Distinct roles of Hsp110 proteins in regulating prion propagation

Abstract

Prion diseases are fatal, transmissible neurodegenerative disorders characterized by the conformational conversion of the cellular prion protein (PrPC) into its pathogenic isoform PrPSc. Propagation of prions depends on the fragmentation of PrPSc aggregates to generate new seeds. However, the factors regulating this process remain incompletely understood. For yeast prions, Hsp104 that has no mammalian homologue, is firmly established as a disaggregase. Hsp110 has emerged as a potential mediator of protein disaggregation and aggregate remodeling in mammalian cells. Although Hsp110 proteins have been implicated in several protein misfolding disorders, their individual contributions to mammalian prion propagation have not been characterized. In this study, we examined the roles of the three Hsp110 proteins -Apg-1, Apg-2, and Hsp105- in cellular prion infection. CRISPR-generated knockout cell lines were established for each Hsp110 and evaluated for their effect on de novo prion infection. PrPSc accumulation was assessed by immunoblot across serial passages and up to 40 days, and prion seeding activity was quantified using real-time quaking-induced conversion (RT-QuIC). Loss of individual Hsp110s produced distinct effects on prion propagation. Apg-1-deficient cells exhibited strongly reduced levels of PrPSc over all passages analyzed, suggesting a role for Apg-1 in establishing persistent prion infection. Interestingly, prion seeding activity was detectable in later passages. Hsp105-deficient cells displayed reduced PrPSc levels during the early stages, indicating that Hsp105 contributes to early prion replication. In contrast, Apg-2 knockout resulted in high prion seeding activity over all passages analyzed and only slightly reduced PrPSc levels in early passages. Collectively, these findings demonstrate that Hsp110 family members perform distinct functions during early phases of prion infection and establishment of persistent infection. This work provides new insight into the role of molecular chaperones in prion biology and identifies Hsp110 proteins as potential modulators of prion replication and potentially therapeutic targets for protein misfolding diseases.