Investigating the mechanism of TNFalpha mediated cell death in oligodendrocytes

描述（由申请人提供）：本提案的目的是探索为什么少突胶质细胞（OGs）倾向于坏死上沉（一种受调节的坏死细胞死亡途径）作为主要细胞死亡机制的分子机制，以及该途径在多发性硬化症（MS）动物模型中对进行性脱髓鞘、炎症和神经退行性变的贡献。多发性硬化症是一种中枢神经系统（CNS）的炎症性脱髓鞘疾病，是年轻人最常见的慢性神经退行性疾病，发生在他们最具生产力的年龄。虽然MS的免疫学基础已被广泛研究，但我们对导致og变性的机制知之甚少，og是一种产生髓磷脂的细胞，在维持神经元轴突的活性和完整性方面起着关键作用。防止OGs死亡可能能够抑制脱髓鞘和轴突变性，这是进行性ms患者不可逆神经功能障碍的主要原因，TNFalpha激活TNFR1最近被证明介导两种可选的细胞死亡途径：caspase依赖性细胞凋亡和caspase非依赖性RIP1激酶依赖性坏死坏死（程序性坏死）。然而，对于迄今为止分析的大多数细胞类型，只有当caspases的激活被化学抑制剂或基因突变抑制时，坏死下垂才会被激活。有趣的是，我们发现og在单独受到TNFalpha刺激时发生坏死下垂，而TNFalpha可被Nec-1或RIP3缺乏有效阻断。我们已经证明，7-Cl-O-necrostatin-1 (7-Cl-O-Nec-1)，一种高度特异性的RIP1激酶抑制剂，在体外和两种MS小鼠模型（铜普利酮模型和实验性自身免疫性脑脊髓炎模型（EAE））中，对tnfalpha诱导的少突胶质细胞死亡具有保护作用。此外，RIP3-/-小鼠也对cuprizone模型产生抗性，RIP3-/- OGs对TNFalpha具有保护作用。我们建议研究为什么OGs倾向于将坏死坏死作为主要的细胞死亡途径，以及RIP1激酶在介导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TNFalpha敏感性的作用和机制。这是为了验证假设

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.