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Abstract: Axon regeneration in the adult central nervous system (CNS) is prevented by inhibitory molecules present in myelin, which bind to a receptor complex that leads to downstream RhoGTP activation and axon growth cone collapse. Here, we compared expression of Citron kinase (Citron-K), a target molecule of RhoGTP in non-regenerating dorsal root ganglion neurons (DRGN) after dorsal column (DC) injury, and in regenerating DRGN after either sciatic nerve (SN) injury or preconditioning SN+DC lesion models. We show by microarray that Citron-K mRNA levels in DRGN of a non-regenerating DC injury model were elevated 2-fold compared to those of intact control DRGN. Conversely, Citron-K levels were reduced by 2 and 2.4-fold at 10days post lesion in the regenerating SN and preconditioning SN+DC lesion models, respectively, compared to levels in control intact DRGN. Western blotting and immunohistochemistry confirmed these observations and localised Citron-K immunostaining to both DRGN and satellite glia. In dissociated, adult rat DRG cell cultures, 80% knockdown of Citron-K, in the presence of inhibitory concentrations of CNS myelin extract (CME), promoted significant disinhibited DRGN neurite outgrowth, only when cells were stimulated with neurotrophic factors. The levels of RhoGTP remained unchanged after Citron-K knockdown in the presence of CME while enhanced cofilin levels correlated with disinhibited DRGN neurite outgrowth. This observation suggests that Citron-K plays a role in axon growth downstream of Rho activation. We conclude that Citron-K regulates actin polymerisation downstream of RhoA and may offer a potentially novel therapeutic approach for promoting CNS axon regeneration. [ABSTRACT FROM AUTHOR]

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]