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Rhodopsin is a G-protein-coupled receptor that is specifically and abundantly expressed in rod photoreceptors. Over 150 rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP). The most common mutation in the United States is the conversion of proline to histidine at position 23 (P23H) in the N-terminal domain of rhodopsin. We previously found that P23H rhodopsin was misfolded, ubiquitinylated, and rapidly degraded. Here, we investigated the role of lysine residues on P23H rhodopsin ubiquitinylation and turnover. We transfected HEK293 cells with a P23H human rhodopsin construct where all 11 lysine residues were mutated to arginine (K-null P23H). We found that the K-null P23H rhodopsin was significantly less ubiquitylated than intact P23H rhodopsin. We found that K-null P23H protein turnover was significantly slower compared to P23H rhodopsin through cycloheximide chase analysis. Finally, we also generated a wild-type rhodopsin construct where all lysines were converted to arginine and found significantly reduced ubiquitylation. Our findings identify ubiquitinylation of lysine residues as an important posttranslational modification involved in P23H rhodopsin protein degradation., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Rhodopsin is essential for phototransduction, and many rhodopsin mutations cause heritable retinal degenerations. The P23H rhodopsin variant generates a misfolded rhodopsin protein that photoreceptors quickly target for degradation by mechanisms that are incompletely understood. To gain insight into how P23H rhodopsin is removed from rods, we used mass spectrometry to identify protein interaction partners of P23H rhodopsin immunopurified from Rho P23H/P23H mice and compared them with protein interaction partners of wild-type rhodopsin from Rho +/+ mice. We identified 286 proteins associated with P23H rhodopsin and 276 proteins associated with wild-type rhodopsin. 113 proteins were shared between wild-type and mutant rhodopsin protein interactomes. In the P23H rhodopsin protein interactome, we saw loss of phototransduction, retinal cycle, and rhodopsin protein trafficking proteins but gain of ubiquitin-related proteins when compared with the wild-type rhodopsin protein interactome. In the P23H rhodopsin protein interactome, we saw enrichment of gene ontology terms related to ER-associated protein degradation, ER stress, and translation. Protein-protein interaction network analysis revealed that translational and ribosomal quality control proteins were significant regulators in the P23H rhodopsin protein interactome. The protein partners identified in our study may provide new insights into how photoreceptors recognize and clear mutant rhodopsin, offering possible novel targets involved in retinal degeneration pathogenesis., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho +/P23H ) mice. Significantly increased rhodopsin protein levels were found in Atf6 -/- Rho +/P23H retinas compared to Atf6 +/- Rho +/P23H retinas at early ages (~ P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6 -/- Rho +/P23H retinas, and photoreceptor layer thickness was unchanged at this early age in Rho +/P23H mice lacking Atf6. By contrast, older Atf6 -/- Rho +/P23H mice developed significantly increased retinal degeneration in comparison to Atf6 +/- Rho +/P23H mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice., (© 2021. The Author(s).)

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

Rhodopsin mutations cause many types of heritable retinitis pigmentosa (RP). Biochemical and in vitro studies have demonstrated that many RP-linked mutant rhodopsins produce misfolded rhodopsin proteins, which are prone to aggregation and retention within the endoplasmic reticulum, where they cause endoplasmic reticulum stress and activate the Unfolded Protein Response signaling pathways. Many vertebrate models of retinal degeneration have been created through expression of RP-linked rhodopsins in photoreceptors including, but not limited to, VPP/GHL mice, P23H Rhodopsin frogs, P23H rhodopsin rats, S334ter rhodopsin rats, C185R rhodopsin mice, T17M rhodopsin mice, and P23H rhodopsin mice. These models have provided many opportunities to test therapeutic strategies to prevent retinal degeneration and also enabled in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death. Here, we examine and compare the contribution of endoplasmic reticulum stress to retinal degeneration in several vertebrate models of RP generated through expression of mutant rhodopsins.

Many mutations in rhodopsin gene linked to retinitis pigmentosa (RP) cause rhodopsin misfolding. Rod photoreceptor cells expressing misfolded rhodopsin eventually die. Identifying mechanisms to prevent rhodopsin misfolding or to remove irreparably misfolded rhodopsin could provide new therapeutic strategies. IRE1, ATF6, and PERK signaling pathways, collectively called the unfolded protein response (UPR), regulate the functions of endoplasmic reticulum, responsible for accurate folding of membrane proteins such as rhodopsin. We used chemical and genetic approaches to selectively activate IRE1, ATF6, or PERK signaling pathways one at a time and analyzed their effects on mutant rhodopsin linked to RP. We found that both artificial IRE1 and ATF6 signaling promoted the degradation of mutant rhodopsin with lesser effects on wild-type rhodopsin. Furthermore, IRE1 and ATF6 signaling preferentially reduced levels of aggregated rhodopsins. By contrast, PERK signaling reduced levels of wild-type and mutant rhodopsin. These studies indicate that activation of either IRE1, ATF6, or PERK prevents mutant rhodopsin from accumulating in the cells. In addition, activation of IRE1 or ATF6 can selectively remove aggregated or mutant rhodopsin from the cells and may be useful in treating RP associated with rhodopsin protein misfolding.

Purpose: Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding., Methods: To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins., Results: ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell., Conclusions: This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.

The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2alpha and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2alpha, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

Many mutations in rhodopsin gene linked to retinitis pigmentosa (RP) cause rhodopsin misfolding. Rod photoreceptor cells expressing misfolded rhodopsin eventually die. Identifying mechanisms to prevent rhodopsin misfolding or to remove irreparably misfolded rhodopsin could provide new therapeutic strategies. IRE1, ATF6, and PERK signaling pathways, collectively called the unfolded protein response (UPR), regulate the functions of endoplasmic reticulum, responsible for accurate folding of membrane proteins such as rhodopsin. We used chemical and genetic approaches to selectively activate IRE1, ATF6, or PERK signaling pathways one at a time and analyzed their effects on mutant rhodopsin linked to RP. We found that both artificial IRE1 and ATF6 signaling promoted the degradation of mutant rhodopsin with lesser effects on wild-type rhodopsin. Furthermore, IRE1 and ATF6 signaling preferentially reduced levels of aggregated rhodopsins. By contrast, PERK signaling reduced levels of wild-type and mutant rhodopsin. These studies indicate that activation of either IRE1, ATF6, or PERK prevents mutant rhodopsin from accumulating in the cells. In addition, activation of IRE1 or ATF6 can selectively remove aggregated or mutant rhodopsin from the cells and may be useful in treating RP associated with rhodopsin protein misfolding.

Rhodopsin mutations cause many types of heritable retinitis pigmentosa (RP). Biochemical and in vitro studies have demonstrated that many RP-linked mutant rhodopsins produce misfolded rhodopsin proteins, which are prone to aggregation and retention within the endoplasmic reticulum, where they cause endoplasmic reticulum stress and activate the Unfolded Protein Response signaling pathways. Many vertebrate models of retinal degeneration have been created through expression of RP-linked rhodopsins in photoreceptors including, but not limited to, VPP/GHL mice, P23H Rhodopsin frogs, P23H rhodopsin rats, S334ter rhodopsin rats, C185R rhodopsin mice, T17M rhodopsin mice, and P23H rhodopsin mice. These models have provided many opportunities to test therapeutic strategies to prevent retinal degeneration and also enabled in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death. Here, we examine and compare the contribution of endoplasmic reticulum stress to retinal degeneration in several vertebrate models of RP generated through expression of mutant rhodopsins.

PurposeEndoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes.MethodsRetinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses.ResultsS334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss.ConclusionsOur study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

Phototransduction is accomplished in the retina by photoreceptor neurons and retinal pigment epithelium (RPE) cells. Photoreceptors rely heavily on the RPE, and death or dysfunction of RPE is characteristic of age-related macular degeneration (AMD), a very common neurodegenerative disease for which no cure exists. RPE replacement is a promising therapeutic intervention for AMD, and large numbers of RPE cells can be generated from pluripotent stem cells. However, questions persist regarding iPSC-derived RPE (iPS-RPE) viability, immunogenicity, and tumorigenesis potential. We showed previously that iPS-RPE prevent photoreceptor atrophy in dystrophic rats up until 24 weeks after implantation. In this follow-up study, we longitudinally monitored the same implanted iPS-RPE, in the same animals. We observed no gross abnormalities in the eyes, livers, spleens, brains, and blood in aging rats with iPSC-RPE grafts. iPS-RPE cells that integrated into the subretinal space outlived the photoreceptors and survived for as long as 2 1/2 years while nonintegrating RPE cells were ingested by host macrophages. Both populations could be distinguished using immunohistochemistry and electron microscopy. iPSC-RPE could be isolated from the grafts and maintained in culture; these cells also phagocytosed isolated photoreceptor outer segments. We conclude that iPS-RPE grafts remain viable and do not induce any obvious associated pathological changes. [ABSTRACT FROM AUTHOR]

Photoreceptors are specialized sensory neurons essential for light detection in the human eye. Photoreceptor cell dysfunction and death cause vision loss in many eye diseases such as retinitis pigmentosa and achromatopsia. Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling have been implicated in the development and pathology of heritable forms of retinitis pigmentosa and achromatopsia. We review the role of ER stress and UPR in retinitis pigmentosa arising from misfolded rhodopsins (RHO) and in achromatopsia arising from genetic mutations in Activating Transcription Factor 6 (ATF6). This article is part of a Special Issue entitled SI:ER stress . [ABSTRACT FROM AUTHOR]