Huntington’s disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HTT gene, which causes an expansion in the polyglutamine tract of the huntingtin protein. In 2018, the cryo-EM structure of the 350 kDa protein huntingtin (Htt) in complex with huntingtin associated protein of 40 kDa (HAP40) was solved, which demonstrated that huntingtin had to be co-translated and complexed with HAP40 to retain structure. However, little is known about HAP40 and thus the biological relevance of this structure. In this project, we transduced cells with fluorescently labelled recombinant apo-Htt or Htt-HAP40 to determine if HAP40 must be complexed with huntingtin in order for huntingtin to have biological activity. This method has not been implemented in HD research and may also improve current fluorescent microscopy models for huntingtin, as it has the advantage of looking at full-length protein rather than small fragments. We also found that with the huntingtin lowering drug branaplam, there is a linear correlation between huntingtin and HAP40 levels, where HAP40 levels will decrease when huntingtin levels are directly decreased as detected by western blot analysis. Furthermore, we found that this lowering effect by branaplam ameliorates DNA repair deficits in HD. With the potential for branaplam to become a treatment for HD, we should continue to test its effect on other HD-associated phenotypes to determine the effect of huntingtin and downstream HAP40 lowering. / Thesis / Master of Health Sciences (MSc)

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by an extended (≥36) CAG repeat in the huntingtin gene. Its hallmark is brain athrophy, but huntingtin is widely deposited in all tissues of the body, most notably in the brain and testes. Its pathogenic effect is conditioned by the formation of cytotoxic forms of aggregates and fragments, which occur in both brain and peripheral tissues. Testicular atrophy has been demonstrated in postmortem samples from human patients with Huntington's disease and in transgenic mouse models. We investigated reproductive decline in a large animal model of Huntington's disease. A transgenic (tgHD) minipig model was created by inserting a lentiviral vector into the genome of a pig. Vector contained a truncated form of the N terminal part of huntingtin gene. Boars of this transgenic line showed a reduced ability to produce offspring from 13 months of age. We confirmed apoptosis of seminiferous epithelial cells and Sertoli cells, and a production of morphologically damaged spermatozoa, which were unable to efficiently fertilize the oocyte under in vitro conditions. We found a reduction of mitochondrial metabolism parameters in the sperm of tgHD boars. These changes were not dependent on the age of the boars., It is directly related to the...

No description available.

Mitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), which are essential for mitochondrial function. Mutations in the mitochondrial fission and fusion machinery lead to neurodegeneration. Thus, whether defective mitochondrial dynamics participates in ALS and HD requires further investigation. ALS is a fatal neurodegenerative disease characterized by upper and lower motor neuron loss. Mutations in Cu/Zn superoxide dismutase (SOD1) cause the most common familiar form of ALS by mechanisms not fully understood. Here, a new motor neuron-astrocyte coculture system was created and live-cell imaging was used to evaluate mitochondrial dynamics. Excessive mitochondrial fission was observed in mutant SOD1G93A motor neurons, correlating with impaired axonal transport and neuronal cell death. Inhibition of mitochondrial fission restored mitochondrial dynamics and protected neurons against SOD1G93A -induced mitochondrial fragmentation and neuronal cell death, implicating defects in mitochondrial dynamics in ALS pathogenesis. iv HD is an inherited neurodegenerative disorder caused by glutamine (Q) expansion in the polyQ region of the huntingtin (HTT) protein. In the current work, mutant HTT caused mitochondrial fragmentation in a polyQ-dependent manner in both primary cortical neurons and fibroblasts from human patients. An abnormal interaction between DRP1 and HTT was observed in mutant HTT mice and inhibition of mitochondrial fission or promotion of mitochondrial fusion restored mitochondrial dynamics and protected neurons against mutant HTT-induced cell death. Thus, mutant HTT may increase mitochondrial fission by elevating DRP1 GTPase activity, suggesting that mitochondrial dynamics plays a causal role in HD. In summary, rebalanced mitochondrial fission and fusion rescues neuronal cell death in ALS and HD, suggesting that mitochondrial dynamics could be the molecular mechanism underlying these diseases. Furthermore, DRP1 might be a therapeutic target to delay or prevent neurodegeneration.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by progressive loss of striatal and cortical neurons. HD is caused by an abnormal polyglutamine (polyQ) expansion in Huntingtin protein (HTT). HTT controls vesicular trafficking along axons in neurons through interaction with components of the molecular motor machinery. Arginine methylation is one of the most abundant post-translational modifications (PTMs) and is catalyzed by protein arginine methyltransferases (PRMTs). Recent evidence supports a key role for arginine methylation in neurodegeneration and particularly in polyglutamine diseases. However, whether HTT is methylated at arginine residues has not been investigated yet and the role of arginine methylation in HD pathogenesis remains to be fully elucidated. In this thesis, I show that vesicle-associated HTT is methylated in vivo at two evolutionarily conserved arginine residues, namely R101 and R118. Methylation of HTT at R118 is catalyzed by Protein Arginine Methyltransferase 6 (PRMT6), which localizes on vesicles together with HTT, whereas further analyses are required to identify the enzyme(s) responsible for R101 methylation. Interestingly, loss of PRMT6-mediated R118 methylation reduces the association of HTT with vesicles, impairs anterograde axonal transport and exacerbates polyQ-expanded HTT toxicity. Conversely, PRMT6 overexpression improves the global efficiency of anterograde axonal transport and rescues cell death in neurons expressing polyQ-expanded HTT. These findings establish a crucial role of arginine methylation as a modulator of both normal HTT function and polyQ-expanded HTT toxicity and identify PRMT6 as a novel modifier of HD pathogenesis. Importantly, defects in HTT methylation may contribute to neurodegeneration in HD and promoting arginine methylation of HTT might represent a new therapeutic strategy for HD.

<p>Huntington’s disease (HD) is a devastating autosomal dominant neurodegenerative disorder for which there are no disease modifying treatments. Owing to this are the multiple biological functions of the huntingtin protein and the lack of understanding of the exact pathways being affected in HD. It is clear that the huntingtin protein normally provides anti-apoptotic support and that there are underlying energetic problems and cell stress defects associated with disease. Work from our group has shown that huntingtin acts as a stress sensor and translocates from the endoplasmic reticulum to the nucleus upon cell stress. We therefore hypothesized that huntingtin has a nuclear function in the cell stress response; which would tie together what is currently known about huntingtin, its pro-apoptotic function and the energetic defects of neurodegeneration. In this thesis we describe huntingtin as having a role in the nuclear cofilin-actin rod stress response. Cofilin is an actin binding protein normally involved in actin treadmilling. During stress, cofilin saturates F-actin leading to rod formation which functions to alleviate ATP. We show that this response is impaired in the presence of mutant huntingtin and that the aberrations in this response can be mediated through the enzyme tissue transglutaminase. Little is known about the physiological role and requirement of the cofilin-actin rod response. Therefore we created a system to test if rod formation was required in cells during stress, which indicates if and how targeting this pathway will be possible. We additionally looked at targeting the nuclear import and export properties of the cofilin protein, which directly affect rod formation and may be targetable in cofilin modifying drug discovery efforts. Overall, this work has described a specific and relevant pathway affected by mutant huntingtin and started the process of assessing this pathway as a therapeutic avenue for Huntington’s disease.</p> / Doctor of Philosophy (PhD)

<p>Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. Recent research demonstrates that post-translational modifications of huntingtin could be an important determinant of mutant huntingtin’s toxicity in HD. In particular, phosphorylation at residues serine 13 and 16 within the first 17 amino acids of huntingtin (N17), have been shown to be critical modulators of mutant huntingtin’s toxicity and localization, and can be triggered by stress. This project aims to study how phosphorylation within N17 alters the interactome at this site and what physiological stress results in the nuclear localization of N17 and huntingtin. The initial search to identify potential interactors was conducted through an affinity chromatography assay using a wild type striatal cell line derived from knock in mouse model of HD. Fluorescent lifetime imaging microscopy (FLIM) to determine Fӧrester resonance energy transfer (FRET), co-immunoprecipitation and co-immunofluorescence assays were then used to validate real interactors of N17. Analysis from this project has validated two previously unidentified interactors of N17; SET, a small nucleo-oncoprotein, and vimentin, a type 3 intermediate filament. Both interactors have suggested two potentially novel roles for N17 within huntingtin, in cell cycle regulation and intermediate filament dynamics. Finally, smart screening techniques using stress-inducing compounds reveal that the sensitivity of N17 to stress and its subsequent nuclear localization can be attributed in part to activation of oxidative stress pathways. Data shown here can be expanded upon to elucidate how huntingtin function and response to cell stress are regulated and mediated via N17 and potentially how this mechanism is disrupted within HD.</p> / Master of Science (MSc)

In healthy cells, huntingtin is primarily found in the cytoplasm; however, upon cellular stress, huntingtin is phosphorylated (phospho-huntingtin) at serines 13 and 16 of the amino-terminal N17 domain and shuttled into the nucleus. Such dynamism in nucleocytoplasmic translocation and post-translational modification suggests an important role for huntingtin in Huntington’s disease (HD) pathogenesis as these phenotypes propose possible mechanisms for disease progression. Huntingtin nuclear import is also facilitated by its proline-tyrosine nuclear localization signal (PY-NLS), which harbours a highly conserved intervening sequence specific to the huntingtin gene. This encouraged a proteome investigation to identify potential protein partners of the PY- NLS. Results of this study revealed that high mobility group box 1 (HMGB1), a cofactor of base excision repair, uniquely bound to the wild-type PY-NLS, but not the PY-NLS KK177/178AA mutant. Immunofluorescence microscopy in human telomerase reverse transcriptase (hTERT) immortalized fibroblast cells using HMGB1- and phospho- huntingtin-specific antibodies revealed a promising association between the two, as changes in nuclear levels of HMGB1 positively correlated with nuclear levels of phospho- huntingtin. This relationship was further confirmed by co-immunoprecipitation of HMGB1 by the PY-NLS and N17 domain. Also, when exogenous oxidative stress was introduced, increased interaction between HMGB1 and huntingtin was observed. This suggests that HMGB1 facilitates the nuclear import of huntingtin in a ROS-dependent manner, and thus, presents a novel avenue to a potential therapeutic target in HD pathogenesis. / Thesis / Master of Science (MSc)

Yes / Objective: This study investigated whether dual tasks make disproportionately high demands in Huntington’s disease (HD) compared with controls, and also tested the Multiple Resources Theory. Method: Thirteen HD participants and 13 controls completed 2 dual task sets that varied in difficulty and complexity: Set 1 paired simple choice reaction time (RT) with digit forward, and Set 2 paired complex choice RT with digit backward. Results: We found that HD participants were overall slower; however, although they maintained similar levels of accuracy in the simple choice RT tasks with controls, their accuracy decreased in the complex choice RT tasks. In addition, we found that HD participants were more susceptible to speed-accuracy trade-offs. Despite that, they did not show greater dual task costs than controls. Conclusions: Overall, our findings do not support the Multiple Resources Theory, but they do provide some support for the Unitary Resource Theory and the attentional impairment hypothesis.

Jeff Carroll's research into Huntington's disease is not only offering hope for the afflicted; it's changing the course of his own genetic destiny.