition pore (MPTP) opening (Kroemer et al., 1997), does not reduce p53 induced gene expression nor ROS production, but significantly inhibited apoptosis (86%). This evidence demonstrats that p53 signalling and production of ROS are upstream of mitochondrial damage (Polyak et al., 1997). Pre-treatment with bongkrekic acid in primary striatal cultures containing mutant htt dramatically reduces NMDA-mediated cell death (Zeron et al, 2004; Tang et al., 2005).  p53 and NF-Kfi compete for binding to CBP/p300 transcription factors  121  P53 mediated transcriptional repression, by p53 binding to the transcriptional cofactors p300 and CBP, may also contribute to dysfunction and apoptosis in HD.  NF-KB,  a  transcription factor involved in survival pathways, and p53 compete for binding to p300/CBP, which is a required component for the proper function of both transcription factors (Zhong et al., 2002;Chen et al., 2002). Cellular stress results in decreased binding of N F - K B to p300 and increased binding of p53 to p300 (Wadgaonkar et al., 1999;Ikeda et al., 2000). P53 mediated activation of programmed cell death and reduced signalling of survival pathways such as N F - K B suggest there may be a reciprocal regulation of p53 and N F K B which decides between activation of survival or cell death pathways (Culmsee et al., 2001).  Nuclear translocation of NF-Kfi is associated with toxicity in an ex vivo model of HD Elevated C a ^ influx, through NMDAR-mediated excitotoxicity, is a major activator of 1-KB kinase (IKK), a signal activated kinase complex which affects  NF-KB  activation (Qin et al., 1999). N F - K B is normally sequestered in the cytoplasm by a family of inhibitory proteins  (IKBS)  (Ghosh et al., 1998). When I K B S are activated by I K K  phosphorylation, proteolytic release and nuclear translocation of N F - K B occurs (Ghosh and Karin, 2002). Mutant htt activates the I K B complex, causing elevated activity and nuclear localization of N F - K B in cultured cells and striatal neurons from H D transgenic mice (Khoshnan et al., 2004). Furthermore, mutant htt interacts directly with IKKy, a regulatory subunit of I K B and promotes aggregation and nuclear localization of mutant htt while inhibition of I K K activity blocks mutant htt induced toxicity in acute striatal slice cultures (Khoshnan et al., 2004). Neurodegenerative models including ischemia, brain trauma and H D lesion models also demonstrate activation of N F - K B and expression of p53 (Grilli et al., 1996;Clemens et al., 1997;Qin et al., 1999;Nakai et al., 2000;Mattson et al., 2000). The observation of nuclear translocation of a prosurvival factor such as in H D may be explained by caspase-mediated degradation of pro-apotpotic fragment. Caspase-6-mediated cleavage of  NF-KB  NF-KB  NF-KB  and formation of a  leads to a  transcriptionally inactive p65 molecule. The p65 truncated fragment acts as a dominant-  122  negative inhibitor of  NF-KB,  promoting apoptosis, whereas caspase-6-resistant p65  protects cells from apoptosis (Levkau et al., 1999). Generation of the dominant-negative fragment of p65 during apoptosis may be an efficient pro-apoptotic feedback mechanism between caspase activation and N F - K B inactivation.  nNos and NO are downstream neurotoxic effects of NMDAR-mediated excitotoxicity  A further, well established consequence of the N M D A R channel opening and Ca  + +  influx is nNos activation, an important mediator of neurotoxicity and cellular  dysfunction (Dawson et al., 1991;Ayata et al., 1997;Eliasson et al., 1999;Sattler et al., 1999). nNos converts 1-arginine into N O (Dawson and Dawson, 1996), a gas involved in several physiological processes in the brain including neuromodulation, neurotransmission, synaptic plasticity and neurodegeneration. A rise in cytosolic C a  + +  triggered by N M D A R activation stimulates calmodulin dependent nNos and N O production in a C a ^ dependent manner (Brenman et al., 1996). N O together with oxygen-free radical species form peroxynitrite, a stable free radical that causes neuronal damage and cell death (Calabrese et al., 2000). The etiology and pathogenesis of neurodegenerative disorders such as H D is unknown. However, there is strong evidence that perturbation of the cellular oxidant/ antioxidant balance and reactive nitrogen species play a significant role (reviewGrunewald and Beal, 1999). When the rate of free radical generation exceeds the capacity of the cells defence mechanism, oxidative stress results triggering toxic signalling pathways and the eventual death of the cell. There are currently two characterized pathways that link htt and NO. These include the htt/HAP-l/calmodulin/Nos link and the CBP/htt/Nos link. Htt is known to bind to HAP-1 (Li et al., 1996) and the htt/HAP-1 complex binds calmodulin, a major regulator of nNos (Bao et al., 1996). Increased calmodulin binding is observed in the presence of mhtt. It is unclear what this effect has on Nos activity, however increased or decreased activity of C A M could result in inhibition of nNos activity. HAP-1 also interacts with the type 1 inositol (l,4,5)-triphosphate receptor (IP3I) forming a IP3IHAPl-htt complex (Tang et al., 2003). In the presence of mutant htt, enhanced sensitivity  123  of IP31 to inositol (1,4,5) triphosphate is observed, resulting in Ca stores which may further contribute to C a  ++  release from E R  overload and cellular stress (Tang et al.,  2003). Pre-treatment of primary striatal neurons containing mutant htt with IP 1 3  inhibitors results in partial protection from glutamate induced cell death (Tang et al., 2005). C a ^ levels affect nNos levels through a promoter on exon 2 of the nNos gene (Sasaki et al., 2000). This promoter is also responsive to C R E B , suggesting CBP is also an important regulator of nNos expression. As htt interacts with CBP, and this is increased in the presence of mhtt, this may also result in altered nNos levels (Steffan et al., 2000;Nucifora, Jr. et al., 2001). Manipulation of nNos levels by inhibitors or genetic deletion results in protection from NMDAR-mediated cell death in vivo (Perez-Severiano et al., 1998) and reduced N M D A neurotoxicity in cortical cultures (Dawson et al., 1994;Gunasekar et al., 1995). Conversely, N O has been shown in some studies to be neuroprotective by downregulation of N M D A R activation, upregulation of heat shock proteins and inhibition of  NFKB.  The dual role of protection and mediator of toxicity observed with N O is similar  to other proteins such as N F K B and suggests that it may rather be a critical balance and/or the intracellular redox state that determines which pathway is triggered. Decreased nNos is observed in truncated H D mouse models (R6/1 and R6/2, N171), which show protection from excitotoxic induced apoptosis (Gordinier and Deckel, 2000;Jarabek et al., 2004; Hansson et al., 1999;2001), while administration of the precursor to NO, 1-arginine, speeds the disease process in H D mice (Deckel et al., 2000). Furthermore, studies of HD lesion models, including 3-NP and QA, show that peroxynitrite is involved in the neurodegeneration, supporting a role for NO/nNos in H D (Galpern et al., 1996;Nishino et al., 2000). nNos m R N A is reduced in the striatum of HD patients and this is more pronounced as the disease state advances (Norris et al., 1996). However, assessment of cerebral brain fluid in early stage HD patients has revealed increases and decreases in vascular activity, an indirect measurement of NO production (Deckel and Duffy, 2000). Whether the decreased blood flow velocity reflects a compensatory mechanism against  124  excitotoxic stress is currently unclear and further studies are required to determine the role of NO/nNos in the pathogenesis of HD.  p53 accumulation  and caspase-6 activation are upstream neuronal toxic insults of  excitotoxic induced cell death  Excitotoxic induced H D lesion models (i.e. K A or QA) also demonstrate, in , addition to p53 accumulation, amplification and activation of caspase-6 (Narkilahti et al., 2005), a transcriptional target of p53 (MacLachlan et al., 2002). The underlying mechanism involves D N A binding of p53 to the third intron of caspase-6 and transactivation. Overexpression of p53 in cell culture has been demonstrated to cause an increase in procaspase-6 levels which is followed by an increase in caspase-6 activity and cleavage of caspase-6 specific substrates (MacLachlan et al, 2002). The work in this thesis supports the crucial involvement of caspase-6 in the pathogenesis of HD. Furthermore, it has been shown that caspase-6 selectively cleaves CBP (Rouaux et al., 2004), suggesting a function for caspase-6 in the altered gene transcription observed in H D through cleavage of transcription factors. In addition, caspase-6 is the major protease which cleaves cleaves Akt (Medina et al., 2005), a serine/theronine kinase that has demonstrated protective effects in a number of model systems (Datta et al., 1999;Noshita et al., 2002). Decreased phosphorylation of mutant htt by Akt has been described in human and mouse models of H D and loss of its protective effect implicated in the pathogenesis of H D (Humbert et al., 2002;Warby et al., 2005). Caspase-6 activation is also observed post kainate-induced epileptogensis (Narkilati, 2004) and, as may be expected, is activated early in response to ischemic induced damage (Harrison et al., 2001; Singh et al., 2002;Nu et al., 2004) further supporting the role of caspase-6 in excitotoxic induced cell death.  Activation of caspase-6 and nuclear translocation of htt fragments coincide wih onset of neuronal dysfunction in the YAC128 model  Activation of caspase-6 and translocation of fragments of htt to the nucleus coincide with onset of motor abnormalites in the Y A C 128 model, suggesting that nuclear  125  translocation of a specific fragment of mutant htt may be responsible for triggering neuronal dysfunction. Nuclear entry of the caspase-6 fragment of htt may have devastating consequences such as permanently altering interactions with htt's cytoplasmic interacting partners, some of which have been hypothesized to contribute to the pathogenesis of H D (Li et al., 1995; Kalchman et al., 1996; Kalchman et al., 1997; Wanker et al., 1997). In addition the nuclear localization of the caspase-6 fragment of htt containing an expanded polyglutamine tract may influence the transcriptional dysregulation observed in HD, which may be mediated by permitting interaction of htt with nuclear proteins that normally do not occur.  Activation of caspase-2 is a downstream consequence of caspase 6 processing  Caspase-6 was originally identified as an executioner caspase due to its role in cytoskeletal alterations of the nuclear envelope through cleavage of nuclear lamins (Srinivasula et al., 1996). However, caspase-6 has since been shown to also function as an initiator caspase through its ability to cleave and activate caspase-2 and -3 (Xanthoudakis et al., 1999;Allsopp et al., 2000; Henshall et al., 2002). Enhanced immunoreactivity of activated caspase-2 in medium sized neurons of Y A C 7 2 striatum and human H D post-mortem caudate has previously been observed (Hermel et al., 2004) and we have shown upregulation of caspase-2 subsequent to caspase-6 activation in human presymptomatic H D striatum. Caspase-2, the most evolutionarily conserved caspase, is an initiator caspase that acts upstream of mitochondria by inducing Bid cleavage and Bax translocation to the mitochondria and cytochrome c release (Lassus et al., 2002;Troy and Shelanski, 2003). Activation of caspase-2 is required for permeablization of mitochondria demonstrating that the initiator caspases-6 and -2 are upstream of mitochondrial changes including loss of mitochondrial membrane potential and cytochrome c release (Miyashita et al, 1998).  Increased interaction with mutant huntingin and caspase-2 is observed in HD and correlates with decreased BDNF  levels  126  Htt interacts with caspase-2 in a polyglutamine repeat dependent manner in vitro and in vivo and recruits caspase-2 into an apoptosome-like complex (Hermel et al, 2004) suggesting that caspase-2 activation may be a critical step in H D cell death. This hypothesis is supported by the observation that co-expression of the catalytically inactive form of caspase-2 and mutant htt in primary striatal neurons reduces cell death post C a  ++  exposure. Upregulation of caspase-2 in Y A C 7 2 striatum and cortex has been demonstrated to correleate with decreasing levels of B D N F (Hermel et al., 2004), a prosurvival factor produced by cortical projections and delivered to striatal neurons, and suggests modulation of caspase-2 expression by B D N F . This correlates with the requirement of caspase-2 for trophic factor deprivation (NGF) - induced death (Troy et al., 2001) and the demonstration that overexpression of B D N F rescues striatal neurons in rats exposed to excessive glutamatergic signals (Perez-Navarro et al., 2000). Interestingly, wild type htt has been shown to regulate transcription of B D N F , and a decrease in B D N F levels is observed in the presence of mutant htt in vivo (Zuccato et al., 2001). Decreased levels of B D N F would cause insufficient neurotrophic support for striatal neurons and a concurrent increase in caspase-2 further contributing to amplification of the apoptotic cascade in H D brain.  Mitochondrial  dysfunction and cytochrome c release trigger caspase-3 and cell death  Initially, functional mitochondrial activity and A T P availability are required for apoptosis induced by glutamate neurotoxicity. However, at a certain point the mitochondria are no longer able to sequester the increasing C a  ++  influx into the cell.  Activation of caspase-2 and bax translocation to the mitochondria results in mitochondrial permeabilization and M P T P opening, disabling the mitochondria and causing mitochondrial dysfunction. Substantial evidence in the literature supports the involvement of mitochondrial dysfunction in HD. Initial clues regarding the role of mitochondria in H D were derived from animal studies which demonstrated that intrastriatal injection of mitochondrial toxins in mice led to neuronal degeneration similar to that observed in human H D  127  (Brouillet et al., 1995). Further investigation has revealed striatal-specific mitochondrial defects in post mortem brains of H D patients (Gu et al., 1996;Browne et al., 1997;Tabrizi et al., 1999). Furthermore, mitochondria isolated from lymphoblast of H D patients are less resistant to induction of the M P T upon C a lower C a  + +  ++  challenge (Panov et al., 2002) and a  threshold is observed to trigger M P T opening in striatal cells containing  mutant htt (Choo et al., 2004). Mutant htt may also directly influence mitochondrial C a  + +  handling by forming ion channels in the mitochondrial membrane (Bezprozvanny and Hayden, 2004). Evidence for a role in mitochondrial dysfunction in potentiating NMDAR-mediated excitotoxicity in H D is also observed in primary striatal cultures derived from H D mouse models. Use of mitochondrial inhibitors such as cyclosporine A and bongkrekic acid substantially diminishes NMDA-mediated cell death (Zeron et al., 2004;Tang et al., 2005). Once the mitochondria reach the threshold for C a ^ handling, MPTP opening and subsequent permeabilization and cytochrome c release would trigger downstream activation of caspase-3. Amplification and activation of caspase-3 is generally described as occurring in the end stages of apototsis and reflects the cell's full commitment to programmed cell death. While activated caspase-3 has been observed in H D cell culture and mouse models (Sawa et al., 1999;Kim et al., 2001;2002;Yu et al., 2003;Toulmond et al., 2004), studies on the time line of caspase-3 involvement in H D are limited as, in general, most studies only focused on caspase-3 and did not assess the contribution of other caspases. The limited studies available which did assess caspase-3 and caspase-6 are the K A / Q A lesion model of HD. In this paradigm caspase-6 is observed prior to caspase-3 (Ferrer et al., 2000; Henshall et al., 2002). The evidence regarding caspase-3 in H D is controversial. Caspase-3 activation is observed in primary MSNs containing mutant htt post N M D A induced excitotoxicity (Zeron et al., 2002;2004) and it has been detected in mouse models of H D (Yu et al., 2003). Although, use of caspase-3 inhibitors did eliminate NMDA-induced death of MSNs containing mutant htt (Zeron et al., 2002) they did not improve cell survival in another in vitro model of H D (Kim et al., 1999). Caspase-3 expression is observed in human post-mortem H D striatum, however it localized to astrocytes and not neurons (Hermel et al., 2004). This is not an insignificant result as  128  caspase-3 activation in astrocytes may stress neighboring neurons through insufficient support and/or release of apoptotic inducing signals (an emerging hypothesis in the apoptosis field is cell murder). Finally, caspase-3 cleaved fragments of htt have been detected in human H D brain (Wellington et al., 2002). Mhtt induced neurotoxicity, including increasing C a ^ load, ROS production, caspase activation and aberrant signaling may all contribute to triggering the final step of caspase-3 activation in the cell death pathway. The work in this thesis coupled with the extensive body of literature on caspase-3, would support the downstream role of this caspase in the neurodegeneration observed in HD. In conclusion, I have delinated a pathway, based on my work and the substantial body of literature regarding HD, apoptosis and excitotoxicity that I believe may highlight important insights into the pathogenesis of HD. It is impossible to include all the many signaling events that may occur as a result of mutant htt induced toxicity. However, I believe there are enough pieces of the puzzle to start to seriously determine key drug targets that may lead to a cure for this devastating and presently terminal illness. I hope that one day HD will be an illness of past and that future generations will no longer go through the agony of watching their family members suffer such a terrible disease.  129  EXCITOTOXICTY  , PSD95/mhtt ^  THAP/mhtt  L  * k ^ER J  Caspase-3  InspF  t  ROS  Ca++ |  cell death  C B P  Caspase 6  i full length htt mhtt /*r———c l e a v a g e * toxic htt fragment  TP53 C ' * / ^CBP/p30qJ / PIDD | WW'  X Caspase-2  INFKB — tp65  cytochrome c release  BID cleavage  TC-2/mhtt  /  Mitochondrial permeabilization  •V  .akt  1  mhtt nuclear localization  J.BDNF  /  /  TCBP/mhtt  /  transcriptional dysregulation altered protein interactions  impaired energy production  Figure 5.1 Model for H D Overactivation o f glutamate receptors and altered P S D 9 5 signalling results in increased C a * * influx into neurons containing mutant htt. triggering upregulation and accumulation o f p53, a direct regulator o f caspase-6. A toxic feedback loop o f caspase-6 activation and cleavage o f mutant htt generates a proapoptotic 586aa fragment o f mutant htt, causing further amplification o f the cycle. Deleterious secondary effects include nuclear translocation o f a proapoptotic NFKB truncated fragment and decreasing C B P and A k t levels through cleavage by caspase-6. Downstream caspase-2 activation, decreased neurotrophic B D N F and B I D translocation to the mitochondria, further contribute to lowering the threshold required for commitment to apoptosis. Additional insults include altered htt protein interactions (pictogram at lower left) and transcriptional dysregulation due to nuclear translocation o f htt and altered p53/htt interactions. Mitochondrial permeability transition pore opening and cytochrome c release cause activation of caspase-3 and full commitment to programmed cell death.  130  5.4 Future directions The results presented in this thesis highlight several avenues of research to further explore the molecular events underlying caspase proteolysis of mutant htt and excitotoxicity in the pathogenesis of HD.  1) What therapeutic targets should be considered? Activated caspases are observed in human and mouse HD brain and caspase cleaved htt present in presymptomatic H D brain, highlighting the role of caspase cleavage of htt in the pathogenesis of HD. The characterization of the C R mice emphasize the importance of specific caspases in the initiation of disease and further studies to assess the Y A C 128 model on a caspase deficient background and/or the use of caspase inhibitors as a therapeutic approach in the Y A C model. This would first establish, without doubt, that the 586aa site in htt is cleaved by caspase-6, and would further determine whether in vivo, complete knockdown of a caspase is a potential therapeutic strategy for HD. Caspases are involved not only in PCD but also play key roles in learning and memory. Complete knockdown of caspase levels in vivo may result in detrimental side effects. The following caspases may be considered: i. Caspase-6 inhibitor - At the present time there is no available caspase-6 knockout. Peptide inhibitors have been developed, however, they do not penetrate the blood brain barrier. Thus, intitally, intrastriatial injections may need to be considered. A further approach would be viral mediated delivery of siRNA against caspase-6. ii. Caspase-2 inhibitor - Transgenic crosses of the caspase-2 knockout with the Y A C 128 model would determine whether complete elimination of caspase-2 alleviates the HD phenotype in the Y A C 128 model. Another approach may be viral-mediated siRNA against caspase-2 and/or transgenic cross of Y A C 128 with mice over expressing X I A P or NIAP. iii. Caspase-1 inhibitor -Transgenic cross of caspase-1 knockout with the Y A C 128 model and/or use of caspase-1 inhibitors would determine whether the H D phenotype in the Y A C 128 model is influenced by caspase -1.  2) Does intrabody therapy, using intrabodies against the 5686aa site in htt, reproduce the neuroprotective phenotype observed in the C6R mice? A further possibility, which would more accurately represent the situation in the C6R study, would be an intrabody approach with antibodies developed to specifically bind the 586aa site in full length htt and inhibit caspase cleavage at that site. The intrabody approach would alleviate the possible adverse effect of down regulation of potentially important proteins (caspases).  3) Are proteins implicated in excitotoxicity altered in the YAC128 model of HD? 131  Substantial evidence supports the involvement of excitotoxicity in the pathogenesis of HD. Furthermore, caspase-6 cleavage of mutant htt influences susceptibility to excitotoxic stress, highlighting caspase 6-mediated proteolysis of htt and excitotoxicity as primary mechanisms of striatal atrophy in HD. i.  ii.  iii.  iv.  p53 - Involvement of p53 in excitotoxicity is well established. Upregulation of p53 is observed in human HD brain and reducing levels of p53 alleviate symptoms in a mouse model of HD. PSD95 - Alteration in PSD95 interaction with N M D A receptors effect downstream signalling and excitotoxicity. Decreased interaction of PSD95 with mutant htt is already established and disruption of the PSD95 interaction with N M D A receptors is therapeutic in mouse models. nNos - N O production, through nNos, is implicated in contributing to neurodegeneration. nNos knockouts demonstrate protection from N M D A mediated excititoxocity and a link between htt/NO is already established in the H D literature. N F K B - Cleavage of I K K and nuclear translocation of N F K B results in altered gene transcription and downstream cell death. Mutant htt directly interacts with I K K y suggesting possible alterations in N F K B surivial and/or apoptotic pathways.  4) Do levels of akt and phoshorylated huntingtin influence the neuroprotective phenotype observed in the C 6 R mice? Caspase-6 is the major protease which cleaves cleaves Akt, a serine/theronine kinase which mediates protective signalling in a number of model systems. Decreased phosphorylation of mutant htt by Akt has been described in human and mouse models of HD and loss of its protective effect implicated in the pathogenesis of HD. Conversely, rat hippocampal neurons exposed to AB undergo neuronal death and do not demonstrate a decrease in p-akt levels (ie tpakt levels associated with cells undergoing apotosis). In addition, interaction of akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in SCA1. (i.e. Phosphatidylinositol 3-kinase/akt signaling and 14-3-3 cooperated to modulate the neurotoxicity of ataxin)  5) Does expression of caspase-6 resistant mutant htt together with caspase-cleavable mutant htt alter the HD phenotype in the Y A C 1 2 8 model? A transgenic cross of the Y A C 128 model and the C6R mice would determine whether, even in the presence of caspase-cleavable mutant htt, C6R mutant htt is able to provide protection and that the therapeutic efficacy of only inhibiting 50% of caspase-6 cleavage of mutant htt results in protection. As mutant htt demonstrates a dosage dependent phenotype it may be expected that at minimum a delay of symptoms will be observed.  6) Do levels of wild type htt influence the phenotype in transgenic mice expressing C R htt?  132  Characterization of transgenic mice expressing CR mutant htt on a htt deficient background would determine whether endogenous, wild type htt influences the protection observed in the C6R mice and lack of protection present in the C3R lines. Overexpression of wild type htt on the YAC 128 model background does alleviate behavioural symptoms however does not substantially affect neuropathology. Furthermore, detailed characterization of these lines would establish whether caspase cleavage of htt plays a role in development and maturation of neurons. 5.5 Conclusion  Considerable advances have been made in the understanding of the pathogenesis of HD. However, to date no effective treatments are available to alter the course of the disease. The findings from the in vivo characterization of mice expressing C6R mutant htt has shed light on the molecular events underlying the pathogenesis of HD and strongly implicates proteolysis of mutant htt and deranged neuronal calcium signalling via excitotoxic pathways in the pathogenesis of HD. These studies highlight caspase-6 as a validated drug target and possible therapeutic approach in the Y A C 128 model.  133  Chapter 6: Bibliography  Bibliography (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. 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