Gene expression profiling studies in regenerating nerves in a mouse model for CMT1X: uninjured Cx32-knockout peripheral nerves display expression profile of injured wild type nerves

Abstract

X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the human gene for Connexin32 (Cx32). This present study uses Ilumina Ref8-v2 BeadArray to examine the expression profiles of injured and uninjured sciatic nerves at 5, 7, and 14 days post-crush injury (dpi) from Wild Type (WT) and Cx32-knockout (Cx32KO) mice to identify the genes and signaling pathways that are dysregulated in the absence of Schwann cell Cx32. Given the assumption that loss of Schwann cell Cx32 disrupts the regeneration and maintenance of myelinated nerve leading to a demyelinating neuropathy in CMT1X, we initially hypothesized that nerve crush injury would result in significant increases in differential gene expression in Cx32KO mice relative to WT nerves. However, microarray analysis revealed a striking collapse in the number of differentially expressed genes at 5 and 7 dpi in Cx32KO nerves relative to WT, while uninjured and 14 dpi time points showed large numbers of differentially regulated genes. Further comparisons within each genotype showed limited changes in Cx32KO gene expression following crush injury when compared to uninjured Cx32KO nerves. By contrast, WT nerves exhibited robust changes in gene expression at 5 and 7 dpi with no significant differences in gene expression by 14dpi relative to uninjured WT nerve samples. Taken together, these data suggest that the gene expression profile in uninjured Cx32KO sciatic nerve strongly resembles that of a WT nerve following injury and that loss of Schwann cell Cx32 leads to a basal state of gene expression similar to that of an injured WT nerve. These findings support a role for Cx32 in non-myelinating and regenerating populations of Schwann cells in normal axonal maintenance in re-myelination, and regeneration of peripheral nerve following injury. Disruption of Schwann cell-axonal communication in CMT1X may cause dysregulation of signaling pathways that are essential for the maintenance of intact myelinated peripheral nerves and to establish the necessary conditions for successful regeneration and remyelination following nerve injury.

Keywords: CMT1X; Differential gene expression; Non-myelinating; Peripheral nerve injury; Regeneration; Schwann cells.

Figure 1

Gene Expression Profile of Cx32KO Nerve Samples Crush Injury Relative to WT. The number of differentially regulated genes in Cx32KO nerves relative to WT is dramatically reduced at 5 and 7dpi. Ratio of all corrected Log2 transformed raw gene expression values for Cx32KO versus WT nerves at 5, 7, and 14 days post injury (dpi).

Figure 2

Venn Diagram Comparing Differentially Expressed Genes in Cx32KO Nerves Relative to WT Following Crush Injury. Venn diagram illustrating the overlap of differentially expressed genes in Cx32KO nerve samples relative to WT at each time point (Figure 2A.). Venn diagrams of up-regulated (Figure 2B.) and down-regulated (Figure 2C.) genes in Cx32KO nerves relative to WT.

Figure 3

Comparison of Shared Genes in Uninjured Cx32KO Nerves and Injured WT. DEG for uninjured Cx32KO (relative to uninjured WT) were compared to the gene lists in WT nerves (relative to uninjured WT) at 5 and 7dpi demonstrate a high degree of overlap between injured WT nerves and uninjured Cx32KO nerves (Figure 3A.); with greater than 75% of DEG shared between uninjured Cx32KO and injured WT at 7dpi versus uninjured WT. Comparison of DEG from WT at 5 and 7dpi relative to uninjured WT (Figure 3B.) and Cx32KO at 5 and 7dpi relative to uninjured Cx32KO (Figure 3C.) shows a high degree of overlap in WT nerves.

Figure 4

Schwann/Myelin GOSlim Functional Analysis of Cx32KO vs WT for Uninjured and Injured Groups at 5, 7, and 14 Days Post-Injury. Gene lists for each experimental time group were analyzed for enrichment of Schwann/Myelin GOSlim functional categories using the GOMiner online tool. Enrichment scores for each GOSlim term were calculated using Fisher’s exact test, with corrections. By convention, a functional category is considered over-expressed or enriched with enrichment scores > 1. Enrichment scores >3 correspond to significant over-expression (p< 0.05).

Figure 5

Immune/Stress Response GOSlim Functional Analysis of Cx32KO vs WT for Uninjured and Injured Groups at 5, 7, and 14 Days Post-Injury. Gene lists for each experimental group were analyzed for enrichment of Immune/Stress Response GOSlim functional categories using the GOMiner online too. Enrichment for Immune/Stress Response terms were observed in uninjured and 14dpi injured Cx32KO nerves relative to WT.

Figure 6

Differential Gene Expression of Genes Associated with Schwann/Myelin GOSlim Terms in Uninjured and Injured Cx32KO Nerves vs WT. After compensating for overlaps across Schwann/Myelin GO gene annotations, a list of 157 Schwann/Myelin GOSlim genes was generated. This list was compared to DEG from uninjured and injured Cx32KO nerves relative to WT. 25 genes were significantly regulated in Cx32KO nerves relative to WT for at least one time point. Figure 6A: Down-regulated genes. Figure 6B: Up-regulated genes.

Figure 7

DEG Associated with Cytokine/Chemokine Signaling Pathways in Uninjured and 14dpi Cx32KO Nerves vs WT. A consensus gene list for the chemokine and cytokine signaling pathway was generated and cross-referenced to the Immune/Stress Response GOSlim gene creating a consensus cytokine/chemokine gene list for analysis. This gene list was used to probe DEG from uninjured and injured Cx32KO nerves relative to WT. Figure 7A: Down-regulated genes. Figure 7B: Up-regulated genes.

Figure 8

c-Jun GOSlim DEG in Uninjured and Injured 14dpi Cx32KO Nerves vs WT. A consensus c-Jun GOSlim gene list was created and used to probe DEG from uninjured and injured Cx32KO relative to WT. 36 significantly regulated genes were identified in uninjured and 14dpi Cx32KO samples relative to WT. Figure 8A: Down-regulated genes. Figure 8B: Up-regulated genes.