Potential contribution of ryanodine receptor 2 upregulation to cGMP/PKG signaling‐induced cone degeneration in cyclic nucleotide‐gated channel deficiency

Abstract

Abbreviations: ATF6, activating transcription factor 6; CaMKII, calmodulin-dependent protein kinase II; cAMP, cyclic adenosine monophosphate; CAR, cone arrestin; cGMP, cyclic guanosine monophosphate; CHOP, CCAAT-enhancer-binding protein homologous protein; CNG, cyclic nucleotide-gated; CREB, cyclic adenosine monophosphate response element-binding protein; ER, endoplasmic reticulum; eIF2α, eukaryotic translation initiation factor 2 alpha; GFAP, glial fibrillary acidic protein; HRGP, human red/green pigment; Iba1, ionized calcium-binding adaptor molecule 1; IP 3 R, inositol-1,4,5trisphosphate receptor; Ire1α, serine/threonine-protein kinase/endoribonuclease 1α; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RetGC1, retinal guanylate cyclase 1; RyR2, ryanodine receptor 2; SERCA, sarco/endoplasmic reticulum Ca 2+ -ATPase; TUNEL, terminal deoxynucleotidyltransferase dUTP nick end-labeling; UPR, unfolded protein response. AbstractPhotoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca 2+ influx in rod and cone photoreceptors. Mutations in cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. Mice lacking functional cone CNG channel show endoplasmic reticulum (ER) stress-associated cone degeneration. The elevated cyclic guanosine monophosphate (cGMP)/cGMPdependent protein kinase (PKG) signaling and upregulation of the ER Ca 2+ channel ryanodine receptor 2 (RyR2) have been implicated in cone degeneration. This work investigates the potential contribution of RyR2 to cGMP/PKG signaling-induced ER stress and cone degeneration. We demonstrated that the expression and activity of RyR2 were highly regulated by cGMP/PKG signaling. Depletion of cGMP by deleting retinal guanylate cyclase 1 or inhibition of PKG using chemical inhibitors suppressed the upregulation of RyR2 in CNG channel deficiency. Depletion of cGMP or deletion of Ryr2 equivalently inhibited unfolded protein response/ER stress, activation of the CCAAT-enhancer-binding protein homologous protein, and activation of the cyclic adenosine monophosphate response element-binding protein, leading to early-onset cone protection. In addition, treatment with cGMP significantly enhanced Ryr2 expression in cultured photoreceptor-derived Weri-Rb1 cells. Findings from this work demonstrate the regulation of cGMP/PKG signaling on RyR2 in the retina and support the role of RyR2 upregulation in cGMP/PKG signaling-induced ER stress and photoreceptor degeneration. K E Y W O R D S cGMP/PKG signaling, CNG channel, cone photoreceptors, retinal degeneration, ryanodine receptor 6336 | YANG et Al.

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia

“…In line with a neuroprotective effect of Prkg2-deletion, the levels of p-eIF2a were reduced to a level similar to Cnga3/Nrl/Gucy2e TKO mice (Figure 5c,d), which are protected from cGMP-mediated cytotoxicity due to the lack of the cGMP-synthesizing retGC enzyme [13]. Previous studies have demonstrated that CNG channel-deficient mice show increased expression/activity of ER Ca 2+ -releasing channels, responsible for Ca 2+ efflux from the ER into the cytosol [20,21]. We, therefore, analyzed the gene expression of Itpr1, which encodes the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), and Ryr2, the gene encoding the type 2 ryanodine receptor (Ryr2).…”

“…As depicted in Figure 5e,f, both genes showed enhanced gene expression levels in the Cnga3 KO context, which were normalized after additional knockout of Prkg2. A similar effect was observed for the unfolded protein response (UPR)-related genes Atf6b (activating transcription factor 6 beta), Bax (Bcl2-associated X protein), Cebpb (CCAAT/enhancer-binding protein beta), Creb3l3 (cAMP-responsive element-binding protein 3-like 3), Derl1 (degradation in ER protein 1), Dnajc3 (DnaJ [Hsp40] homolog, subfamily C, member 3), Ern2 (ER to nucleus signaling 2), Ganc (glucosidase, alpha; neutral C), Srebf2 (sterol regulatory element-binding factor 2), and Uggt2 (UDPglucose glycoprotein glucosyltransferase 2; Figure 5e-g), which are upregulated in Cnga3deficient mice [19,21]. This suggests that Prkg2 knockout counteracts ER stress and UPR and normalizes ER homeostasis.…”

“…This suggests that, besides cGMP-Prkg2-mediated cytotoxicity, additional cell death mechanisms contribute to the degeneration of cones. Mechanistically, deletion of Prkg2 dampened the levels of ER stress and UPR markers, suggesting that cGMP-Prkg2 signalling contributes to both phenomena and crucially impacts viability of CNG channel-deficient cone photoreceptors [18,21,31].…”

“…Retinal protein preparation, SDS-PAGE, and western blot analysis were performed as described previously [21,35]. A Li-Cor Odyssey machine and Li-Cor software (Li-Cor Biosciences, Lincoln, NE, USA) were used for detection and densitometric analysis.…”

“…Total RNA preparation and reverse transcription were per-formed as described previously [19,21]. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were performed with a real-time PCR detection system (iCycler; Bio-Rad, Hercules, CA, USA).…”