Defective trafficking of cone photoreceptor CNG channels induces the unfolded protein response and ER-stress-associated cell death

Abstract

SYNOPSIS Mutations that perturb the function of photoreceptor cyclic nucleotide-gated (CNG) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the endoplasmic reticulum (ER) is known to cause ER stress and trigger the unfolded protein response (UPR), an evolutionarily conserved cellular program that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared to wild type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones (TUDCA, 4PBA, and the cGMP analog CPT-cGMP) differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization defective CNG channels, and may represent a contributing factor for photoreceptor degeneration.

Rescue of Enzymatic Function for Disease-associated RPE65 Proteins Containing Various Missense Mutations in Non-active Sites

“…The rescue of enzyme activity of the mutant RPE65s at 30 °C suggests that chemical chaperones that have been shown to help proper folding of other retinal mutant proteins (36, 52–55) can also rescue the non-active site mutant RPE65s. We therefore first tested the effect of PBA or glycerol on expression levels of WT and mutant RPE65s in ARPE-19 cells.…”

The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis

“…In photoreceptor-derived cell lines with mutations in CNG channel subunits, the administration of 4-PBA improved maturation, trafficking and localization of defective CNG channel subunits, thereby attenuating associated cytotoxicity (Duricka et al, 2012). GammaD-crystallin is another important protein for vision as it plays a major role in the crystalline structure and transparency of the lens.…”

Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency

“…It is worth mentioning that though lack of cone function in the absence of CNG channel (such as in CNGA3 Ϫ/Ϫ mice) causes opsin mislocalization, subunit mislocalization arising from diseaselinked missense mutations in CNGA3 has been reported extensively in heterologous expression system. Enhanced cytosolic aggregation and activation of ER stress were observed in many CNGA3 mutants, including those with mutations located in the subunit transmembrane S1 and S4 domains (45,46) and at the carboxyl terminals (47)(48)(49). Thus, the UPR and ER stress in CNGA3 missense (and loss of function) mutations could be caused not only by opsin mislocalization (due to lack of normal channel function/phototransduction) but also by mislocalization of mutant CNGA3 subunits, and the ER stress might be more severe than the situation of lack of CNGA3.…”