ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells (original) (raw)
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ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS
Cell death & disease, 2014
The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increas...
Molecular and Cellular Neuroscience, 2009
A b s t r a c t CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.
Journal of Neurochemistry, 2005
Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signalregulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogenactivated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment. Abbreviations used: AAV, adeno-associated virus; BDNF, brain-derived neurotrophic factor; CTb, cholera toxin b subunit; Erk1/2, extracellular signal-regulated kinases 1/2; GFP, green fluorescent protein; HA, hemagglutinin; MEK1, mitogen-activated protein kinase kinase 1; MEKca, constitutively active MEK1 mutant; MEK-wt, wild-type MEK1; PI3K, phosphatidylinositol 3-kinase; RGCs, retinal ganglion cells.
Glia, 2012
It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentrationdependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.
Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates
Molecular and Cellular Neuroscience, 2003
Several molecules inhibit axonal growth cones and may account for the failure of central nervous system regeneration, including myelin proteins and various chondroitan sulfate proteoglycans expressed at the site of injury. Axonal growth inhibition by myelin and chondroitan sulfate proteoglycans may in part be controlled by Rho-GTPase, which mediates growth cone collapse. Here, we tested in vitro whether pharmacological inhibition of a major downstream effector of Rho, Rho-kinase, promotes axonal outgrowth from dorsal root ganglia grown on aggrecan. Aggrecan substrates stimulated Rho activity and were inhibitory to axonal growth. Y-27632 treatment promoted the growth of axons by 5-to 10-fold and induced "steamlined" growth cones with longer filopodia and smaller lamellipodia. Interestingly, more actin bundles reminiscent of stress fibers in the central domain of the growth cone were observed when grown on aggrecan compared to laminin. In addition, Y-27632 significantly promoted axonal growth on both myelin and adult rat spinal cord cryosections. Our data suggest that suppression of Rho-kinase activity may enhance axonal regeneration in the central nervous system.
Axonal Regeneration: Underlying Molecular Mechanisms and Potential Therapeutic Targets
Biomedicines
Axons in the peripheral nervous system have the ability to repair themselves after damage, whereas axons in the central nervous system are unable to do so. A common and important characteristic of damage to the spinal cord, brain, and peripheral nerves is the disruption of axonal regrowth. Interestingly, intrinsic growth factors play a significant role in the axonal regeneration of injured nerves. Various factors such as proteomic profile, microtubule stability, ribosomal location, and signalling pathways mark a line between the central and peripheral axons’ capacity for self-renewal. Unfortunately, glial scar development, myelin-associated inhibitor molecules, lack of neurotrophic factors, and inflammatory reactions are among the factors that restrict axonal regeneration. Molecular pathways such as cAMP, MAPK, JAK/STAT, ATF3/CREB, BMP/SMAD, AKT/mTORC1/p70S6K, PI3K/AKT, GSK-3β/CLASP, BDNF/Trk, Ras/ERK, integrin/FAK, RhoA/ROCK/LIMK, and POSTN/integrin are activated after nerve injury...
Experimental Neurology, 2007
We investigated the effect of a novel ROCK inhibitor, Y-39983, on neurite regeneration in vitro and axonal regeneration in the crushed cat optic nerve in vivo. To determine the effective dose for neurite regeneration, retinal pieces were cultured with ROCK inhibitors, Y-39983 or Y-27632, a well-characterized ROCK inhibitor, and the number and length of TUJ-1-positive neurites were evaluated. The greatest number of neurites protruded at a dose of 3-10 μM Y-39983 and at a dose of 10-100 μM Y-27632, respectively. The neurite number at maximum effect of Y-39983 was greater than that of Y-27632. No significant difference was observed between values of neurite length with the inhibitors. Based on this finding, we examined the effect of Y-39983 on axonal regeneration in the crushed optic nerve in vivo. Immediately after crushing the left optic nerve, Y-39983 was injected into the vitreous and the crushed site. An injection of 10 μM Y-39983 induced the crushed axons to regenerate and pass over the crush site. In contrast, very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection. The second injection of 10 μM Y-39983 on day 7 doubled the number of regenerated axons, suggesting that new axons may have entered into the optic nerve after day 7 and that a continuous supply of the drug may make more axons to regenerate.