The mammalian brain and spinal cord do not regenerate after injury. It is believed that a predominance of inhibitory molecules, particularly in myelin, is responsible for this effect and that blocking these molecules would allow regeneration. Until recently a precise molecule(s) in myelin that could account for this inhibition had not been identified. Myelin associated glycoprotein (MAG), a glycoprotein specific to both CNS and PNS myelin, has been shown by the applicants to potently inhibit axonal outgrowth from all post-natal cerebellar and adult dorsal root ganglion (DRG) neurons but to promote outgrowth from newborn DRG neurons. In addition, MAG can account for a majority of the inhibitory properties of CNS myelin in vitro and has been shown to inhibit regeneration in vivo. Furthermore, a soluble form of MAG, MAG-Fc (the extracellular domain of MAG fused to the Fc portion of IgG) binds specifically to both cerebellar and DRG neurons via a sialo-glycoprotein and can inhibit axonal regeneration. Importantly, desialyation of neurons reverses inhibition, but because MAG mutated at its sialic acid recognition site, Arg118, still inhibits outgrowth when expressed by cells, implies there are two inter-dependent yet distinct epitopes on MAG; a sialic acid binding epitope and neurite inhibition epitope. The overall goals of this proposal are to characterize the interaction of MAG with its receptor and, using this information, test agents likely to block/disrupt the interaction and so prevent MAG's inhibition of spinal cord regeneration. First, using mutated forms of MAG and a panel of MAG monoclonal antibodies, the inhibition site will be precisely mapped. Second, MAG mutants and antibodies, identified in this way, will be tested for their ability to reverse the inhibition of wildtype MAG first in culture (MAG expressed by CHO cells) and then in vivo (by addition of the antibodies/mutated MAG to injured spinal cord). Also, in transgenic mice the effect of mis-expressing MAG in the PNS during normal regeneration will be assessed. Third, using MAG-Fc, MAG-binding proteins from neurons that are inhibited and that are promoted will be identified by molecular weight and compared. Antibodies will be raised to these proteins and tested for their ability to reverse the inhibition/promotion of neurite outgrowth. Accomplishment of these specific aims will advance our understanding of, not only inhibition of neurite outgrowth with a view to preventing such an inhibition after injury, but also of how the same molecule can, depending on the age and type of neuron, have opposite effects.
