ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells

Abstract

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.

Boosting Central Nervous System Axon Regeneration by Circumventing Limitations of Natural Cytokine Signaling

“…5,[16][17][18][19][20] In the future, this approach could be combined with an inducible system (e.g., Tet on/off) to enable transient hIL-6 expression only during the regenerative phase. 51 Alternatively, hIL-6 delivery by encapsulated and therefore removable cells 52 might represent a promising therapeutic concept to promote axon regeneration upon nerve injuries in human patients.…”

“…5,16 Similarly, application of exogenous CNTF is neuroprotective and stimulates axon regeneration in the injured optic nerve, particularly upon continuous release from virally transduced cells. [16][17][18][19][20] However, cytokine-induced signaling is normally attenuated via intracellular negative feedback loops. For instance, PTEN inhibits PI3K activity, while SOCS3 compromises JAK/ STAT3 pathway activation.…”

Oncomodulin links inflammation to optic nerve regeneration

“…As we have discussed elsewhere (24), however, CNTF has only weak axon-promoting effects on mature RGCs (present study and refs. 7,25,26), and its ability to induce RGCs to regenerate axons in vivo is highly variable across laboratories, and requires concentrations many orders of magnitude above its known ED 50 . Of note, CNTF was recently shown to act in part through an indirect mechanism in inducing RGCs to regenerate axons through a peripheral nerve graft.…”

Hyper-IL-6: a potent and efficacious stimulator of RGC regeneration

“…16,27 Similarly, application of exogenous CNTF, LIF, and IL-6 mediates neuroprotection and stimulates axon regeneration in RGC cultures as well as the injured optic nerve in vivo, particularly upon continuous release from virally transduced cells. 26,[28][29][30][31] CNTF, LIF, and IL-6 belong to the family of interleukin-6 (IL-6)-type cytokines activating the signal transducing receptor glycoprotein 130 (gp130) (Figure 1), 32 which in the adult retina is mainly expressed in RGCs. 33 LIF requires direct interaction with the LIF-receptor (LIFR) to form a signaling complex with gp130.…”