Decrease of intracellular calcium to restrain rod cell death in retinitis pigmentosa

Abstract

Purpose : Retinitis Pigmentosa (RP), an inherited degenerative disease causing blindness in later life, is highly genetically heterogeneous. Recent gene-specific therapies have been developed, however only few patients can benefit from such highly personalized medicines. Targeting common features of the degenerative process may overcome this limitation. In murine models of RP, caused by mutations in the Rho and Pde6b genes, similar cell death pathways have been identified and linked to excess intracellular calcium. Ca²⁺ can enter photoreceptor cells through cGMP gated channels (CNGC) and is extruded through several pumps and Plasma Membrane Calcium ATPase (PMCA). The purpose of this study was to define common and different molecular mechanisms causing increased intracellular Ca²⁺ in different models of RP and new molecules targeting them.

Methods : We generated two in vitro 661W models for dominant and recessive mutations causing RP: 1) genetically modified cells for inducible expression of the toxic mutant P23H RHO protein; 2) block of PDE6 function by treatment with zaprinast. We evaluated intracellular calcium by cytofluorimetric analysis. Activation of calpains and PKG were evaluated by fluorescent substrates or antibodies. We treated cells with cGMP analogues to block CNGC and PEDF to affect PMCA activity.

Results : Cytofluorimetric analyses of intracellular Ca²⁺ confirmed increases at early times after induction of degeneration either by activating expression of P23H mutant RHO or by blocking PDE6 function. In both systems, we detected activation of calpains. Treatments with molecules reinforcing PMCA activity decreased intracellular Ca²⁺ and cell death, confirming the key role of high intracellular Ca²⁺ in retinal degeneration. By blocking CNGC function with cGMP inhibiting analogs we defined the contribution of Ca²⁺ influx through CNGC in these two models of the disease.

Conclusions : Increased intracellular Ca²⁺ is a common early event during photoreceptor degeneration. Here, we showed that photoreceptor cell death can be decreased by blocking CNGC with cGMP analogues or by boosting PMCA activity. Affecting calcium pumps is a promising approach to target photoreceptor degeneration.
Acknowledgements: European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 765441

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.