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NEUROLOGY Brake on Axon Regrowth DiscoveredJamming Mechanism May Help Reverse Brain, Cord Injury In two separate studies, researchers at HMS have identified a novel inhibitor of axon regeneration and discovered that the protein and two other known inhibitors function by binding to the same receptor. The research may facilitate development of drugs to block the receptor, allowing axons to regenerate and forming new therapies for people who have suffered injury to their central nervous system.
Zhigang He (front center) hopes that his research will help develop drugs to treat central nervous system injuries. His team includes (clockwise from left) Jieun Kim, Vuk Koprivica, Kevin Wang, and Rajeev Sivasankaran. (Photo by Steve Gilbert) Since nerve cell axons in the mature central nervous system do not regrow, neurologists have no way of fully treating paralysis due to injury. The only available treatment is to administer corticosteroids so undamaged axons do not suffer secondary injury. The Stop Signals"About a hundred years ago, people started asking why it was impossible to get the axon to regenerate upon injury," said Zhigang He, HMS assistant professor of neurology at Children's Hospital, who led the two studies. In earlier research on the mechanisms that prevent regeneration, scientists found that the local environment played a role. Later, experiments showed that myelin contained compounds that inhibited axon regrowth. In the past 10 years, researchers have identified two compounds--Nogo-A and myelin-associated glycoprotein (MAG) as axon regeneration inhibitors. And recently, researchers have identified the Nogo-A receptor, dubbed NgR.While Nogo-A is specifically expressed in the central nervous system, MAG is expressed in both the central and peripheral systems, with expression 10 times higher in the first. He and his team were convinced that other inhibitors exist because in a study on mutant mice with a deletion in the mag gene, when Nogo-A was blocked, there was only a five percent regeneration of axons, suggesting other inhibitors present in myelin. Guided by Path FindingIn the past, researchers have identified proteins that guide axon path finding during development, classifying them as soluble, transmembrane, or glycosylphosphatidylinositol (GPI)-linked. He's team believed that mechanisms similar to those of axon guidance could come into play during inhibition of axon regeneration. They decided to test for GPI-linked myelin inhibitors since none had ever been found before; Nogo-A and MAG are transmembrane proteins.Kevin Wang, Vuk Koprivica, Jieun Kim, and Rajeev Sivasankaran of He's lab conducted simple experiments using purified myelin from bovine white matter. They treated the myelin with phosphotidylinositol-specific phospholipase C (PI-PLC), an enzyme that specifically cleaves all GPI-linked proteins. They found that the released proteins induced axonal collapse and prevented neurite outgrowth, demonstrating the properties of a regeneration inhibitor. The released protein was identified as oligodendrocyte-myelin glycoprotein (OMgp). The researchers used expression cloning to identify OMgp-binding proteins on the cell surface, finding that the Nogo-A receptor, NgR, also was the receptor for OMgp. To test if Nogo-A and OMgp competed for binding to NgR, they systematically chopped up the receptor and identified the domains to which Nogo-A and OMgp attach. They found that the inhibitors competed for overlapping regions of the receptor, with OMgp requiring a larger portion of NgR than Nogo-A. This study is published online in the June 16 Nature. Receptor DemandIn the second study, appearing in the July 18 Neuron, Sivasankaran and Wang collaborated with Marie Filbin's team at the City University of New York and identified NgR as the receptor for MAG, as well. A few years ago, Filbin had identified several cell surface proteins that MAG possibly binds to, and one was the same size as NgR. Using NgR-specific antibodies, Sivasankaran and Wang confirmed that NgR was also the receptor for MAG and that Nogo-A and MAG competed for binding to the receptor.Though it is not clear whether OMgp and MAG compete to bind NgR, He said that since the three inhibitors bind with comparably high affinity, they are expected to compete. There is still the possibility that other inhibitors exist besides these three, according to He. But when his team performed a biochemical analysis they found that Nogo-A, MAG, and OMgp accounted for most of the inhibitory activity in myelin. In fact, each of the three proteins is by itself enough to inhibit axon regeneration. The discovery that multiple inhibitors bind to the same receptor is crucial in understanding the signaling events that prevent axon regeneration. "In the next year and a half, we are going to get a handle on a lot of molecular players involved in this process so that we can begin understanding how signaling takes place," said Sivasankaran, a postdoctoral fellow. "We need to find out why so many inhibitors have the same receptor," said Wang, a graduate student. The single receptor for all three inhibitors is an interesting phenomenon from a medical point of view, said He. Drugs, like peptides, might be developed to block the receptor and therefore the pathways of all three inhibiting proteins. --Sena Desai |
