Investigating the mechanism of TNFalpha mediated cell death in oligodendrocytes

描述(申请人提供)：这项建议的目的是探索为什么少突胶质细胞(OG)更喜欢坏死性下垂(一种受调控的坏死细胞死亡途径)作为主要的细胞死亡机制的分子机制，以及这一途径在多发性硬化症(MS)动物模型中对进行性脱髓鞘、炎症和神经变性的贡献。MS是一种中枢神经系统(CNS)的炎症性脱髓鞘疾病，是年轻人在最具生产力的年龄最常见的慢性神经退行性疾病。虽然MS的免疫学基础已经得到了广泛的研究，但我们对导致OGs退变的机制仍然知之甚少。OGs是一种产生髓鞘的细胞，在维持神经元轴突的活性和完整性方面发挥着关键作用。阻止OGs的死亡可能能够抑制脱髓鞘和轴突变性，这是进行性MS患者不可逆的神经功能障碍的主要原因。最近研究表明，TNFR1的激活介导了两种不同的细胞死亡途径：caspase依赖的细胞凋亡和caspase非依赖的RIP1激酶依赖的坏死症(程序性坏死)。然而，对于到目前为止所分析的大多数细胞类型，只有当caspase的激活被化学抑制剂或基因突变抑制时，坏死性下垂才被激活。有趣的是，我们发现OGs在单独受到TNFpha刺激时会发生坏死性下垂，这可以被NEC-1或RIP3缺乏有效地阻断。我们发现，7-氯-O-NEC-1(7-氯-O-NEC-1)是一种高度特异的RIP1激酶抑制剂，在体外对TNFα诱导的少突胶质细胞死亡和体内两种多发性硬化小鼠模型[铜必宗模型和实验性自身免疫性脑脊髓炎模型(EAE)]具有保护作用。此外，RIP3-/-小鼠也对铜试剂耐药，RIP3-/-OGs对肿瘤坏死因子α有保护作用。我们建议研究为什么OGs更倾向于使用坏死性下垂作为主要的细胞死亡途径，以及RIP1激酶在OGs死亡中的作用和机制。具体目的1是研究细胞代谢和氧化还原状态控制OGs对肿瘤坏死因子α敏感性的作用和机制。这是为了检验这样一个假设，即 细胞间相互作用调节OGs的高代谢活性为控制OGs的氧化还原状态和对肿瘤坏死因子α介导的坏死性下垂的敏感性提供了关键机制。目的2：研究S亚硝化在调节OGs对肿瘤坏死因子α诱导的细胞死亡敏感性中的作用。这是为了验证一种假设，即在TNFα刺激的OGs中，增加的硝化应激导致caspase的抑制和OGs对坏死性下垂的敏化。目的3：利用RIP1激酶死亡敲击突变小鼠，在体内和体外研究RIP1激酶在介导OGS坏死性下垂中的作用。我们的研究可能为开发RIP1激酶抑制剂作为治疗MS的OG保护性策略，以及开发口服可用、高度特异和无毒的RIP1激酶抑制剂7-Cl-O-NEC-1作为先导化合物提供强有力的理论基础。

DESCRIPTION (provided by applicant): The goal of this proposal is to explore the molecular mechanism as to why oligodendrocytes (OGs) prefer necroptosis, a regulated necrotic cell death pathway, as the primary cell death mechanism and the contribution of this pathway to progressive demyelination, inflammation and neurodegeneration in animal models of multiple sclerosis (MS). MS, an inflammatory demyelinating disease of the central nervous system (CNS), is the most common chronic neurodegenerative disease for young adults during their most productive ages. While the immunological basis of MS has been studied extensively, we still know very little about the mechanism that leads to the degeneration of OGs, the myelin producing cells that play a critical role in the maintenance of activity and integrity of neuronal axons. Preventing the death of OGs might be able to inhibit demyelination and axonal degeneration, the major cause of irreversible neurological disability in patients with progressive MS. Activation of TNFR1 by TNFalpha has recently been shown to mediate two alternative cell death pathways: caspase-dependent apoptosis and caspase-independent RIP1 kinase-dependent necroptosis (programmed necrosis). However, for most cell types analyzed so far, necroptosis is only activated when the activation of caspases is inhibited by chemical inhibitors or by genetic mutation. Interestingly, we found that OGs undergo necroptosis upon stimulation by TNFalpha alone which can be effectively blocked by Nec-1 or by RIP3 deficiency. We have shown that 7-Cl-O-necrostatin-1 (7-Cl-O-Nec-1), a highly specific inhibitor of RIP1 kinase, protects against TNFalpha-induced oligodendrocyte death in vitro and two mouse models of MS in vivo [cuprizone model and experimental autoimmune encephalomyelitis model (EAE)]. In addition, RIP3-/- mice are also resistant to cuprizone model and RIP3-/- OGs are protected against TNFalpha. We propose to investigate as to why OGs prefer to use necroptosis as the primary cell death pathway and the role and mechanism of RIP1 kinase in mediating the death of OGs. Specific Aim 1 is to investigate the role and mechanism by which cellular metabolism and redox state control the sensitivity of OGs to TNFalpha. This is to test the hypothesis that the cell- cell interaction regulated high metabolic activity in OGs provides a critical mechanism that controls redox state and the sensitivity of OGs to TNFalpha mediated necroptosis. Specific Aim 2 is to investigate the role of S-nitrosylation in regulating the sensitivity of OGs to TNFalpha induced cell death. This is to test the hypothesis that elevated nitrosylation stress in TNFalpha stimulated OGs leads to the inhibition of caspases and sensitization of OGs to necroptosis. Specific Aim 3 is to examine the involvement of RIP1 kinase in mediating necroptosis of OGs in vivo and in vitro using RIP1 kinase dead knockin mutant mice. Our study may provide a strong rationale for the development of RIP1 kinase inhibitors as an OG protective strategy for the treatment of MS, and an orally available, highly specific and nontoxic RIP1 kinase inhibitor, 7-Cl-O-Nec-1, as a lead compound.