[P2.47]: Dissection of specific genetic signals from a background of tissue repair and inflammatory response noise during optic nerve regeneration in Danio rerio

Abstract

Neurite outgrowth upon extracellular cues is an essential event in early neuronal differentiation. Rac1 and Cdc42, members of Rho family GTPases, are involved in this process as regulators of actin cytoskeletal reorganization. We previously showed that NGF/TrkA signaling drives the cycling of a positive feedback loop comprised of PI3-kinase, Vav2/Vav3, Rac1/Cdc42, and actin cytoskeleton at neurite tips of PC12 cells using FRET imaging and RNAi techniques. In this study, we demonstrate that SHIP2 is a critical component of the negative feedback on PIP 3 in NGF-induced neurite outgrowth. This negative feedback loop has been unexpected previously. Acute inhibition of TrkA transiently decreased Rac1/Cdc42 activity and the PIP 3 level to below basal levels, indicating the presence of NGF-dependent negative regulation. With the help of in silico simulation, we speculated that the activation of PI-5-phosphatase for PIP 3 was involved. In agreement with this model, depletion of SHIP2 by RNA interference attenuated the inhibitor-induced super-suppression of Rac1/Cdc42 activity and the level of PIP 3 . The critical role of SHIP2 in neurite outgrowth of PC12 cells was further supported by the finding that depletion of SHIP2 and PTEN, phosphatases for PIP 3 , markedly potentiated NGF-induced Rac1/ Cdc42 activation and PIP 3 accumulation, and significantly increased the number and the length of neurites. This increase in neurite number is possibly due to that the depletion of SHIP2 and PTEN disturbs the proper localization of PIP 3 and Rac1/ Cdc42 activities. Further refinement of the computational model predicted a negative feedback from Rac1 to SHIP2, which was validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP 3 coordinately works with the PI3kinase-mediated positive feedback to form an initial protrusive pattern determined by Turing's reaction-diffusion system, and later, to punctuate the PIP 3 accumulation to maintain proper neurite outgrowth.