Multiple sclerosis as a cytodegenerative proteopathy

项目总结/摘要 MS传统上被认为是CNS的自身免疫性脱髓鞘疾病。然而，有一个 许多与MS发病机制的"由外向内"假设不一致的地方，导致我们提出 相反，MS主要是一种"由内而外"疾病，其中原发性变性主要针对髓磷脂， 少突胶质细胞，其次在易感宿主中引起自身免疫反应。 假设：MS是一种主要的蛋白质错误折叠疾病，靶向髓鞘形成单位，由 可传递蛋白聚集体的积累。具体来说，这种蛋白质病是由一种 促进髓鞘和少突胶质细胞慢性毒性的病理性朊病毒（一般意义上）， 导致髓鞘完整性的破坏，导致继发性自身免疫，从而编程广泛的 炎症和进行性MS表型谱。 目的1：MS样病理的脑内传播。这一目标将进行详细的审查，我们的 从人脑匀浆（来自死亡对照和 进行性MS受试者）至野生型和人源化转基因小鼠，通过直接i.c.接种。这一目标将 测试人类MS大脑含有有毒错误折叠蛋白质聚集体的假设， 病理并在受体宿主中传播。将使用高场MRI、（免疫）组织学和行为学 来监控病理发展我们期望找到类似于"正常出现的白色物质"的病理学 人MS脑的异常（轻微髓鞘病、轴突损伤、微小和星形胶质细胞增生），并且值得注意的是， 我们确实期望指示适应性免疫的显著的淋巴细胞应答。阴性对照（非MS 人脑匀浆）以证明MS特异性。传代（即感染 转基因小鼠脑匀浆并重新接种到幼稚小鼠中）将表明， 继续传输。朊病毒蛋白是最初假设的病原体，因此实验将 相应地使用PrP转基因进行定制，认识到PrP和/或其相互作用蛋白之一可以 也将发挥关键作用。 目的2：MS样病理的腹膜内传播。虽然直接i.c.接种是最有效的 传递朊病毒样错误折叠蛋白的方法，它具有诱导非特异性病理的风险， 特别是在对各种损伤更敏感的菌株中。这一目标将试图通过传播病理学 一种侵入性较小的腹腔注射途径将使用类似的源材料。优点包括潜在的 更早和更敏感的行为读数（运动缺陷，感觉异常），如果预期 诱发脊髓病。此外，用i.p. 相比于I.C.注射，可能会增加传播的机会。最终，相似 将进行（免疫）组织学检查。我们还将检查脾脏是否存在错误折叠的蛋白质 沉积作为线索，这个器官是否参与加工和扩大可传播的种子。 目的3：人MS脑和传代小鼠CNS的生化分析。一个关键的观察， 我们的初步生化研究表明，朊病毒蛋白与 髓鞘碱性蛋白，主要的髓鞘蛋白之一。这一目标将更详细地探讨这一潜在的 非常重要的相互作用可能是维持髓鞘完整性的关键。免疫沉淀，Western， 将进行LC-MS和生化分析，以探测这种相互作用的破坏。重点将是 这两种蛋白质的瓜氨酸，因为这种翻译后修饰改变了净电荷，因此 相互作用效率，对生理蛋白质-蛋白质产生潜在的重要不利影响 交互.

Project Summary/Abstract MS is traditionally considered to be an autoimmune demyelinating disorder of the CNS. However there are a number of inconsistencies with this “outside-in” hypothesis of MS pathogenesis which lead us to propose instead that MS is primarily an “inside-out” disease, where a primary degeneration mainly targeting the myelin- oligodendrocyte, secondarily entrains an auto-immune reaction in the predisposed host. HYPOTHESIS: MS is a primary protein misfolding disorder targeting the myelinating unit, driven by accumulation of transmissible protein aggregates. Specifically, this proteopathy is underpinned by a pathological prion (in the generic sense) which promotes chronic toxicity of myelin and oligodendrocytes, resulting in disruption of myelin integrity, leading to secondary autoimmunity, thus programming the broad spectrum of inflammatory and progressive MS phenotypes. Aim 1: Intracerebral transmission of MS-like pathology. This aim will conduct a detailed examination of our ability to transmit an MS-like pathology from human brain homogenate (from deceased controls and progressive MS subjects) to wild-type and humanized transgenic mice, via direct i.c. inoculation. This aim will test the hypothesis that human MS brain contains toxic misfolded protein aggregates that can transmit pathology and propagate in the recipient hosts. High- field MRI, (immuno)histology and behavior will be used to monitor developing pathology. We expect to find pathology similar to “normal appearing white matter” abnormalities in human MS brain (subtle myelinopathy, axonal damage, micro- and astrogliosis), and notably, we do expect a significant lymphocytic response indicative of adaptive immunity. Negative controls (non-MS human brain homogenate) will be performed to demonstrate MS specificity. Passaging (ie taking infected transgenic mouse brain homogenate and re-inoculating into naïve mice) will show that the offending agent continues to transmit. Prion protein is the etiological agent originally hypothesized, therefore experiments will be tailored accordingly using PrP transgenics, recognizing that PrP and/or one of its interacting proteins could also be playing a key role. Aim 2: Intraperitoneal transmission of MS-like pathology. While direct i.c. inoculation is the most robust method of transmitting prion-like misfolded proteins, it carries the risk of inducing non-specific pathology, especially in strains that are more susceptible to various insults. This aim will attempt to transmit pathology via a less invasive route using i.p. injections. Similar source material will be used. Advantages include potentially earlier and more sensitive behavioural readouts (locomotor deficits, sensory abnormalities) if the expected myelopathy is induced. Moreover, larger volumes of inocula and more frequent injections are possible with i.p. compared to i.c. injection, potentially increasing the chances of transmission. Terminally, similar (immuno)histology will be performed. We will also examine the spleen for evidence of misfolded protein deposition as a clue to whether this organ participates in processing and amplification of transmissible seeds. Aim 3: Biochemical analysis of human MS brain and passaged mouse CNS. A key observation made during our preliminary biochemical studies showed a striking alteration in the interaction between prion protein and myelin basic protein, one of the major myelin proteins. This aim will explore in greater detail this potentially very important interaction that could be key for maintenance of myelin integrity. Immunoprecipitation, Westerns, LC-MS and biochemical analysis will be performed to probe disruptions in this interaction. Focus will be on citrullination of both proteins as this post- translational modification alters net charge, and therefore the interaction efficiency, exerting a potentially important adverse influence on physiological protein-protein interactions.