Molecular Genetics Of Scrapie Pathogenesis

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include sporadic Creutzfeldt-Jakob disease (sCJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. Prions can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding prion pathogenesis and developing effective therapeutics. The infectious agent of prion 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. Susceptibility to infection can be influenced by amino acid homology between PrPC and PrPSc while structural differences between PrPSc molecules are believed to encode strain phenotypes. My laboratory addresses different aspects of prion diseases at both the molecular and pathogenic level including: 1) identifying the earliest events which occur during prion infection, 2) defining the molecular pathways involved in prion-associated neurodegeneration, 3) determining the molecular basis of prion strains, 4) determining how PrPC sequence and post-translational modifications influence PrPSc formation and disease phenotype and, 5) development of effective prion therapeutics. Although there is an increasing body of work suggesting that mitochondrial dysfunction is important in neurodegenerative protein misfolding diseases such as Alzheimers disease (AD) and Parkinsons disease (PD), the role of mitochondria in prion disease is poorly understood. We have found that mitochondrial pathways of apoptosis are implicated in non-amyloid forms of prion disease (Annual Report 2014) and were the first to show that mitochondrial respiration is impaired in late-stage prion disease (Annual Report 2017). Consistent with PrPC potentially having an effect on mitochondrial function and/or health, we have also published data showing that PrPC is present in brain mitochondria from healthy wild-type and transgenic mice (Annual Report 2017). More recently, we have published data suggesting that regulation of mitochondrial energy production by the molecule SARM1 may help to reduce levels of reactive oxygen species produced by mitochondria and thus slow prion disease progression (Annual Report 2022). Changes in mitochondrial energy production and dynamics can lead to changes in mitochondrial health and the initiation of mitophagy, the process whereby damaged mitochondria are destroyed by the cell. In 2023, we completed studies looking at prion infection in mice which were ablated for two proteins, PINK1 and Parkin, that are involved in a well-described pathway of mitophagy. We finished the analysis of prion pathology, including PrPSc levels and neuropathology, and completed western blot analysis of multiple proteins involved in mitochondrial respiration and mitochondrial dynamics. Our results show that both molecules exert a protective effect on prion infection. A manuscript describing this work has been submitted and reviewed and is currently undergoing revision at PLoS One (Ward et al., The PINK1/Parkin pathway of mitophagy exerts a protective effect during prion disease, 2023). A part of this study utilized a Seahorse XF Analyzer to measure mitochondrial respiration and viability and was done in collaboration with Dr. Catharine Bosios laboratory. In 2023, we continued a project studying prion infection in a line of mice where axonal degeneration is delayed and some types of neurons in the retina are protected from degeneration. The protein involved in this process helps to regulate the availability of a substrate that the mitochondria need to produce energy. We are continuing to use western blot analysis and immunohistochemistry to determine whether the pathological phenotype of prion disease in these mice is altered when compared to wild-type controls. These studies will help to clarify the mechanisms underlying axonal loss in brain and retina during prion disease. The retinal portion of this project is a collaboration with Dr. Bruce Chesebros laboratory. Our studies on the role of mitochondria in prion disease implicate regulation of mitochondrial energy production as a mediating factor for prion disease progression. In 2023, Dr. Jason Hollister in the lab initiated an in vivo study to study whether or not changes in energy production by the cell can slow prion disease progression by reducing cellular stress and triggering autophagy, a mechanism by which the cells degrade prions. The results of this study will have significant implications for the impact of diet on neurodegenerative diseases and could lead to diet-based approaches to slowing disease progression. In 2023, the post-doctoral fellow in the lab, Dr. Daniel Shoup, continued his project with a cell-based system that he developed to study the redox state of mitochondria during the initial stages of prion infection. His data show that there is a prion strain-dependent change in the mitochondrial redox state that is modified by the presence or absence of PrPC. He further showed that the alteration in the mitochondrial redox state correlates with the presence of PrPSc and with cell-dependent changes in PrPSc structure during the initial stages of prion infection. Currently, he is initiating experiments to see if degradation of PrPSc by the cell leads to the release of toxic PrPSc byproducts that trigger the change in redox state. These studies will help us to understand not only how PrPSc may directly or indirectly influence mitochondrial function but also how mitochondria respond to prion infection. Two major structural forms of PrPSc, Type 1 and Type 2, have been identified in sporadic CJD (sCJD), the most common form of human CJD. It is known that prions in many cases of sCJD are mixtures of Type 1 and Type 2 PrPSc, suggesting that PrPSc may differ in different regions of the brain. In 2023, we completed an analysis of the N-termini of PrPSc from multiple brain regions of Type 1 and Type 2 CJD brains. We found that different PrPSc conformations can be present in different regions of a sCJD infected brain. The results are being written up in a manuscript and should be submitted before the end of the year. The ultimate goal of this study is to determine whether certain structural populations of PrPSc correlate with specific sCJD phenotypes. The prion agent is notoriously difficult to inactivate with the routine sterilization protocols used in hospitals, where iatrogenic transmission of CJD is an ongoing concern. Thus, prions remain a significant public health issue. We have published a study demonstrating that a common technique used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity (Annual Report 2018). We have expanded our study to determine the amount of prion infectivity in sCJD and hamster prion infected brain samples processed for mass spectrometry using several other common methods. The animal studies were completed last year and, in 2023, we completed the biochemical analysis of the brains from inoculated mice. We are currently in the process of assessing prion-related pathology in the brain. In addition, in 2023 we collaborated with Dr. Brent Race on a study demonstrating that the disinfectant Wex-cide can be used for the inactivation of certain strains of prions. The results of these studies will be of use to regulators, biosafety specialists, and researchers tasked with determining how prion-infected samples can be inactivated and/or safely analyzed outside of BSL-2 containment.