Molecular Genetics Of Scrapie Pathogenesis

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

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 several 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 (Moore et al. J. Proteome Res. 13: 4620 (2014), Annual Report 2014) and were the first to show that mitochondrial respiration is impaired in late-stage prion disease (Faris et al., J. Virol. 91: e00524-17 (2017), Annual Report 2017). We have also published data showing that PrPC is present in brain mitochondria from healthy wild-type and transgenic mice (Faris et al., Sci. Rep. 7: 41556 (2017), Annual Report 2017) suggesting that, as has been proposed for other proteins associated with neurodegenerative disorders, PrPC may play a role in mitochondrial function. In 2021, we continued studies looking at prion disease progression and mitochondrial dysfunction in mice with known mitochondrial defects. These studies utilize a Seahorse XF Analyzer to measure mitochondrial respiration and viability and are part of a collaboration with Dr. Catharine Bosios laboratory. Our results show that prion disease is more rapid and mitochondrial respiration increased in prion infected mice lacking a gene involved in axonal degeneration. In 2021, in order to understand the molecular basis of these changes in prion disease incubation time and mitochondrial oxygen consumption, we analyzed the expression levels of multiple proteins involved in mitochondrial respiration, mitochondrial dynamics, and regulation of oxidative stress. We are currently writing up the results which will be submitted for publication before the end of 2021. In 2021, we completed Seahorse XF Analyzer studies measuring mitochondrial respiration and viability in 3 different lines of prion infected transgenic mice, each of which had a different gene involved in mitochondrial dynamics ablated. Our results show that mitochondrial respiration was minimally impacted in these mice, even though prion disease incubation times were more rapid. In order to understand why prion disease in more rapid in these mice, our next step is to analyze the expression levels of multiple proteins involved in mitochondrial respiration, mitochondrial dynamics, and the regulation of oxidative stress. In 2021, we also initiated studies to assess prion infection in a line of mice where axonal degeneration is delayed. These studies will help to clarify the role that axonal loss plays in the brain and retinal pathology associated with prion disease. Different proteinase K (PK) cleavage sites in the N-terminus of PrPSc are indicative of differences in its structure. Based on the PK cleavage sites, two major structural forms of PrPSc have been identified in sCJD: Type 1 and Type 2. Recently, it has been found that prions in many cases of sCJD are mixtures of Type 1 and Type 2 PrPSc. This suggests that there may be a complex population of PrPSc molecules present, many of which have different secondary structures (Brain 132: 2643 (2009)). Our project involves using LC-MS/MS Mass Spectrometry (MS) to precisely map the N-termini of PrPSc molecules associated in different brain regions from neurological subtypes of CJD. In 2020, this project was halted due to insurmountable design flaws in our Agilent 6550 iFunnel Q-TOF mass spectrometer. In 2021, we obtained a replacement Q-TOF mass spectrometer from Agilent. An Orbitrap mass spectrometer was also installed at RML for use by all laboratories. In 2021, we were able to use these new instruments to resume and largely complete the initial sample analysis on this project. The N-termini of PrPSc from multiple brain regions of Type 1 and Type 2 CJD brains were determined. In addition, we also determined the PrPSc allotype ratio for samples that were heterozygous for methionine and valine at codon 129 in PrPC. We are currently in the process of analyzing these data to determine the distribution of different PrPSc conformations and allotypes in a CJD infected brain. The ultimate goal of this study is to determine whether certain structural populations of PrPSc correlate with specific CJD phenotypes and PrPSc allotypes. 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. The extreme resistance of prions to inactivation and their ability to persist in the environment for decades thus remain significant public health issues. Similar concerns apply to the laboratory setting. It is often necessary to analyze prion samples using advanced analytical techniques that are frequently only available outside of biosafety level 2 (BSL-2) containment, the minimum biosafety level required for studying infectious prions. However, the remarkable resistance of prions to inactivation can make it difficult to produce and analyze prion samples free of infectivity that still retain sufficient sample integrity for research purposes We recently published a study demonstrating that a straightforward denaturation and in-gel protease digestion protocol used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity (Moore et al., Biochim Biophys Acta Proteins Proteom. 1866: 1174 (2018), Annual Report 2018). In 2021, we continued in vivo studies to determine the amount of prion infectivity in CJD and hamster prion infected brain samples that had been processed for mass spectrometry using several other common methods. These transmission experiments will be completed by the end of the year, after which we will begin the histopathological and biochemical analysis of the inoculated mice. The results of this study will be of use to regulators, biosafety specialists, and researchers tasked with determining if prion-infected samples can be safely analyzed outside of BSL-2 containment.