Molecular Genetics Of Scrapie Pathogenesis

传染性海绵状脑病（tse或朊病毒疾病）是一组罕见的神经退行性疾病，包括人类散发的克雅氏病（sCJD）、绵羊的痒病、牛海绵状脑病（BSE）和长尾鹿和麋鹿的慢性消耗性疾病（CWD）。朊病毒可以跨越物种屏障。BSE在英国已经感染了人类，而CWD可能在美国也有类似的情况，这一事实强调了了解朊病毒发病机制和开发有效治疗方法的重要性。朊病毒疾病的传染因子被称为朊病毒，主要由正常的、对蛋白酶敏感的朊病毒蛋白（PrPC）的一种异常折叠的、蛋白酶抗性形式（PrPSc）组成。PrPC和PrPSc之间的氨基酸同源性可能影响对感染的易感性，而PrPSc分子之间的结构差异被认为编码菌株表型。我的实验室在分子和致病水平上解决朊病毒疾病的不同方面，包括：1)确定在朊病毒感染期间发生的最早事件，2)定义与朊病毒相关的神经变性有关的分子途径，3)确定朊病毒菌株的分子基础，4)确定PrPC序列和翻译后修饰如何影响PrPSc的形成和疾病表型，5)开发有效的朊病毒疗法。

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 differences in structure 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 2020, we continued studies looking at prion disease progression and mitochondrial dysfunction using mice with known mitochondrial defects. These studies are part of a collaboration with Dr. Catharine Bosios laboratory which has a Seahorse XF Analyzer to measure mitochondrial respiration and viability. We have found that, in mice in which a gene involved in axonal degeneration has been knocked out, more rapid prion disease progression is associated with an unexpected increase in mitochondrial respiration. We are currently investigating the basis of this increase in mitochondrial oxygen consumption. In 2020 we inoculated prions into three different lines of transgenic mice, each of which had a different gene involved in mitochondrial dynamics ablated. Our results show that prion disease incubation times are differentially affected depending upon the gene that has been knocked out. We are in the process of analyzing mitochondrial function in these mice in order to determine how alterations in mitochondrial dynamics may influence prion disease progression. In 2020 post-doctoral fellow Dr. Daniel Shoup began testing how cell lines he derived in 2019, which express a reporter gene to measure mitochondrial redox stress, responded to exposure to prions. His preliminary results suggest that prion infection affects the redox state of the cell in a prion-strain dependent manner. These studies are ongoing, and the results will provide insights into the mechanisms involved in cellular loss during prion infection. Amino acid mismatches between PrPC and PrPSc influence the rate at which PrPSc forms. Thus, heterozygosity at key residues has the potential to significantly slow or even prevent disease transmission (J Gen Virol 93: 2749-2756 (2012)). In human PrPC, methionine/valine heterozygosity at codon 129 influences prion transmission and is a known resistance factor to sCJD (ACTA Neuropathol 130: 159-170 (2015)). We hypothesized that the PrPSc allotype ratio, i.e. the ratio of PrPSc with methionine at codon 129 (PrPSc-M129) to PrPSc with valine at codon 129 (PrPSc-V129), determines how efficiently prions from heterozygous cases of CJD transmit disease. Consistent with the possibility that PrPSc allotype could impact disease transmission, we found that the PrPSc allotype ratio varied in codon 129 MV heterozygous cases of sCJD (Moore et al. PLoS Pathog. 12: e1005416 (2016), Annual Report 2016), with some cases having a greater amount of PrPSc-M129 than others. Based on these data, we further hypothesized that those cases with higher levels of PrPSc-M129 would more efficiently transmit disease to mice expressing human PrPC with a methionine at codon 129 (PrPC-M129). In 2020, we published the results of transmission of 10 cases of codon 129 MV heterozygous CJD into two different lines of susceptible transgenic mice overexpressing PrPC-M129. Our data showed that, while PrPSc allotype was not predictive of disease incubation time, it did appear to correlate with the type of PrPSc deposited. Our data also showed that, within a single brain, prions with different infectious properties were present in brain regions with different PrPSc allotypes. This latter observation may help to explain why multiple prion strains can be isolated from a single brain. Cumulatively, our data demonstrated that there was more heterogeneity in the transmission properties of CJD neurological subtypes than has been previously described and suggest a complex picture of CJD transmission where CJD subtype, PrPSc conformation, PrPSc allotype, and the host likely all contribute to the final disease phenotype. 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 2020, we continued in vivo studies to determine the amount of prion infectivity in CJD samples that were processed for mass spectrometry using several other common methods. Additionally, we expanded these studies in 2020 to include hamster prions. Hamster prion titers are 4-5 logs higher than in humans and will enable us to determine if the mass spectrometry techniques used inactivate very high titer prions. These experiments will take at least another year to complete but the results will be of use to regulators, biosafety specialists, and researchers tasked with determining whether or not prion-infected samples can be safely analyzed outside of BSL-2 containment.