The gap junction (GJ) protein connexin32 (Cx32) is expressed by myelinating Schwann cells and oligodendrocytes and is mutated in X-linked Charcot-Marie-Tooth disease. In addition to a demyelinating peripheral neuropathy, some Cx32 mutants are associated with transient or chronic CNS phenotypes. To investigate the molecular basis of these phenotypes, we generated transgenic mice expressing the T55I or the R75W mutation and an IRES-EGFP, driven by the mouse Cnp promoter. The transgene was expressed in oligodendrocytes throughout the CNS and in Schwann cells. Both the T55I and the R75W mutants were localized in the perinuclear cytoplasm, did not form GJ plaques, and did not alter the expression or localization of two other oligodendrocytic GJ proteins, Cx47 and Cx29, or the expression of Cx29 in Schwann cells. On wild type background, the expression of endogenous mCx32 was unaffected by the T55I mutant, but was partly impaired by R75W. Transgenic mice with the R75W mutation and all mutant animals with Gjb1-null background developed a progressive demyelinating peripheral neuropathy along with CNS myelination defects. These findings suggest that Cx32 mutations result in loss of function in myelinated cells without trans-dominant effects on other GJ proteins. Loss of Cx32 function alone in the CNS causes myelination defects.

X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the gene that encodes the gap junction protein connexin32 (Cx32). Cx32 is expressed by myelinating Schwann cells and forms gap junctions in non-compact myelin areas but axonal involvement is more prominent in X-linked compared to other forms of demyelinating Charcot-Marie-Tooth disease. To clarify the cellular and molecular mechanisms of axonal pathology in CMT1X, we studied Gjb1-null mice at early stages (i.e. 2- to 4-month-old) of the neuropathy, when there is minimal or no demyelination. The diameters of large myelinated axons were progressively reduced in Gjb1-null mice compared to those in wild type littermates. Furthermore, neurofilaments were relatively more dephosphorylated and more densely packed starting at 2 months of age. Increased expression of β-amyloid precursor protein, a marker of axonal damage, was also detected in Gjb1-null nerves. Finally, fast axonal transport, assayed by sciatic nerve ligation experiments, was slower in distal axons of Gjb1-null vs. wild type animals with reduced accumulation of synaptic vesicle-associated proteins. These findings demonstrate that axonal abnormalities including impaired cytoskeletal organization and defects in axonal transport precede demyelination in this mouse model of CMT1-X.