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