Biologic and biochemical features of prion pathogenesis

Includes bibliographical references.2016 Fall.Prions are the causative agents of a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. Prions are unique in that disease is initiated when the normal prion protein (PrPC) undergoes a conformational change and propagates through a process of templated conversion to an infectious, misfolded, isoform (PrPRES, PrPCWD, or PrPSc) which can assemble into oligomers and amyloid fibrils. Disease is associated with prion accumulation in the central nervous system, causing the pathologic lesions of neurodegeneration, white matter spongiosis, and a reactive astrogliosis. Previous work has demonstrated the process of prion propagation and disease pathogenesis can be influenced by conversion cofactors, inhibitors, and biologic systems. Heat shock proteins have been shown to protect against the toxic disease effects of denatured and aggregated proteins in several models of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and spinocerebellar ataxia. In this dissertation, I investigated if heat shock protein 72 (HSP72) expression in neurons could protect against prion disease-associated pathology through a cell culture and mouse model of murine-adapted scrapie strain RML. In contrast to the role in other neurodegenerative diseases, HSP72 did not alter the prion disease course or amount of prion conversion in either disease model. Chronic wasting disease (CWD) is a naturally occurring, horizontally transmitted prion disease affecting wild and captive cervid populations that is rapidly expanding into new states and countries. Studies investigating the distribution of PrPCWD during early subclinical CWD infection have detected prions in the oropharyngeal lymphoid tissues as early as 1.5 months; however, the complete tissue distribution of PrPCWD immediately following prion exposure and the chronological progression of prion tissue accumulation remains unknown. Here, I show prions initially accumulate in the oropharyngeal lymphoid tissues following mucosal exposure and rapidly disseminate to all systemic lymphoid tissues prior to neuroinvasion. These findings will help better understand the early pathogenesis of CWD prior to clinical disease and potentially identify therapeutic targets. Prion disease diagnosis relies on demonstration of the misfolded isoform by immunodetection, amyloid seeding assays, or animal bioassays, all assays which may require separate sample preparations precluding examination by multiple tests. To address this limitation, I developed a new technique to detect PrPCWD amyloid seeding in fixed paraffin-embedded (FPE) tissues by real-time quaking induced conversion (RT-QuIC). FPE RT-QuIC proved to be more sensitive than IHC for prion detection and the use of RT-QuIC amyloid formation kinetics yielded a semi-quantitative estimate of the prion burden in samples without the cost and time of animal bioassays. The normal cellular prion protein resides in cell membrane lipid rafts, which has been shown to be a site of pathogenic conversion. Previous in vitro assays have highlighted the ability of lipids to promote prion formation but knowledge is limited regarding the capacity of lipids to inhibit prion formation. Here, I show endogenous polar brain lipids directly inhibit prion amyloid formation in RT-QuIC in a dose-dependent manner. This work is the first to identify an inhibitory role of lipids and suggests the prion conversion process is influenced by a balance of pro-conversion and inhibitory molecules