Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. In humans, the most common type of prion disease is Creutzfeldt-Jakob disease (CJD) which can occur in several forms. Sporadic CJD (sCJD) makes up the majority of the cases and occurs randomly at an incidence of 1-2 per million worldwide. Iatrogenic CJD (iCJD) is associated with exposure to prion contaminated medical instruments or products while familial CJD (fCJD) is associated with mutations in the prion protein gene. 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, in 2015 we have generated the protein profiles of PrPSc isolated from over 20 cases of CJD where PrPSc is deposited as diffuse, amyloid negative deposits and/or as amyloid plaques. Our results show that the protein profiles differ depending upon whether PrPSc is derived from sCJD or iCJD suggesting pathogenic differences between the two different disease types. A manuscript describing this work is in preparation. In 2015 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. Multiple different CJD isolates have been inoculated into these mice and, in some cases, multiple brain regions from the same patient have also been inoculated. While we completed transmission data for some of these isolates in 2015, mice inoculated with multiple isolates are still being monitored for disease. This experiment represents another approach to delineate the mechanisms underlying amyloid and non-amyloid forms of prion disease. These studies will 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 2015, we continued 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. In particular, we have looked at how proteolytic processing may influence PrP fibril formation. These studies will help us to understand the cellular environments required for PrP fibril formation and provide insight into why amyloid or fibrillar forms of human PrPSc tend to be associated with low prion infectivity. Thus, they directly address the issue of what is required for a misfolded protein to be infectious.
