Neogenin-RGMa Signaling at the Growth Cone Is Bone Morphogenetic Protein-independent and Involves RhoA, ROCK, and PKC

Abstract

The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMainduced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin ؊/؊ mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected. Furthermore, preincubation of DRG axons with the bone morphogenetic protein (BMP) antagonist noggin had no effect on RGMa-mediated growth cone collapse, implying that the role of RGM in axonal guidance is neogenin-and not BMP receptordependent. Pretreatment with 1) C3-transferase, a specific inhibitor of the Rho GTPase; 2) Y-27632, a specific inhibitor of Rho kinase; and 3) Gö6976, the general PKC inhibitor, strongly inhibited the collapse rate of PC12 neurites. Growth cone collapse induced by RGMa was abolished by the expression of dominant negative RhoA, but not by dominant negative Rac1. In contrast to RGMa, netrin-1 induced no growth cone retraction but instead reduced RGMa-mediated growth cone collapse. These results suggest that activation of RhoA, Rho kinase, and PKC are physiologically relevant and important elements of the RGMa-mediated neogenin signal transduction pathway involved in axonal guidance.During the development of the central and peripheral nervous system, target-derived axon extension is guided by attractive and repulsive diffusible or membrane-bound factors acting over short and long distances (1). During axonal chemorepulsion, repulsive guidance cues hinder neurite outgrowth, turn growth cones away from a guidance cue, and in the case of growth cone collapse induce a dramatic retraction of the growth cone. Although the repulsive guidance molecules (RGM) 2 and their receptor neogenin have been identified as guidance factors (2-6), the cytoplasmic signaling mechanisms responsible for triggering neogenin-mediated and -directed growth cone collapse remain to be clarified. Recently Hata et al.(7) demonstrated that neurite extension of cerebellar granule neurons was inhibited when these cells were plated on a monolayer of RGMa overexpressing cells and that this effect can be blocked with RhoA and Rho kinase inhibitors. Furthermore, increased amounts of GTP-RhoA were observed in these primary neurons after the addition of soluble RGMa.Neogenin, which is expressed by growing nerve cells in the developing vertebrate brain, consists of four immunoglobulinlike domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain (4, 8 -10). Neogenin is closely related to the netrin-1 receptor deleted in colorectal carcinoma (DCC); it binds RGMa and, to a lesser extent, netrin-1 directly (4, 11). Although its functional role in neuronal development has not been elucidated in detail, hi...

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The BMP Coreceptor RGMb Promotes While the Endogenous BMP Antagonist Noggin Reduces Neurite Outgrowth and Peripheral Nerve Regeneration by Modulating BMP Signaling

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