The Role of Connexin32 in the Pathogensis of CMTX

描述(申请人提供)：编码Cx47的人类基因GJC2的隐性突变导致Pelizaeus-Merzbacher样病(PMLD)和遗传性痉挛麻痹(HSP)，推测与它们在少突胶质细胞中缺乏Cx47功能有关。Cx47功能缺失如何导致PMLD的临床表现尚不清楚。核磁共振成像描绘了严重的髓鞘异常，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是必不可少的。在这一竞争更新中，我们将确定新的Cx47突变体是否影响GJ连接，探索O：O连接是否是少突胶质细胞的一般特征，以及O：O连接的功能意义。1.研究导致PMLD或家族性淋巴水肿的hCx47突变体的分子缺陷。在上一个授予周期中，我们发现与PMLD相关的3个隐性Cx47突变体具有缺陷的运输，并且不能与Cx47或Cx43形成功能通道。我们将通过研究另外两个错义突变、两个影响Cx47 C末端的移码突变、PDZ结合结构域的缺失(del433-437；相关的但不是自然发生的突变)和导致完全不同的表型-家族性淋巴水肿的6个显性突变来建立这一观察结果。将通过免疫染色、刮除负载、光漂白后荧光恢复(FRAP)和电生理来研究每个突变体形成功能性GJ斑块的能力。显性突变是否对WT Cx47有显性-负效应也将被研究(通过共染色、共免疫沉淀和电生理学)。我们还将产生表达Cx47del433-437或R257C的转基因小鼠系，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突变体恶化Gjc2杂合(Gjc2+/-)小鼠表型的能力。2.O：O偶联是少突胶质细胞的普遍特征吗？在这项资助的最后一个资助周期中，我们发现O：A偶联在缺乏Cx32和Cx47的小鼠中丢失。我们还显示，不出所料，O：O偶联在胼胝体中显著，在缺乏Cx32和Cx47的小鼠中也丢失了O：O偶联，GJS直接连接到束内的少突胶质细胞。这一目的的目的是确定是否在其他白质束中发现O：O偶联，因此这是束内少突胶质细胞的一般特征。为此，我们将测量磺胺荷丹明-B(SR-B)的扩散，检查O：O连接的超微结构，并检测神经胶质连接蛋白(Cx30、Cx32、Cx43、Cx47)在视神经和颈髓腹索的表达。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。到目前为止的实验结果支持两个非互斥的功能--K+的空间缓冲和代谢协作。在星形胶质细胞缺乏Cx43和Cx30的小鼠急性脑片中，K+清除量略有下降。这些小鼠也为代谢偶联提供了最好的证据，证明了Cx30和Cx43是荧光葡萄糖类似物(2-NDBG)在细胞内扩散到神经元活性增强的神经纤维层区域所必需的。我们推测O：O偶联可能对有髓轴突起到类似的作用，并在这一目标中提出了对这种可能性的实验分析。我们将确定(A)活动是否增加了2-NDBG扩散的程度，以及(B)向单个少突胶质细胞中注入葡萄糖是否可以在缺糖条件下“挽救”轴突传导。

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.