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.
Charcot-Marie-Tooth disease (CMT) is a common and debilitating disorder, affecting about 130,000 people in the United States and several million throughout the world. This proposal seeks to understand why patients with the second most common form of CMT develop muscle weakness. These studies should lead to a better general understanding of the role of connexins in the peripheral nerve and should elucidate treatments for patients with CMT and other genetic and acquired nerve diseases.
