It is well established that mutations in GJB1, the gene encoding the gap junction forming protein connexin32 (Cx32), cause CMT1X, the X-linked form of Charcot-Marie-Tooth disease (CMT1X). However, in spite of more than 20 years of investigation, it is still not known how these mutations cause neuropathy. A simple hypothesis would be that all CMT1X mutations are pathogenic as a result of the failure to localize Cx32 to non-compact myelin. This idea is partially supported by the observation that a majority of clinically well characterized Cx32 mutants are abnormally localized when studied in exogenous expression systems; however, some mutants are targeted normally. One possibility is that mutations found to localize normally in exogenous cells will not do so when expressed in vivo. A failure of these mutants to localize properly in Schwann cells would support the notion that pathogenicity is simply a matter of incorrect localization. On the other hand, finding that these mutants also target normally in myelinating Schwann cells, would be an important finding because it would imply that normal localization of Cx32 does not prevent development of neuropathy; therefore, the neuropathy in these cases would have to be caused by more subtle alteration in the function of Cx32. In summary, the work proposed will test whether clinically well characterized mutations lead to neuropathy in spite of being normally localized in the myelinating Schwann cell. In addition to providing an important insight into the mechanism of CMT1X pathogenesis, the animal models developed for this proposal will become a valuable resource for the CMT1X research community, since currently the best available model of CMT1X, the Cx32 null mouse (Cx32 KO) models only a small fraction of patients with CMT1X.
In Aim one, we will use CRISPR/Cas9 technology to produce two new ?knockin? models of CMT1X ? p.V139M and p.E102G. One of the two mutants (p.E102G) is capable of forming functional channels; the other one (p.V139M) does not. Both, however, traffic normally in exogenous systems.
In Aim two we will examine the subcellular localization of Cx32 in peripheral nerve fibers to examine whether localization of Cx32 in the myelinated fiber is normal. We will double label with an antibody to Cx32 and Golgi complex protein, GRP94 (ER marker). E-Cadherin or Cx29 to identify whether Cx32 labeled protein colocalizes with these markers. In addition, for each mutation (p.E102G, p.V139M , and Gjb1 deletion) as well as WT mice, we will sacrifice 5 mice at 4 and 6 months, and examine caudal and femoral motor nerves to (i) document that the particular mutation leads to peripheral neuropathy and (ii) quantify axonal loss and other pathologies in comparison to the wild-type and Cx32KO.
Charcot-Marie-Tooth disease (CMT) is a common and debilitating disorder, affecting about 130,00 people in the United States and several million throughout the world. This proposal seeks to understand why a subset of patients with the second most common form of CMT, CMT1X, develop debilitating limb weakness. These studies should lead to a better general understanding of the role of connexins in nerve function and should facilitate the development of treatments for patients with CMT1X.