Molecular Mechanisms of Prion Protein Amyloid Formation

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Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. PrPSc can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unclear whether or not prion diseases where PrPSc is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrPSc is primarily deposited as non-amyloid. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) understanding the pathways of PrP amyloid formation and, 2) studying how mutations in PrP influence PrPSc amyloid formation in familial forms of prion disease. Using LC-MS/MS Nanospray Ion Trap Mass Spectrometry, we have generated the proteomes of prion infected mouse brain tissue which had accumulated PrPSc in either amyloid or non-amyloid forms. Our results suggest that the brain has a similar, general neuroinflammatory response to deposition of amyloid and non-amyloid forms of prions. However, cell death pathways appeared to differ, with mitochondrial pathways of apoptosis implicated only in the non-amyloid form of prion disease. Metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. This work is in press at the Journal of Proteome Research. In 2014 we continued long term in vivo work to study the pathogenesis of different forms of amyloid and non-amyloid human prion disease in transgenic mice expressing either mutant or wild-type human PrP. This represents another approach to delineate the mechanisms underlying amyloid and non-amyloid forms of prion disease. These studies will likely take several years to complete but will provide important information in several poorly understood areas of human prion disease including 1) the contribution of the host versus the contribution of the prion strain to different in vivo disease phenotypes and 2) the mechanisms of amyloid versus non-amyloid prion formation. In 2014, we have initiated a new series of in vitro fibrillization studies to study how different PrPC mutations associated with inherited forms of familial human prion disease dictate the formation of PrP amyloid. These studies will help us to understand why amyloid or fibrillar forms of human PrPSc tend to be associated with low prion infectivity and thus directly address the issue of what is required for a misfolded protein to be infectious. In 2014, we have begun to use LC-MS/MS Nanospray Ion Trap Mass Spectrometry to study brain tissue from human cases of amyloid and non-amyloid forms of prion disease. This work will help us begin to understand how PrPSc and other proteins differ between the two disease states and should provide important insights into their pathogenesis.