Molecular Mechanisms of Prion Protein Amyloid Formation

传染性海绵状脑病(TSE或Prion病)是一组罕见的神经退行性疾病，包括绵羊瘙痒病、牛海绵状脑病(BSE)和马鹿、麋鹿慢性衰弱病(CWD)。在人类中，最常见的Pron病是克雅氏病(CJD)，它可以以几种形式发生。散发性CJD(SCJD)占CJD病例的大多数，在世界范围内以每百万人1-2的发病率随机发生。医源性CJD(ICJD)与接触普恩病毒污染的医疗器械或产品有关，而家族性CJD(FCJD)与Prion蛋白基因突变有关。TSE疾病的感染源被称为Prion，主要由正常的、对蛋白酶敏感的Prion蛋白PrPC的异常折叠、蛋白酶抵抗形式(PrPSc)组成。PrPSc可以以弥漫性淀粉样阴性沉积或致密淀粉样阳性沉积的形式沉积在大脑中。由于目前尚不清楚的原因，淀粉样蛋白病的传播性似乎比非淀粉样蛋白更低。此外，PrPSc主要以淀粉样蛋白沉积的Prion疾病是否遵循与PrPSc主要以非淀粉样蛋白沉积的Prion疾病相同的致病过程尚不清楚。 多项研究表明，由淀粉样β(A)蛋白、α突触核蛋白和tau形成的淀粉样蛋白可以通过类病毒机制在转基因小鼠模型中在细胞间传播(例如Science 313：1781-1784(2006)，NAT Cell Biol 11：909-913(2009)，J Exp Med 209：975-986(2012))。基于这些数据，有人认为阿尔茨海默病(AD)和帕金森氏病(PD)等神经退行性蛋白质病中淀粉样蛋白的形成是通过普恩病毒样机制发生的，而与AD相关的A蛋白也可能是可传播的、传染性的普恩病毒。在神经退行性变过程中错误折叠的蛋白的共同沉积，例如在某些sCJD病例中PrPSc和A共同定位于斑块(美国神经病理学杂志96：116-122(1998))，也表明这些蛋白之间的相互作用可能在疾病发病机制中起作用。 我们对了解PrP淀粉样蛋白形成的分子机制很感兴趣，并已开始使用体外和体内模型系统来探讨这一问题。本项目的重点是：1)了解PrP淀粉样蛋白的形成和扩散的途径；2)了解细胞如何控制淀粉样蛋白的聚集和解聚；3)研究PrP基因的突变和氨基酸多态如何影响PrPSc淀粉样蛋白的形成。由于PrPSc的形成和扩散似乎在机制上类似于其他神经退行性疾病中淀粉样蛋白的形成和扩散，我们的PrPSc研究结果很可能广泛适用于其他蛋白质错误折叠和沉积的疾病。 PrPSc N端不同的蛋白水解酶K(PK)裂解位点决定了其结构的差异。基于PK裂解位点，在sCJD中发现了两种主要的PrPSc结构形式：类型1和类型2。最近，人们发现许多sCJD病例是类型1和类型2的PrPSc的混合物，这表明可能存在一组具有不同二级结构的PrPSc分子(Brain 132：2643(2009))。我们的项目包括使用LC-MS/MS纳米螺旋离子陷阱质谱仪(MS)来精确定位与不同神经学亚型CJD相关的PrPSc分子的N-末端。我们在2018年完成了大约三分之一的实验样本，希望在2019年完成剩余的样本。该项目的目标是确定PrPSc的某些结构群体是否与特定的CJD表型相关。 2019年，我们继续与Pedro Piccardo博士合作，使用MS研究感染BSE的非人类灵长类动物(NHP)。这些动物发生一种神经退行性疾病，其特征是PrPSc、过度磷酸化的tau和α突触核蛋白在某些脑区积累(J Gen Virol 95：1612-16-18(2014))，但不是其他脑区。我们使用MS进行了一项蛋白质组学研究，试图确定在大脑两个不同区域观察到的不同疾病发病机制的潜在分子机制。2019年，对蛋白质组学数据的统计分析显示，一些数据集的健壮性不如其他数据集，我们目前正在对这些样本重复MS。然后，我们将对数据进行统计分析，任何观察到的蛋白质表达差异将使用非MS技术进行确认。这一实验模型将使我们能够更好地理解复杂蛋白质病中神经变性背后的分子机制。 PrPSc、A或其他淀粉样蛋白在神经退变过程中的有序聚集被认为是蛋白质错误折叠疾病(如普里恩病和阿尔茨海默病)的关键。然而，这些聚集体形成的过程以及细胞可以降解这些聚集体的机制仍然知之甚少。在早期对普恩如何与细胞相互作用的研究中，我们表明普恩的摄取和解聚因菌株不同而不同(J。87：11552-61(2013年)，2013年年度报告；上午J.帕索尔。184：3299-3307(2014)，《2014年年度报告》)表明PrPSc聚集体的组成因菌株而异。2019年，IRTA的一位新成员Daniel Shoup博士加入了该实验室，并发起了一个项目，研究使用基于细胞的系统和无细胞系统的PrPSc聚合和解聚过程。这些研究不仅将为PrP如何聚集以及为什么聚集提供重要的见解，而且还将提供为什么一些细胞对普恩病毒感染高度敏感，而另一些细胞则不是。

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 CJD cases and occurs randomly at an incidence of 1-2 per million people 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. For reasons that are not yet clear, amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unknown 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. Multiple studies have shown that amyloid formed from amyloid beta (A) protein, alpha synuclein and tau can propagate via a prion-like mechanism and spread from cell-to-cell in transgenic mouse models (e.g. Science 313: 1781-1784 (2006), Nat Cell Biol 11: 909-913 (2009), J Exp Med 209: 975-986 (2012)). Based on these data, it has been suggested that amyloid formation in neurodegenerative proteinopathies such as Alzheimers Disease (AD) and Parkinsons disease (PD) occurs via prion-like mechanisms and that proteins such as AD-associated A may also be transmissible, infectious prions. Co-deposition of misfolded proteins during neurodegeneration, such as the co-localization of PrPSc and A to plaques in some cases of sCJD (ACTA Neuropathol 96:116-122 (1998)), also suggest that interactions between these proteins could contribute to disease pathogenesis. 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 spread, 2) understanding how amyloid aggregation and disaggregation are controlled by the cell and, 3) studying how mutations and amino acid polymorphisms in PrP influence PrPSc amyloid formation in familial forms of prion disease. Since PrPSc formation and spread appear to be mechanistically similar to the formation and spread of amyloid in other neurodegenerative diseases, the results of our prion studies will likely be broadly applicable to other diseases of protein misfolding and deposition. 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 many cases of sCJD are mixtures of Type 1 and Type 2 PrPSc suggesting that there may be a complex population of PrPSc molecules present with different secondary structures (Brain 132: 2643 (2009)). Our project involves using LC-MS/MS Nanospray Ion Trap Mass Spectrometry (MS) to precisely map the N-termini of PrPSc molecules associated with different neurological subtypes of CJD. We completed about one-third of our experimental samples in 2018 and in 2019 hope to complete the remaining samples. The goal of this project is to determine whether certain structural populations of PrPSc correlate with specific CJD phenotypes. In 2019, we continued a collaboration with Dr. Pedro Piccardo using MS to study BSE-infected non-human primates (NHP). These animals develop a neurodegenerative disease characterized by accumulation of PrPSc, hyper-phosphorylated tau, and alpha synuclein (J Gen Virol 95:1612-16-18 (2014)) in some brain regions but not others. We have used MS to do a proteomics study to try to determine the potential molecular mechanisms underlying the different disease pathogenesis observed in two different regions of the brain. In 2019, statistical analysis of the proteomics data revealed that some of the data sets were less robust than others and we are currently in the process of repeating the MS on those samples. We will then re-analyze the data statistically and any observed differences in protein expression will be confirmed using non-MS based techniques. This experimental model will enable us to better understand the molecular mechanisms behind neurodegeneration in complex proteinopathies. The ordered aggregation of PrPSc, A, or other amyloid proteins during neurodegeneration is thought to be critical to the pathogenesis of protein misfolding diseases such as prion disease and AD. However, the processes by which these aggregates form and the mechanisms by which the cell can degrade these aggregates remains poorly understood. In earlier studies of how prions interact with cells, we showed that the uptake and disaggregation of prions varied by strain (J. Virol. 87: 11552-61 (2013), Annual Report 2013; Am. J. Pathol. 184: 3299-3307 (2014), Annual Report 2014) suggesting that the composition of PrPSc aggregates differed between strains. In 2019 a new IRTA fellow, Dr. Daniel Shoup, joined the lab and initiated a project to study the processes involved in PrPSc aggregation and disaggregation using both cell-based and cell-free systems. These studies will provide important insights not only into how and why PrP aggregates but also why some cells are highly susceptible to prion infection while others are not.