Altered trafficking of connexin32: A myelin-related protein mutated in CMTX

X-linked Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy caused by mutations in the gap junction gene, connexin32 (Cx32). Although Cx32 is expressed in many different cell types, the clinical manifestations of CMTX are restricted to the myelinating Schwann cells in the peripheral nervous system (PNS). The work presented in this thesis was designed to investigate the role of Cx32 in peripheral nerve in two ways. The first approach involved characterizing the expression of wild-type rat Cx32 relative to other myelin genes during the development of the nervous system, following nerve injury, and in cultured Schwann cells. We found that Cx32 protein was coordinately expressed with proteolipid protein in several brain regions and was enriched in myelin fractions from two of these regions. Similarly, the expression patterns of Cx32 and myelin protein zero coincided in crushed and transected rat sciatic nerve. Expression of Cx32 protein was not detectable in forskolin-stimulated, cultured rat Schwann cells. The second approach focused on elucidating the pathogenesis of CMTX by stably transfecting communication-incompetent PC 12J cells with nine different mutant Cx32 cDNAs and examining the cellular localization of the protein products. We found that Cx32 mutants R15Q, V63I, V139M, and R220Stop were localized both at the cell surface and in the cytoplasm, whereas mutants G12S, R142W, E186K, and E208K were retained intracellularly, either in the Golgi apparatus or in a more diffuse cytoplasmic compartment. A frameshift mutant at codon 175 did not produce any detectable protein, although it was appropriately transcribed. These studies showed that Cx32 protein is coordinately regulated with other myelin proteins. Furthermore, Cx32 mutants can be classified into three groups based on their cellular localization. The transmembrane domains and amino-terminus of Cx32 may be involved in proper Cx32 trafficking. Finally, I provide independent support for a common pathophysiology, altered protein trafficking, among diseases caused by mutations in myelin genes