Excess Nitric Oxide as a Mechanism of Glial Dysfunction in CMT1X

一氧化氮 (NO) 与神经损伤和周围神经病变的关系已有充分记录。然而，直到 最近几乎没有证据表明髓鞘神经胶质细胞本身会产生一氧化氮。示威活动 雪旺细胞中一氧化氮合酶 1 和 3（NOS-1 和 NOS-3）的作用，提高了内在 NO 的潜力 这些细胞的功能障碍。在这里，我们提出并检验了施万中 NO 反应放大的假设 细胞是 CMT1X 病理学的根本原因，CMT1X 是一种相对常见的遗传性外周血（有时 由连接蛋白 32 (Cx32) 突变引起的中枢神经系统疾病，连接蛋白表达于 髓鞘神经胶质细胞。我们假设的关键是数据表明 Cx32 是参与蛋白质复合物的一部分 无信号。由连接蛋白形成的间隙连接提供了耦合细胞之间的通讯途径。 然而，仅有缺陷的间隙连接通讯并不能充分解释间隙连接所发挥的作用。 神经胶质细胞中的 Cx32 或其他细胞类型中的连接蛋白。这里概述的工作将利用细胞培养、离体和体内 体内模型研究 Cx32 缺失或突变的生理和病理后果。我们 表明在 Cx32 缺陷型雪旺细胞中，相互作用的自我强化正反馈循环涉及 NO 增加、线粒体功能障碍和 Ca2 稳态受损是由 Cx32 和 NO 途径成分之间的相互作用。这些实验应该阐明目标 用于 CMT1X 的治疗干预，这可能也适用于表现出与疾病相关的其他疾病 连接蛋白表达的改变。我们将比较我们在野生型小鼠中的发现与在缺乏 Cx32 的小鼠中的发现 (Cx32KO) 和在 Cx32KO 背景上表达 CMT1X 突变体 Cx32T55I 的小鼠 (T55ITg/32KO)。目标 1 将检验以下假设：Cx32 的破坏会使雪旺细胞容易出现一氧化氮功能障碍，并且 问： Cx32 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测量 和周围神经？我们将检查 WT、32KO 和 T55ITg/32KO 中 NO 水平的相对差异 基线时的雪旺细胞以及用 siRNA 急性敲低 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-亚硝基化、酪氨酸硝化和线粒体功能也将受到影响。 评估。目标 2 将问：Cx32 KO 雪旺细胞和神经中出现的 NO 失调是否是由于 Cx32 和 NO 途径中元件之间正常发生的相互作用消失？ Cx32 似乎是一部分 膜相关蛋白复合物，包括 eNOS 和至少一种在 NO 中重要的酶 (ASS) ASS 已被证明可以直接与肝脏中的 Cx32 相互作用；此外，至少表达 一种连接蛋白已被证明可以与 eNOS 相互作用并降低其活性。我们将使用 LC-MS/MS 来 检查 Cx32 是否直接或间接与 NOS 或 NO 合成途径的其他元件相互作用。 我们还将对数据进行公正的分析，以捕获其他潜在的相关交互。

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.