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Abstract: Adeno-associated viral vectors (AAV) are increasingly used to deliver therapeutic genes to the central nervous system (CNS) where they promote transgene expression in post mitotic neurones for long periods with little or no toxicity. In adult rat dorsal root ganglia (DRG), we investigated the cellular tropism of AAV8 containing the green fluorescent protein gene (gfp) after either intra-lumbar DRG or intrathecal injection and showed that transduced DRG neurones (DRGN) expressed GFP irrespective of the delivery route, while non-neuronal cells were GFP−. After intra-DRG delivery of AAV8 gfp , the mean DRGN transduction rate was 11%, while intrathecal delivery transduced a mean of 1.5% DRGN. After intra-DRG injection, 2% of small DRGN (<30μm in diameter) were GFP+ compared with 32% of large DRGN (>60μm in diameter). Axons of transduced DRGN were also GFP+; no intra-spinal neurones were transduced. A small number of contralateral DRGN were transduced after intra-DRG injection, suggesting that AAV8 may diffuse from injected DRG into the spinal canal. Microglia and astrocytes were highly ramified with increased GFAP+ immunoreactivity (i.e. activated) in the neuropil around GFP+ DRG axon projections within the cord after intra-DRG injection. This study showed that after both intra-DRG and intrathecal delivery, strong preferential AAV8 tropism exists for large DRGN unassociated with cell death, but GFP+ axons projecting in the spinal cord induced local glial activation. These results open up opportunities for targeted delivery of therapeutics such as neurotrophic factors to the injured spinal cord. [Copyright &y& Elsevier]

Abstract: Lesioned central nervous system (CNS) axons fail to regenerate because of limited availability of neurotrophic factors (NTF) to promote neuron survival and drive axon regeneration through an environment rich in multiple myelin- and non myelin-derived axon growth inhibitory ligands that initiate growth cone collapse through the Rho/Rho kinase (ROCK) signalling pathway. However, pharmacological inhibition of Rho and ROCK promotes neurite outgrowth in PC12, Ntera-2 cells and embryonic/early postnatal neurons in culture. We have used our well-characterised CNS myelin-inhibited adult rat retinal culture model to show that Y27632 only promotes disinhibited neurite outgrowth if RGC are co-stimulated with ciliary neurotrophic factor (CNTF). Y27632 in CNTF-stimulated retinal cultures promotes optimal RGC neurite outgrowth at 10 μM concentrations, while higher concentrations negatively correlate with RGC neurite outgrowth and survival. Raising the levels of cAMP in Y27632-treated retinal cultures also promotes significant RGC neurite outgrowth, an effect that is potentiated by the further inclusion of CNTF. Our results suggest that Y27632-induced ROCK inhibition promotes robust disinhibited axon regeneration of adult neurons only when growth promoting factors are added and/or cAMP levels are raised. [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]

Abstract: Adenovirus (Ad) is an efficient and safe vector for CNS gene delivery since it infects non-replicating neurons and does not cause insertional mutagenesis of host cell genomes. However, the promiscuous Ad CAR receptor targets cells non-specifically and activates a host immune response. Using Ad5 containing an expression cassette encoding the gene for green fluorescent protein, gfp, regulated by the neuron specific promoter synapsin-1 and the woodchuck post-transcriptional regulatory element (WPRE), we demonstrate efficient, prolonged and promoter-restricted gfp expression in neurons of mixed primary adult rat dorsal root ganglion (DRG) and retinal cell cultures. We also demonstrate restricted gfp expression in DRG neurons after direct injections of Ad5 containing the synapsin-1 gfp /WPRE construct into L4 DRG in vivo, while Ad5 CMV gfp transfected both DRG glia and neurons. Moreover, since the effective titres of delivered Ad5 are reduced with this neuron specific promoter/WPRE expression cassette, the viral immune challenge should be attenuated when used in vivo. [Copyright &y& Elsevier]

Abstract: The presence of multiple axon growth inhibitors may partly explain why central nervous system axons are generally incapable of regenerating after injury. Using RNA interference (RNAi) in dorsal root ganglia neurons (DRGN), we demonstrate siRNA-mediated silencing of components of the inhibitory signalling cascade, including p75NTR, NgR and Rho-A mRNA, of 70%, 100% and 100% of the relevant protein, respectively, while changes in neither protein levels nor cellular immunoreactivity were detected using the relevant scrambled siRNA control sequences. Importantly, after 48 h in culture after siRNA-mediated knockdown of Rho-A, neurite outgrowth was enhanced by 30% compared to that after p75NTR and 50% after NgR silencing. By 3 days, a 5-, 3.5- and 6.5-fold increase in βIII-tubulin protein levels were observed compared to controls without siRNA after knockdown of p75NTR, NgR and Rho-A, respectively. Together, these results suggest that Rho-A knockdown might be the most effective target for a disinhibition strategy to promote CNS axon regeneration in vivo. [Copyright &y& Elsevier]

Abstract: After injury to the central nervous system, a glial/collagen scar forms at the lesion site, which is thought to act as a physicochemical barrier to regenerating axons. We have shown that scar formation in the transected optic nerve (ON) is attenuated when robust growth of axons is stimulated. Matrix metalloproteases (MMP), modulated by tissue inhibitors of MMP (TIMP), degrade a wide variety of extracellular matrix components (ECM) and may be activated by growing axons to remodel the ECM to allow regeneration through the inhibitory environment of the glial or collagen scar. Here, we investigate whether MMP levels are modulated in a nonregenerating (scarring) versus a regenerating (nonscarring) model of ON injury in vivo. Western blotting and immunohistochemistry revealed that MMP-1, -2, and -9 levels were higher and TIMP-1 and TIMP-2 levels were lower in regenerating compared to nonregenerating ON and retinae. In situ zymography demonstrated significantly greater MMP-related gelatinase activity in the regenerating model, mainly colocalized to astrocytes in the proximal ON stump and around the lesion site. These results suggest that activation of MMP and coincident down-regulation of TIMP may act to attenuate the inhibitory scarring in the regenerating ON, thus transforming the ON into a noninhibitory pathway for axon regrowth. [Copyright &y& Elsevier]

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]