Molecular Genetics Of Scrapie Pathogenesis

痒病发病机制的分子遗传学

• 批准号: 10692051
• 负责人: SUZETTE Alise PRIOLA
• 金额: $ 116.48万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692051
• 关键词: Acute; Address; Affect; Alzheimer' s Disease; Amino Acids; Annual Reports; Apoptosis; Axon; Biochemical; Bovine Spongiform Encephalopathy; Brain; Brain region; Cells; Chronic Wasting Disease; Codon Nucleotides; Collaborations; Complex; Containment; Creutzfeldt-Jakob Syndrome; Data; Data Analyses; Deer; Development; Digestion; Disease; Disease Progression; Endopeptidase K; Environment; Equine mule; Event; Gel; Genes; Genus Hippocampus; Goals; Great Britain; Hamsters; Hospitals; Human; Iatrogenesis; Immunohistochemistry; Impairment; Infection; Infectious Agent; Laboratories; Maps; Mass Spectrum Analysis; Measures; Methionine; Methods; Mitochondria; Molecular; Molecular Conformation; Molecular Genetics; Mouse Strains; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurons; Oxidation-Reduction; Oxidative Regulation; Oxidative Stress; Parkinson Disease; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Peptide Hydrolases; Phenotype; Play; Population; Post-Translational Protein Processing; Postdoctoral Fellow; PrP; PrPSc Proteins; Predisposition; Prion Diseases; Prions; Process; Prophylactic treatment; Proteins; Proteome; Protocols documentation; Public Health; Publishing; Research; Research Personnel; Resistance; Respiration; Retina; Role; Sampling; Scrapie; Sheep; Site; Specialist; Sterilization; Structure; System; Techniques; Therapeutic; Transgenic Mice; Vaccine Therapy; Valine; Western Blotting; Wild Type Mouse; advanced analytics; axonal degeneration; base; disease phenotype; experimental study; in vivo; in vivo Bioassay; mitochondrial dysfunction; protein misfolding; therapeutically effective; transmission process

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 2022, we published studies looking at prion disease progression and mitochondrial dysfunction in prion infected mice. These studies utilized a Seahorse XF Analyzer to measure mitochondrial respiration and viability and were 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 called SARM1. In 2022, we continued our studies on the role of mitochondria in prion infection using 3 different lines of prion infected transgenic mice, each of which had a different gene involved in mitochondrial dynamics ablated. Our results from 2021 showed that mitochondrial respiration was altered in one strain of these mice. In 2022, we analyzed the expression levels of multiple proteins involved in mitochondrial respiration, mitochondrial dynamics, and the regulation of oxidative stress in these mice. These results will help us understand how these processes are affected by prion infection and should be written up and submitted this year. In 2022, the post-doctoral fellow in the lab, Dr. Daniel Shoup, initiated and optimized a cell-based system to study the redox state of mitochondria during the initial stages of prion infection. His preliminary data suggest that there is a prion strain-dependent change in the mitochondrial redox state that is modified by the presence or absence of PrPC. He is currently pursuing experiments to determine if this effect is related to the breakdown of PrPSc during the initial stages of prion infection. These studies will help us understand whether or not PrPSc can directly or indirectly influence mitochondrial function as well as how mitochondria respond to prion infection. In 2022, we completed in vivo bioassay experiments 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. We are currently in the process of using 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. 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. It is known 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 with different brain regions from different neurological subtypes of CJD. In 2022, 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 completing analysis of 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 have 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 2022, we completed 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. In 2022, we plan to complete immunohistochemical and biochemical analysis of the brains from inoculated mice to confirm the presence or absence of prion infection. 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.

可传播的海绵状脑病（TSE或PRION疾病）是一组罕见的神经分支性疾病，包括人类中零星的克鲁特兹菲特 - jakob病（SCJD），绵羊中的scrapie，牛皮，牛海绵状的肾上腺病（BSE）（BSE）和富氏病（Cornocine）和病态的疾病（C.王室可以越过物种障碍。 BSE在英国感染了人类，并且担心CWD可能在美国采取类似的行动强调了理解王室发病机理和发展有效疗法的重要性。 Prion疾病的感染因子称为prion，主要由正常的，蛋白酶敏感的prion蛋白PRPC的异常重折叠的抗蛋白酶抗蛋白酶抗性形式（PRPSC）组成。 PRPC和PRPSC之间的氨基酸同源性可以影响感染的敏感性，而PRPSC分子之间的结构差异被认为是编码应变表型的。 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有效的毒疗法。 尽管越来越多的工作表明线粒体功能障碍在几种神经退行性蛋白质误后疾病（如阿尔茨海默氏病（AD）和帕金森氏病（PD）中很重要，但线粒体在prion病中的作用很众所周知。 我们发现，凋亡的线粒体途径与非淀粉样蛋白疾病的非淀粉样形式有关（Moore等人J. ProteomeRes。13：4620（2014），2014年年度报告，2014年年度报告，并首次表明线粒体呼吸是在晚期疾病后期疾病中受到了损害（Faris et earlion et ailor.jerol。，2017年7月7日）。 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. 在2022年，我们发表了研究，研究了受prion感染小鼠的prion病疾病进展和线粒体功能障碍。这些研究利用Seahorse XF分析仪来测量线粒体呼吸和生存能力，并且是与Catharine Bosios实验室合作的一部分。我们的结果表明，在缺乏参与轴突变性的基因的prion感染小鼠中，prion病更快，线粒体呼吸增加了称为SARM1。 在2022年，我们使用3种不同的受prion感染的转基因小鼠的线粒体在病毒感染中的作用进行了研究，每个小鼠都有不同的基因参与线粒体动力学的消融。 我们从2021年开始的结果表明，在这些小鼠的一种菌株中，线粒体呼吸发生了改变。在2022年，我们分析了参与线粒体呼吸，线粒体动力学和这些小鼠中氧化应激的调节的多种蛋白的表达水平。这些结果将有助于我们了解这些过程如何受到prion感染的影响，并应在今年提交并提交。 2022年，实验室的博士后研究员丹尼尔·舒普（Daniel Shoup）博士启动并优化了基于细胞的系统，以研究prion感染的初期阶段，研究线粒体的氧化还原状态。他的初步数据表明，线粒体氧化还原状态存在prion菌株依赖性变化，该变化通过PRPC的存在或不存在而改变。他目前正在寻求实验，以确定这种效果是否与PRPSC在prion感染的初期阶段的崩溃有关。这些研究将有助于我们了解PRPSC是否可以直接或间接影响线粒体功能以及线粒体如何应对prion感染。 在2022年，我们完成了研究轴突变性延迟并保护视网膜中某些类型的神经元的小鼠中研究prion感染的体内生物测定实验。目前，我们正在使用蛋白质印迹分析和免疫组织化学来确定与野生型对照相比，这些小鼠中的病毒疾病的病理表型是否改变了。这些研究将有助于阐明病毒疾病期间大脑和视网膜中轴突丧失的机制。 PRPSC N末端中不同蛋白酶K（PK）切割位点表明其结构差异。 基于PK裂解位点，在SCJD：1型和2型中已经确定了PRPSC的两种主要结构形式。众所周知，在许多情况下，SCJD的prions是1型和2型PRPSC的混合物。这表明可能存在复杂的PRPSC分子，其中许多分子具有不同的二级结构（Brain 132：2643（2009））。 我们的项目涉及使用LC-MS/MS质谱法（MS）来精确绘制与CJD不同神经系统亚型不同大脑区域相关的PRPSC分子的N末端。 在2022年，确定了来自1型和2型CJD大脑多个大脑区域的PRPSC的N末端。此外，我们还确定了PRPC中蛋氨酸和瓣膜杂合的样品的PRPSC同种型比。 目前，我们正在完成对这些数据的分析，以确定CJD感染大脑中不同PRPSC构象和同型的分布。这项研究的最终目的是确定PRPSC的某些结构种群是否与特定的CJD表型和PRPSC同型相关。 众所周知，对医院中使用的常规灭菌方案灭活毒品，在医院使用的常规灭菌方案，在该方案中，CJD的医源性传播是一个持续关注的问题。王室对失活的极端抵抗及其在数十年中持续在环境中的能力仍然是重大的公共卫生问题。类似的担忧适用于实验室环境。通常有必要使用经常仅在生物安全2级（BSL-2）遏制之外可用的先进分析技术来分析prion样品，这是研究传染病所需的最低生物安全水平。但是，毒品对失活的显着抗药性可能使得难以生产和分析无感染性的prion样品，这些样本仍然保留了足够的样本完整性，以进行研究目的 我们发表了一项研究，该研究表明，一种直接的变性和凝胶蛋白酶消化方案用于制备质谱质谱的样品，导致至少7种prion感染感染的日志（Moore等人，Biochim Biophys Acta Acta proteins Proteom。在2022年，我们完成了体内研究，以确定CJD和仓鼠prion感染的脑样品的prion感染量，这些样本已使用其他几种常见方法进行了质谱。 在2022年，我们计划对接种小鼠的大脑完成免疫组织化学和生化分析，以确认存在或不存在prion感染。这项研究的结果将用于监管机构，生物安全专家和负责确定是否可以在BSL-2遏制之外安全分析的研究人员。