The role of connexin32 in the pathogenesis of CMTX

编码人类连接蛋白32(HCx32)、hCx43和GJA12的基因GJB1、GJA1和GJA12的突变 HGx47，导致X连锁的Charcot-Marie-Tooth病(CMT1X)，眼球缺失。异型增生 (Oddd)和PeHzaeus-Merzbacher-ijke病(PMLD)，所有这些疾病都会引起重要的CNS 似乎与少突胶质细胞功能异常有关的异常。的中心主题 这项授权是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) 偶联，被GJB1/Cx32、GJA1/Cx43或GJA12/Cx47突变破坏。 1.研究导致PMLD的hCx47突变体的分子缺陷。 我们将进一步鉴定这些缺陷的性质，并确定野生型(WT)hCx47或 这些hOx47突变体可以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 jmyants的分子缺陷。 我们将研究突变蛋白的分子性质，确定细胞是否表达 Oddd突变体可以单独或与表达WT hCx43或WT的celJ形成功能通道 1oddd突变体是否对-wt hCx43有显性负效应。 3-确定hCx32“中枢神经系统突变体”是否对hCx47具有显性效应。“、”..“ 这些“中枢神经系统突变体”聚集在HeLa细胞的内质网(ER)或Gblgi。我们的 最新证据表明，这些“中枢神经系统突变体”与WT hCx47共表达会导致部分 Wt hCx47在内质网或高尔基体中的滞留，表明这些Cx32突变体对Wt起显性作用 HCx47。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，并确定其定位 他们提议的合作伙伴的身份发生了变化。我们还将通过基因注射星形细胞来研究A/O偶联 Gjb1/cx32和Gja12/cx47急性脊髓切片绿色荧光蛋白标记 具有小分子的小鼠可以通过GJS。这样，我们就可以确定相对分子 两种异型通道在A/O耦合中的重要性。

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), oculodehtodigital. dysplasia (ODDD), and PeHzaeus-Merzbacher-ijke 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 heterptypic 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 causingi PMLD. We will characterize further the nature.ofthese defects, .arid determine whether wild type (WT) hCx47 or these hOx47 mutants can form functional channels with hCx43 by dye transfer and electrophysiblogy. 2. Investigate the molecular defects of hCx43 jmytants causing ODDD. We will investigate the molecular nature of the mutaiit proteins, determine whether cells expressing an ODDD mutant can form functional channels by themselves, or with celJs expressing either WT hCx43 or WT iGx47, and determine.1whether ODDD mutants'have dominant-negative effects on-WT hCx43. 3-Determine whether hCx32 "CNS mutants" have dominant effects on hCx47. ¿ ,.'..' n HeLa cells these "CNS mutants" accumulatefiithef in.the endoplasniic reticujum (ER) or in the Gblgi. Our Dreliminary evidenceindicates that co-expression of these."CNS mutants" with WT hCx47 results in partial retention of Wt hCx47 in the ER or Golgi, indicating .tn.atthese 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 astrpcytes genetically abeled with green fluorescent protein (GFP) in acute spinal cord slices from Gjb1/cx32 and Gja12/cx47 double hull" mice with small molecules that can cross GJs. In this way, we will determine the relative mportance of the two kinds of heterotypic channels in A/0 coupling.