Mechanisms of Neurodegeneration & Neuroprotection in EAE

描述(申请人提供)：多发性硬化症(MS)是一种中枢神经系统(CNS)脱髓鞘和退行性疾病。大多数MS患者经历复发缓解症状，然后是疾病的次级进展期(24)。免疫调节疗法在复发-缓解期部分改变了MS的病程；然而，该病的二次进展期仍然对此类治疗具有抵抗力(25)。这种二分法的一个可能的解释是，在疾病的第二阶段，中枢神经系统遭受了髓鞘和轴突的不可逆转的损害，这种损害不再能通过代偿机制来纠正。最近的几项研究表明，在MS及其动物模型实验性自身免疫性脑脊髓炎(EAE)中都存在轴突损伤，并可能导致固定的神经功能缺陷(26，27)。因此，改善轴突损伤的策略可能会预防多发性硬化症的慢性残疾。Wlds小鼠是一种自发发生的突变，具有对抗多种形式的轴突损伤的独特表型。该模型的神经保护机制目前尚不清楚，但可能与Ube4b/Nmnat嵌合基因的功能有关(28)。我们发现WID小鼠的EAE病程减弱，轴突损伤和脱髓鞘比野生型小鼠更少。此外，我们还发现，在EAE期间，这些小鼠在中枢神经系统中CD200的表达增加，这与巨噬细胞/小胶质细胞在中枢神经系统的积累减少有关。已知CD200在神经元上表达，其受体CD200R的连接对巨噬细胞/小胶质细胞的激活具有抑制作用。CD200在中枢神经系统的表达增加可能会促进EAE和MS的神经保护。在这次修订的拨款申请中，我们的目标是2倍。首先，我们将探讨CD200在减弱中枢神经系统免疫反应和神经病理中的作用。巨噬细胞/小胶质细胞被认为是轴突损伤和脱髓鞘的主要介质。下调其激活的策略，特别是在中枢神经系统，可能会预防慢性残疾。CD200激活CD200R就是这样一种策略。为此，我们将更详细地研究CD200在幼稚Wlds和野生型小鼠以及EAE期间中枢神经系统中的表达动态和分布。我们将探讨CD200R结扎在体外对小胶质细胞和巨噬细胞激活的影响，以及在小胶质细胞诱导的神经毒性模型上的作用。最后，我们将研究CD200 CD200R通路激活在EAE效应(慢性)和启动阶段的作用。我们的第二个目标是了解Wlds嵌合基因与CD200等底物的关系。根据我们最近的初步结果，显示CD200在WLD脊髓中的泛素化减少，我们假设WLD基因通过改变某些底物的泛素蛋白酶体途径(UPS)而发挥作用，从而导致神经保护表型。UPS的改变导致了几种神经退行性疾病/模型。我们将探索嵌合蛋白与CD200的关系。此外，我们将尝试确定在Wlds模型中可能在轴突保护中发挥作用的其他泛素化改变的底物。最后，我们将探讨抑制UPS可能减少炎症介导的轴索病变的可能性。我们预计，这一最终目标的结果可能为未来研究UPS通路在EAE和MS中的轴突保护作用提供基础。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a demyelinating and degenerative disease of the central nervous system (CNS). The majority of MS patients experience relapsing-remitting symptoms, followed by a secondary progressive phase of disease (24). Immunomodulatory therapies partially alter the disease course of MS during the relapsing-remitting phase; however the secondary progressive phase of the disease remains resistant to such treatments (25). A possible explanation for this dichotomy is that in the secondary phase of disease, the CNS has sustained irreversible damage to both myelin and axons, which can no longer be corrected by compensatory mechanisms. Several studies have recently shown that axonal damage is present in both MS and its animal model experimental autoimmune encephalomyelitis (EAE) and may contribute to fixed neurological deficits (26, 27). Thus, strategies that ameliorate axon damage may prevent chronic disability in MS. The Wlds mouse is a spontaneously occurring mutant with the unique phenotype of protection against several forms of axonal injury. The mechanisms of neuroprotection in this model is currently unknown, however likely relates to the function of the Ube4b/Nmnat chimeric gene (28). We have found that WId mice have an attenuated course of EAE, and have less axonal damage and demyelination than wild-type mice. In addition, we have found that these mice have increased expression of CD200 in the CNS during EAE, correlating with decreased accumulation of macrophages/microglia in the CNS. CD200 is known to be expressed on neurons, and ligation of its receptor CD200R is inhibitory for macrophage/microglial activation. Increased expression of CD200 in the CNS may promote neuroprotection in EAE and potentially in MS. In this revised grant application, our goals are 2 fold. Firstly, we will explore the role of CD200 in attenuating the immune response and neuropathology in the CNS. Macrophages/microglia have been implicated as the primary mediators of axonal damage and demyelination. Strategies to down-regulate their activation, particularly in the CNS may prevent chronic disability. Activation of CD200R by CD200 is 1 such strategy. In this Aim, we will study in more detail the dynamics and distribution of CD200 expression in the CNS in naive Wlds and wild-type mice, as well as during EAE. We will explore the effects of CD200R ligation on microglia and macrophage activation in vitro, and on a model of microglia-induced neurotoxicity. And lastly we will study the effects of CD200 CD200R pathway activation during the effector (chronic) as well as priming stages of EAE. Our second goal focuses on understanding the relationship of the Wlds chimeric gene and substrates such as CD200. Based on our recent preliminary results, which demonstrate decreased ubiquitination of CD200 in Wld spinal cord, we hypothesize that the Wld gene exerts its effects by altering the ubiquitin proteasome pathway (UPS) degradation of certain substrates, leading to the neuroprotective phenotype. Alterations in the UPS are responsible for several neurodegenerative diseases/models. We will explore the relationship between the chimeric protein and CD200. In addition, we will attempt to identify other substrates subject to altered ubiquitination that may play a role in axon protection in the Wlds model. Lastly, we will explore the possibility that inhibition of the UPS may reduce inflammation-mediated axonopathy. We anticipate that results from this final Aim may provide us with the basis for future studies in the role of the UPS pathway in axon-protection in EAE, and potentially in MS.