Inhibition of Inositol 1,4,5-Trisphosphate Receptor Activity Reduces Endoplasmic Reticulum 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. CNG channel deficiency accounts for about 80% of achromatopsia patients and is associated with progressive cone dystrophies. Our previous studies demonstrated endoplasmic reticulum (ER) stress-associated apoptotic cone death in CNG channel deficiency. We also showed enhanced phosphorylation of the ER calcium channel inositol 1,4,5-trisphosphate receptor (IP₃R) in CNG channel-deficient mice. This work investigated whether IP₃R activity contributes to ER stress and apoptotic cone death in CNG channel deficiency.

 

Methods

 

Cnga3^-/-/Nrl^-/-mice (CNGA3 deficiency on a cone-dominant background) were used to determine whether inhibiting IP₃R activity reduces ER stress and cone death. Expression levels of IP₃R isoforms were analyzed by qRT-PCR. Two IP₃R inhibitors, 2-APB and U-73122, were used to suppress IP₃R activity by daily treatment (intraperitoneal injection) for 8 days, beginning at postnatal (P) day 7, and TUNEL labeling and western blot analysis were performed to assess cone death and ER stress protein markers, respectively. In addition, cellular localization of cone opsin in Cnga3^-/-mice treated with 2-APB and U-73122 for 20 days, beginning at P7, was evaluated by immunofluorescence labeling and high magnification confocal microscopy/quantitative image analysis.

 

Results

 

Expression levels of IP₃R isoforms were significantly increased in Cnga3^-/-/Nrl^-/- retinas compared with Nrl^-/- controls, with IP₃R1 expression increased by about 3-fold. Inhibition of IP₃R activity via 2-APB and U-73122 treatment decreased TUNEL labeling by approximately 30% and 38%, respectively, compared with vehicle-treated controls. Levels of ER stress marker p-eIF2-alpha decreased by about 10% and 30% following 2-APB and U-73122 treatment, respectively. Opsin localization to the cone outer segment (OS) was significantly enhanced following 2-APB and U-73122 treatment.

 

Conclusions

 

In CNG channel-deficient mice, we demonstrate that IP₃R activity plays a role in ER stress, opsin localization, and cone degeneration. Inhibiting IP₃R activity protects cones, reduces ER stress, and enhances opsin OS localization. The findings of this study provide insight into the mechanisms of cone degeneration and potential therapeutic targets in CNG channel deficiency.