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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.

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 p75 NTR (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 p75 NTR , which blinds axons to myelin-derived axon growth-inhibitory ligands present in optic nerve grafts., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Purpose: We investigate if the BMP4/Smad1 intracellular signaling pathway is neuroprotective and axogenic in adult rodent retinal ganglion cells (RGC) in vivo and in vitro., Methods: Adult retinal cultures were prepared from intact and after optic nerve crush (ONC) injured rats that have been stimulated to survive and regenerate using an intravitreal peripheral nerve (PN) graft. Laser capture microdissection (LCM) then was used to isolate RGC with and without neurites. Quantitative RT-PCR determined changes in BMP4/Smad1 signaling pathway mRNA. Immunohistochemistry confirmed localization of BMP4 and activation of Smad1 in ONC+PN-stimulated RGC in vivo. BMP4 peptide was used to stimulate RGC survival and neurite/axon regeneration in vitro and in vivo. Finally, the rapamycin sensitivity of the effects was determined in BMP4-stimulated RGC in vitro and in vivo., Results: In retinal cultures prepared from intact and ONC+PN-stimulated rats, RGC with neurites had upregulated regeneration-related and BMP4/Smad1 signaling pathway mRNA levels, while low levels of these mRNAs were present in RGC isolated without neurites. An optimal dose of 200 ng/mL BMP4 peptide in vitro promoted approximately 30% RGC survival and disinhibited RGC neurite outgrowth, despite the presence of inhibitory CNS myelin extracts. BMP4 also promoted approximately 30% RGC survival in vivo and stimulated significant RGC axon regeneration at 100, 200, and 400 μm beyond the lesion site. Finally, the response of RGC to BMP4 treatment in vitro and in vivo was rapamycin-insensitive., Conclusions: Activation of the BMP4/Smad1 pathway promotes survival and axon regeneration independent of mTOR and, therefore, may be of therapeutic interest.

Purpose: To investigate, using in vivo and in vitro models, retinal ganglion cell (RGC) neuroprotective and axon regenerative effects and underlying mechanisms of siRTP801, a translatable small-interfering RNA (siRNA) targeting the mTOR negative regulator RTP801., Methods: Adult rats underwent optic nerve (ON) crush (ONC) followed by intravitreal siRTP801 or control siRNA (siEGFP) every 8 days, with Brn3a+ RGC survival, GFAP+ reactive gliosis, and GAP43+ regenerating axons analyzed immunohistochemically 24 days after injury. Retinal cultures, prepared from uninjured animals or 5 days after ONC to activate retinal glia, were treated with siRTP801/controls in the presence/absence of rapamycin and subsequently assessed for RGC survival and neurite outgrowth, RTP801 expression, glial responses, and mTOR activity. Conditioned medium was analyzed for neurotrophin titers by ELISA., Results: Intravitreal siRTP801 enabled 82% RGC survival compared to 45% with siEGFP 24 days after ONC, correlated with greater GAP43+ axon regeneration at 400 to 1200 μm beyond the ONC site, and potentiated the reactive GFAP+ Müller glial response. In culture, siRTP801 had a direct RGC neuroprotective effect, but required GFAP+ activated glia to stimulate neurite elongation. The siRTP801-induced neuroprotection was significantly reduced, but not abolished, by rapamycin. The siRTP801 potentiated the production and release of neurotrophins NGF, NT-3, and BDNF, and prevented downregulation of RGC mTOR activity., Conclusions: The RTP801 knockdown promoted RGC survival and axon elongation after ONC, without increasing de novo regenerative sprouting. The neuroprotection was predominantly direct, with mTORC1-dependent and -independent components. Enhanced neurite/axon elongation by siRTP801 required the presence of activated retinal glia and was mediated by potentiated secretion of neurotrophic factors.

The article discusses a study which showed that targeting of the central ataxia telangiectasia-mutated-checkpoint kinase-2 (ATM-Chk2) pathway regulating the response to double-strand breaks slows neural decline in Drosophila models of chronic neurodegeneration. Results show that Chk2 inhibitor, prexasertib, represents a treatment option to promote functional recovery after spinal cord or optic nerve injury. Also cited are the neuroprotective effects of prexasertib and the experimental design.