Expression Profiling in a Model of X-Linked Charcot-Marie-Tooth Disease, Type 1

Abstract

Mutations and deletions in gene GJB1, which encodes for the Cx32 protein, lead to X-linked Charcot-Marie-Tooth disease, type 1(CMT1X), an inherited demyelinating peripheral neuropathy, resulting in axonal loss and demyelination. Regeneration in response to injury is a unique feature of the peripheral nervous system that can lead to recovery of nerve function. The current study investigates the expression profile of injured and uninjured sciatic nerves and Schwann cells to identify genes and proteins dysregulated in the absence of Cx32. The gene expression profile of sciatic nerves from Cx32KO and C57BL/6 (WT) mice were characterized and show differential expression in 501 genes in the uninjured nerves from Cx32KO vs. WT. At 5 and 7 days post injury differential expression is seen in only 24 and 10 genes respectively. Differences return at 14 days post injury with differential expression observed in 278 genes. These findings suggest that Schwann cells in intact Cx32KO nerve behave as though axon-Schwann cell communication has been disrupted, as is the case during Wallerian degeneration. Alternatively, the gene expression profile of Schwann cells from Cx32 KO nerve may reflect that Cx32KO Schwann cells are in an injury state. Ingenuity pathway analysis software showed that 196 signaling pathways and numerous biological functions were affected. Immunocytochemistry experiments on cultured Schwann cells showed protein expression of NGFR, a marker of the dedifferentiated/non-myelinating state, was higher in cells from the Cx32KO while expression of myelin protein zero (P0), a marker of the pro-myelinating/myelinating Schwann cell, was lower in the Cx32KO cells compared to WT. These findings suggest that Schwann cells lacking Cx32 assume a non-myelinating or dedifferentiated phenotype. Western blot analysis comparing protein isolates from Cx32KO and WT sciatic nerves showed no significant differences. Expression of c-Jun, p-c-Jun and c-Fos was evaluated in cultured Schwann cells and intact sciatic nerve. c-Fos staining in Cx32KO cells was lower than in WT cells. Staining for c-Jun and p-c-Jun proteins was similar in the Cx32KO and WT cells. Results of western blot analysis showed no significant differences between genotypes. Co-expression of c-Jun and p-c- Jun, c-Fos and c-Jun and c-Fos and p-c- Jun was also evaluated by immunocytochemistry. Co-expression of c-Jun and c-Fos showed less co-expression in the Cx32KO Schwann cells as did co-expression of p-c-Jun and c-Fos and co-expression of c-Jun and p-c-Jun. These findings suggest that lack of Cx32 in Schwann cells is associated with reduced levels of c-Fos and c-Jun related complexes, which may lead to significant alterations in transcriptional regulation.