Inherited demyelinating neuropathies, also known as Charcot-Marie-Tooth (CMT) disease type 1, are characterized by demyelination and remyelination of peripheral nerve axons. We were the first to show that mutations in connexin32 (Cx32), a gap junction protein, cause X-linked dominant CMT (CMTX), and that Cx32 is localized to the paranodes and incisures of the Schwann cell myelin sheath. We hypothesize that there are both loss-of-function and toxic gain-of-function effects of CMTX mutations, and that both may lead to demyelination because they interfere with the formation or function of gap junctions in the myelin sheath, although other explanations are possible. In this proposal, we will examine the function of Cx32 in the myelin sheath as well as the effects of CMTX mutations in vitro and in vivo, to determine the normal function of Cx32 as well as how CMTX mutations cause demyelination.
Our Specific Aims are (l) to determine whether there are functional gap junctions in myelinating Schwann cells, by demonstrating that small, but not large, molecular weight, markers can diffuse radially across the myelin sheath through incisures; (2) to study the effects of CMTX mutations on the synthesis and function of Cx32 in cultured mammalian cells, by examining the intracellular processing of different Cx32 mutants and their electrophysiological properties; (3) to determine whether different CMTX mutations can cause dominantly inherited demyelinating neuropathy in transgenic mice. By systematically evaluating the function of different CNN mutations in vitro and in vivo, we will determine whether different CMTX mutations cause a loss-of-function or a toxic gain-of-function. In addition, we will determine whether CMTX mutations disrupt the function of the gap junctions that are found at incisures and paranodes in the myelin sheath, and explore the relationship between the disruption of these gap junctions to the pathogenesis of demyelination. The other members of this Program Project will enable us to accomplish our goals. Dr. Rutkowski will establish myelinating co-cultures, so that we can determine whether Cx32 mutations affect the ability of Schwann cells to form and maintain myelin sheaths. Drs. Barchi and Kallen will help us to develop a spatial model of the Cx32 protein, so that we can better predict the consequence of CMTX mutations.
