Gene Deficiency in CMTX May Limit Nerve Injury, Mouse Study Suggests

Sciatic motor nerves in mice lacking the GJB1 gene — defective in people with X-linked Charcot-Marie-Tooth (CMTX) disease — regenerated more after external injury than was seen in healthy mice, a study has revealed. 

These findings suggested that GJB1 deficiency may provide some protection limiting the impact of additional nerve injury, the scientists said. 

The study, “Effects of Early Crush on Aging Wild Type and Connexin 32 Knockout Mice: Evidence for a Neuroprotective State in CMT1X Mouse Nerve,” was published in the Journal of the Peripheral Nervous System. 

CMT is a genetic disease characterized by damage to the peripheral nerves, which connect the spinal cord to muscles and nerve cells that detect sensations such as heat, touch, and pain.

Mutations in the GJB1 gene, which codes for the connexin-32 (Cx32) protein and is found on the X-chromosome, were identified as a cause of CMTX, accounting for at least 10% of all CMT cases. 

To support CMTX research, a mouse model lacking the GJB1 gene (Cx32KO) was bred that showed extensive nerve damage by three months of age, affecting nerve fibers responsible for motion (motor) more than sensory fibers. These mice had fewer nerve fibers (axons) and reduced or absent myelin — the fatty coating that surrounds axons allowing electrical impulses to transmit efficiently. 

Also in Cx32KO mice, the speed of electrical impulses traveling through the sciatic nerve — the large nerve extending from the spinal cord through the legs — was different than in healthy mice. 

The sciatic nerve crush experiment is a well-defined model to investigate peripheral nerve regeneration, in which the sciatic nerve is surgically damaged. 

In this study, a team led by scientists at the University of Illinois in Chicago investigated the short- and long-term effects of sciatic nerve crush injury on the regenerated nerve in Cx32KO mice and their normal counterparts to understand molecular mechanisms further. Cx32KO mice and healthy mice without injury were also tested. 

Electrophysiology techniques measured the speed of electrical impulses along the sciatic nerve in two groups of mice: an early post-crush period between 14 and 27 days (short-term) and an aging period between 18 and 20 months (long-term). 

An examination of mice with short-term injury found no evidence of impaired regeneration in motor nerve fibers of Cx32KO mice compared to injured normal mice. In all measures, both types of mice showed sustained recovery towards complete regeneration. Unexpectedly, Cx32KO nerves demonstrated a significantly better performance than healthy nerves by showing shorter distal latency, which refers to response to stimulation just above the ankle.

As anticipated, in the long-term group, nerve impulses were slower in uninjured Cx32KO sciatic nerves than in the uninjured normal nerves, consistent with peripheral nerve damage seen in CMTX. 

While in normal mice, nerve impulses were slower after injury, the speed of electrical impulses in Cx32KO sciatic nerves was the same as those seen in uninjured Cx32KO nerves.

When examining sensory nerve fibers that connect the sciatic nerve to feet, the researchers found a loss of function in normal and Cx32KO mice that was similar to their uninjured counterparts. Also, uninjured sensory nerves of both types of animals had the same level of function, consistent with studies showing that “sensory nerves appear to be less affected by loss of Cx32,” the researchers wrote.  

Before injury, “whatever protection is afforded by the Cx32KO nerve environment appears to be restricted to those axons whose function is more severely affected,” they added. 

A large-scale analysis of gene activity in the sciatic nerve had found a high degree of difference between normal and Cx32KO sciatic nerves before injury, with those differences disappearing at five to seven days post-crush, then reappearing at 14 days. 

Many of the same genes showed increased or decreased activity in both uninjured Cx32KO and normal nerves during the week after crush. However, 14 days after sciatic nerve damage, most genes in normal nerves return to previous levels, whereas those in Cx32KO nerves maintained altered activity. 

“This pattern suggests that many of the transcripts [RNA] whose expression in [normal mice] is transiently altered during the week post-injury, are in fact permanently altered in the Cx32KO nerves,” the team wrote. 

“Together with our electrophysiologic data, these results suggest that Cx32 KO nerves are in a more sustained pro-regenerative state,” they added.

“Early nerve injury has no negative electrophysiologic effect on the Cx32 KO motor nerves,” the scientists concluded. “The lack of an additional effect of crush on Cx32KO motor nerve parameters suggests that Cx32 knockout [loss] may implement a form of neuroprotection that limits the effects of subsequent injury.”