Excess Nitric Oxide as a Mechanism of Glial Dysfunction in CMT1X

过量一氧化氮是 CMT1X 胶质细胞功能障碍的机制

• 批准号: 9462471
• 负责人: CHARLES K ABRAMS
• 金额: $ 19.99万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9462471
• 关键词: 3-nitrotyrosine; Acute; Affect; Antibodies; Back; Binding; Biological Assay; Biological Sciences; Biotin; Buffers; Calcium; Cell Culture Techniques; Cell Death; Cell physiology; Cells; Central Nervous System Diseases; Charcot-Marie-Tooth Disease; Communication; Connexin 43; Connexins; Coupled; Data; Data Set; Disease; Elements; Enzymes; Event; Exhibits; Feedback; Functional disorder; Gap Junctions; Generations; Genus Hippocampus; Homeostasis; Impairment; Inherited; Lead; Liver; Measures; Membrane Microdomains; Membrane Potentials; Membrane Proteins; Mitochondria; Mus; Muscle Weakness; Mutation; Nerve; Nerve Tissue; Neuroglia; Nitrates; Nitric Oxide; Nitric Oxide Pathway; Nitric Oxide Synthase Type I; Other Genetics; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Peripheral; Peripheral Nerves; Peripheral Nervous System Diseases; Peroxonitrite; Physiological; Play; Preparation; Process; Production; Protein Isoforms; Protein S; Proteins; Resistance; Respiration; Role; Schwann Cells; Series; Signal Transduction; Small Interfering RNA; Technology; Testing; Therapeutic Intervention; Toxic effect; Tyrosine; United States; Western Blotting; Wild Type Mouse; Work; argininosuccinate lyase; argininosuccinate synthase; caveolin 1; cell growth; cell injury; cell type; connexin 32; experimental study; in vivo Model; inhibitor/antagonist; knock-down; live cell imaging; mitochondrial dysfunction; mitochondrial membrane; mutant; nerve injury; nitration; protein complex; response; targeted treatment

The involvement of nitric oxide(NO) in nerve injury and peripheral neuropathy is well documented. However, until recently there was little or no evidence of generation of NO in myelinating glia themselves. The demonstration of nitric oxide synthases 1 and 3 (NOS-1 and NOS-3) in Schwann cells, raises the potential for intrinsic NO dysfunction in these cells. Here we propose and test the hypothesis that an amplified NO response in Schwann cells is an underlying cause of pathology in CMT1X, a relatively common inherited peripheral (and sometimes central) nervous system disorder caused by mutations in connexin 32 (Cx32) a connexin expressed in myelinating glia. Key to our hypothesis is data suggesting that Cx32 is part of a complex of proteins involved in NO signaling. Gap junctions formed by connexins provide communication pathways between coupled cells. However, defective gap junctional communication alone does not account for the full extent of the role played by Cx32 in glial cells or by connexins in other cell types. Work outlined here will utilize cell culture, ex vivo, and in vivo models to investigate the physiologic and pathological consequences of loss of or mutation in Cx32. We suggest that in Cx32-defective Schwann cells, a self-reinforcing positive feedback loop of interactions involving NO increases, mitochondrial dysfunction, and impaired Ca2+ homeostasis is triggered by disruption or loss of interactions between Cx32 and components of the NO pathway. These experiments should elucidate targets for therapeutic intervention in CMT1X which will likely apply also to other disorders exhibiting disease-related alterations in connexin expression. We will compare our findings in wild-type mice to those in mice lacking Cx32 (Cx32KO) and in mice expressing the CMT1X mutant Cx32T55I on a Cx32KO background (T55ITg/32KO). Aim 1 will examine the hypothesis that disruption of Cx32 predisposes Schwann cells to nitric oxide dysfunction and ask: Does the absence of or mutation in Cx32 affect measures related to nitric oxide function in Schwann cells and peripheral nerve? We will examine the relative difference in NO levels in WT, 32KO and T55ITg/32KO Schwann cells at baseline and whether acute knockdown of Cx32 with siRNA causes changes in NO production. Peroxynitrite production, protein S-nitrosylation, tyrosine nitration and mitochondrial function will also be assessed. Aim 2 will ask: Does the NO dysregulation seen in Cx32 KO Schwann cells and nerve arise due to loss of normally occurring interactions between Cx32 and elements in the NO pathway? Cx32 appears to be part of a membrane associated protein complex including eNOS and at least one enzyme (ASS) important In NO synthesis, and ASS has been shown to directly interact with Cx32 in liver; furthermore, expression of at least one connexin has been shown to both interact with and reduce activity of eNOS. We will use LC-MS/MS to examine whether Cx32 directly or indirectly interacts with a NOS or other elements of the NO synthesis pathway. We will also perform an unbiased analysis of our data to capture other potentially relevant interactions.

一氧化氮（NO）参与神经损伤和周围神经病变是有据可查的。然而直到 最近，很少或没有证据表明在髓鞘神经胶质细胞本身中产生NO。示范 一氧化氮合酶1和3（NOS-1和NOS-3）的雪旺细胞，提高了潜在的内源性NO 这些细胞的功能障碍。在这里，我们提出并测试的假设，一个放大的NO反应，在雪旺氏 细胞是CMT 1X病理学的根本原因，CMT 1X是一种相对常见的遗传性外周（有时 由连接蛋白32（Cx 32）突变引起的中枢）神经系统疾病， 髓鞘神经胶质。我们的假设的关键是数据表明Cx 32是一种蛋白质复合物的一部分， 没有信号。由连接蛋白形成的间隙连接提供了偶联细胞之间的通讯途径。 然而，有缺陷的缝隙连接通讯本身并不能解释所有的作用， Cx 32在神经胶质细胞中或通过连接蛋白在其他细胞类型中。这里概述的工作将利用细胞培养，离体， 研究Cx 32缺失或突变的生理和病理后果的体内模型。我们 这表明，在Cx 32缺陷的雪旺细胞，一个自我加强的正反馈回路的相互作用， NO增加、线粒体功能障碍和受损的Ca 2+稳态是由线粒体内分泌的破坏或丧失引发的。 Cx 32与NO通路的组分之间的相互作用。这些实验应该能阐明 对于CMT 1X的治疗性干预，这可能也适用于表现出疾病相关性的其他病症， 连接蛋白表达的改变。我们将比较我们在野生型小鼠和缺乏Cx 32的小鼠中的发现。 在Cx 32 KO背景下表达CMT 1X突变体Cx 32 T55 I的小鼠中（T55 ITg/32 KO）。目的 1将检验Cx 32的破坏使雪旺细胞易于发生一氧化氮功能障碍的假设， 问：Cx 32的缺失或突变是否会影响雪旺细胞中一氧化氮功能相关的测量 和外周神经我们将检查WT、32 KO和T55 ITg/32 KO中NO水平的相对差异 雪旺细胞在基线和是否急性敲低Cx 32与siRNA引起的变化，在NO的生产。 过氧亚硝酸盐的产生，蛋白质S-亚硝基化，酪氨酸硝化和线粒体功能也将被研究。 评估。目的2将问：在Cx 32 KO雪旺细胞和神经中观察到的NO失调是否由于 Cx 32和NO通路中的元件之间正常发生的相互作用的丧失？Cx 32似乎是一部分 包括eNOS和至少一种在NO中重要的酶（ASS）的膜相关蛋白复合物 已显示ASS与肝脏中的Cx 32直接相互作用;此外，至少 一种连接蛋白已显示既与eNOS相互作用又降低eNOS的活性。我们将使用LC-MS/MS 检查Cx 32是否直接或间接与NOS或NO合成途径的其他元件相互作用。 我们还将对我们的数据进行无偏见的分析，以捕获其他潜在的相关交互。