Inhibition of Endoplasmic Reticulum Calcium Channels Reduces Endoplasmic Reticulum Stress and Cone Death in CNG Channel-Deficient Mice

Abstract

Purpose : The photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransduction and cellular calcium homeostasis. We previously showed that the cone CNG channel-deficient mice display endoplasmic reticulum (ER) stress-associated cone death, increased cellular cyclic guanosine monophosphate (cGMP), and increased phosphorylation of the ER calcium channel 1,4,5-inositol triphosphate receptor (IP₃R). The present work investigates the relationship between CNG channel deficiency in the outer segment and ER calcium regulation within the inner segment, via IP₃R and ryanodine receptors (RYR), in order to define their possible roles in ER stress and cone death.

Methods : We used pharmacological inhibitors to block ER calcium channels in Cnga3^-/-/Nrl^-/- mice (CNGA3 deficiency on a cone-dominant background). Postnatal day 7 (P7) mice received 2-APB or U-73122 at dose of 10.0 and 8.0 μg/g/day, respectively, by daily intraperitoneal injection to block IP₃R, and received tetracaine by intravitreal injection to inhibit RYR. Mice were analyzed at P15 for cone death using TUNEL labeling on retinal cross sections and for expression levels of ER stress markers by immunoblotting. Cellular calcium levels were measured in vitro from isolated Cnga3^-/-/Nrl^-/- and Nrl^-/- photoreceptors loaded with Fura-2 (10µM).

Results : Treatment with ER calcium channel inhibitors resulted in a 30% reduction of TUNEL positive cells in the outer nuclear layer of Cnga3^-/-/Nrl^-/- retinas. The levels of the ER stress marker proteins, including phospho-IRE1α, phospho-eIF2α, and cleaved form of ATF-6, were reduced by about 40-50% in the drug-treated mice, compared with vehicle-treated controls. Baseline [Ca²⁺]_i levels in P14/15 Cnga3^-/-/Nrl^-/- photoreceptors were nearly half that of photoreceptors from age-matched Nrl^-/- mice (59.9±5.69 and 111.1±16.2, respectively, p<0.01).

Conclusions : We show that the inhibition of the ER calcium channels reduces ER stress and cone death, supporting the view that ER calcium depletion/cytosolic calcium reduction modulates ER stress/cone death in CNG channel deficiency. Our findings show how outer segment dysfunction regulates cGMP-dependent signals in the downstream inner segment and suggest that suppressing ER calcium channels may represent a novel strategy for photoreceptor preservation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.