The Role of Connexin32 in the Pathogensis of CMTX

Connexin32在CMTX发病机制中的作用

• 批准号: 8186867
• 负责人: STEVEN Simon Scherer
• 金额: $ 35万
• 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/8186867
• 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. PUBLIC HEALTH RELEVANCE: Mutations in GJB1 (encodes Cx32) and GJC2 (encodes Cx47) cause demyelinating diseases in humans. To devise rational treatments for these diseases, it is imperative to determine how and why these mutations cause demyelination. The investigations proposed in this grant will examine how recessive and dominant GJC2 mutations affect the ability of the mutant proteins to form functional gap junctions, including their effects on the ability of gap junction coupling between glial cells in vivo. These data will enhance our understanding of the biology and pathobiology of oligodendrocytes, and may elucidate important aspects of other demyelinating diseases such as multiple sclerosis, and may lead to novel therapeutic approaches.

描述（由申请人提供）：GJC 2（编码Cx47的人类基因）的隐性突变导致Pelizaeus-Merzbacher样疾病（PMLD）和遗传性痉挛性轻瘫（HSP），可能与少突胶质细胞中Cx47功能的缺乏有关。Cx47功能的丧失如何导致PMLD的临床表现尚不清楚。核磁共振成像描绘了严重的髓鞘形成障碍，但这还有待尸检或活检证实。在细胞和动物模型中，与PMLD相关的突变体损害了GJ通讯，但我们还不知道为什么GJ通讯对少突胶质细胞的正常功能至关重要。在这种竞争性更新中，我们将确定新的Cx47突变体是否影响GJ偶联，探索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或家族性水肿的hCx 47突变体的分子缺陷。在本研究的最后一个研究周期中，我们发现与PMLD相关的3个Cx47隐性突变体具有缺陷性运输，并且与Cx47或Cx43均不形成功能性通道。我们将通过研究2个额外的错义突变、2个影响Cx47 C端的移码突变、PDZ结合结构域缺失（del 433 -437;相关但不是自然发生的突变）和6个导致完全不同表型的显性突变（家族性淋巴水肿）来建立这一观察结果。将通过免疫染色、刮片加载、光漂白后荧光恢复（FRAP）和电生理学研究每种突变体形成功能性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）。我们还将产生表达Cx47 del 433 -437或R257 C的转基因小鼠品系，Cx47 del 433 -437或R257 C是引起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”Gjc 2缺失（Gjc 2-/-）小鼠表型的能力，以及R257 C突变体恶化Gjc 2杂合（Gjc 2 +/-）小鼠表型的能力。2. O：O偶联是少突胶质细胞的一个普遍特征吗？在本研究的最后一个研究周期中，我们发现O：A偶联在缺乏Cx 32和Cx47的小鼠中丢失。我们还发现，在胼胝体中，O：O偶联是突出的，在缺乏Cx 32和Cx47的小鼠中，O：O偶联也丢失，GJ直接加入束内少突胶质细胞。本研究的目的是确定O：O偶联是否存在于其他白色物质束中，因此是束内少突胶质细胞的一般特征。为此，我们将测量磺酰罗丹明-B（SR- B）的扩散，检查O：O连接的超微结构，并检查两个束-视神经和颈脊髓腹索中胶质连接蛋白（Cx 30，Cx 32，Cx43，Cx47）的表达。3. O：O偶联的功能意义。O：A和O：O偶联的作用尚不确定。迄今为止的实验结果支持两个非相互排斥的功能-K+的空间缓冲和代谢合作。在星形胶质细胞缺乏Cx43和Cx 30的小鼠的急性脑切片中，K+清除率适度降低。这些小鼠还提供了代谢偶联的最佳证据，证明Cx 30和Cx43是荧光葡萄糖类似物（2-NDBG）向神经元活性增加的神经元区域的细胞内扩散所需的。我们推断O：O偶联可能为有髓轴突提供类似的目的，并提出了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）葡萄糖注入单个少突胶质细胞是否可以在葡萄糖剥夺条件下“拯救”轴突传导。 公共卫生相关性：GJB 1（编码Cx 32）和GJC 2（编码Cx47）的突变导致人类脱髓鞘疾病。为了设计合理的治疗方法，必须确定这些突变如何以及为什么会导致脱髓鞘。这项研究将研究隐性和显性GJC 2突变如何影响突变蛋白形成功能性间隙连接的能力，包括它们对体内胶质细胞之间间隙连接偶联能力的影响。这些数据将增强我们对少突胶质细胞的生物学和病理学的理解，并可能阐明其他脱髓鞘疾病如多发性硬化症的重要方面，并可能导致新的治疗方法。