The role of connexin32 in the pathogenesis of CMTX

GJB 1、GJA 1和GJA 12中的突变，编码人连接蛋白32（hCx 32）、hCx 43和hCx 44的基因， hGx 47，导致X连锁型腓骨肌萎缩症（CMT 1X），眼指关节。发育不良 （ODDD）和PeHzaeus-Merzbacher-ijke病（PMLD），所有这些疾病都引起重要的CNS疾病。 这些异常似乎与少突胶质细胞的异常功能有关。的中心主题 这表明Cx 30：Cx 32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，其被GJB 1/Cx 32、GJA 1/Cx43或GJA 12/Cx47的突变破坏。 1.研究引起PMLD的hCx 47突变体的分子缺陷。 我们将进一步描述这些缺陷的性质，并确定野生型（WT）hCx 47或 这些hOx 47突变体可以通过染料转移和电泳与hCx 43形成功能通道。 2.研究引起ODDD的hCx 43突变体的分子缺陷。 我们将研究突变蛋白的分子性质，确定表达突变蛋白的细胞是否具有突变蛋白的功能。 ODDD突变体可以自身形成功能性通道，或者与表达WT hCx 43或WT hCx 43的细胞形成功能性通道。 iGx 47，并确定ODDD突变体是否对WT hCx 43具有显性负效应。 3-确定hCx 32“CNS突变体”是否对hCx 47具有显性效应。是的。'.. ' 在HeLa细胞中，这些“CNS突变体”在内质网（ER）或Gblgi中积累。我们 初步证据表明这些基因的共同表达。具有WT hCx 47的“CNS突变体”导致部分的 野生型hCx 47在ER或高尔基体中的保留，表明这些Cx 32突变体对野生型发挥显性作用 hCx 47。我们将进一步描述这些缺陷的性质。. . 4.确定Cx 32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Cx 32和/或Cx47的小鼠的大脑进行免疫染色， 他们提议的合作伙伴被改变了。我们还将通过基因注射星形细胞来研究A/O偶联 用绿色荧光蛋白（GFP）标记Gjb 1/cx 32和Gja 12/cx 47的急性脊髓切片 “双船体”小鼠的小分子，可以通过GJ。这样，我们就可以确定相对 两种异型通道在A/0偶联中的重要性。

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.