Researchers Identify Proteins that Inhibit Post-Traumatic CNS Nerve Regeneration

A proteomic study of nerves in the central nervous system showed that proteins present in CNS axonal nodes prevent new nerve fibers from sprouting, which is necessary for nerve regeneration following CNS injury.

The study, if confirmed, could have implications for future treatments in spinal cord injury.

"If we could therapeutically remove the inhibitory nature of nodal membranes after injury, we might be able to encourage collateral axon outgrowth from nodes of Ranvier from neighboring unlesioned axons to grow out in place of the injured nerve, and possibly restore functional neurocircuitry," said Jeff Huang, the first author of the study, which was published in the online version of Science on Nov. 17.

Huang, who is now a postdoc at the Cambridge Center for Brain Repair at the University of Cambridge in the UK, noted that contrary to the CNS, the peripheral nervous system has the ability to regenerate nerves following injury. Nerve fibers in the PNS sprout from axon terminals, as well as from nodes of Ranvier, which are regions along the axon without a myelin sheath.

"It is well known that axon regeneration is highly limited in the CNS, due to inhibitory factors from myelin membranes," said Huang. "However, we asked, 'Why is it that nodes of Ranvier from the CNS do not normally sprout upon injury, even though it is not a myelinated region?' We hypothesized that there might be non-myelin derived inhibitory factors present or deposited in the nodal region."

Huang and his colleagues decided to use a proteomic approach to catalog proteins in the CNS nodes of Ranvier.

"The nodes of Ranvier are a region that has been more or less mysterious," said Huang. "Proteomics is a good, rapid, very effective way to get a profile of proteins."

Huang and his PhD advisor, David Colman, who is the director of the Montreal Neurological Institute, collaborated with John Yates III of the Scripps Research Institute to perform multi-dimensional protein identification technology, or MudPIT, analysis on nodal membranes which were isolated via subcellular fractionation. MudPIT was chosen over a 2D gel approach because Colman and Huang felt that gels are not as good for detecting low-abundance proteins such as adhesion and signaling molecules.

"With MudPIT, you just run the fraction through two columns, and when the fraction drips out, it gets identified," said Huang. "The technique is fairly rapid, highly sensitive, and the nice thing about this approach is it doesn't have any bias for isoelectric point or protein size."

The researchers identified more than 300 proteins from a region of CNS axons that contains nodes of Ranvier. They cataloged the proteins into various categories, including signaling proteins, cytoplasmic proteins, adhesion proteins and transmembrane proteins. They knew that several of the identified proteins inhibit axon outgrowth, including oligodendrocyte myelin glycoprotein, or OMgp.

Huang and his colleagues decided to further characterize OMgp by using an antibody to stain its location within the CNS axons. They found that the protein appeared to surround the nodes of Ranvier.

"OMgp happened to have a very striking and novel localization," said Huang. "It's strange because the node of Ranvier under electron microscopy appears to be bare, but in fact it's surrounded by OMgp."

To further investigate the function of OMgp, Huang obtained OMgp knockout mice from the Cambridge, Mass.-based branch of Biogen. They used molecular markers to examine the murine CNS nodes of Ranvier and found that the nodes were abnormally wide, and there was an increase in axon outgrowth.

The increase in axon outgrowth was significant, but not dramatic, Huang said. He speculated that other inhibitory factors, aside from OMgp, probably prevented the axon from sprouting more nerve fibers.

Huang said that he would like to look next at whether the mouse axons sprout more nerve fibers under CNS injury conditions.

"Spinal cord injury is definitely something that we will look at," said Huang. "We want to see under injury conditions if there might be an increase in nerve regeneration."

In addition, Huang would like to investigate what role OMgp plays in the formation of nodes of Ranvier, which are flanked on one side by sodium channels and on another side by potassium channels. The channels are key for the conduction of nerve signals.

"How these sodium and potassium domains are formed on the axons is really unclear," said Huang. "OMgp may play some sort of role in the establishment of nodes of Ranvier, or in its maintenance. The OMgp mouse model might be useful for looking at known proteins at the nodes of Ranvier, or at the sodium and potassium channels."

Huang noted that the OMgp knockout mice did not appear to have any behavioral abnormalities, but it is unclear if their nerve conduction is compromised by their lack of OMgp.

Aside from investigating OMgp, Huang would also like to further characterize some of the other axon outgrowth inhibitors that were found to be present in the nodes of Ranvier, including myelin-associated glycoprotein, or MAG, and NG2.

"For me, personally, it would be nice to look at some of these other molecules in the future," said Huang. "I'm hoping also that other people might want to work on these molecules."

— Tien Shun Lee ([email protected])