The Role of Endoplasmic Reticulum (ER) Calcium Channels in ER Stress and Cone Death in CNG Channel Deficiency

Abstract

Purpose : The cone cyclic nucleotide-gated (CNG) channel is a ligand-gated calcium/sodium ion channel required for proper phototransduction. Mutations in cone CNG channel account for about 80% of achromatopsia patients and are associated with progressive cone dystrophies. Our previous studies have demonstrated endoplasmic reticulum (ER) stress-associated cone death in CNG channel deficiency. We also showed enhanced expression and activity of the ER calcium channels inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) and ryanodine receptor 2 (RyR2) in CNG channel-deficient mice. This work investigated the contributions of IP₃R1 and RyR2 to ER stress and cone death in CNG channel deficiency.

Methods : CNG channel-deficient mice (Cnga3^-/- and Cnga3^-/-/Nrl^-/- with CNG channel deficiency on a cone-dominant background) with cone specific deletion of Ryr2 (encoding RyR2) or Itpr1 (encoding IP₃R1) were generated by cross mating. Photoreceptor function was evaluated by electroretinography (ERG). Cone survival and death were evaluated by peanut agglutinin lectin (PNA) staining and TUNEL labeling. Glial fibrillary acidic protein (GFAP) labeling was performed to evaluate retinal stress and Müller cell activation. ER stress markers were analyzed by immunoblotting.

Results : Deletion of Ryr2 or Itpr1 did not exert negative effects on cone function/survival in wild-type mice. Deletion of Ryr2 or Itpr1 increased cone density by about 30% in Cnga3^-/- mice and reduced the number of TUNEL-positive cells by about 40%. Expression levels of the ER stress markers phospho-eIF2α and phospho-IRE1α were increased by 0.6-1.3-fold in Cnga3^-/-/Nrl^-/- retinas, and these elevations were abolished by deletion of Itpr1 or Ryr2. Deletion of Ryr2 or Itpr1 reversed changes in GFAP labeling in Cnga3^-/- mice to that seen in the control mice.

Conclusions : In CNG channel-deficient mice, we demonstrate that ER calcium channels RyR2 and IP₃R1 play a significant role in ER stress and cone death. Deletion of Ryr2 or Itpr1 protects cones, reduces ER stress, and decreases activation of Müller cells. Our findings provide insight into the mechanisms of cone degeneration and potential therapeutic strategies in CNG channel deficiency.

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