Combined inhibition of Cdk5 and ROCK additively increase cell survival, but not the regenerative response in regenerating retinal ganglion cells (original) (raw)
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Brain, 2007
Functional regeneration in the CNS is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. A principal, yet unresolved question is the interaction between these two major factors. We thus evaluated the role of pharmacological inhibition of rho kinase (ROCK), a key mediator of myelinderived axonal growth inhibition and CNTF, a potent neurotrophic factor for retinal ganglion cells (RGC), in models of retinal ganglion cell apoptosis and neurite outgrowth/regeneration in vitro and in vivo. Here, we show for the first time that the ROCK inhibitor Y-27632 significantly enhanced survival of RGC in vitro and in vivo. In vitro, the co-application of CNTF and Y-27632 potentiated the effect of either substance alone. ROCK inhibition resulted in the activation of the intrinsic MAPK pathway, and the combination of CNTF and Y-27632 resulted in even more pronounced MAPK activation. While CNTF also induced STAT3 phosphorylation, the additional application of ROCK inhibitor surprisingly diminished the effects of CNTF on STAT3 phosphorylation. ROCK activity was also decreased in an additive manner by both substances. In vivo, both CNTF and Y-27632 enhanced regeneration of RGC into the non-permissive optic nerve crush model and additive effects were observed after combination treatment. Further evaluation using specific inhibitors delineate STAT3 as a negative regulator of neurite growth and positive regulator of cell survival, while MAPK and Akt support neurite growth. These results show that next to neurotrophic factors ROCK inhibition by Y-27632 potently supports survival of lesioned adult CNS neurons. Co-administration of CNTF and Y-27632 results in additive effects on neurite outgrowth and regeneration. The interaction of intracellular signalling pathways may, however, attenuate more pronounced synergy and has to be taken into account for future treatment strategies.
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.
Targeting Cdk5 Activity in Neuronal Degeneration and Regeneration
Cellular and Molecular Neurobiology, 2009
The major priming event in neurodegeneration is loss of neurons. Loss of neurons by apoptotic mechanisms is a theme for studies focused on determining therapeutic strategies. Neurons following an insult, activate a number of signal transduction pathways, of which, kinases are the leading members. Cyclin-dependent kinase 5 (Cdk5) is one of the kinases that have been linked to neurodegeneration. Cdk5 along with its principal activator p35 is involved in multiple cellular functions ranging from neuronal differentiation and migration to synaptic transmission. However, during neurotoxic stress, intracellular rise in Ca 2? activates calpain, which cleaves p35 to generate p25. The long half-life of Cdk5/p25 results in a hyperactive, aberrant Cdk5 that hyperphosphorylates Tau, neurofilament and other cytoskeletal proteins. These hyperphosphorylated cytoskeletal proteins set the groundwork to forming neurofibrillary tangles and aggregates of phosphorylated proteins, hallmarks of neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Amyotropic Lateral Sclerosis. Attempts to selectively target Cdk5/p25 activity without affecting Cdk5/p35 have been largely unsuccessful. A polypeptide inhibitor, CIP (Cdk5 inhibitory peptide), developed in our laboratory, successfully inhibits Cdk5/p25 activity in vitro, in cultured primary neurons, and is currently undergoing validation tests in mouse models of neurodegeneration. Here, we discuss the therapeutic potential of CIP in regenerating neurons that are exposed to neurodegenerative stimuli.
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...
Proceedings of Annual Meeting of the Physiological Society of Japan Proceedings of Annual Meeting of the Physiological Society of Japan, 2006
Neurons in the CNS can regenerate their axons in an environment of the peripheral nervous system, but this ability is limited. Here we show that an anti-glaucoma drug, nipradilol, at low concentration led to a four-fold increase in the number of cat retinal ganglion cells regenerating their axons into a transplanted peripheral nerve 4 and 6 weeks after axotomy. Nipradilol also increased the number of three main regenerating retinal ganglion cell types (alpha, beta, not alpha/beta), and enhanced the rate of axonal regeneration of these retinal ganglion cells. Nipradilol is a donor of nitric oxide and an antagonist of alpha-1, beta-1 and-2 adrenoreceptors, and we therefore examined whether one of these pharmacological effects might be more important in promoting axon regeneration. A nitric oxide donor increased the number of regenerating retinal ganglion cells, but not the rate of axonal regeneration. Denitro-nipradilol (nitric oxide-deprived nipradilol) or a nitric oxide scavenger injected before nipradilol increased the number of regenerating retinal ganglion cells but did not promote regeneration rate. Blockade of individual alpha-and beta-adrenoreceptors did not increase the number of regenerating retinal ganglion cells or the rate of regeneration. From these results, it is suggested that nitric oxide plays a crucial role in mediating the effects of nipradilol on axon regeneration and neuroprotection, and the metabolite of nipradilol supports the effects.
The effect of PKC activation on the survival of rat retinal ganglion cells in culture
Brain Research, 2000
Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated Ž . protein kinase C PKC , an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept ''in vitro'' for 48 h. For Ž . this purpose, we used the phorbol 12-myristate 13-acetate PMA , a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ngrml produced the Ž . maximal effect a two-fold increase on ganglion cells survival compared with 48 h control . This effect was totally abolished by 1.25 mM Ž . Ž . chelerythrine chloride an inhibitor of PKC and 30 mM genistein an inhibitor of tyrosine kinase enzymes . Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 mM 5-fluoro-2 X -deoxyuridine, an inhibitor of cell proliferation, or 25 mM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells ''in vitro'' may be mediated by a mechanism that involves PKC activation. q
Journal of Neuroscience, 2010
A major barrier to regeneration of central nervous system (CNS) axons is the presence of growthinhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced 4 "hit compounds", which act with nM potency on several different neuronal types, and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions, and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.
Journal of Neurochemistry, 2011
Apoptotic cell death of retinal ganglion cells (RGC) has been identified as a pathologic hallmark in several hereditary retinal diseases, senile maculopathy and glaucoma. In many other neurodegenerative disorders, such as Parkinson's or Alzheimer's disease, neuronal apoptosis represents a hallmark and histological endpoint of disease progression. Clearly, inhibition of cell death interferes only with a late process in the pathogenesis. It is commonly acknowledged that axonal degeneration may precede the later apoptotic degeneration of the cell body. Thus restorative strategies have to address axonal pathology as well (Coleman and Perry 2002; Knöferle et al. 2010). After controlling the initial insult, a therapeutic approach will have to foster the axonal regenerative response by overcoming myelin derived outgrowth inhibitors and molecules that are present in scar tissue as well as to increase the limited intrinsic regenerative capacity of adult neurons (Filbin 2003). In order to increase both cellular survival and axonal regeneration at the same time, we have chosen different in part combinatorial
Effects of a CDK inhibitor on lens regeneration
Wound Repair and Regeneration, 2004
Lens regeneration in adult newts is always initiated from the dorsal iris by transdifferentiation of the pigment epithelial cells. One of the most important early events should be the ability of pigment epithelial cells to dedifferentiate and re-enter the cell cycle. As a first step in an attempt to study this event, we have decided to examine the effects of a cyclin-dependent kinase-2 inhibitor on lens regeneration. At the appropriate concentration, this inhibitor completely abolished the ability of pigment epithelial cells to form a new lens, but it did not stop them from dedifferentiating and forming a small lens vesicle. The effects of this inhibitor seem to be mediated by its opposite effects on cell proliferation and apoptosis. The inhibitor significantly reduced cell proliferation and enhanced apoptosis of pigment epithelial cells both in vitro and in vivo and of the regenerating lens in vivo.
Protein kinases selectively modulate apoptosis in the developing retina in vitro
Neurochemistry International, 1997
In the retina of newborn rats there is evidence for two mechanisms of programmed cell death[ Apoptosis of ganglion cells "RGCs# following axotomy depends on protein synthesis[ In contrast\ inhibition of protein synthesis leads to apoptosis in the neuroblastic layer "NBL#[ The induction of apoptosis following translational arrest suggests that post!translational modi_cations of apoptosis!associated proteins may be crucial to the cell death programs in the developing retina[ We investigated the possible role of protein kinases upon apoptosis in retinal explants in vitro[ An increase in the intracellular concentration of cAMP produced either by the adenylyl!cyclase activator forskolin "09 mM# or by 7!Br!cAMP "0 mM#\ prevented apoptosis induced in the NBL by inhibition of protein synthesis\ but had no statistically signi_cant e}ect upon RGC death[ In contrast\ neither 7!Br!cGMP "0 mM# nor the speci_c cGMP!phosphodiesterase inhibitor zaprinast "09Ð099 mM# had signi_cant e}ects on apoptosis in the retina[ The cAMP! phosphodiesterase inhibitors isobutylmethylxantine "IBMX\ 9[0Ð0 mM# and Ro!190613 "49Ð199 mM# also prevented apoptosis in the NBL[ The isoquinolinesulfonamide H78 "19 mM#\ a speci_c cAMP!dependent protein kinase inhibitor\ partially reverted the protective e}ect of either forskolin or IBMX within the NBL[ Neither 01!O!tetradecanoyl phorbol!02!acetate "TPA\ 09 nM# nor bisindolylmaleimide "9[1Ð9[4 mM#\ respectively an activator and an inhibitor of protein kinase C had signi_cant e}ects upon the retinal explants[ The protein kinase inhibitor 1!aminopurine "1!AP\ 09 mM# prevented apoptosis of axotomized ganglion cells and induced apoptosis in the NBL[ Forskolin prevented the apoptosis induced by 1!AP in the NBL\ whereas TPA had no e}ect[ The e}ects of 1!AP were\ however\ not dependent on inhibition of protein synthesis[ The data indicate that modulation of the activity of both cAMP!dependent protein kinase and several protein kinases sensitive to 1!aminopurine selectively a}ect apoptosis in distinct cell layers of the developing retina[ Þ 0886 Elsevier Science Ltd