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The establishment of a flow cytometry system to study HTT regulation and expression Kosior, Natalia Athina

Abstract

Huntington’s disease (HD) is a late-onset neurodegenerative disorder characterized by motor deficits, behavioral abnormalities and psychiatric symptoms. HD is caused by a CAG trinucleotide repeat expansion in the HTT gene resulting in expression of a mutant polyglutamine stretch in the huntingtin protein. HD affects up to 14 per 100,000 people in British Columbia. This devastating disorder is characterized by relatively selective neuronal loss in the caudate and putamen, regions of the brain referred to collectively as the striatum. Numerous mechanisms leading to this selective neurodegeneration have been proposed but the pathways involved are still not well understood. The huntingtin protein (HTT) is ubiquitously expressed, meaning that it is present in all cell types in the central nervous system, prompting investigations into potential pathogenic mechanisms outside of the characteristic neuronal loss. The brain is composed of microglia and astrocyte cell populations that together, make up the central immune system. Immune system activation has been implicated in the disease pathogenesis of various neurodegenerative diseases, including HD. The purpose of this thesis was to establish a flow cytometry system to investigate potential HTT regulatory mechanisms as well as increase knowledge of immune cell dysfunction in HD. Following the establishment of the flow cytometry system I found that HTT expression does not vary across the cell cycle. Using an adapted technique, I identified robust but not absolute genetic knock-down in two microglia-specicfic conditional knock out mouse models. Adding to the neuroinflammation focus, I identified mutant-huntingtin specific changes in protein phosphorylation expression in microglia as a means of identifying potential signaling cascades involved in exaggerated cytokine release. Lastly, I investigated transcriptional dysregulation in HD microglia and astrocyte populations and the effect of a candidate therapeutic on gene expression in these cell types. My research provides additional insight into potential protein phosphorylation and genetic pathways involved in immune dysfunction in HD and will focus future experiments aimed at understanding the role of central inflammation in HD.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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