CONNEXIN32 MUTATIONS IN CHARCOT-MARIE-TOOTH-X DISEASE

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DESCRIPTION (From the Applicant's Abstract): The X-linked form of type I Charcot-Marie-Tooth disease (CMTX) is the second most common hereditary peripheral neuropathy in humans. CMTX is caused by mutations in the gap junction channel protein connexin32 (Cx32) that are thought to affect its ability to mediate the radial diffusion of substances through the Schwann cell myelin sheath. CMTX is genetically as well as phenotypically heterogeneous: over 200 different defects in the coding region of the Cx32 gene have been found in CMTX patients, whose clinical symptoms range from very mild/asymptomatic to eventually becoming wheelchair-bound. Studies in transfected tissue culture cells and transgenic mice indicate that different mutations interfere with Cx32 function by distinct mechanisms: some mutants are translated inefficiently and/or degraded very quickly; others appear to affect channel function at the cell surface; and many are detected only intracellularly and act as dominant negative inhibitors of wild-type connexins. How different Cx32 mutations result in these diverse phenotypes is unknown. Our initial characterization of three CMTX-linked Cx32 point mutants in PCI2 cell transfectants revealed that each mutant was defective in folding and oligomeric assembly and underwent a distinct intracellular fate: E208K Cx32 accumulated in the endoplasmic reticulum whereas both the E186K and R142W mutants were transported to the Golgi region from which they trafficked either to lysosomes (RI42W Cx32) or back to the ER (EI86K Cx32). The goal of the proposed studies is to elucidate the molecular mechanisms by which CMTX-linked mutations affect the assembly, intracellular transport, and degradation of Cx32. These studies will be conducted in transfected tissue culture cells as well as in peripheral nerve tissue from mice expressing Cx32 mutants. Specifically, we will use molecular and cellular biological techniques to: (1) define the intracellular trafficking routes and molecular chaperone interactions of CMTX-linked Cx32 mutants; (2) identify, and test the correctability of, conformational defects in Cx32 induced by different CMTX-linked mutations; (3) elucidate the mechanisms whereby Cx32 mutants gain access to, and are degraded by, cytosolic proteasomes; and (4) determine the molecular basis for the dominant-negative activity of some CMTX-linked mutants and identify the in vivo target of this activity in myelinating Schwann cells. These studies will elucidate the causes of phenotypic diversity in CMTX as well as provide the first molecular insights into the quality control mechanisms that govern the fidelity of connexin assembly into gap junctional channels.