Inhibition of cGMP/PKG Signaling Suppresses Endoplasmic Reticulum Stress and Alters Inositol Trisphosphate Receptor Activity in CNG Channel Deficiency

Abstract

Purpose: Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown the characteristics of endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER calcium channel inositol trisphosphate receptor (IP₃R) in CNG channel deficient mice. We also showed a remarkable elevation of the cellular cyclic guanosine monophosphate (cGMP) level and an increased activity of the cGMP-dependent protein kinase (PKG) in CNG channel-deficient retinas, and have demonstrated a role of cGMP accumulation in cone degeneration. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R activity in CNG channel deficiency by inhibiting PKG activity and deleting Gucy2e, the gene encoding retinal guanylate cyclase-1.

Methods: Two cone CNG channel-deficient mouse lines, Cnga3^-/-/Nrl^-/- (Cnga3 deficiency on a cone-dominant background) and Cnga3^-/-/Nrl^-/-/Gucy2e^-/-, were used in this study. Inhibition of PKG activity was achieved by treating mice with the PKG inhibitors KT5823 and Rp-8-Br-cGMPS. Retinal cGMP level and PKG activity were analyzed by ELISA, TUNEL labeling was performed to assess cone death, glial fibrillary acid protein (GFAP) labeling was performed to evaluate Müller glial cell activation, and levels of phospho-eIF2α and phospho-IP₃R were analyzed to evaluate ER stress and IP₃R activity, respectively.

Results: We found that treatment with PKG inhibitor or deletion of Gucy2e effectively reduced PKG activity and apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells in CNG channel deficient mice. Furthermore, inhibition of PKG activity and deletion of Gucy2e significantly reduced ER stress, manifested as reduced levels of phospho-eIF2α, and increased phosphorylation of IP₃R.

Conclusions: This work demonstrates that inhibition of cGMP/PKG signaling suppresses ER stress and alters IP₃R activity in CNG channel deficiency. The cGMP/PKG signaling may contribute to cone death via eliciting an alteration of ER calcium homeostasis, which leads to ER stress and apoptotic cell death.