Phospholipase Cδ3 Regulates RhoA/Rho Kinase Signaling and Neurite Outgrowth

Phospholipase C delta 3 (PLC delta 3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLC delta 3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLC delta 3 in neuronal migration and outgrowth. PLC delta 3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLC delta 3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLC delta 3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLC delta 3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLC delta 3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLC delta 3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLC delta 3KD cells. We also found that exogenous expression of PLC delta 3 down-regulated RhoA protein, and constitutively active PLC delta 3 promotes the RhoA down-regulation more significantly than PLC delta 3 upon differentiation. These results indicate that PLC delta 3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.