The Role of Connexin32 in the Pathogensis of CMTX

Connexin32在CMTX发病机制中的作用

• 批准号: 8534290
• 负责人: STEVEN Simon Scherer
• 金额: $ 33.78万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534290
• 关键词: Acute; Affect; Animal Model; Area; Astrocytes; Autopsy; Axon; Binding; Biology; Biopsy; Brain; Buffers; Cell membrane; Cell model; Central Nervous System Diseases; Cervical spinal cord structure; Clinical; Co-Immunoprecipitations; Communication; Connexin 43; Connexins; Connexon; Corpus Callosum; Coupling; Data; Defect; Demyelinating Diseases; Demyelinations; Diffusion; Disease; Dominant-Negative Mutation; Electrophysiology (science); Fluorescence Recovery After Photobleaching; Frameshift Mutation; Gap Junctions; Gene Family; Gene Mutation; Genes; Glucose; Goals; Grant; Hereditary Spastic Paraparesis; Human; Integral Membrane Protein; Investigation; Ions; Knockout Mice; Lead; Lymphedema; Magnetic Resonance Imaging; Measures; Metabolic; Missense Mutation; Molecular; Multiple Sclerosis; Mus; Mutation; Myelin; Neuroglia; Neurons; Neuropil; Oligodendroglia; Optic Nerve; Paint; Patients; Phenotype; Proteins; Proteolipids; Role; Slice; Staining method; Stains; Transgenic Mice; Vertebrates; dysmyelination; experimental analysis; gap junction channel; glucose analog; in vivo; molecular mass; mutant; novel; novel therapeutic intervention; small molecule; trafficking; white matter

DESCRIPTION (provided by applicant): Recessive mutations in GJC2, the human gene that encodes Cx47, cause Pelizaeus-Merzbacher-like disease (PMLD) and hereditary spastic paraparesis (HSP), presumably related their lack of Cx47 function in oligodendrocytes. How the loss of Cx47 function results in the clinical picture of PMLD is unknown. MRI paints the picture of profound dysmyelination, but this has yet to be confirmed in an autopsy or biopsy. In cellular and animal models, the mutants associated with PMLD impair GJ communication, but we do not yet know why GJ communication is essential for the proper functioning of oligodendrocytes. In this competing renewal, we will determine whether novel Cx47 mutants affect GJ coupling, explore whether O: O coupling a general feature of oligodendrocytes, and the functional significance of O: O coupling. 1. Investigate the molecular defects of hCx47 mutants causing PMLD or familial lymphedema. In the last grant cycle of this grant, we showed that 3 recessive Cx47 mutants associated with PMLD have defective trafficking and do not form functional channels with either Cx47 or Cx43. We will build on this observation by investigating 2 additional missense mutations, 2 frameshift mutations that affect the C-terminus of Cx47, deletion of the PDZ-binding domain (del433-437; a relevant, but not a naturally-occurring mutation), and 6 dominant mutations that cause a completely different phenotype, Familial Lymphedema. The ability of each mutant to form functional GJ plaques will be investigated - by immunostaining, scrape loading, fluorescence recovery after photobleaching (FRAP), and electrophysiology. Whether the dominant mutants have dominant-negative effects on WT Cx47 will also be investigated (by co-staining, co-immunoprecipitation, and electrophysiology). We will also generate lines of transgenic mice that express the Cx47del433-437 or R257C, one of the dominant mutations causing Familial Lymphedema. We will assess the ability of these mutants to "rescue" the phenotype of Gjc2-null (Gjc2-/-) mice, and of the R257C mutant to worsen the phenotype of Gjc2-heterozygous (Gjc2+/-) mice. 2. Is O: O coupling a general feature of oligodendrocytes? In the last grant cycle of this grant, we showed that O: A coupling in lost in mice lacking both Cx32 and Cx47. We also showed, expectedly, O: O coupling was prominent in the corpus callosum, that O: O coupling was also lost in mice lacking both Cx32 and Cx47, that the GJs directly join intrafascicular oligodendrocytes. The goal of this aim is to determine whether O: O coupling is found in other white matter tracts, and hence is a general feature of intrafascicular oligodendrocytes. To that end, we will measure the diffusion of sulforhodamine-B (SR- B), examine the ultrastructure of O: O junctions, and examine the expression of glial connexins (Cx30, Cx32, Cx43, Cx47) in two tracts - the optic nerve and the ventral funiculus of the cervical spinal cord. 3. The functional significance of O: O coupling. The function of O: A and O: O coupling is uncertain. The experimental results to date support two, non-mutually exclusive, functions - spatial buffering of K+ and metabolic cooperation. K+ clearance is modestly decreased in acute brain slices from mice in which astrocytes lack both Cx43 and Cx30. These mice also provide the best evidence for metabolic coupling, demonstrating that Cx30 and Cx43 are required for the intracellular diffusion of a fluorescent glucose analogue (2-NDBG) to areas of neuropil with increased neuronal activity. We reasoned that O: O coupling might serve a similar purpose for myelinated axons, and propose an experimental analysis of this possibility in this Aim. We will determine whether (a) activity increases the extent of 2-NDBG diffusion, and (b) whether glucose infused into a single oligodendrocyte can "rescue" axonal conduction in glucose-deprived conditions.

描述（由申请人提供）：编码 Cx47 的人类基因 GJC2 的隐性突变导致 Pelizaeus-Merzbacher 样疾病 (PMLD) 和遗传性痉挛性截瘫 (HSP)，可能与少突胶质细胞中 Cx47 功能的缺乏有关。 Cx47 功能丧失如何导致 PMLD 的临床表现尚不清楚。 MRI 描绘了严重的髓鞘发育不良的情况，但这一点尚未在尸检或活检中得到证实。在细胞和动物模型中，与 PMLD 相关的突变体会损害 GJ 通讯，但我们还不知道为什么 GJ 通讯对于少突胶质细胞的正常功能至关重要。在这次竞争性更新中，我们将确定新的 Cx47 突变体是否影响 GJ 偶联，探讨 O: O 偶联是否是少突胶质细胞的一般特征，以及 O: O 偶联的功能意义。 1. 研究导致 PMLD 或家族性淋巴水肿的 hCx47 突变体的分子缺陷。在本次资助的最后一个资助周期中，我们发现与 PMLD 相关的 3 个隐性 Cx47 突变体具有运输缺陷，并且不与 Cx47 或 Cx43 形成功能通道。我们将在此观察的基础上，研究 2 个额外的错义突变、2 个影响 Cx47 C 末端的移码突变、PDZ 结合域的删除（del433-437；相关但不是自然发生的突变）以及 6 个导致完全不同表型（家族性淋巴水肿）的显性突变。将通过免疫染色、刮擦负载、光漂白后荧光恢复 (FRAP) 和电生理学来研究每个突变体形成功能性 GJ 斑块的能力。还将研究显性突变体是否对 WT Cx47 具有显性负效应（通过共染色、免疫共沉淀和电生理学）。我们还将培育表达 Cx47del433-437 或 R257C 的转基因小鼠品系，Cx47del433-437 或 R257C 是导致家族性淋巴水肿的显性突变之一。我们将评估这些突变体“拯救”Gjc2缺失（Gjc2-/-）小鼠表型的能力，以及R257C突变体使Gjc2杂合（Gjc2+/-）小鼠表型恶化的能力。 2. O: O偶联是少突胶质细胞的普遍特征吗？在本次资助的最后一个资助周期中，我们表明 O: A 偶联在缺乏 Cx32 和 Cx47 的小鼠中丢失。我们还发现，如预期的那样，O: O 偶联在胼胝体中很突出，在同时缺乏 Cx32 和 Cx47 的小鼠中，O: O 偶联也消失了，GJ 直接连接束内少突胶质细胞。该目的的目的是确定 O: O 偶联是否存在于其他白质束中，因此是束内少突胶质细胞的一般特征。为此，我们将测量磺胺罗丹明-B (SR-B) 的扩散，检查 O: O 连接的超微结构，并检查两个神经束（视神经和颈脊髓腹索）中神经胶质连接蛋白（Cx30、Cx32、Cx43、Cx47）的表达。 3、O:O偶联的功能意义。 O:A和O:O耦合的作用是不确定的。迄今为止的实验结果支持两种非互斥的功能——K+的空间缓冲和代谢合作。在星形胶质细胞同时缺乏 Cx43 和 Cx30 的小鼠的急性脑切片中，K+ 清除率略有下降。这些小鼠还为代谢偶联提供了最好的证据，证明荧光葡萄糖类似物 (2-NDBG) 细胞内扩散到神经元活性增加的神经毡区域需要 Cx30 和 Cx43。我们推断 O: O 耦合可能对有髓轴突起到类似的作用，并在本目标中提出了对这种可能性的实验分析。我们将确定（a）活性是否增加 2-NDBG 扩散的程度，以及（b）注入单个少突胶质细胞的葡萄糖是否可以在葡萄糖剥夺条件下“拯救”轴突传导。