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There are contradictory reports on the role of the serine/threonine kinase isoform glycogen synthase kinase-3β (GSK3β) after injury to the central nervous system (CNS). Some report that GSK3 activity promotes axonal growth or myelin disinhibition, whilst others report that GSK3 activity prevents axon regeneration. In this study, we sought to clarify if suppression of GSK3β alone and in combination with the cellular-stress-induced factor RTP801 (also known as REDD1: regulated in development and DNA damage response protein), using translationally relevant siRNAs, promotes retinal ganglion cell (RGC) survival and neurite outgrowth/axon regeneration. Adult mixed retinal cell cultures, prepared from rats at five days after optic nerve crush (ONC) to activate retinal glia, were treated with siRNA to GSK3β (siGSK3β) alone or in combination with siRTP801 and RGC survival and neurite outgrowth were quantified in the presence and absence of Rapamycin or inhibitory Nogo-A peptides. In in vivo experiments, either siGSK3β alone or in combination with siRTP801 were intravitreally injected every eight days after ONC and RGC survival and axon regeneration was assessed at 24 days. Optimal doses of siGSK3β alone promoted significant RGC survival, increasing the number of RGC with neurites without affecting neurite length, an effect that was sensitive to Rapamycin. In addition, knockdown of GSK3β overcame Nogo-A-mediated neurite growth inhibition. Knockdown of GSK3β after ONC in vivo enhanced RGC survival but not axon number or length, without potentiating glial activation. Knockdown of RTP801 increased both RGC survival and axon regeneration, whilst the combined knockdown of GSK3β and RTP801 significantly increased RGC survival, neurite outgrowth, and axon regeneration over and above that observed for siGSK3β or siRTP801 alone. These results suggest that GSK3β suppression promotes RGC survival and axon initiation whilst, when in combination with RTP801, it also enhanced disinhibited axon elongation. [ABSTRACT FROM AUTHOR]

CNS neurons are generally incapable of regenerating their axons after injury due to several intrinsic and extrinsic factors, including the presence of axon growth inhibitory molecules. One such potent inhibitor of CNS axon regeneration is Reticulon (RTN) 4 or Nogo-A. Here, we focused on RTN3 as its contribution to CNS axon regeneration is currently unknown. We found that RTN3 expression correlated with an axon regenerative phenotype in dorsal root ganglion neurons (DRGN) after injury to the dorsal columns, a well-characterised model of spinal cord injury. Overexpression of RTN3 promoted disinhibited DRGN neurite outgrowth in vitro and dorsal column axon regeneration/sprouting and electrophysiological, sensory and locomotor functional recovery after injury in vivo. Knockdown of protrudin, however, ablated RTN3-enhanced neurite outgrowth/axon regeneration in vitro and in vivo. Moreover, overexpression of RTN3 in a second model of CNS injury, the optic nerve crush injury model, enhanced retinal ganglion cell (RGC) survival, disinhibited neurite outgrowth in vitro and survival and axon regeneration in vivo, an effect that was also dependent on protrudin. These results demonstrate that RTN3 enhances neurite outgrowth/axon regeneration in a protrudin-dependent manner after both spinal cord and optic nerve injury. [ABSTRACT FROM AUTHOR]