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Recent research has suggested that the growth of central nervous system (CNS) axons during development is mediated through the PI3K/Akt/mammalian target of rapamycin (mTOR) intracellular signalling axis and that suppression of activity in this pathway occurs during maturity as levels of the phosphatase and tensin homologue (PTEN) rise and inhibit PI3K activation of mTOR, accounting for the failure of axon regeneration in the injured adult CNS. This hypothesis is supported by findings confirming that suppression of PTEN in experimental adult animals promotes impressive axon regeneration in the injured visual and corticospinal motor systems. This review focuses on these recent developments, discussing the therapeutic potential of a mTOR-based treatment aimed at promoting functional recovery in CNS trauma patients, recognising that to fulfil this ambition, the new therapy should aim to promote not only axon regeneration but also remyelination of regenerated axons, neuronal survival and re-innervation of denervated targets through accurate axonal guidance and synaptogenesis, all with minimal adverse effects. The translational challenges presented by the implementation of this new axogenic therapy are also discussed. [ABSTRACT FROM AUTHOR]

Optic nerve injuries cause death of retinal ganglion cells and degeneration of their axons. Vigneswara et al. show that combined delivery of caspase-2 and caspase-6 inhibitors into the eye promotes retinal ganglion cell survival and axon regeneration in a rodent model, an effect that is mediated through the JAK/STAT pathway.We have previously shown that crushing the optic nerve induces death of retinal ganglion cells by apoptosis, but suppression of CASP2, which is predominantly activated in retinal ganglion cells, using a stably modified short interfering RNA CASP2, inhibits retinal ganglion cell apoptosis. Here, we report that combined delivery of short interfering CASP2 and inhibition of CASP6 using a dominant negative CASP6 mutant activates astrocytes and Müller cells, increases CNTF levels in the retina and leads to enhanced retinal ganglion cell axon regeneration. In dissociated adult rat mixed retinal cultures, dominant negative CASP6 mutant + short interfering CASP2 treatment also significantly increases GFAP+ glial activation, increases the expression of CNTF in culture, and subsequently increases the number of retinal ganglion cells with neurites and the mean retinal ganglion cell neurite length. These effects are abrogated by the addition of MAB228 (a monoclonal antibody targeted to the gp130 component of the CNTF receptor) and AG490 (an inhibitor of the JAK/STAT pathway downstream of CNTF signalling). Similarly, in the optic nerve crush injury model, MAB228 and AG490 neutralizes dominant negative CASP6 mutant + short interfering CASP2-mediated retinal ganglion cell axon regeneration, Müller cell activation and CNTF production in the retina without affecting retinal ganglion cell survival. We therefore conclude that axon regeneration promoted by suppression of CASP2 and CASP6 is CNTF-dependent and mediated through the JAK/STAT signalling pathway. This study offers insights for the development of effective therapeutics for promoting retinal ganglion cell survival and axon regeneration. [ABSTRACT FROM PUBLISHER]

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]

Inhibition of central nervous system axon growth is reportedly mediated in part by calcium-dependent phosphorylation of axonal epidermal growth factor receptor, with local administration of the epidermal growth factor receptor kinase inhibitors AG1478 and PD168393 to an optic nerve lesion site promoting adult retinal ganglion cell axon regeneration. Here, we show that epidermal growth factor receptor was neither constitutively expressed, nor activated in optic nerve axons in our non-regenerating and regenerating optic nerve injury models, a finding that is inconsistent with phosphorylated epidermal growth factor receptor-dependent intra-axonal signalling of central nervous system myelin-related axon growth inhibitory ligands. However, epidermal growth factor receptor was localized and activated within most glia in the retina and optic nerve post-injury, and thus an indirect glial-dependent mechanism for stimulated retinal ganglion cell axon growth by epidermal growth factor receptor inhibitors seemed plausible. Using primary retinal cultures with added central nervous system myelin extracts, we confirmed previous reports that AG1478/PD168393 blocks epidermal growth factor receptor activation and promotes disinhibited neurite outgrowth. Paradoxically, neurites did not grow in central nervous system myelin extract-containing cultures after short interfering ribonucleic acid-mediated knockdown of epidermal growth factor receptor. However, addition of AG1478 restored neurite outgrowth to short interfering ribonucleic acid-treated cultures, implying that epidermal growth factor receptor does not mediate AG1478-dependent effects. TrkA-/B-/C-Fc fusion proteins and the kinase blocker K252a abrogated the neuritogenic activity in these cultures, correlating with the presence of the neurotrophins brain derived neurotrophic factor, nerve growth factor and neurotrophin-3 in the supernatant and increased intracellular cyclic adenosine monophosphate activity. Neurotrophins released by AG1478 stimulated disinhibited retinal ganglion cell axon growth in central nervous system myelin-treated cultures by the induction of regulated intramembraneous proteolysis of p75NTR and Rho inactivation. Retinal astrocytes/Müller cells and retinal ganglion cells were the source of neurotrophins, with neurite outgrowth halved in the presence of glial inhibitors. We attribute AG1478-stimulated neuritogenesis to the induced release of neurotrophins together with raised cyclic adenosine monophosphate levels in treated cultures, leading to axon growth and disinhibition by neurotrophin-induced regulated intramembraneous proteolysis of p75NTR. These off-target effects of epidermal growth factor receptor kinase inhibition suggest a novel therapeutic approach for designing treatments to promote central nervous system axon regeneration. [ABSTRACT FROM PUBLISHER]

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]

This review will describe the unique advantages that are offered by the visual system of mammals and other vertebrates for studying the regenerative responses of the central nervous system (CNS) to injury, and recent insights provided by such studies. In the mouse and rat visual system a variety of experimental paradigms promote survival of retinal ganglion cells (RGC) and optic nerve regeneration, probably through stimulation by neurotrophic factors (NTF) either directly, or indirectly through retinal astrocyte/Müller cell intermediary activation. NTF induce disinhibition of axon growth through regulated intramembranous proteolysis of p75^{NTR}, and the inactivation of RhoA and EGFR signalling. The concomitant release of metalloproteinases (MMP) and plasminogen activators from RGC axons, and tissue inhibitors of metalloproteinases from optic nerve glia repress scarring and thereby reduce titres of scar-derived inhibitory ligands expressed in the wound. MMP also degrade myelin-derived inhibitory ligands along regenerating axon trajectories after regulated release from glia at the growing front of regenerating RGC axons. Optic nerve transection induces apoptosis of RGC which is blocked by anti-apoptotic regimes and thus, in combination with blockers of axon-growth inhibitory signalling and promoters of axon growth may be a therapeutic formula for promoting sustained axon regeneration. All these findings in the visual system are translatable to the CNS as a whole and thus strategies that successfully promote visual axon regeneration will be equally effective elsewhere in the CNS. Future developments likely to advance the field of regenerative research include a greater understanding of phylogenetic differences in the response of the CNS to injury, the role of NTF, cAMP, EGFR, glia/neuron interactions in disinhibiting and promoting axon growth, the control of neuron death, and effective drug delivery. [ABSTRACT FROM AUTHOR]

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]

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]

Axotomised retinal ganglion cells (RGCs) die rapidly by apoptosis and fail to regenerate because of the limited availability of neurotrophic factors and a lack of axogenic stimuli. However, we have recently showed that pigment epithelium-derived factor (PEDF) promotes RGC survival and axon regeneration after optic nerve crush injury. PEDF has multiple fragments of the native peptide that are neuroprotective, anti-angiogenic and anti-inflammatory. Here we investigated the neuroprotective and axogenic properties of a fragment of PEDF, PEDF-34, in retinal neurons in vitro and when delivered by intravitreal injection and eye drops in vivo . We found that PEDF-34 was 43% more neuroprotective and 52% more neuritogenic than PEDF-44 in vitro . Moreover, in vivo , intravitreal delivery of 1.88 nM PEDF-34 was 71% RGC neuroprotective at 21 days after optic nerve crush compared to intact controls, whilst daily eye drops containing 1.88 nM PEDF-34 promoted 87% RGC survival. After topical eye drop delivery, PEDF-34 was detected in the vitreous body within 30 min and attained physiologically relevant concentrations in the retina by 4 h peaking at 1.4 ± 0.05 nM by 14 days. In eye drop- compared to intravitreal-treated PEDF-34 animals, 55% more RGC axons regenerated 250 μm beyond the optic nerve lesion. We conclude that daily topical eye drop application of PEDF-34 is superior to weekly intravitreal injections in promoting RGC survival and axon regeneration through both direct effects on retinal neurons and indirect effects on other retinal cells. [ABSTRACT FROM AUTHOR]