P23H Rhodopsin Transgenic Rats Research Articles

Role of Curcumin in Retinal Diseases-A review.

PurposeTo review the role of curcumin in retinal diseases, COVID era, modification of the molecule to improve bioavailability and its future scope.MethodsPubMed and MEDLINE searches were pertaining to curcumin, properties of curcumin, curcumin in retinal diseases, curcumin in diabetic retinopathy, curcumin in age-related macular degeneration, curcumin in retinal and choroidal diseases, curcumin in retinitis pigmentosa, curcumin in retinal ischemia reperfusion injury, curcumin in proliferative vitreoretinopathy and curcumin in current COVID era.ResultsIn experimental models, curcumin showed its pleiotropic effects in retinal diseases like diabetic retinopathy by increasing anti-oxidant enzymes, upregulating HO-1, nrf2 and reducing or inhibiting inflammatory mediators, growth factors and by inhibiting proliferation and migration of retinal endothelial cells in a dose-dependent manner in HRPC, HREC and ARPE-19 cells. In age-related macular degeneration, curcumin acts by reducing ROS and inhibiting apoptosis inducing proteins and cellular inflammatory genes and upregulating HO-1, thioredoxin and NQO1. In retinitis pigmentosa, curcumin has been shown to delay structural defects of P23H gene in P23H-rhodopsin transgenic rats. In proliferative vitreoretinopathy, curcumin inhibited the action of EGF in a dose- and time-dependent manner. In retinal ischemia reperfusion injury, curcumin downregulates IL-17, IL-23, NFKB, STAT-3, MCP-1 and JNK. In retinoblastoma, curcumin inhibits proliferation, migration and apoptosis of RBY79 and SO-RB50. Curcumin has already proven its efficacy in inhibiting viral replication, coagulation and cytokine storm in COVID era.ConclusionCurcumin is an easily available spice used traditionally in Indian cooking. The benefits of curcumin are manifold, and large randomized controlled trials are required to study its effects not only in treating retinal diseases in humans but in their prevention too.

Hepatic oxidative stress in pigmented P23H rhodopsin transgenic rats with progressive retinal degeneration

Retinitis pigmentosa (RP) comprises a group of inherited retinal degenerative conditions characterized by primary degeneration of the rod photoreceptors. Increased oxidative damage is observed in the retina, aqueous humor, and plasma of RP animal models and patients. The hepatic oxidative status may also be affected in RP due to oxidative damage influencing soluble macromolecules exiting the retina or to alterations in the melanopsin system resulting in chronic circadian desynchronization that negatively alters the oxidative stress defense system.P23H rats were crossed with pigmented Long Evans rats to produce offspring exhibiting the clinical conditions of RP. We measured hepatic malondialdehyde and 4-hydroxyalkenal concentrations as oxidative stress markers; nitrite level as a total nitrosative damage marker; total antioxidant capacity; and the activities of catalase, superoxide dismutase (SOD), and glutathione S-transferase. Retinal visual function was assessed based on optomotor and electroretinogram responses.P23H transgenic rats exhibited diminished visual acuity, contrast sensitivity, and electroretinographic responses according to the level of retinal degeneration. P23H rats at 30 days of age already demonstrated only 47% of the hepatic total antioxidant capacity of wild-type animals. Hepatic catalase and SOD activities were also reduced in P23H rats after 120 days, but we detected no difference in glutathione S-transferase activity. P23H rats had increased hepatic oxidative and nitrosative damage markers. GSH/GSSG ratio showed a significant diminution in P23H rats at P120 compared to WT.We conclude that the liver is under increased oxidative stress in P23H rats. Further studies are required, however, to clarify the contribution of systemic oxidative damage to the pathogenesis of RP.

Gene Therapy Strategies to Restore ER Proteostasis in Disease.

Proteostasis alterations are proposed as a transversal hallmark of several pathological conditions, including metabolic disorders, mechanical injury, cardiac malfunction, neurodegeneration, and cancer. Strategies to improve proteostasis aim to reduce the accumulation of specific disease-related misfolded proteins or bolster the endogenous mechanisms to fold and degrade abnormal proteins. Endoplasmic reticulum (ER) stress is a common pathological signature of a variety of diseases, which engages the unfolded protein response (UPR) as a cellular reaction to mitigate ER stress. Pharmacological modulation of the UPR is challenging considering the physiological importance of the pathway in various organs. However, local targeting of ER stress responses in the affected tissue using gene therapy is emerging as a possible solution to overcome side effects. The delivery of ER chaperones or active UPR components using adeno-associated virus (AAV) has demonstrated outstanding beneficial effects in several disease models (e.g., neurodegenerative conditions, eye disorders, and metabolic diseases). Here, we discuss current efforts to design and optimize gene therapy strategies to improve ER proteostasis in different disease contexts.

Allele-Specific Inhibition of Rhodopsin With an Antisense Oligonucleotide Slows Photoreceptor Cell Degeneration.

To preserve photoreceptor cell structure and function in a rodent model of retinitis pigmentosa with P23H rhodopsin by selective inhibition of the mutant rhodopsin allele using a second generation antisense oligonucleotide (ASO). Wild-type mice and rats were treated with ASO by intravitreal (IVT) injection and rhodopsin mRNA and protein expression were measured. Transgenic rats expressing the murine P23H rhodopsin gene (P23H transgenic rat Line 1) were administered either a mouse-specific P23H ASO or a control ASO. The contralateral eye was injected with PBS and used as a comparator control. Electroretinography (ERG) measurements and analyses of the retinal outer nuclear layer were conducted and correlated with rhodopsin mRNA levels. Rhodopsin mRNA and protein expression was reduced after a single ASO injection in wild-type mice with a rhodopsin-specific ASO. Transgenic rat eyes that express a murine P23H rhodopsin gene injected with a murine P23H ASO had a 181 ± 39% better maximum amplitude response (scotopic a-wave) as compared with contralateral PBS-injected eyes; the response in control ASO eyes was not significantly different from comparator contralateral eyes. Morphometric analysis of the outer nuclear layer showed a significantly thicker nuclear layer in eyes injected with murine P23H ASO (18%) versus contralateral PBS-injected eyes. Allele-specific ASO-mediated knockdown of mutant P23H rhodopsin expression slowed the rate of photoreceptor degeneration and preserved the function of photoreceptor cells in eyes of the P23H rhodopsin transgenic rat. Our data indicate that ASO treatment is a potentially effective therapy for the treatment of retinitis pigmentosa.

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.

Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats.

Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision. J. Comp. Neurol. 522:2928–2950, 2014. © 2014 Wiley Periodicals, Inc.

XPORT-Dependent Transport of TRP and Rhodopsin

TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing, and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G protein-coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step toward solving a longstanding problem inthe successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.

Rescue of Photoreceptor Degeneration by Curcumin in Transgenic Rats with P23H Rhodopsin Mutation

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.

Circadian dysfunction in P23H rhodopsin transgenic rats: effects of exogenous melatonin

This study focuses on the effects of retinal degeneration on the circadian patterns of P23H rats, as well as on the effect of exogenous melatonin administration. To this end, the body temperature of P23H and Sprague-Dawley rats was continuously monitored and their retinas examined at different stages of degeneration, by means of histological labeling and electroretinogram recordings. Melatonin (2 mg/kg BW/day) was supplied ad libitum throughout the experiment to a subset of animals. The body temperature recordings from wild-type and mutant animals showed no differences in the periodogram and the pattern of the mean waveform. However, a progressive decrease in the relative amplitude of the rhythm (RA), a decline in the coupling strength of the rhythm to environmental zeitgebers (interdaily stability, IS) and increased rhythm fragmentation (intradaily variability, IV) were observed in P23H rats, when compared to wild-type animals. The P23H animals showed a progressive decrease in light-induced retinal responses until reaching 18 months of age. By this age, all photoreceptors had already disappeared, and no responses were found in the EGRs. Exogenous administration of melatonin improved the visual response of P23H rats. In fact, the maximum b-wave recorded at 14 months of age was significantly higher in melatonin-treated P23H rats than in the control animals. Furthermore, the maximum b-wave recorded for P23H rats at the age of 14 months significantly correlated with RA, IS, and IV. This leads us to conclude that vision loss in P23H rats is correlated with a progressive fragmentation of their circadian patterns. Both effects are partially reversed by melatonin administration.

Transplantation of Photoreceptor and Total Neural Retina Preserves Cone Function in P23H Rhodopsin Transgenic Rat

BackgroundTransplantation as a therapeutic strategy for inherited retinal degeneration has been historically viewed to restore vision as a method by replacing the lost retinal cells and attempting to reconstruct the neural circuitry with stem cells, progenitor cells and mature neural retinal cells.Methods and FindingsWe present evidence for an alternative strategy aimed at preventing the secondary loss of cones, the most crucial photoreceptors for vision, by transplanting normal photoreceptors cells into the eye of the P23H rat, a model of dominant retinitis pigmentosa. We carried out transplantation of photoreceptors or total neural retina in 3-month-old P23H rats and evaluated the function and cell counts 6 months after surgery. In both groups, cone loss was significantly reduced (10%) in the transplanted eyes where the cone outer segments were found to be considerably longer. This morphological effect correlated with maintenance of the visual function of cones as scored by photopic ERG recording, but more precisely with an increase in the photopic b-wave amplitudes by 100% and 78% for photoreceptor transplantation and whole retinal transplantation respectively.ConclusionsWe demonstrate here that the transplanted tissue prevents the loss of cone function, which is further translated into cone survival.

Restoration of visual function in P23H rhodopsin transgenic rats by gene delivery of BiP/Grp78

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.

Photoreceptor Protection by Adeno-Associated Virus–Mediated LEDGF Expression in the RCS Rat Model of Retinal Degeneration: Probing the Mechanism

Lens epithelium-derived growth factor (LEDGF) is upregulated in response to stress and enhances the survival of neurons in the retina and optic nerve, as well as a wide range of other cells, such as fibroblasts and keratinocytes. Photoreceptor protection was investigated in the RCS rat retinal degeneration model after Ledgf delivery with an adeno-associated virus (AAV) and the mechanism of protection explored. Thirty-six RCS and nine P23H rats had bilateral subretinal injections of AAV-Ledgf in one eye and buffer in the contralateral eye as the control. Retinal function was evaluated 8 weeks later by the electroretinogram and compared with photoreceptor cell layer count. LEDGF mRNA and protein levels and mRNA levels of known stress-related factors were compared in treated and control retinas to explore the mechanism of LEDGF protection. Nine RCS rats were treated with adenovirus-heat shock protein 27 (Ad-HSP27) and examined for protection. Significant photoreceptor protection was evident functionally and morphologically in 65% to 100% of the RCS rats treated at early ages of up to 7 weeks. Cell protection was more prominent in the superior retinal hemisphere which has a slower natural degeneration rate in untreated eyes. Although many of the heat shock proteins and other stress-related genes showed significant elevation in the AAV-Ledgf-treated eyes, all increases were approximately twofold or less. Transduction of retinal cells with Ad-HSP27 also resulted in photoreceptor protection. AAV-Ledgf elicited no photoreceptor functional protection in P23H rhodopsin transgenic rat retina. Chronic LEDGF treatment via AAV-Ledgf administration gave successful protection of photoreceptors in the RCS rat retina and retarded cell death by about 2 weeks. Induction of heat shock proteins also gave photoreceptor protection. However, compelling evidence was not found that LEDGF protection was associated with upregulation of heat shock proteins.