Charles K. Abrams - US grants

X-linked Charcot Marie-Tooth Disease (CMTX) is an inherited peripheral neuropathy associated with mutations in the gap junction protein connexins 32 (Cx32). The central tenet of this proposal is that alterations in the functional properties of the ion channel formed by Cx32 can cause CMTX. Further, we propose that to understand the link between mutations and the disorder, we must explore the specific nature of the induced deficits in channel gating, permeability and control of formation. Although an association between a specific mutation in an ion channel and a neuromuscular disease is often compelling, the mechanism that underlies the deficit can remain unrevealed by clinical measures, morphologic examination or epidemiology. The studies )roposed in this grant are a direct and logical continuation of the applicant's previous work exploring the link )etween CMTX and the mutations in the genes controlling gap junction proteins; they build on previous efforts to correlate functional deficits at the cellular and subcellular level with specific mutations and with phenotypic variability. The first two specific aims will use electrophysiologic techniques to examine the loss of Cx32 function resulting from alterations in gating and permeability of mutant forms of Cx32. The third specific aim will again use electrophysiologic techniques to evaluate the possibility that mutations in Cx32 may turn this protein into a suicide channel, leading to disrupted function in cells in which it is expressed. The fourth specific aim utilizes targeted gene replacement to examine the differential effects of two mutant forms of Cx32. The support of a K02 Independent Scientist Award will insure that the applicant is able to maintain at least 75% effort in basic science research while at the same time benefiting from the unique scientific environment of the Department of Neuroscience of the Albert Einstein College of Medicine. These factors will provide an opportunity for continued intellectual and technical development of the PI during this critical early period of development as a physician- scientist engaged in an independent program of translational research.

DESCRIPTION (provided by applicant): A fundamental principle underlying this grant is that connexin 32 (Cx32) is required for normal function of Schwann cells. Though mutations in Cx32 cause alterations in channel function and are clearly associated with the X-Linked Charcot-Marie-Tooth Disease (CMTX), the pathogenesis of this disorder remains to be elucidated. The localization of Cx32 to the paranodes and Schmidt-Lantermann incisures of the myelinating Schwann cell has lead to the hypothesis that Cx32 forms reflexive gap junctions within noncompact myelin and provides a "short circuit" pathway between the ab- and adaxonal cytoplasm of the myelinating Schwann cell. However, data suggest that: 1) mice lacking Cx32 show reduced capacity for regeneration associated myelination; 2) Cx32 is expressed and regulated in cultures of primary Schwann cells; 3) Schwann cells expressing two different mutant forms of Cx32 have strikingly different effects on regeneration in a xenograft model and 4) Schwann cells cultured from Cx32 knockout mice show increased death. These findings lead to the hypothesis that Cx32 is required for normal function of non-myelin-associated Schwann cells, especially those that are proliferating in response to nerve injury and participating in nerve regeneration. Aim 1 will use morphometry, whole animal electrophysiology and behavioral and biochemical assessments to examine the hypothesis that loss of Cx32 is detrimental to normal regenerative Capacity. Aim 2 will use Schwann cell culture and the dual patch clamp technique to examine the hypothesis that: 1) Cx32 is functionally expressed in proliferating adult Schwann cells in primary culture; 2) its expression levels are regulated by GGF (which is thought to induce Schwann cell proliferation during Wallerian degeneration); and 3) loss of Cx32 mediated cell-cell channels leads to increased Schwann cell death. Aim 3 will use primary Schwann cells in culture and the nerve transection model to examine the hypotheses that expression of Cx32 is required to prevent apoptotic cell death and/or limit proliferation of Schwann cells. The experiments outlined in this proposal should play a key role in understanding the role of Cx32 in the Schwann cell and how mutations in Cx32 lead to inherited peripheral neuropathy.

DESCRIPTION (provided by applicant): The connexins are integral membrane proteins that form gap junction channels. However, recent data suggest that connexins may have roles in: 1) regulation of cell growth and proliferation;2) resistance to both apoptotic and necrotic cell death;and 3) regulation of transcription, which are independent of functional channel formation. Mutations in human connexin 47 (CX47) lead to either Pelizaeus Merzbacher Like Disease (PMLD), a severe disorder, or a milder Hereditary Spastic Paraparesis (HSP) phenotype. However, neither the cellular mechanisms by which these mutations cause human disease nor the bases for their differing effects are well understood. Because both PMLD and HSP mutants are predicted to be incompetent to form a functioning junctional pathway, we propose that the greater severity of disease caused by PMLD mutations is not based on loss of junctional coupling. Rather, we hypothesize that the difference is related to a mechanism independent of functional channel formation. We propose to use Illumina Beadchip technology to evaluate and compare the patterns of gene expression produced when WT CX47 or PMLD/HSP mutants are expressed in oligodendrocytes in primary culture obtained from the Cx47 knockout mice. This will allow for the identification of cellular pathways disrupted when disease causing CX47 mutants are expressed in oligodendrocytes lacking CX47WT. We will pay particular attention to transcriptional sequelae of activation of the UPR or disorganization of the junctional complex. The experiments proposed here should provide an understanding of 1) the pathogenesis of PMLD and HSP;and 2) the basis for the differences between these two disorders. Though not the primary goals of this proposal, these studies may also provide insight into the roles of Cx47WT in oligodendrocytes and mechanisms of pathogenesis of human diseases caused by mutations in other connexins. PUBLIC HEALTH RELEVANCE: Connexins are gap junction proteins which provide for intercellular communication, but may have other roles as well. This proposal studies the effects of mutations in a connexin gene which lead to disease of the central nervous system. These studies should lead to a better general understanding of how mutations in connexin genes lead to human disease.

The involvement of nitric oxide(NO) in nerve injury and peripheral neuropathy is well documented. However, until recently there was little or no evidence of generation of NO in myelinating glia themselves. The demonstration of nitric oxide synthases 1 and 3 (NOS-1 and NOS-3) in Schwann cells, raises the potential for intrinsic NO dysfunction in these cells. Here we propose and test the hypothesis that an amplified NO response in Schwann cells is an underlying cause of pathology in CMT1X, a relatively common inherited peripheral (and sometimes central) nervous system disorder caused by mutations in connexin 32 (Cx32) a connexin expressed in myelinating glia. Key to our hypothesis is data suggesting that Cx32 is part of a complex of proteins involved in NO signaling. Gap junctions formed by connexins provide communication pathways between coupled cells. However, defective gap junctional communication alone does not account for the full extent of the role played by Cx32 in glial cells or by connexins in other cell types. Work outlined here will utilize cell culture, ex vivo, and in vivo models to investigate the physiologic and pathological consequences of loss of or mutation in Cx32. We suggest that in Cx32-defective Schwann cells, a self-reinforcing positive feedback loop of interactions involving NO increases, mitochondrial dysfunction, and impaired Ca2+ homeostasis is triggered by disruption or loss of interactions between Cx32 and components of the NO pathway. These experiments should elucidate targets for therapeutic intervention in CMT1X which will likely apply also to other disorders exhibiting disease-related alterations in connexin expression. We will compare our findings in wild-type mice to those in mice lacking Cx32 (Cx32KO) and in mice expressing the CMT1X mutant Cx32T55I on a Cx32KO background (T55ITg/32KO). Aim 1 will examine the hypothesis that disruption of Cx32 predisposes Schwann cells to nitric oxide dysfunction and ask: Does the absence of or mutation in Cx32 affect measures related to nitric oxide function in Schwann cells and peripheral nerve? We will examine the relative difference in NO levels in WT, 32KO and T55ITg/32KO Schwann cells at baseline and whether acute knockdown of Cx32 with siRNA causes changes in NO production. Peroxynitrite production, protein S-nitrosylation, tyrosine nitration and mitochondrial function will also be assessed. Aim 2 will ask: Does the NO dysregulation seen in Cx32 KO Schwann cells and nerve arise due to loss of normally occurring interactions between Cx32 and elements in the NO pathway? Cx32 appears to be part of a membrane associated protein complex including eNOS and at least one enzyme (ASS) important In NO synthesis, and ASS has been shown to directly interact with Cx32 in liver; furthermore, expression of at least one connexin has been shown to both interact with and reduce activity of eNOS. We will use LC-MS/MS to examine whether Cx32 directly or indirectly interacts with a NOS or other elements of the NO synthesis pathway. We will also perform an unbiased analysis of our data to capture other potentially relevant interactions.

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.