ϒ-secretase and LARG mediate distinct RGMa activities to control appropriate layer targeting within the optic tectum.

While a great deal of progress has been made in understanding the molecular mechanisms that regulate retino-tectal mapping, the determinants that target retinal projections to specific layers of the optic tectum remain elusive. Here we show that two independent RGMa-peptides, C- and N-RGMa, activate two distinct intracellular pathways to regulate axonal growth. C-RGMa utilizes a Leukemia-associated RhoGEF (LARG)/Rho/Rock pathway to inhibit axonal growth. N-RGMa on the other hand relies on ϒ-secretase cleavage of the intracellular portion of Neogenin to generate an intracellular domain (NeICD) that uses LIM-only protein 4 (LMO4) to block growth. In the developing tectum (E18), overexpression of C-RGMa and dominant-negative LARG (LARG-PDZ) induced overshoots in the superficial tectal layer but not in deeper tectal layers. In younger embryos (E12), C-RGMa and LARG-PDZ prevented ectopic projections toward deeper tectal layers, indicating that C-RGMa may act as a barrier to descending axons. In contrast both N-RGMa and NeICD overexpression resulted in aberrant axonal-paths, all of which suggests that it is a repulsive guidance molecule. Thus, two RGMa fragments activate distinct pathways resulting in different axonal responses. These data reveal how retinal projections are targeted to the appropriate layer in their target tissue.

Uncoupling Neogenin association with lipid rafts promotes neuronal survival and functional recovery after stroke.

The dependence receptor Neogenin and its ligand, the repulsive guidance molecule a (RGMa), regulate apoptosis and axonal growth in the developing and the adult central nervous system (CNS). Here, we show that this pathway has also a critical role in neuronal death following stroke, and that providing RGMa to neurons blocks Neogenin-induced death. Interestingly, the Neogenin pro-death function following ischemic insult depends on Neogenin association with lipid rafts. Thus, a peptide that prevents Neogenin association with lipid rafts increased neuronal survival in several in vitro stroke models. In rats, a pro-survival effect was also observed in a model of ocular ischemia, as well as after middle cerebral artery occlusion (MCAO). Treatments that prevented Neogenin association with lipid rafts improved neuronal survival and the complexity of the neuronal network following occlusion of the middle artery. Toward the development of a treatment for stroke, we developed a human anti-RGMa antibody that also prevents Neogenin association with lipid rafts. We show that this antibody also protected CNS tissue from ischemic damage and that its application resulted in a significant functional improvement even when administrated 6 h after artery occlusion. Thus, our results draw attention to the role of Neogenin and lipid rafts as potential targets following stroke.

The repulsive guidance molecule, RGMa, promotes retinal ganglion cell survival in vitro and in vivo.

The repulsive guidance molecule, RGMa, and its receptor Neogenin, regulate neuronal cell death during development, but little is known about their expression and roles in the adult CNS. Here, we show that Neogenin is expressed in the adult rodent retina, particularly on retinal ganglion cells. To determine whether the Neogenin/RGMa pathway is important in the fully developed retina, we examined its contribution to damage-induced neurodegeneration. The effects of RGMa on survival of retinal ganglion cells (RGCs) were examined in vitro and in vivo. Using cultured whole-mount retinal explants, we showed that the addition of RGMa increased RGC survival and that this effect was mediated by the Neogenin receptor. Immunohistochemical analysis indicated that the inhibition of cell death by RGMa resulted from reduced caspase-3 activation. Then, using an in vivo model of RGC apoptosis after optic nerve transection, we demonstrated that intraocular injection of RGMa at 3 and 7 days after axotomy greatly reduced RGC death 14 days postaxotomy. This study provides the first evidence that RGMa is a molecular target for neuroprotection in retinal pathologies, and suggests that targeting "dependence receptors" such as Neogenin has therapeutic potential for the treatment of neuropathologies in the adult CNS.