Retinoschisin is associated with the retinal Na/K-ATPase signaling complex - impact on disease pathology of X-linked juvenile retinoschisis

Abstract

Purpose : Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a degenerative disease of the central retina. Still, the molecular processes underlying XLRS pathogenesis are not fully understood. We have recently shown that retinoschisin, the protein encoded by RS1, is a regulator of Erk signaling and apoptosis in retinal cells. In this study, we wanted to clarify the role of retinoschisin for the functionality of the Na/K-ATPase, its interaction partner in retinal membranes.

Methods : Retinoschisin binding to specific Na/K-ATPase subunits was investigated in Hek293 cells heterologously expressing different Na/K-ATPase subunit combinations. Ion transport activity of the Na/K-ATPase was analyzed in murine retinal explants from retinoschisin-deficient (Rs1h^-/Y) mice as well as in Xenopus leavis oocytes applying enzymatic assays or atomic absorption spectroscopy, respectively. Signaling was followed in Rs1h^-/Y murine retinal explants by immunoblotting analysis for Erk, Ip3/Akt and Ca²⁺ signaling. Co-immunoprecipitation and immunohistochemistry was done to to elucidate intracellular signal transmitters.

Results : Our findings demonstrate that the β2-subunit of the Na/K-ATPase is required for membrane binding of retinoschisin whereas the α-subunit is exchangeable. Our investigation revealed no influence of retinoschisin on ion pump activity or substrate affinity of the retinal Na/K-ATPase. However, we identified an influence of retinoschisin on Na/K-ATPase regulated signaling cascades: Retinoschisin treatment decreased activation of Src and CamKII (markers for Erk and Ca²⁺ signaling) in Rs1h^-/Y retinal explants. Co-immmunoprecipitations from murine retinae suggested a direct interaction between retinoschisin, the Na/K-ATPase, and signaling mediators caveolin and Phospholipase C (Plc). Furthermore, we show co-localization of the retinoschisin-Na/K-ATPase complex with intracellular signal tranducers Src, Plc, and the IP3 receptor.

Conclusions : Together, our data suggest that the Na/K-ATPase signalosome complex is reacting to retinoschisin binding. Disturbances in this interaction likely represent an initial step in XLRS pathogenesis.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.