Defects of Neurotransmission in Ischemia and Reperfusion

描述（由申请人提供）：中风是一种病理性血流量减少，导致脑组织不可逆损伤、脑功能长期中断，并经常导致患者死亡。缺血研究的主要焦点是细胞死亡的机制和寻找挽救脆弱神经细胞的药理学方法。对与细胞死亡无关的神经细胞功能的长期损害的关注要少得多。然而，现有的文献表明，即使是相对轻微的缺血也会导致突触通讯的长期抑制。由于未知的原因，只有突触前的释放受到影响，而突触后的反应仍然保留。在目前的研究中，我们假设，突触传递的抑制发生N-乙基马来酰亚胺敏感的融合蛋白（NSF）的半胱氨酸残基的亚硝化一氧化氮（NO）和NO相关的活性氮物种。NSF蛋白是三聚体ATP酶，其对于连续突触囊泡对接/融合至关重要。通过N-乙基马来酰亚胺修饰NSF中的关键半胱氨酸残基导致囊泡对接和融合的不可逆抑制。类似地，NSF巯基的NO依赖性修饰可能导致囊泡神经递质释放的长期抑制。为了验证我们的假设，我们提出了以下具体目标。(1)我们将测试缺血后24小时内缺血组织中巯基修饰的增加，并将进一步证明从缺血脑免疫沉淀的NSF蛋白中的巯基修饰。(2)使用突触体制备，我们将测试是否一氧化氮和相关的活性氮物种修改NSF，并通过这种机制抑制囊泡神经递质的释放。我们将进一步尝试通过应用硫醇还原剂来逆转NSF亚硝化和神经递质释放的缺陷。该项目将阐明缺血中突触传递长期中断的分子机制，并可能为中风和短暂性脑缺血发作后的患者治疗和康复提供额外的方法。

DESCRIPTION (provided by applicant): Stroke is a pathological reduction in blood flow which causes irreversible damage of brain tissue, long-term disruptions of brain functions, and frequently patient death. The major focus of ischemia research is on the mechanisms of cell death and the search for pharmacological approaches to salvage vulnerable neural cells. Substantially less attention is paid to long-term impairment of neural cell function that is not associated with cell death. Existing literature suggest, nonetheless, that even relatively mild ischemia causes long-lasting suppression of synaptic communication. For unknown reasons, only presynaptic release is affected, while postsynaptic responses remain preserved. In the current study we hypothesize that suppression of synaptic transmission occurs due to nitrosation of cysteine residues in N-ethylmaleimide sensitive fusion protein (NSF) by nitric oxide (NO) and NO-related reactive nitrogen species. The NSF protein is a trimeric ATPase which is crucial for continuous synaptic vesicle docking/fusion. Modification of a critical cysteine residues in the NSF by N-ethylmaleimide causes irreversible inhibition of vesicle docking and fusion. Similarly, NOdependent modification of NSF thiols may cause long-term inhibition of vesicular neurotransmitter release. To test our hypothesis we propose the following specific aims. (1) We will test for the increased modification of thiols in the ischemic tissue up to 24 hours after ischemia and will further demonstrate thiol modification in the NSF protein immunoprecipitated from the ischemic brain. (2) Using synaptosomal preparation we will test whether nitric oxide and related reactive nitrogen species modify NSF and via this mechanism inhibit vesicular neurotransmitter release. We will further attempt to reverse NSF nitrosation and defects in neurotransmitter release by applying thiol reducing agents. This project will clarify the molecular mechanisms of less understood long-term disruption of synaptic transmission in ischemia and may suggest additional approaches for patient treatment and rehabilitation after stroke and transient ischemic attacks.