Michael Hayden

Huntington disease (HD) is a devastating neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Early disrupted cortico-striatal (CS) transmission contributes to neuronal spine and synapse dysfunction primarily in striatal medium spiny neurons (MSNs), the most vulnerable cell type in HD, as well as in cortical neurons. Irreversible neurodegeneration of MSNs occurs with disease progression, leading to motor, cognitive, and psychiatric symptoms. However, synaptic dysfunction and spine loss are hypothesized to be therapeutically reversible before neuronal death occurs. One of the earliest alterations to occur in HD mouse models is enhanced striatal extrasynaptic (ex) N-methyl-D-aspartate receptor (NMDAR) expression and activity. This activity is mediated primarily through GluN2B subunit-containing receptors and is linked to increased activation of cell death pathways, inhibition of survival signaling, and greater susceptibility to excitotoxicity. Death-associated protein kinase 1 (DAPK1) is a pro-apoptotic kinase highly expressed in neurons during development. DAPK1 becomes re-activated and recruited to exNMDARs during ischemia where it phosphorylates GluN2B at S1303, amplifying receptor function. Approaches to reduce DAPK1 activity have demonstrated benefit in animal models of stroke, Alzheimer disease, Parkinson disease, and chronic stress, indicating that DAPK1 may be a novel target for neuroprotection. The overall hypothesis of this thesis was that aberrant DAPK1 activity contributes to exGluN2B dysfunction in HD, leading to early CS synaptic instability. An in vitro CS co-culture model was extensively optimized to allow rapid and robust detection of HD-like CS synaptic phenotypes, including MSN spine loss. Early dysregulation of DAPK1 was observed in the YAC128 HD mouse model, which was associated with elevated exGluN2B pS1303. Inhibition of DAPK1 normalized exGluN2B phosphorylation and surface expression, and completely prevented YAC128 MSN spine loss in CS co-culture. DAPK1 silencing restored nuclear CREB activation and partially rescued age-associated dendritic spine loss in vivo, thus validating DAPK1 as a target for synaptic protection in HD and warranting development of DAPK1-targeted therapies for neurodegeneration.

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Huntington disease (HD) is a devastating neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). A repeat of ≥36 CAG defines the HD mutation and is diagnostic in the presence of motor or psychiatric phenotypes. All patients have an expanded CAG repeat, usually inherited from an affected parent and heterozygous with a normal (≤35) CAG repeat allele. This thesis addresses numerous gaps in our understanding of the HD mutation at the population level, including heterozygote frequency, penetrance, ancestry, the de novo mutation rate, and haplotypes useful for therapeutic gene silencing. Dense SNP genotyping across HTT in HD patients and relatives from Canada, Sweden, France, and Italy allowed definition of the three most common HD mutation haplotypes in populations of European ancestry. All common defining alleles of these haplotypes were identified from the 1000 Genomes Project reference data and validated by direct genotyping of HD patients and their families. Haplotypes of the HD mutation provide mutually exclusive sets of target variants for allele-specific HTT silencing in the maximum number of European ancestry patients. Haplotypes of the HD mutation were investigated in additional HD patient populations worldwide. The HD mutation in Peru occurs most frequently on the A1 HTT haplotype, as in Europe, but on a distinct A1 variant found in Amerindian controls, supporting an indigenous origin of the HD mutation in Latin America. The general population frequency of the HD mutation was estimated from CAG repeat genotyping of 7315 individuals in Canada, the United States, and Scotland, revealing that approximately 1 in 400 people (0.246%) has a CAG repeat of 36 or greater. This frequency of the HD mutation in the general population is higher than expected from clinical prevalence estimates. The normal CAG repeat distribution was characterized in a range of ethnically distinct populations, allowing comparative estimates of the HD new mutation rate. Estimated as a function of intermediate CAG repeat frequencies below the pathogenic range (27-35 CAG), 1 in 5372 births is a new mutation for HD in European ancestry populations, representing 7.1% of HD mutations (≥36 CAG) in the general population.

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Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. HD is caused by a CAG repeat expansion in the huntingtin (HTT) gene leading to the production of the mutant huntingtin protein (mHTT). Caspase-6 (C6) is a cysteine aspartyl protease that plays a central role in apoptosis and has been postulated to play a role in inflammation. Increased C6 activation is observed in human HD brains and mouse models and the inhibition of C6-mediated cleavage of mHTT protects against neuropathology and behavioural deficits in the YAC128 mouse model of HD. Additionally, alterations in inflammation are a feature of many neurodegenerative diseases, including HD. Hyperactive inflammatory responses are observed in both HD patients and mouse models and C6 has been postulated to play a role in mediating inflammation. Constitutive deletion of the Casp6 gene (denoted as C6) in YAC128 mice results in a partial rescue of some features of HD; however, the continued presence of the 586 cleavage fragment in the absence of C6 suggests possible compensation by other proteases. The goal of this thesis was to investigate the impact of modulating C6 in the adult YAC128 mouse and to further characterize the role of C6 in inflammation. To that end, the C6 gene was partially deleted in the adult YAC128 mouse and characterization of these mice reveals no amelioration in motor or cognitive phenotypes but a modest improvement in certain psychiatric behaviours. Neuropathological assessment shows no attenuation in canonical brain pathology but peripherally, the loss of C6 attenuates the overactive inflammatory response observed in YAC128 mice. These data suggest that partial loss of C6 in the brain is not sufficient to improve most behavioural and neuropathological phenotypes but implicate C6 in the regulation of inflammation. Furthermore, loss of C6 results in a blunted inflammatory response characterized by reduced cytokine release. As the presence of elevated cytokine levels have been suspected to cause psychiatric behaviours such as depression, this finding provides a possible mechanistic link between C6 activity and the onset of affective behaviours.

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Huntington disease (HD) is caused by a CAG expansion in HTT characterized by motor, cognitive, and psychiatric disturbances. Huntingtin Interacting Protein 14 (HIP14) and HIP14-like (HIP14L) are palmitoyl acyltransferases (PATs) that mediate the post-translational addition of fatty acids to proteins (palmitoylation). They palmitoylate HTT and have reduced interaction with and palmitoylation of mutant HTT (mHTT), leading to increased mHTT inclusion formation and toxicity. HTT is essential for full enzymatic activity of HIP14 and loss of either of these genes leads to HD-like phenotypes. The goal was to determine the role of palmitoylation in the pathogenesis of HD. The overall hypothesis is that disturbed HIP14- and HIP14L-HTT interaction in HD reduces PAT function leading to the under-palmitoylation and mislocalization of HTT and key HIP14 and HIP14L substrates. Multiple putative PAT binding sites in HTT were identified, one around aa224 and one around aa427, that are required for full interaction but aa1-548 are required for the structural integrity of the binding sites. Loss of both Hip14 and Hip14l leads to embryonic lethality between day 10 and 11 in utero, due to failed placenta formation. Intriguingly, the extraembryonic tissue of Hip14-/-;Hip14l-/- embryos share many features with that of Htt-/- embryos and palmitoylation of HTT was decreased by 25% in Hip14-/-;Hip14l-/- mouse embryonic fibroblasts. Palmitoylation of mHTT, SNAP25, and PSD-95 was decreased in the YAC128, BACHD, and Hu97/18 mouse models of HD. The HD-like phenotype of the Hip14-/- mice is developmental and non-progressive, unlike the adult-onset, progressive phenotype of the YAC128 mice. Mice in which Hip14 deficiency is induced in adulthood show reduced survival, motor deficits, anhedonia, increased escape response, increased forebrain weight and cortical volume, and decreased corpus callosum volume. This indicates that loss of Hip14 from conception allows for developmental compensation that cannot occur if Hip14 deficiency occurs in the adult.

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Intermediate alleles (IAs) for Huntington disease (HD) have between 27–35 CAGrepeats. While they usually do not confer the HD phenotype, they are prone togermline CAG repeat instability. Consequently, offspring are at-risk of inheriting anexpanded allele in the HD range (≥36 CAG). Currently there are numerous gaps inour molecular and clinical knowledge on IAs despite their characterization almost 20years ago. This thesis utilized a unique mixed-method design of molecular andqualitative techniques in order to generate new knowledge on the frequency,haplotype, and CAG repeat instability of IAs and explored current geneticcounselling practices and patient understanding and interpretation of an IA predictivetest results (PTR).In the Huntington Disease Biobank at the University of British Columbia, 30%(n=54/181) of IA familial transmissions demonstrated intergenerational CAG repeatinstability. Of these unstable transmissions, 14% were repeat expansions into thedisease-associated range. In a sample of British Columbia’s general population, withno known association to HD, 5.8% (n=92/1594) of individuals were found to have anIA. Of the IAs ascertained in this general population sample, 60% were onhaplotypes associated with a high-risk of CAG repeat instability. Paternal CAG-sizespecific risk estimates for repeat instability, including repeat expansion into the HDrange, were established using sperm (n=18763) from 31 males with an IA. Alleles atthe upper limits of the intermediate CAG size range (34-35 CAG) had the mostsignificant risk (i.e. 2.5-21.0%) of expanding into the disease range. Interviews withmedical genetics service providers and individuals who received an IA-PTR revealedpre-test genetic counselling practices vary based on the individuals’ family historyand that clients struggled to understand the clinical implications and significance oftheir IA-PTR.This thesis substantially contributes to our knowledge of IAs for HD. Collectively thecomprehensive findings have important implications for genetic counselling and willhelp ensure individuals undergoing predictive testing receive appropriate support,education, and counselling on IAs.

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Huntingtin Interacting Protein 14 (HIP14) is a palmitoyl acyl transferase (PAT)that was first identified due to altered interaction with mutant huntingtin, theprotein responsible for Huntington Disease. HIP14 palmitoylates a specific set ofneuronal substrates critical at the synapse, and downregulation of HIP14 bysiRNA in vitro results in increased cell death in neurons. Recent findings haverevealed that mice lacking murine Hip14 (Hip14-/-) demonstrate a Huntington-Disease-like phenotype. In the current study, we have generated andcharacterized human HIP14 BAC transgenic mice. We generated humanizedHIP14 transgenic mice by crossing the HIP14 BAC mouse to the Hip14-/- model.Rescue of the Hip14-/- phenotype indicates that the defects seen in Hip14-/- miceare in fact due to loss of HIP14. In addition, our findings indicate human HIP14can compensate for the loss of the murine ortholog, and that very low levels ofHIP14 are sufficient to rescue the Hip14-/- phenotype. Finally, we assesspatterns of HIP14 expression in early development. Our findings further ourunderstanding of HIP14 in vivo, and point to several potential avenues for futurestudies.

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Epidemiological studies consistently demonstrate an inverse relationship between HDL levels and cardiovascular disease (CVD), independent of LDL and triglyceride levels. Due to the crucial role ABCA1 plays in HDL biogenesis, increasing ABCA1 expression is considered an attractive strategy to increase plasma HDL levels. In this thesis we attempt to identify novel post-transcriptional and post-translational mechanisms that regulate ABCA1 expression and/or function. Prior to translation, ABCA1 protein expression is regulated by non-coding RNA molecules known as microRNAs which bind and inhibit translation of mature mRNA transcripts in the cytoplasm. In this study we used bioinformatic prediction programs to identify potential microRNA regulators of ABCA1. Using reporter constructs, protein expression analysis by immunoblotting, and cholesterol efflux assays, we validated microRNA-145 as a novel repressor of ABCA1 translation. The inhibition of endogenous microRNA-145 in HepG2 cells increases both ABCA1 protein levels and cholesterol efflux activity. The inhibition of this microRNA in the liver is a potential strategy to increase HDL levels. Following translation, numerous post-translational modifications and protein-protein interactions are required for the ABCA1 protein to function properly. In this study we identified palmitoylation as a novel post-translational modifier of ABCA1. The majority of ABCA1-mediated cholesterol efflux and HDL biogenesis occurs at the cell surface. We show that palmitoylation is a crucial lipid addition for proper ABCA1 plasma membrane localization. We also identify a number of enzymes that mediate the incorporation of radio-labeled palmitate onto ABCA1, and demonstrate that the overexpression of the palmitoyl transferase enzyme DHHC8 increases ABCA1 palmitoylation and cholesterol efflux activity. The increase of ABCA1 palmitoylation in the liver is a novel strategy to increase HDL levels. In this thesis, we have contributed to the understanding of ABCA1 biology by the identification of two novel regulators of ABCA1 expression and/or function.

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Genetic knowledge holds great promise in terms of health benefits, yet also raises challenges regarding the delivery of beneficial testing and services. Addressing this challenge is especially important in rural areas where lack of access to clinical genetics is pervasive, resulting in considerable inequities in service availability. The purpose of this research study is to explore the hypothesis that a novel telehealth strategy for delivering predictive testing (PT) for Huntington disease (HD) can address the potential inequity that exists in access to PT in rural communities in British Columbia. To address the hypothesis, the project employed a three part, highly structured, mixed method sequential exploratory approach. The first part of the research involved: a) a mapping study; b) a qualitative interview study of 33 at-risk individuals; c) a survey of 102 individuals at-risk for HD; and d) an 11-person expert workshop. The second part of the research built on results from the prior work and was to develop a telehealth PT protocol and a HD PT dedicated website for individuals considering testing. The final component of the research involved a pilot project that compared the novel telehealth protocol with the standard, Vancouver-based PT protocol. Evaluation of the pilot project was conducted by quantitative survey with 28 participants and was subject to statistical analysis. Results revealed that PT rates are lower in rural areas and that access is a significant issue due to distance related factors and the inflexible nature of the PT process. The pilot project demonstrated that providing PT via telehealth is not only possible, but is also warranted. There were no significant differences in terms of quality of care, information, counseling and support during the PT process between the Vancouver-tested and telehealth-tested groups. Overall, the pilot study reveals that providing PT via telehealth can improve access to PT while maintaining high quality of care and support. The work adds to a growing body of literature on the utility of telehealth services in genomic medicine in an age of increased technological innovation and comfort with such communication mechanisms.

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Huntington disease (HD) is a progressive disorder characterized by involuntary movements, emotional disturbances, and memory loss. There is currently no cure for HD. Accumulating evidence has implicated excitotoxicity, a process in which excessive signaling via the glutamate receptors results in neurotoxicity, in HD. The main aim of the studies presented was to evaluate the potential of small molecules known to target excitotoxicity-related pathways in the YAC128 mouse model of HD. We examined whether treatment with memantine, a clinically well-tolerated NMDA receptor antagonist, can improve the phenotype of YAC128 mice. We demonstrated that treatment with memantine results in improvements in motor function and rescues the striatal deficits in a dose-specific manner. Rasagiline is a selective inhibitor of monoamine oxidase type B (MAO-B) clinically approved for the treatment of Parkinson’s disease that has been shown to protect against a number of neurotoxic stimuli. We demonstrate that rasagiline protects against striatal lesioning in acute models of excitotoxicity and improves the motor function of the YAC128 mice. We next examined the effect of treatment with a combination of memantine and rasagiline on the phenotype of the YAC128 mice. We demonstrate that treatment with a combination of memantine and rasagiline provides early and sustained improvements in motor function and rescues striatal deficits in the YAC128 mice. Induction of a heat shock protein (HSP) response has been shown to be neuroprotective in models of excitotoxicity and polyglutamine-induced neurodegenerative disease. We examined whether treatment with arimoclomol, a compound shown to enhance the HSP response, can improve the phenotype of the YAC128 mice. Our findings demonstrate that treatment with arimoclomol does not lead to up-regulation of an HSP response or rescue of the behavioural and striatal deficits in the YAC128 mice. Finally, we characterize psychiatric disturbances in YAC128 mice, demonstrating that YAC128 mice exhibit depressive-like symptoms as assessed by the Porsolt forced swim test and the sucrose consumption test of anhedonia. These measures may be employed in assessing any anti-depressive effects of candidate treatments in preclinical therapeutic trials. Our findings suggest that targeting excitotoxicity may be a viable therapeutic approach in HD.

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No abstract available.

In neurons, modification by the lipid palmitate regulates trafficking and function of signaling molecules, neurotransmitter receptors and associated synaptic scaffolding proteins. HIP14 (huntingtin interacting protein 14) is the first identified and characterized mammalian palmitoyl transferase that regulates this process. I have shown that HIP14 has striking effects on modulating trafficking and function of many proteins important for synapse formation and plasticity such as PSD-95, a postsynaptic scaffolding molecule.The importance of the finding that HIP14 is a neuronal palmitoyl transferase is further emphasized by our recent discovery that huntingtin protein folding, trafficking and function are regulated by the enzyme HIP14. Expansion of the polyglutamine tract in huntingtin as seen in Huntington Disease (HD) results in reduced association with HIP14 and decreased palmitoylation of huntingtin, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity. By manipulating HIP14 levels through expression or knockdown, we can manipulate the number of huntingtin inclusion bodies and neuronal cell viability. Overall, these discoveries offer novel mechanism for HD pathogenesis and provide new approaches to therapy for HD.The tight association of HIP14 with wild-type huntingtin, which differs from other known enzyme-substrate interactions, indicates that huntingtin serves other functions beyond being a substrate of HIP14. I have discovered that, in vitro, wild-type huntingtin may facilitate activity of HIP14 to palmitoylate other neuronal substrates such as SNAP25, PSD95 and GAD65. By contrast, mutant htt does not act this way, probably due to lack of interaction with HIP14. Furthermore, immunoprecipitated HIP14 from huntingtin+/- mice also exhibits less enzyme activity in palmitoylating GST-SNAP25 in vitro, suggesting that decreased huntingtin expression compromises HIP14 activity. In vivo, using Acyl Biotin Exchange assay, I have also found that palmitoylation of a number of presynaptic and postsynaptic proteins that are involved in neurotransmission are reduced in huntingtin+/- mice. This study not only ascribes an important biochemical function to wild-type huntingtin, but also suggests that defects in protein palmitoylation in general due to mutant huntingtin lack of ability to facilitate HIP14 activity may contribute to the pathogenesis of HD.

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Huntington disease (HD), the “Dancing Mania” of the Middle Ages, has always been aparticular target of social stigma and discrimination. With the discovery of a polymorphic DNAmarker linked to HD in 1983, individuals at-risk for HD were able to learn whether or not theyhad inherited the causative HD mutation and possibly escape its stigma and discrimination. Forthose who had inherited the HD mutation increased discrimination became a real possibility.Genetic discrimination (GD) refers to the differential treatment of asymptomaticindividuals or their family based on genetic differences. It has been over twenty years since theintroduction of predictive testing (PT) for HD, yet little is known about the nature and extent ofGD and whether PT actually results in increased levels of GD. The objective of this dissertationwas to use qualitative and quantitative methods to investigate the nature and extent of GDamong persons at-risk for HD.Qualitative findings provide insight into how individuals interpret, personalize andmanage GD. Results from the national survey indicate that 40% of respondents reported atleast one experience of GD. Reported experiences occurred most often in reference to life anddisability insurance, and among family and friends. Surprisingly, there were few reports of GD inemployment, health care and government settings. Experiences were not significantlyassociated with PT. However, the proportion of respondents who reported GD was 16% higheramong persons who have the HD mutation than among those that do not and untestedrespondents. Interestingly, respondents’ family history (FH), rather than their PT result, was themajor reason given for their experiences as well as an important predictor of GD. Psychologicaldistress was a health outcome of GD.This is the first study to investigate the nature and extent of GD among an asymptomatictested and untested population. This dissertation provides evidence that GD is a frequentlyreported experience and a source of distress for persons at-risk for HD. These findings provideinsight for policy, identify areas where more education and support is needed, and providedirection to genetic professionals supporting their clients as they confront issues of GD.

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