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（OG）偏好坏死性凋亡（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，以及该途径对多发性硬化症（MS）动物模型中进行性脱髓鞘、炎症和神经变性的贡献。MS是一种中枢神经系统（CNS）的炎性脱髓鞘疾病，是年轻人在其最具生产力的年龄期间最常见的慢性神经退行性疾病。虽然MS的免疫学基础已被广泛研究，但我们仍然对导致OG变性的机制知之甚少，OG是在维持神经元轴突的活性和完整性方面发挥关键作用的髓鞘产生细胞。预防OG的死亡可能能够抑制脱髓鞘和轴突变性，这是进行性MS患者不可逆神经功能障碍的主要原因。最近已显示TNF α激活TNFR 1介导两种替代细胞死亡途径：半胱天冬酶依赖性细胞凋亡和半胱天冬酶非依赖性RIP 1激酶依赖性坏死性凋亡（程序性坏死）。然而，对于迄今为止分析的大多数细胞类型，坏死性凋亡仅在半胱天冬酶的激活被化学抑制剂或基因突变抑制时才被激活。有趣的是，我们发现OG在TNF α单独刺激后发生坏死性凋亡，这可以被Nec-1或RIP 3缺陷有效阻断。我们已经证明，7-Cl-O-necrostatin-1（7-Cl-O-Nec-1），一种高度特异性的RIP 1激酶抑制剂，在体外和体内两种MS小鼠模型[铜腙模型和实验性自身免疫性脑脊髓炎模型（EAE）]中保护TNF α诱导的少突胶质细胞死亡。此外，RIP 3-/-小鼠也对cuprizone模型具有抗性，并且RIP 3-/-OG被保护免于TNF α。因此，我们拟探讨为什么OGs更喜欢使用坏死性凋亡作为主要的细胞死亡途径，以及RIP 1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OG对TNF α敏感性的作用和机制。这是为了验证假设， 细胞-细胞相互作用调节OG的高代谢活性提供了控制氧化还原状态和OG对TNF α介导的坏死性凋亡的敏感性的关键机制。具体目标2是研究S-亚硝基化在调节OG对TNF α诱导的细胞死亡的敏感性中的作用。这是为了检验TNF α刺激的OG中升高的亚硝基化应激导致半胱天冬酶抑制和OG对坏死性凋亡的敏化的假设。具体目标3是使用RIP 1激酶死亡敲入突变小鼠在体内和体外检查RIP 1激酶介导的OG坏死性凋亡的参与。我们的研究可能为RIP 1激酶抑制剂作为治疗MS的OG保护策略的开发提供了强有力的理论基础，以及口服的，高度特异性和无毒的RIP 1激酶抑制剂，7-Cl-O-Nec-1，作为先导化合物。

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.