The role of connexin32 in the pathogenesis of CMTX

描述(申请人提供)：编码人类连接蛋白32(HCx32)、hCx43和hGx47的基因GJB1、GJA1和GJA12的突变分别导致X连锁形式的Charcot-Marie-Tooth病(CMT1X)、眼齿数字发育不良(Oddd)和Pelizaeus-Merzbacher样病(PMLD)，所有这些都会导致重要的中枢神经系统异常，似乎与少突胶质细胞的异常功能有关。这项资助的中心主题是Cx30：Cx32和Cx43：Cx47异型通道介导星形胶质细胞/少突胶质细胞(A/O)结合，这种结合被GJB1/Cx32、GJA1/Cx43或GJA12/Cx47突变破坏。 1.研究引起PMLD的hCx47突变体的分子缺陷。我们将进一步确定这些缺陷的性质，并确定野生型(WT)hCx47或这些hCx47突变体是否能够通过染料转移和电生理与hCx43形成功能通道。 2.研究引起ODD的hCx43突变体的分子缺陷。 我们将研究突变蛋白的分子性质，确定表达oddd突变的细胞是否可以自己形成功能通道，或者是否可以与表达wt hCx43或wt hCx47的细胞形成功能通道，并确定oddd突变是否对-wt hCx43具有显性-负向影响。 3.确定hCx32“中枢神经系统突变体”对hCx47是否具有显性效应。 在HeLa细胞中，这些“CNS突变体”聚集在内质网(ER)或高尔基体中。我们的初步证据表明，这些“CNS突变体”与WT hCx47共表达导致Wt hCx47在内质网或高尔基体中部分保留，表明这些Cx32突变体对WT hCx47起显性作用。我们将进一步描述这些缺陷的性质。 4.确定Cx32和Cx47在星形胶质细胞/少突胶质细胞偶联中的作用。 我们将对缺乏Cx32和/或Cx47的小鼠的大脑进行免疫染色，并确定其拟议伴侣的定位是否发生改变。我们还将通过将绿色荧光蛋白(GFP)标记的星形胶质细胞注射到Gjb1/cx32和Gja12/cx47双零小鼠的急性脊髓切片中，研究A/O偶联，从而确定这两种异型通道在A/O偶联中的相对重要性。

DESCRIPTION (provided by applicant): Mutations in GJB1, GJA1, and GJA12, the genes that encode human connexin32 (hCx32), hCx43, and hGx47, cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X), oculodentodigital dysplasia (ODDD), and Pelizaeus-Merzbacher-like disease (PMLD), respectively, all of which cause important CNS abnormalities that appear to be related to abnormal functioning of oligodendrocytes. The central theme of this grant is that Cx30:Cx32 and Cx43:Cx47 heterotypic channels mediate astrocyte/oligodendrocyte (A/O) coupling, which is disrupted by mutations of GJB1/Cx32, GJA1/Cx43, or GJA12/Cx47. 1. Investigate the molecular defects of hCx47 mutants causing PMLD. We will characterize further the nature of these defects, and determine whether wild type (WT) hCx47 or these hCx47 mutants can form functional channels with hCx43 by dye transfer and electrophysiology. 2. Investigate the molecular defects of hCx43 mutants causing ODDD. We will investigate the molecular nature of the mutants proteins, determine whether cells expressing an ODDD mutant can form functional channels by themselves, or with cells expressing either WT hCx43 or WT hCx47, and determine whether ODDD mutants' have dominant-negative effects on-WT hCx43. 3. Determine whether hCx32 "CNS mutants" have dominant effects on hCx47. In HeLa cells these "CNS mutants" accumulate either in the endoplasmic reticulum (ER) or in the Golgi. Our preliminary evidence indicates that co-expression of these "CNS mutants" with WT hCx47 results in partial retention of Wt hCx47 in the ER or Golgi, indicating that these Cx32 mutants exert a dominant effect on WT hCx47. We will characterize further the nature of these defects. 4. Determine the role of Cx32 and Cx47 in astrocyte/oligodendrocyte coupling. We will immunostain the brains of mice that lack Cx32 and/or Cx47, and determine whether the localization of their proposed partners is altered. We will also investigate A/O coupling by injecting astrocytes genetically labeled with green fluorescent protein (GFP) in acute spinal cord slices from Gjb1/cx32 and Gja12/cx47 double null" mice with small molecules that can cross GJs. In this way, we will determine the relative importance of the two kinds of heterotypic channels in A/0 coupling.