Brain Advance Access originally published online on December 5, 2007
Brain 2008 131(1):250-263; doi:10.1093/brain/awm284
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ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells
1Department of Neurology, Georg-August-University Göttingen, University Medicine, Waldweg 33, 37073 Göttingen, 2DFG Research Center for Molecular Physiology of the Brain (CMPB), Göttingen, Germany and 3European Research Training Network (RTN) ‘Nervous System Repair’
Correspondence to: Paul Lingor, MD, Department of Neurology, Georg-August-University Göttingen, University Medicine, Waldweg 33, 37073 Göttingen, Germany. E-mail: plingor{at}gwdg.de
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 myelin-derived 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.
Key Words: retinal ganglion cells; CNTF; rho kinase; axotomy; regeneration
Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; CNTF, ciliary neurotrophic factor; CTB, choleratoxin subunit-B; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EBSS, Earle's balanced salt solution; FCS = fetal calf serum; GAP = growth associated protein; HRP = horse radish peroxidase; JAK, janus kinase; MAG, myelin-associated glycoprotein; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; OMgp, oligodendrocyte-myelin glycoprotein; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PKC, protein kinase C; PKN, protein kinase N; RGC, retinal ganglion cell; ROCK, rho kinase; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; STAT, signal transducer and activator of transcription
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Received July 5, 2007. Revised October 18, 2007. Accepted October 26, 2007.
*These authors contributed equally to this work.
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