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 RGMa-induced 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 receptor-dependent. 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.
Highlights
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]
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
RGMa Induces Growth Cone Collapse in Neogenin-expressing Primary Dorsal Root Ganglion Cells—Nonradioactive in situ hybridization showed that neogenin is expressed by dorsal root ganglia at E14.5, but RGMa is not (Fig. 1A)
Summary
Neogenin Mutants—Mice homozygous for neogenin were kindly provided by Dr M. RGMa-Fc was stably transfected into HEK293 cells, and the supernatants with the fusion protein were further processed for collapse experiments. The proteins were clustered with an anti-human IgG1-Fc antibody (Calbiochem) to induce a stronger collapsing activity of the RGMa fusion protein. RGMa-pI ϩ IgG or pI ϩ IgG was added 5 min after the beginning of the collapse assay, and every experiment was observed and recorded for 30 min with a Live Cell Imaging System (Zeiss) at a magnification of ϫ20. For C3-transferase experiments, 500 l of medium containing 15 l of C2–C3 (300 ng/ml) was added to the PC12 cell cultures 5 h before collapse induction by RGMa-pI ϩ IgG. Rho kinase was inhibited by adding 100 nM Y-27632 Rho kinase inhibitor (Calbiochem) to the cultures 30 min before the RGMa-pI ϩ IgG collapse experiments. Images were collected with the Zeiss Live Cell Imaging System and AxioVision analysis software
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