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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]

Neurotrophic factors (NTF) secreted by Schwann cells in a sciatic nerve (SN) graft promote retinal ganglion cell (RGC) axon regeneration after either transplantation into the vitreous body of the eye or anastomosis to the distal stump of a transected optic nerve. In this study, we investigated the neuroprotective and growth stimulatory properties of SN grafts in which Schwann cells had been killed (acellular SN grafts, ASN) or remained intact (cellular SN grafts, CSN). We report that both intravitreal (ivit) implanted and optic nerve anastomosed CSN promote RGC survival and when simultaneously placed in both sites, they exert additive RGC neuroprotection. CSN and ASN were rich in myelin-associated glycoprotein (MAG) and axon growth-inhibitory ligand common to both the central nervous system (CNS) and peripheral nervous system (PNS) myelin. The penetration of the few RGC axons regenerating into an ASN at an optic nerve transection (ONT) site is limited into the proximal perilesion area, but is increased >2-fold after ivit CSN implantation and increased 5-fold into a CSN optic nerve graft after ivit CSN implantation, potentiated by growth disinhibition through the regulated intramembranous proteolysis (RIP) of p75NTR (the signalling trans-membrane moiety of the nogo-66 trimeric receptor that binds MAG and associated suppression of RhoGTP). Mϋller cells/astrocytes become reactive after all treatments and maximally after simultaneous ivit and optic nerve CSN/ASN grafting. We conclude that simultaneous ivit CSN plus optic nerve CSN support promotes significant RGC survival and axon regeneration into CSN optic nerve grafts, despite being rich in axon growth inhibitory molecules. RGC axon regeneration is probably facilitated through RIP of p75NTR, which blinds axons to myelin-derived axon growth-inhibitory ligands present in optic nerve grafts. [ABSTRACT FROM AUTHOR]