Ouabain-Na+/K+-ATPase Signaling Regulates Retinal Neuroinflammation and ROS Production Preventing Neuronal Death by an Autophagy-Dependent Mechanism Following Optic Nerve Axotomy In Vitro.

Ouabain is a classic Na+K+ATPase ligand and it has been described to have neuroprotective effects on neurons and glial cells at nanomolar concentrations. In the present work, the neuroprotective and immunomodulatory potential of ouabain was evaluated in neonatal rat retinal cells using an optic nerve axotomy model in vitro. After axotomy, cultured retinal cells were treated with ouabain (3 nM) at different periods. The levels of important inflammatory receptors in the retina such as TNFR1/2, TLR4, and CD14 were analyzed. We observed that TNFR1, TLR4, and CD14 were decreased in all tested periods (15 min, 45 min, 24 h, and 48 h). On the other hand, TNFR2 was increased after 24 h, suggesting an anti-inflammatory potential for ouabain. Moreover, we showed that ouabain also decreased Iba-1 (microglial marker) density. Subsequently, analyses of retrograde labeling of retinal ganglion cells (RGC) were performed after 48 h and showed that ouabain-induced RGC survival depends on autophagy. Using an autophagy inhibitor (3-methyladenine), we observed a complete blockage of the ouabain effect. Western blot analyses showed that ouabain increases the levels of autophagy proteins (LC3 and Beclin-1) coupled to p-CREB transcription factor and leads to autophagosome formation. Additionally, we found that the ratio of cleaved/pro-caspase-3 did not change after ouabain treatment; however, p-JNK density was enhanced. Also, ouabain decreased reactive oxygen species production immediately after axotomy. Taken together, our results suggest that ouabain controls neuroinflammation in the retina following optic nerve axotomy and promotes RGC neuroprotection through activation of the autophagy pathway.

Ouabain and BDNF Crosstalk on Ganglion Cell Survival in Mixed Retinal Cell Cultures.

Brain-derived neurotrophic factor (BDNF) is a well-known and well-studied neurotrophin. Most biological effects of BDNF are mediated by the activation of TrkB receptors. This neurotrophin regulates several neuronal functions as cell proliferation, viability, and differentiation. Ouabain is a steroid that binds to the Na(+)/K(+) ATPase, inducing the activation of several intracellular signaling pathways. Previous data from our group described that ouabain treatment increases retinal ganglion cells survival (RGC). The aim of the present study was to evaluate, if this cardiac glycoside can have a synergistic effect with BDNF, the classical trophic factor for retinal ganglion cells, as well as investigate the intracellular signaling pathways involved. Our work demonstrated that the activation of Src, PLC, and PKCδ participates in the signaling cascade mediated by 50 ng/mL BDNF, since their selective inhibitors completely blocked the trophic effect of BDNF. We also demonstrated a synergistic effect on RGC survival when we concomitantly used ouabain (0.75 nM) and BDNF (10 ng/mL). Moreover, the signaling pathways involved in this synergistic effect include Src, PLC, PKCδ, and JNK. Our results suggest that the synergism between ouabain and BDNF occurs through the activation of the Src pathway, JNK, PLC, and PKCδ.

Ouabain induces an increase of retinal ganglion cell survival in vitro: the involvement of protein kinase C.

Ouabain is a steroid derivative that can regulate many cellular events such as growth and proliferation. It modulates Na+,K+-ATPase activity leading to the activation of different intracellular pathways through protein-protein interactions that have been characterized during the last few years. The aim of this work was to study the role of ouabain in rat retinal ganglion cell survival after 48 h in culture. Our results demonstrated that ouabain significantly induced an increase in retinal ganglion cell survival. The effect was dose-dependent and was maximal with 3.0 nM. The blockade of protein kinase C activity by 1.25 microM chelerythrine chloride abolished the ouabain effect, indicating an involvement of this intracellular pathway. None of the protein kinase inhibitors usually employed in the study of ouabain-driven intracellular pathways (PD98059, Ly294002, herbimycin, and genistein) was able to influence neuronal survival induced by ouabain. The data presented suggest that ouabain may be the trigger of an intracellular pathway responsible for neuronal survival.