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

基本信息

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
项目类别：
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

项目摘要

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。但是，直到最近，几乎没有或没有证据表明骨髓神经胶质本身无产生。演示一氧化氮合酶1和3（NOS-1和NOS-3）在雪旺细胞中，提高了内在no的潜力这些细胞功能障碍。在这里，我们提出并检验以下假设：Schwann中没有放大的响应细胞是CMT1X病理学的根本原因，CMT1X是一种相对常见的遗传外周（有时中枢神经系统疾病由连接蛋白32（CX32）中的突变引起髓质神经胶质。我们假设的关键是数据表明CX32是参与的蛋白质复合物的一部分没有信号。连接蛋白形成的间隙连接提供了耦合细胞之间的通信途径。但是，仅缺陷差距交流并不能说明胶质细胞中的CX32或其他细胞类型中的连接素。这里概述的工作将利用细胞培养，离体和 VIVO模型研究CX32中丧失或突变的生理和病理后果。我们建议在CX32缺陷的Schwann细胞中，一种自我强化的互动反馈回路涉及没有增加，线粒体功能障碍和CA2+稳态受损是由中断或丢失触发的 CX32与NO途径的组件之间的相互作用。这些实验应阐明目标用于在CMT1X中进行治疗干预，这可能还适用于其他与疾病有关的疾病连接素表达的改变。我们将在野生型小鼠中与缺乏CX32的小鼠中的发现进行比较（CX32KO）和在CX32KO背景（T55ITG/32KO）上表达CMT1X突变体CX32T55I的小鼠中。目的 1将检查以下假设：CX32破坏使雪旺细胞易受一氧化氮功能障碍和问：CX32中没有或突变会影响与一氧化氮功能有关的措施和周围神经？我们将检查WT，32KO和T55ITG/32KO中NO水平的相对差异基线时的Schwann细胞以及用siRNA急性敲低CX32会导致无生产变化。过氧亚硝酸盐产生，蛋白质S-硝基化，酪氨酸硝化和线粒体功能也将是评估。 AIM 2会问：是否在CX32 KO Schwann细胞中看到的NO失调是否会出现，并且由于 CX32与NO途径中元素之间正常发生相互作用的丧失？ CX32似乎是一部分膜相关的蛋白质复合物，包括eNOS和至少一种酶（ASS）合成和ASS已显示出与肝脏中CX32直接相互作用。此外，至少表达已显示一种连接素可以与ENOS的活性相互作用并减少。我们将使用LC-MS/MS到检查CX32是直接还是间接与NO合成途径的NOS或其他元素相互作用。我们还将对数据进行公正的分析，以捕获其他潜在的相关相互作用。