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)

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Research into the molecular pathology of prevalent public health epidemics such as neurodegenerative diseases including frontotemporal dementia (FTD) and Alzheimer’s Disease (AD), non-medical and illicit opioid use (OU), and Human Immunodeficiency Virus-1 (HIV-1) has been hindered by a lack of systems that allow for rapid and high-throughput modeling of the complex multifactorial conditions in a human context. In this thesis we have addressed this challenge using a multi-pronged approach that encompasses single cell RNA sequencing (scRNA-seq) of three-dimensional (3D) human induced pluripotent stem cell (hiPSC) assembloid culture models and patient derived peripheral blood mononuclear cell (PBMC) samples. We describe the development of an iPSC derived neuron-astrocyte assembloid model of tauopathies, including FTD and AD (AstTau), that rapidly recapitulates propagation of toxic human oligomeric tau (oTau) and cell type specific pathology including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. scRNA-seq identified vulnerable excitatory neuron specific inflammatory pathways and a heat shock response in astrocytes, recapitulating transcriptomic signatures of adult neurodegeneration and supporting a hypothesis of cell type specific neuroinflammation in tau pathogenesis. To more completely model AD, we incorporated amyloid precursor protein (APP) mutant iPSCs into the assembloid model. These iPSCs contained the familial AD APP V717I mutation or the isogenic CRISPR corrected control, and were used to derived neurons, astrocytes, and microglia. This advanced combinatorial system (AstAD and MAstAD) enabled selective microglial incorporation, APP mutation expression, and oTau seeding allowing us to identify discrete contributions to AD pathogenesis. Ast/MAstAD developed extracellular amyloid-β (Aβ) and microglial activation in addition to the pathology observed in AstTau. scRNA-seq identified divergent microglial activation in response to Aβ and oTau pathology, with APP V717I mutation and oTau seeding synergistically exacerbating AD phenotypes. These assembloid models enable study of the cellular and molecular inflammatory mechanisms in multifactorial neurodegenerative diseases. To better understand disease signatures at the crossroads of multifactorial OU, HIV-1, and antiretroviral (ART) viral suppression we also produced a scRNA-seq data set of more than 100,000 peripheral blood mononuclear cells (PBMCs) from 75 study participants. We identified chronic immune activation and T cell activation dysfunction driven by interferon transcriptomic signatures that were elevated in people with HIV (PWH) with opioid use as compared to PWH without OU. We also identified a functional natural killer cell subtype that was depleted with OU in PWH. Cessation of OU reduced these potentially deleterious inflammatory transcriptomic profiles, supporting the hypothesis that OU in PWH amplifies a state of chronic immune activation. Taken together, single cell transcriptomic resolution has enabled the identification of cell type specific disease signatures in complex pathophysiologies. These data demonstrate the dynamic range of inflammatory signaling across multifactorial disease states and emphasize the need for disease- and cell- type specific approaches to therapeutic development. / 2025-02-05T00:00:00Z

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of nigral dopaminergic (DA) neurons and the formation of Lewy bodies. Despite most cases being idiopathic, mutations in several genes have been implicated in familial forms of PD. In particular, recessive mutations in Parkin gene (PARK2) are the most common cause of young-onset inherited parkinsonism. Parkin is an E3 ubiquitin ligase involved both in the control of mitochondrial turnover and in the proteasome-dependent degradation of proteins, two pathways that have been causally linked to PD development. Although initially described as a recessive disorder, experimental evidence suggests that heterozygous Parkin mutations can exert dominant toxic effects causing neurodegeneration. In 2012, Ruffmann and colleagues identified the first pure heterozygous R275W Parkin patient with clinical features of typical late-onset PD and a diffuse Lewy body pathology. To assess the impact of R275W Parkin, we generated the first mouse line carrying Parkin R274W mutation, which corresponds to the human R275W substitution. Unlike Parkin deficient mouse models, both homo- and heterozygous R274W mice show an age-related motor impairment, degeneration of dopaminergic neurons and neuroinflammation. We detected structural and functional mitochondrial abnormalities related to PARIS-PGC-1α axis impairment in R274W+/+ mice brain and skeletal muscle. Strikingly, we noticed signs of protein aggregation in both R274W+/- and +/+ mice, while we identified bona fide Lewy bodies only in the midbrain of heterozygous mice. Additionally, in the brains of R274W mice we discovered overt abnormalities of the glymphatic system, the main route for brain waste clearance. Our preliminary observations suggest that Parkin influences aquaporin-4 (AQP4) localization. Altogether, our data suggest that R274W Parkin substitution behaves both as a loss ofand a gain of toxic function, highlighting a link between Parkin dominant toxicity and age-dependent motor impairment, neuroinflammation, DA neurons loss, glymphatic system dysfunctions and α-synuclein aggregation in vivo. Hence, our study provides a new robust mouse model to explore PD pathogenesis and glymphatic dysfunctions, offering the possibility to test novel therapeutic strategies with great predictivity.

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The immune response is a complex series of cell-mediated reactions by which an organism combats infection, responds to injury, external stresses, or disease. In both Drosophila melanogaster and vertebrates, aging is associated with progressive declines in physiological functions as well as susceptibility to stress and disease. Naturally, the immune activity is increased with age, yet the efficacy of this response is reduced with age. Conversely, when the immune activation is artificially-induced by Lipopolysaccharide, aging is accelerated. Like aging, neurodegeneration is also associated with increased immune activity. The Blood-Brain barrier (BBB) is a physical barrier with highly selective permeability that isolates the brain from the rest of the body. This barrier is essential for ion homeostasis, and exclusion or efflux of exogenous chemicals. The exclusion properties of the Dm BBB are facilitated by paracellular septate junctions of subperineural glia (SPG), which prevent diffusion into or out of the brain. Using the GAL/UAS system in Drosophila, we found that activation of a glial-specific immune response in either immunodeficiency (IMD) or Toll pathways led to reductions in lifespan and age-dependent negative geotaxis. These reductions were also correlated with an early sensitivity towards oxidative and thermal stresses. Furthermore, we found that a SPG-specific immune response of the Toll pathway or disruption of the paracellular BBB itself was sufficient to show the same reductions as pan-glial activation. In short, we found that flies with CNS-specific immune activation showed an inability to cope with long-term and acute forms of stress, and that SPG-specific Toll Activation was sufficient to show these effects. This implicates chronic immune response as a negative factor during aging, neurodegenerative disease, and brain homeostasis. / Thesis / Master of Science (MSc)

Lysosomal storage disorders are a group of rare neurodegenerative diseases that are collectively common, sharing many aspects with other neurodegenerative disorders, including substrate build-up and neuroinflammation. The GM2 Gangliosidoses, Tay-Sachs disease and Sandhoff disease, are pathologically overlapping lysosomal storage disorders, with high prevalence within specific ethnicities. Their effects are neurologically devastating and often fatal at young ages. Current treatments only slow or stall an inevitable decline in health. Novel treatment targets are needed for these disorders, and others with similar pathologies. In these works we demonstrate the negative effect the inflammatory cytokine tumour necrosis factor-alpha has on survival of a model of Sandhoff disease. We demonstrate its role in the upregulation of astrogliosis, and apoptosis, and we present evidence that this effect on astrogliosis occurs through an upregulation of the JAK-2/STAT3 pathway. Though fruitful, a singular focus on inflammation/gliosis in these diseases has left a vacuum in the research into neuron specific molecular processes. We observe the development of inflammation, astrogliosis and neuronal processes in our model, and demonstrate a bi-phasic disease progression, in which early onset microgliosis precedes terminal astrogliosis, apoptosis, and a decline in excitatory glutamate receptors, suggesting neuron-specific malfunction. Furthermore, we show that knockout of the synaptic protein neuronal pentraxin 1 retards neurodegeneration and extends the lifespan of Sandhoff disease mice, independent of inflammation or astrogliosis. Through electrophysiology, we provide evidence of dysregulation of glutamate receptors in Sandhoff disease, and show that knockout of neuronal pentraxin 1 provides rescue from this dysregulation. This work expands on research into gliosis in GM2 gangliosidoses, presents the finding of a novel protein isoform, and presents a new focus on non-glial disease mechanisms and treatments for these and other neurodegenerative disorders. / Thesis / Doctor of Philosophy (PhD) / Lysosomal storage disorders are a group of neurological diseases that are debilitating, and often fatal at a young age. Two diseases of this group- Tay-Sachs disease and Sandhoff disease – are similar in their causes and symptoms. Current treatments for these diseases only slow or stall an inevitable decline in health. New targets for treatment are required, and we provide data suggesting several proteins that may fit this criterion. We also provide evidence of the discovery of a new form of one of these proteins, which is found in high levels in the disease, indicating it may be important in these and other neurodegenerative diseases. Finally, we provide findings indicating that a certain cell type, which is largely ignored in current research for these diseases, may be important in the disease progress. These findings increase our knowledge of Tay-Sachs disease and Sandhoff disease, and open new avenues for medicinal intervention.

Purpose: The purpose of this study was to investigate the role of the vasculature and immune system in models of glaucoma. Vascular changes have been implicated in glaucoma. As well there is mounting evidence that the immune system plays a role in the disease. It is my hypothesis that the vasculature and immune system play a role in the retinal response to injury in models of glaucoma. Methods: Immunohistochemistry, in vivo retinal imaging (Bright field, fluorescent, optical coherence tomography), Slit2 injections and Evan’s blue labeling were used to investigate vascular and immune changes associated with retinal ganglion cell death after optic nerve crush up to 28 days after injury. Histology, immunohistochemistry, and intravascular labeling were utilized to investigate the role of the vascular degeneration and the systemic immune response to elevated intraocular pressure in 8-16 week old AP-2β Neural Crest Cell Knockout (AP-2β NCC KO) mice. Results: The vascular and immune responses to optic nerve crush were not found to play a significant role in the response to retinal ganglion cell death. Conversely the role of vascular degeneration and immune cell recruitment to the retinas of AP-2β NCC KO mice demonstrated that these factors played a significant role in the retinal response to injury. Conclusion: The vasculature and immune system play a varied role in the response to retinal injury and neurodegeneration depending on the model being studied. The vascular and immune changes were of minimal significance in acute optic nerve crush injury. On the other hand, the chronic injury associated with elevated intraocular pressure in AP-2β NCC KO mice was associated with significant vascular degeneration and systemic immune cell infiltration. / Thesis / Doctor of Philosophy (PhD)

Neurodegenerative disorders including Parkinson’s disease (PD), Huntington’s disease (HD) and the most frequent, Alzheimer’s disease (AD), represent one of the most urgent medical needs worldwide. Despite a significantly developed understanding of disease development and pathology, treatments that stop AD progression are not yet available. The recent approval of sodium oligomannate (GV-971) for AD treatment in China emphasized the potential value of natural products for the treatment of neurodegenerative disorders. Many current clinical studies include the administration of a natural compound as a single and combination treatment. The most prominent mechanisms of action are anti-inflammatory and anti-oxidative activities, thus preserving cellular survival. Here, we review current natural products that are either approved or are in testing for a treatment of neurodegeneration in AD. In addition to the most important compounds of plant origin, we also put special emphasis on compounds from algae, given their neuroprotective activity and their underlying mechanisms of neuroprotection.