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Abstract: The reasons for the failure of central nervous system (CNS) axons to regenerate include the presence of myelin- and non-myelin derived inhibitory molecules, neuronal apoptosis and the absence of a potent neurotrophic stimulus. Transactivation of the epidermal growth factor receptor (EGFR) has been implicated in signalling inhibition of axon growth in the CNS. Small molecule EGFR inhibitors such as AG1478 and PD168393 promote CNS axon growth after optic nerve transection despite the presence of inhibitory molecules in the environment of the regenerating axon. However, our results demonstrate that phosphorylated EGFR (pEGFR) is not present on regenerating axons and that the majority of pEGFR is present in glia, suggesting that EGFR cannot play a direct intra-axonal role in signalling inhibition and thus disinhibited CNS axon growth must be indirectly mediated by glia. We argue that EGFR may not have a role in signalling axon growth inhibition since AG1478 and PD168393 promotes neuronal neurite outgrowth in CNS myelin-inhibited cultures after EGFR knockdown. This review discusses the current evidences for and against the involvement of EGFR in signalling myelin inhibition. [Copyright &y& Elsevier]

Abstract: Lesioned central nervous system (CNS) axons fail to regenerate because of limited availability of neurotrophic factors (NTF) to promote neuron survival and drive axon regeneration through an environment rich in multiple myelin- and non myelin-derived axon growth inhibitory ligands that initiate growth cone collapse through the Rho/Rho kinase (ROCK) signalling pathway. However, pharmacological inhibition of Rho and ROCK promotes neurite outgrowth in PC12, Ntera-2 cells and embryonic/early postnatal neurons in culture. We have used our well-characterised CNS myelin-inhibited adult rat retinal culture model to show that Y27632 only promotes disinhibited neurite outgrowth if RGC are co-stimulated with ciliary neurotrophic factor (CNTF). Y27632 in CNTF-stimulated retinal cultures promotes optimal RGC neurite outgrowth at 10 μM concentrations, while higher concentrations negatively correlate with RGC neurite outgrowth and survival. Raising the levels of cAMP in Y27632-treated retinal cultures also promotes significant RGC neurite outgrowth, an effect that is potentiated by the further inclusion of CNTF. Our results suggest that Y27632-induced ROCK inhibition promotes robust disinhibited axon regeneration of adult neurons only when growth promoting factors are added and/or cAMP levels are raised. [Copyright &y& Elsevier]

Abstract: In investigating the consequences of gene silencing in axon growth disinhibition strategies in cultured retinal ganglion cells (RGC), we conducted experiments designed to silence RhoA signalling in PC12 and primary adult rat retinal cell cultures (containing RGC) by siRNA-mediated RhoA mRNA knockdown. We demonstrate wide differences in the levels of RhoA mRNA knockdown, dose-dependent cell toxicity, and induction of endogenous inflammatory cytokine and interferon responses to siRNA therapy. Toxicity effects observed with RhoA-siRNA was significantly reduced with “Stealth” chemical modification of the sequence, promoting ∼50% and 70% knockdown of RhoA mRNA and protein in retinal cells, respectively, while promoting significant disinhibited RGC neurite outgrowth in the presence of inhibitory CNS myelin. Our results highlight differential responsiveness of cell lines compared to primary cultured cells, and demonstrate the efficacy of the “Stealth” modification to reduce siRNA-induced interferon responses, thereby increasing target cell viability and reducing off-target effects of the delivered nucleic acids. [Copyright &y& Elsevier]