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Role of lysosomal pathways in prion toxicity

URL to cite or link to: http://hdl.handle.net/1802/35929

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PDF of dissertation
Thesis (Ph. D.)--University of Rochester. Department of Biology, 2020.
Prion diseases are infectious and fatal neurological disorders that afflict a number of mammalian species. In humans, these diseases have an incidence of one per million individuals. Prion diseases are characterized by extended incubation periods before a rapid onset of neurological symptoms and decline in cognitive function. During the course of these diseases, cytotoxic aggregates of the prion protein (PrPSc) accumulate and cause the conversion of the non-toxic cellular form of the prion protein (PrPC) into additional aggregates in a self-seeding fashion. While the formation of PrPSc generally results in cell death and neurodegeneration, a few mammalian cell lines have been identified that are capable of accumulating and propagating PrPSc aggregates without succumbing to their cytotoxic effects. This thesis utilizes these cell lines as a disease model to identify cellular pathways that are impacted by the presence of PrPSc aggregates. In our first study, we investigated the effects of intracellular accumulations of PrPSc aggregates on cellular degradation pathways. Intracellular PrPSc aggregates primarily accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrPSc aggregates has the potential to globally influence protein degradation kinetics within an infected cell. In order to study this phenomenon, we measured the proteome-wide effects of prion infection on protein degradation rates in N2a neuroblastoma cells by dynamic stable isotopic labeling with amino acids in cell culture (dSILAC) and bottom-up proteomics. The results of these experiments revealed that the majority of the proteome is degraded more rapidly in cells infected with PrPSc aggregates. We showed that this effect occurs concurrently with increases in the cellular activities of autophagy and some lysosomal hydrolases. The enhancement of lysosomal degradative flux may play a role in survival of N2a cells upon prion infection. In our second study, we investigated genetic factors that contribute to cellular tolerance of prion toxicity. A genome-wide CRISPR screen was performed to identify genes that have synthetically lethal interactions with PrPSc in two viable prion-infected cell lines. The screen provided a global survey of how the expression of individual genes in the genome influences cell survival in the presence of prion aggregates. The results identified members of the homotypic fusion and vacuole protein sorting (HOPS) tethering complex as key prion tolerance factors in prion-infected cell lines. The HOPS complex is responsible for facilitating vesicle fusion events with the lysosome, revealing another potentially important link between PrPSc aggregates and lysosomal pathways. In the final study, small molecule drugs that act on lysosomal degradation pathways were tested for prion clearance activities. Both STF-62247 and bafilomycin, known lysosomal inhibitors, are toxic to prion-infected cells, suggesting that a functional lysosomal pathway is required for cell survival during prion infection. Together, these data point towards the lysosome as playing a pivotal role in cell viability during prion infection
Contributor(s):
Charles Richard Hutti - Author
ORCID: 0000-0002-4570-092X

Sina Ghaemmaghami - Thesis Advisor

Primary Item Type:
Thesis
Identifiers:
Local Call No. AS38.669
Language:
English
Subject Keywords:
Cell culture; CRISPR; Dynamic SILAC; Genome-wide CRISPR screen; Mass spectrometry; Prion
Sponsor - Description:
National Science Foundation (NSF) - CAREER Award (MCB-1350165)
National Institutes of Health (NIH) - R35 GM119502 awarded to Professor Ghaemmaghami
University of Rochester - Robert L. and Mary L. Sproull University Fellowship
First presented to the public:
10/19/2020
Originally created:
2020
Original Publication Date:
2020
Previously Published By:
University of Rochester
Place Of Publication:
Rochester, N.Y.
Citation:
Extents:
Number of Pages - xii, 149 pages
Illustrations - illustrations (some color)
License Grantor / Date Granted:
Marcy Strong / 2020-10-19 10:54:17.836 ( View License )
Date Deposited
2020-10-19 10:54:17.836
Submitter:
Marcy Strong

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