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Role of gangliosides in extracellular vesicle secretion in wild-type and Huntington's disease cell models
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- Author / Creator
- Kadam, Vaibhavi
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Gangliosides are sialic acid containing glycosphingolipids highly enriched in the brain that play vital roles in intercellular communication, cell signalling and calcium homeostasis. A decrease in the ganglioside levels in the brain has been associated with several neurodegenerative diseases. Studies in the Sipione lab have previously demonstrated that the levels of gangliosides, especially ganglioside GM1, are decreased in Huntington’s disease (HD). HD is a monogenic neurodegenerative disease characterized by motor impairment and cognitive and psychiatric decline. It is caused by an abnormal expansion in the CAG trinucleotide repeat in the exon 1 region of the huntingtin (HTT) gene, which in turn encodes for an abnormally elongated polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein. As a result, the mutant (mHTT) protein misfolds, acquires toxic conformations and aggregates, eventually compromising neuronal function and viability. Past work in the Sipione lab has also shown that restoring normal levels of GM1 by its exogenous administration in HD mouse models has profound therapeutic effects and reduces mHTT levels in the brain. Because GM1 does not affect the transcription of the Htt gene, the mechanism underlying its effects on mHTT burden may be at the proteostatic level. Proteomics and Gene Onthology analysis of both wild-type and HD mouse brains treated with GM1 in vivo (compared to untreated controls) revealed that the treatment affects the abundance of various proteins of the extracellular vesicle (EV) pathway. EVs are cell-derived, membrane-enclosed particles (30-1000nm) which are involved in intercellular communication and cell signaling and have been found to carry misfolded proteins.
In this thesis, I investigated the hypothesis that cell treatment with exogenous GM1 increases the secretion of EVs containing mHTT. I further investigated the effects of decreasing endogenous ganglioside levels - as observed in HD and other neurodegenerative conditions – on EV and mHTT secretion. My studies demonstrate that cell treatment with GM1 promoted EV release in both normal and HD cells of both neuronal and peripheral origin, without affecting particle size. Furthermore, GM1 restored normal EV secretion in a neuronal cell line stably expressing mHTT (N2a 72Q) that displays reduced GM1 levels compared to wild-type control cells and lower levels of EV secretion. In N2a 72Q cells, GM1 treatment increased the export of mHTT via secreted EVs. On the other hand, pharmacological inhibition of ganglioside synthesis in neuronal cells resulted in impairment of EV secretion and reduced levels of mHTT in EVs. To confirm the effects of endogenous gangliosides on EV secretion, I used a neuronal cell line where B4galnt4, the gene that encodes for a major ganglioside biosynthetic enzyme, was deleted by CRISPR-Cas9. The resulting decrease in cellular levels of complex gangliosides in this model resulted in decreased secretion of EVs, a phenotype that could be attenuated by GM1 administration. Thus, overall, my data suggest that cellular ganglioside levels positively correlate with EV release, highlighting a novel role of neuronal gangliosides in EV and misfolded protein secretion. Of note, modulation of cellular gangliosides was found to influence the GM1 content of EVs. As GM1 is known to mediate cell-cell communication and inflammation, future studies will determine whether the abundance of GM1 on the surface of EVs carrying mHTT may determine their fate, by functioning as a ‘code’ to facilitate EV shuttling to microglia cells for uptake and degradation. -
- Graduation date
- Fall 2020
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.