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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: Intravitreal inflammation, induced by either lens injury, or intravitreal injection of zymosan (IVZ), protects RGC from apoptosis and stimulates axon regeneration after optic nerve transection. Here, we investigate the differential effects of intra-optic nerve zymosan (ONZ) and IVZ injections on RGC neuroprotection and axogenesis. After both IVZ and ONZ injection, zymosan-induced inflammation promoted a similar 4-/5-fold enhancement in RGC survival, compared to optic nerve transected controls, but only IVZ promoted RGC axon regeneration. IVZ was the most effective in activating retinal astrocyte/Müller cells while regulated intramembraneous proteolysis (RIP) of p75NTR and inactivation of Rho (key components of the axon growth inhibitory signalling cascade) occurred in both ONZ and IVZ, but only in the latter did RGC axons regenerate. We suggest that neuroprotective factors may be transported to RGC somata by retrograde transport after ONZ and diffuse into the retina after IVZ injection, but an axogenic agent is required to initiate and maintain disinhibited RGC axon regeneration that may be an exclusive property of a Müller cell-derived factor released after IVZ. [Copyright &y& Elsevier]

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]

Abstract: Administration of epidermal growth factor receptor (EGFR) inhibitors (e.g. AG1478/PD168393) promotes central nervous system (CNS) axon regeneration in vivo by an unknown mechanism. Here, we show that EGFR activation is not required for AG1478-/PD168393-induced neurite outgrowth in cultures of dorsal root ganglion neurons (DRGN) with added inhibitory CNS myelin extract (CME), but is mediated by the paracrine and autocrine actions of the glia-/neuron-derived neurotrophins (NT) NGF, BDNF and NT-3 through Trk signalling in DRGN potentiated by elevated cAMP levels. The DRGN neurite growth seen in CME-inhibited cultures treated with AG1478 is eradicated by blocking Trk signalling but undiminished after siRNA knockdown of >90% EGFR. Moreover, addition of the combined triplet of NT restores neurite outgrowth in CME-inhibited cultures, when cAMP levels are raised. Accordingly, we suggest that chemical EGFR inhibitors act independently of EGFR, inducing glia and neurons to secrete NT and raising cAMP levels in DRG cultures, leading to Trk-dependent disinhibited DRGN neurite outgrowth. [Copyright &y& Elsevier]

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]

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]

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]