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的研究已有文献记载。然而，直到 最近，很少或根本没有证据表明髓鞘胶质细胞本身产生了NO。演示 雪旺细胞中一氧化氮合酶1和3(NOS-1和NOS-3)的表达增加了内源性NO的可能性 这些细胞的功能障碍。在这里，我们提出并检验了一个假设，即在Schwann中放大的NO反应 细胞是CMT1X的潜在病理原因，CMT1X是一种相对常见的遗传性外周疾病(有时 中枢)神经系统疾病：一种表达于 髓鞘胶质细胞。我们假设的关键是有数据表明，Cx32是参与 没有信号。连接蛋白形成的缝隙连接提供了连接细胞之间的通讯途径。 然而，仅有有缺陷的缝隙连接沟通并不能充分说明 Cx32在胶质细胞中表达，或在其他类型的细胞中表达。这里概述的工作将利用细胞培养、体外和体内 研究Cx32基因缺失或突变的生理和病理后果的活体模型。我们 提示在Cx32缺陷的雪旺细胞中，一个自我强化的正反馈相互作用环路涉及 NO增加、线粒体功能障碍和钙稳态受损是由细胞外信号转导系统的破坏或丢失触发的 Cx32与NO途径组分的相互作用。这些实验应该能阐明靶标 用于CMT1X的治疗干预，这可能也适用于其他与疾病相关的疾病 缝隙连接蛋白表达的改变。我们将在野生型小鼠和缺乏Cx32的小鼠身上比较我们的发现 (Cx32KO)和在Cx32KO背景下表达CMT1X突变体Cx32T55I的小鼠(T55ITg/32KO)。目标 1将检验一种假设，即Cx32的破坏使雪旺细胞易患一氧化氮功能障碍和 问：Cx32的缺失或突变是否会影响雪旺细胞中与一氧化氮功能相关的指标 那周围神经呢？我们将检查WT、32KO和T55ITg/32KO中NO水平的相对差异 雪旺细胞在基线水平，以及用siRNA急性敲除Cx32是否会导致NO产生的变化。 过氧亚硝酸盐的产生，蛋白质S亚硝化，酪氨酸硝化和线粒体功能也将 评估过了。目标2将问：在Cx32 KO雪旺细胞和神经中没有看到的失调是不是由于 Cx32和NO途径中的元素之间正常发生的相互作用的丧失？Cx32似乎是 包括eNOS和至少一个在NO中重要的酶(ASS)的膜相关蛋白复合体 合成，ASS已被证明在肝脏中直接与Cx32相互作用；此外，至少表达 一种连接蛋白既能与eNOS相互作用，又能降低eNOS的活性。我们将使用LC-MS/MS来 检查Cx32是否直接或间接地与一氧化氮合酶或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不偏不倚的分析，以捕获其他潜在的相关交互。