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)

Yes

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...

Huntington's disease (HD) is a neuropsychiatric disorder characterized by choreiform movement of the limbs, cognitive disability, psychosis and dementia. It is untreatable, incurable, and ultimately fatal. HD is invariably associated with an abnormally long CAG expansion within the IT15 gene on human chromosome 4. Although the mutant huntingtin protein (mHtt) is ubiquitously expressed in HD patients, cellular degeneration occurs only in neurons within the striatum and cerebral cortex. The Ras homolog Rhes is expressed very selectively in the precise brain areas affected by HD. Recent work using cultured cells suggests that Rhes may be a co-factor with mHtt in cell death. However, there is controversy as to whether cell death underlies the symptoms of HD. We used a validated transgenic mouse model of HD crossed with Rhes knockout mice to show that the behavioral symptoms of HD are regulated by Rhes. HD/Rhes-/- mice showed greatly delayed expression of HD-like symptoms in this in vivo model. Drugs that block or inhibit the actions of Rhes may be useful as the first treatments for HD.

A defining characteristic of neurodegenerative disease is the accumulation of mutant or misfolded proteins within neurons. Selective macroautophagy of aggregates, or aggrephagy, is a lysosome-mediated protein degradation pathway implicated in the turnover of disease-relevant accumulated proteins, but its specific function in vivo in the mammalian nervous system is poorly understood. The large PI3P-binding protein Alfy (Autophagy-linked FYVE protein) is an adaptor required for selective macroautophagy of aggregated proteins in cellular model systems. We sought to address Alfy-mediated aggrephagy in the mammalian brain in mouse models of Huntington’s disease (HD). HD is a neurodegenerative disorder caused by autosomal dominant inheritance of an expanded CAG repeat within the IT15, or huntingtin (htt) gene. The mutation causes an expansion of a polyglutamine (polyQ) tract in the protein Huntingtin (Htt), which results in psychiatric, cognitive, and motor symptomology. A pathological hallmark of HD is the accumulation of intracellular deposits of mutant Htt and ubiquitin. The exact relevance of these deposits remains unclear, but their elimination, hypothesized to occur via macroautophagy, correlates with behavioral improvements in mouse models of HD. The selective mechanisms of this phenomenon are largely unexplored in vivo. We have created two mouse models to address the role of Alfy-mediated selective macroautophagy in mammalian HD brain. First, we created tamoxifen-inducible Alfy knockout mice (Alfy iKO) and crossed them with a redesigned inducible HD mouse (HD103Q) that uses a tetracycline-regulated system to control reversible expression of mutant exon-1 Htt. Western blot, in situ, and PCR analysis confirm that Alfy can be eliminated from brain in adult Alfy iKO mice. A timecourse of Htt aggregation and clearance reveals that HD103Q mice accumulate huntingtin deposits, which clear in a linear manner upon transgene suppression over the course of four months. The loss of Alfy significantly impedes the removal of these deposits. Second, an Alfy knockout mouse was created using gene-trap technology, and mice hemizygous for Alfy knockout were crossed with BACHD mice expressing full-length human mutant Htt. We find that 50% Alfy depletion in the BACHD leads to increased insoluble Htt aggregate deposition along with accelerated decline in motor behavioral performance. Furthermore, inducible knockout of Alfy alone has a severe and age-dependent motor behavioral phenotype. This work reveals an in vivo role for Alfy in turnover of mutant Htt deposits, suggests that the accumulation of detergent-insoluble mutant Htt species contributes to behavioral pathogenesis, and supports an important function for Alfy at the intersection of HD and aging.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.