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Huntington's disease (HD) is an inherited autosomal-dominant neurodegenerative disease caused by the abnormal expansion of CAG repeats in exon 1 of the huntingtin gene located on chromosome 4. This disease is characterized by the premature loss of medium spiny neurons in the striatum and behavioural deficits that typically manifest at middle-age. Despite the identification of its cause decades ago, there is still no disease modifying treatment available for HD patients. Current evidence indicates that exacerbated glutamate signalling in the striatum plays a key role in the pathophysiology of HD. Within the striatum, metabotropic glutamate receptor (mGluR) 2/3 are predominantly expressed on presynaptic terminals, whereas mGluR5 is predominantly localized to postsynaptic terminals. Here, we show that both the activation of mGluR2/3 and the inhibition of mGluR5 can improve HD symptoms in the zQ175 HD mouse model. Specifically, treating zQ175 HD mice with either the mGluR2/3 agonist LY379268 or the mGluR5 negative allosteric modulator (NAM) CTEP rescues motor deficits, reduces mutant huntingtin aggregate formation, improves neuronal survival and alleviates microglia activation. We also provide evidence that shows sex can influence the progression of HD symptoms and the efficacy of therapeutic agents. We found that chronic administration of LY379268 differentially activated and inactivated cell signalling pathways in male and female zQ175 mice. Furthermore, female zQ175 mice required a longer treatment duration with CTEP than male mice to show improvement in their rotarod performance. Using FDNQ175 mice, a newer HD mouse model derived from the zQ175 line, we demonstrated that female FDNQ175 mice were less susceptible to decline in limb function than male mice but showed higher levels of insoluble mutant huntingtin aggregates at a younger age.

The huntingtin N17 domain is the master regulator of huntingtin intracellular localization. N17 is post-translationally modified, and phosphorylation of N17 serines 13 and 16 facilitate the stress dependent nuclear translocation of huntingtin by inhibiting CRM1 binding and nuclear export. In Huntington’s disease (HD), mutant huntingtin is hypo-phosphorylated and increasing N17 phosphorylation has been shown to be protective in HD mouse models. N17 phosphorylation is therefore a valid therapeutic sub-target of huntingtin. The ER stresses that have been previously characterized to affect huntingtin phosphorylation are broad, likely activating a plethora of response pathways. Thus, in this study, we sought to define a specific stress that could affect huntingtin phosphorylation and nuclear localization. Here we show that huntingtin localization and phosphorylation can be specifically affected by reactive oxygen species (ROS). We identify a highly conserved methionine at position 8 (M8) as the specific sensor of oxidative species within N17 and show the capacity of oxido-mimetic M8 point mutations to affect N17 structure, localization and phosphorylation. We also define a specific molecular mechanism whereby N17 oxidation promotes membrane dissociation, thus increasing kinase accessibility and subsequent phosphorylation. These results define a precise molecular mechanism for the normal biological regulation of huntingtin phosphorylation by oxidative signalling. This ability of huntingtin to sense ROS levels at the ER provides a link between age-associated stress and altered huntingtin function. It suggests that ROS stress due to aging may be a critical molecular trigger of HD that could explain the age-onset nature of disease. / Thesis / Master of Science (MSc)

<p>The primary cilium is a singular signaling organelle found on most mammalian cell types. Dysfunction of the primary cilium or associated structures form a group of genetic disorders called ciliopathies. Recently, Huntington’s disease (HD), a monogenetic neurodegenerative disorder, was classified, at least in part, as a ciliopathy. How the primary cilium contributes to the pathogenesis of HD is the focus of this work. We demonstrate that huntingtin localization to the basal body or primary cilium is dependent on the phosphorylation status of serine residues 13 and 16. Furthermore, we demonstrate that, compared to controls, HD cell models have an increased number of cells with a primary cilium and that these cells have higher presence of huntingtin within the ciliary compartment. The primary cilia that form in HD cell lines demonstrate abnormal dynamics and morphology with bulging tips, characteristic of defective retrograde trafficking. We also demonstrate that alpha tubulin acetyltransferase 1 (αTAT1) expression and localization is increased in the primary cilium of HD cell lines. Subsequently, the primary cilium of HD cell lines are highly acetylated when compared to controls. These data support that primary cilia structure, ciliogenesis and ciliome are altered in HD.</p> / Master of Science (MSc)

Neurodegenerative diseases are characterized by selective neuronal vulnerability and subsequent degeneration in specific areas of the brain. Huntington’s Disease (HD) is inherited as an autosomal dominant mutation that primarily affects the cells of the striatum and the cerebral cortex, leading to a triad of symptoms that include the progressive loss of motor function, defects in cognitive ability and psychiatric manifestations. HD is caused by a CAG repeat expansion that exceeds 37 repeats in Exon1 of the ​HTT​ gene, manifesting as a pathogenic polyglutamine (polyQ) amino acid tract expansion in the huntingtin protein. HD is a late onset disorder, with disease onset around 40-50 years of age and symptoms that worsen over 10-20 years. Only a few symptomatic treatments are available and there is currently no cure for the disease. Therapeutics to target the huntingtin gene itself have only been in clinical trial in the past 2 years. The length of the expansion has an inverse relationship with the age of disease onset. Most patients that have repeats between 40-45 CAG, however, have varying age of disease onset. Recent genome-wide association studies (GWAS) have implicated DNA handling and repair pathways as modifiers of age of disease onset up to 6 years. Therapeutic approaches to modify and delay onset indefinitely through other genetic targets will require identification of pathological mechanisms that precede disease onset. Several hallmark phenotypes have been identified in cell and animal models, including pathogenic aggregate formation. These models are not reflective of human biology, using excessively large CAG repeats (>100) associated with the more aggressive, juvenile HD, overlooking the importance of GWAS results and the progression of disease with lower pathogenic CAG repeats (40-50 CAG). We have therefore generated novel, clinically-relevant human patient fibroblast cell lines and have characterized several disease phenotypes. My thesis presents a culmination of several projects that focus on disease modelling, primarily outlining phenotypic differences between wildtype and HD cells that will benefit our understanding of disease pathogenesis. / Thesis / Doctor of Philosophy (PhD)

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This study is a multi-method, two-city, intensive, in-depth qualitative study of Huntington's disease (HD) caregivers (HDCs) in Colombia. I explored the Colombia HDCs' experience through the Hispanic culture of caring. I develop the theory of the subrogate agency based on 5 functional stages of HD from Shoulson and Fanh (1979). This study was conducted in two different regions of Colombia, Medellin and Juan de Acosta, in which high rates of HD cases have been identified. The data were collected through three methods: (a) 56 interviews with HDCs and 8 with physicians; (b) 28 participant observations of Huntington's disease sufferers (HDSs); (c) 4 interviews and 8 focus groups of 6 members each with HDCs of late HDSs. Human agency is the ability to monitor one's own action. This study showed that the gradual and serious loss of all capabilities in HDSs has a social effect on the HDSs' agency. HDSs' survival depends on the subrogation that the HDC offers to the HDS. The HDS retains self-hood, i.e. agency, through the HDC's action. This subrogation causes a paradoxical consequence, resulting in both negative and positive effects on the caregiver. The theory of surrogate agency is supported by the data. Through the progressive phases, the capacity of the sufferer for expressing suffering, and social embarrassment, as well as the capacity to fight against the illness and provide reciprocity to their caregivers, deceases. The reason is that physical and cognitive impairments, as well as depression and anger, continue to increase. The study also documented important socio-cultural differences among the study regions. For instance, HDC's solidarity was based on blood and friendship; in larger cities, HD fragmented families. The study also found that HD is taking an immense toll on caregivers, sufferers and families because they are excluded from the Colombia's Basic Health Plan.

Huntington's disease (HD) is a devastating autosomal dominantly inherited neurological disorder caused by an abnormal expansion of CAG trinucleotide repeats in the gene coding for the Nterminal region of the huntingtin (Htt) protein, which leads to the formation of a polyglutamine stretch. The greater the CAG repeats, the earlier the onset of the disease. The polyglutamine stretch destabilizes the Htt protein leading to misfolding, abnormal processing, aggregation, and inclusion formation. Mutant Htt protein is believed to damage and kill neurons in the striatum by a mechanism involving increased oxidative and metabolic stress, and impaired adaptive cellular stress responses. A large number of abnormalities have been reported in HD, including transcription deficits, energy impairment, excitotoxicity, and lack of trophic support. Reduced trophic support contributes importantly to striatal degeneration in human HD. Specifically, brainderived neurotrophic factor (BDNF) expression is reduced in patients with HD. BDNF is also decreased in brain tissue from mice transgenic for mutant Htt. BDNF levels influences the onset and the severity of motor dysfunction in HD mice. In addition to BDNF, levels of the molecular chaperones heat shock proteins (Hsp40 and 70) decrease progressively in HD brain. Hsp70 is a highly stress-inducible member of a chaperone family of proteins that functions to prevent misfolding and aggregation of newly synthesized mutant proteins and stress-denatured proteins. Hsps appear to play a critical role in HD since expression of active heat shock factor HSF1, a transcription factor responsible for the induction of Hsps, markedly reduces polyglutamine aggregate formation in both cell and mouse models. Many efforts have been made to develop preventive treatments for HD because of the strong genetic link and a freely available genetic test to identify individuals at risk. At present, only symptomatic therapy is available and effective therapeutic approaches to slow the disease iv process have yet to be developed. Previous studies have shown that electroconvulsive shock (ECS) induces the production of growth factors including BDNF and the molecular chaperones HSP40 and HSP70. Because ECS can stimulate the production of neuroprotective proteins, we determined whether ECS treatment could slow the progressive nature of the disease process and provide a therapeutic benefit in a mouse model of HD. ECS or sham treatment was administered to male N171-82Q Htt mutant mice. End points measured included motor function, striatal and cortical pathology, and levels of neurotrophic factors, protein chaperones, and proteins involved in synaptic plasticity. ECS treatment delayed the onset of motor symptoms, reduced body weight loss and extended the survival of HD mice. Striatal neurodegeneration was attenuated and levels of neurotrophic factors, protein chaperones and mitochondria-stabilizing protein were elevated in striatal cells of ECS-treated compared to sham-treated HD mice. Our findings suggest that ECS can increase the resistance of neurons to mutant huntingtin resulting in improved functional outcome and extended survival. The potential of ECS as a treatment for HD patients merits further consideration.

Huntington's Disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the Huntingtin gene IT15. In this study we demonstrated that Bcl-2 interacting mediator of cell death Extra Long (BimEL) protein expression was significantly increased in cells expressing mutant Huntingtin (mHtt). Moreover, striatal BimEL expression remained high in an R6/2 HD mouse model throughout the disease progression. Utilizing novel BimEL phospho-mutants we demonstrated the phosphorylation of Ser65 to be important for the stabilization of BimEL. We provided evidence that impaired proteasome function, increased JNK activity and reduced striatal BDNF lead to changes in the phosphorylation of BimEL, thereby promoting its stabilization specifically within the striatum of R6/2 mice. Furthermore, knocking down BimEL expression prevented mHtt-induced cell death in a HD cell culture. Taken together, these findings suggest that BimEL may contribute to the selective neurodegeneration and pathogenesis of HD. / by Rebecca Leon. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Huntington's disease, an autosomal dominant neurodegenerative disease, affects the cell in several toxical ways. One of them is accumulation of protein aggregates in cytoplasma, which could become a serious problem especially for long-lived cells such as neurons. Autophagy (macroautophagy) is an important catabolic pathway, crucial for cell survival. If fully functional, it should eliminate protein aggregates and reduce the toxic effect on the cell. However, recent works show that this pathway might be defective, most probably in the cytoplasmic cargo recognition. In my work I used a transgenic miniature pig model of Huntington's disease to verify the hypothesis of autophagical dysfunction in individuals suffering from Huntington's disease. I studied levels of autophagosomal markers - LC3 and p62 in mesenchymal stem cells after different autophagy stimulation treatments, and ammonium chloride was found the most effective. In addition I evaluated the effect of age of the animals on autophagic function, but no significant changes were identified, even if animal genotype was considered. Moreover I had an opportunity to study proteins levels in three porcine brain tissues - cortex, cerebellum and striatum. Even though there is no significant diference, we can observe a trend of LC3 II and p62 increase in...