MECHANISMS OF NO-MEDIATED PROTEIN S-NITROSYLATION IN EAE

描述(由申请人提供)：我们的长期目标是识别和描述炎症性脱髓鞘障碍中导致髓鞘破坏的机制。一氧化氮(NO)参与了多发性硬化症(MS)和实验性变态反应性脑脊髓炎(EAE)的病理生理过程，因为在各种促炎细胞因子的诱导下，巨噬细胞/小胶质细胞释放出大量的一氧化氮(NO)。然而，NO导致髓鞘破坏的机制还远不清楚。根据最近关于亚硝化性损伤在其他系统中的分子和细胞后果的研究，以及我们实验室的一些重要发现，我们假设NO可以在距离炎症损伤相当远的地方扩散，导致广泛的髓鞘解压。我们认为，这一过程的基础是N203对重要的髓鞘蛋白进行S亚硝化，N203是一种强大的亚硝化剂，在富含脂肪的环境中(如髓鞘)，N203可以很容易地通过NO自氧化形成。本建议中描述的实验将详细描述与非渗透性NO供体孵育的大鼠脊髓切片中的髓鞘蛋白S亚硝化的机制，其产生的NO水平与EAE和MS中发现的NO水平相当。这些实验还将(1)检查是否存在其他NO诱导的与硫醇相关的修饰(S-硫基化和蛋白质二硫键的形成)，(2)通过质谱鉴定主要的S亚硝化髓鞘蛋白，以及(3)测试几种化合物防止/逆转这些有害蛋白质修饰的能力。此外，还将研究EAE过程中脊髓中S-硝基蛋白质的存在情况，并使用蛋白质组学方法确定这些物种的身份。最后，将通过免疫细胞化学来评估S硝化蛋白在受影响组织中的分布，以直接检验这一假说，即游离NO诱导的亚硝化蛋白损伤可以发生在距离炎性病变一段距离的地方，并且与过氧亚硝酸盐的产生无关。拟议的研究将产生关键信息，支持在炎性脱髓鞘过程中NO介导的髓鞘破坏的新途径。阐明蛋白质修饰的机制，确定髓鞘中受NO影响的分子，以及在广泛使用的MS动物模型中这些修饰在中枢神经系统中的发生，对于理解这种毁灭性疾病的病理生理学至关重要。

DESCRIPTION (provided by applicant): Our long-term goal is to identify and characterize the mechanisms that lead to the destruction of myelin in inflammatory demyelinating disorders. Nitric oxide (NO) has been implicated in the pathophysiology of both multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), as high levels of this gas are released by macrophages/microglia following induction of nitric oxide synthetase by various proinflammatory cytokines. However, the mechanism by which NO leads to myelin breakdown is far from clear. Based on recent investigations regarding the molecular and cellular consequences of nitrosative damage in other systems, and a number of important findings from our laboratory, we hypothesize that NO can spread at considerable distances from inflammatory lesions to cause extensive myelin decompaction. We propose that underlying this process is the S-nitrosylation of important myelin proteins by N203, a strong nitrosylating agent that can be readily formed by NO autooxidation in a lipid-rich environment like the myelin sheath. The experiments described in this proposal will characterize in detail the mechanism of S-nitrosylation of myelin proteins in rat spinal cord slices incubated with a non-permeable NO-donor, which generates levels of NO comparable to those found in EAE and MS. These experiments will also (1) examine the existence of other NO-induced thiol-related modifications (S-thiolation and formation of protein disulfides), (2) identify the major S-nitrosylated myelin proteins by mass-spectrometry, and (3) test several compounds for their ability to prevent/revert these deleterious protein modifications. In addition, the occurrence of S-nitrosylated proteins in spinal cord during the course of EAE will be investigated and the identity of these species determined using a proteomic approach. Finally, the distribution of S-nitrosylated proteins in the affected tissue will be assessed by immunocytochemistry to directly test the hypothesis that nitrosative protein damage induced by free NO can take place at some distance from the inflammatory lesions and independently of peroxynitrite generation. The proposed studies will generate crucial information that could support a new pathway for the NO-mediated destruction of myelin during inflammatory demyelination. The elucidation of the mechanism of protein modification, the identification of the molecules in myelin that are affected by NO, and the occurrence of these modifications in the CNS of a widely-used animal model of MS are essential for understanding the pathophysiology of this devastating disorder.