Degeneration Of Retinal Photoreceptor Cells Research Articles

Sulforaphane and ophthalmic diseases

AbstractSulforaphane (SFN) is an organosulfur compound categorized as an isothiocyanate (ITC), primarily extracted from cruciferous vegetables like broccoli and cabbage. The molecular formula of sulforaphane (SFN) is C6H11NOS2. SFN is generated by the hydrolysis of glucoraphanin (GRP) through the enzyme myrosinase, showing notable properties including anti‐diabetic, anti‐inflammatory, antimicrobial, anti‐angiogenic, and anticancer attributes. Ongoing clinical trials are investigating its potential in diseases such as cancer, neurodegenerative diseases, diabetes‐related complications, chronic kidney disease, cardiovascular disease, and liver diseases. Several animal carcinogenesis models and cell culture models have shown it to be a very effective chemopreventive agent, and the protective effects of SFN in ophthalmic diseases have been linked to multiple mechanisms. In murine models of diabetic retinopathy and age‐related macular degeneration, SFN delays retinal photoreceptor cell degeneration through the Nrf2 antioxidative pathway, NF‐κB pathway, AMPK pathway, and Txnip/mTOR pathway. In rabbit models of keratoconus and cataract, SFN has been shown to protect corneal and lens epithelial cells from oxidative stress injury by activating the Keap1‐Nrf2‐ARE pathway and the Nrf‐2/HO‐1 antioxidant pathway. Oral delivery or intraperitoneal injection at varying concentrations are the primary strategies for SFN intake in current preclinical studies. Challenges remain in the application of SFN in eye disorders due to its weak solubility in water and limited bioavailability because of the presence of blood–ocular barrier systems. This review comprehensively outlines recent research on SFN, elucidates its mechanisms of action, and discusses potential therapeutic benefits for eye disorders such as age‐related macular degeneration (AMD), diabetic retinopathy (DR), cataracts, and other ophthalmic diseases, while also indicating directions for future clinical research to achieve efficient SFN treatment for ophthalmic diseases.

AAV-Mediated Gene Augmentation Therapy Restores Critical Functions in Mutant PRPF31+/− iPSC-Derived RPE Cells

Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild-type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSCs) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and it partially restored structure and barrier function. These results suggest that AAV-based gene therapy targeting RPE cells holds therapeutic promise for patients with PRPF31-related RP.

A Combination of G Protein-Coupled Receptor Modulators Protects Photoreceptors from Degeneration.

Degeneration of retinal photoreceptor cells can arise from environmental and/or genetic causes. Since photoreceptor cells, the retinal pigment epithelium (RPE), neurons, and glial cells of the retina are intimately associated, all cell types eventually are affected by retinal degenerative diseases. Such diseases often originate either in rod and/or cone photoreceptor cells or the RPE. Of these, cone cells located in the central retina are especially important for daily human activity. Here we describe the protection of cone cells by a combination therapy consisting of the G protein-coupled receptor modulators metoprolol, tamsulosin, and bromocriptine. These drugs were tested in Abca4-/-Rdh8-/- mice, a preclinical model for retinal degeneration. The specificity of these drugs was determined with an essentially complete panel of human G protein-coupled receptors. Significantly, the combination of metoprolol, tamsulosin, and bromocriptine had no deleterious effects on electroretinographic responses of wild-type mice. Moreover, putative G protein-coupled receptor targets of these drugs were shown to be expressed in human and mouse eyes by RNA sequencing and quantitative polymerase chain reaction. Liquid chromatography together with mass spectrometry using validated internal standards confirmed that metoprolol, tamsulosin, and bromocriptine individually or together penetrate the eye after either intraperitoneal delivery or oral gavage. Collectively, these findings support human trials with combined therapy composed of lower doses of metoprolol, tamsulosin, and bromocriptine designed to safely impede retinal degeneration associated with certain genetic diseases (e.g., Stargardt disease). The same low-dose combination also could protect the retina against diseases with complex or unknown etiologies such as age-related macular degeneration.

Screening for mutations in RPGR and RP2 genes in Jordanian families with X-linked retinitis pigmentosa.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disease causing progressive degeneration of retinal photoreceptor cells. X-linked RP (XLRP), in which photoreceptor degeneration begins in early childhood and complete blindness often occurs by the fourth decade of life, constitutes the most severe form of this disease. Two genes commonly associated with XLRP have previously been cloned: retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 (RP2). We sought to identify mutations in these genes in Jordanian families suffering from this disease. Five unrelated Jordanian families with confirmed XLRP were screened for such mutations using direct sequencing. Three mutations were identified in the ORF15 exon of RPGR. The silent g.ORF15+470G>A substitution and the g.ORF15+1822insA insertion in the 3ꞌ-untranslated region were found in both normal and affected male family members at comparable frequencies, and thus were considered normal variants. The third mutation, g.ORF15+588G>A, in which alanine is substituted by threonine, was found in all affected men and one unaffected man in the two families harboring this variant. Thus, this mutation may be pathogenic, but with incomplete penetrance. No RP2 mutations were found among the examined families. Mutation screening of RP patients is essential to understand the mechanism behind this disease and develop treatments. A complete family history is required to identify its inheritance pattern and provide genetic counseling for patients and their families.

Identification of putative SNPs in progressive retinal atrophy affected Canis lupus familiaris using exome sequencing.

Progressive retinal atrophy (PRA) is one of the major causes of retinal photoreceptor cell degeneration in canines. The inheritance pattern of PRA is autosomal recessive and genetically heterogeneous. Here, using targeted sequencing technology, we have performed exome sequencing of 10 PRA-affected (Spitz=7, Cocker Spaniel=1, Lhasa Aphso=1 and Spitz-Labrador cross breed=1) and 6 normal (Spitz=5, Cocker Spaniel=1) dogs. The high-throughput sequencing using 454-Roche Titanium sequencer generated about 2.16 Giga bases of raw data. Initially, we have successfully identified 25,619 single nucleotide polymorphisms (SNPs) that passed the stringent SNP calling parameters. Further, we performed association study on the cohort, and the highly significant (0.001) associations were short-listed and investigated in-depth. Out of the 171 significant SNPs, 113 were previously unreported. Interestingly, six among them were non-synonymous coding (NSC) SNPs, which includes CPPED1 A>G (p.M307V), PITRM1 T>G (p.S715A), APP G>A (p.T266M), RNF213 A>G (p.V1482A), C>A (p.V1456L), and SLC46A3 G>A (p.R168Q). On the other hand, 35 out of 113 unreported SNPs were falling in regulatory regions such as 3'-UTR, 5'-UTR, etc. In-depth bioinformatics analysis revealed that majority of NSC SNPs have damaging effect and alter protein stability. This study highlighted the genetic markers associated with PRA, which will help to develop genetic assay-based screening in effective breeding.

Functional and morphological study of retinal photoreceptor cell degeneration in transgenic rabbits with a Pro347Leu rhodopsin mutation.

To investigate the process of retinal degeneration by analyzing the functional and morphological findings in transgenic rabbits with a Pro347Leu rhodopsin mutation. Wild-type (WT) and transgenic (Tg) rabbits at ages 4, 8 and 12 months were used. We conducted functional evaluation by recording the changes in the pupil response to red and blue light stimulation and the amplitude of the electroretinography (ERG). Morphologically, rod and cone distribution was examined using light and electron microscopy. Immunostaining for the identification of retinal ganglion cells (RGCs) was also confirmed by injecting a TUJ-1 monoclonal antibody. Pupil constriction for infrared pupillography and the a- and b-waves for ERG in Tg rabbits decreased with increasing age; the differences were compared to the age-matched WT rabbits. The subnormal ERG in the Tg rabbits, especially the a-wave decrease and pupil constriction with a long latency time, was induced only during exposure to blue light stimulation at 12 months. Light and electron microscopic findings showed a progressive loss of photoreceptor cells over time manifesting by 8 months in the peripheral retina. Moreover, pyknotic nuclei of the outer nuclear layer in the center of the visual streak were observed. At 12 months, there was disappearance of the rods and ballooning degeneration of the cones. Some remaining RGCs had large cell bodies with long branching dendrites. The changes in the pupil light response and amplitude of the ERG could be used to predict the state of retinal degeneration in the Tg rabbit.

Molecular Genetics of FAM161A in North American Patients with Early-Onset Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a hereditary disease that leads to the progressive degeneration of retinal photoreceptor cells and to blindness. It is caused by mutations in several distinct genes, including the ciliary gene FAM161A, which is associated with a recessive form of this disorder. Recent investigations have revealed that defects in FAM161A represent a rather prevalent cause of hereditary blindness in Israel and the Palestinian territories, whereas they seem to be rarely present within patients from Germany. Genetic or clinical data are currently not available for other countries. In this work, we screened a cohort of patients with recessive RP from North America to determine the frequency of FAM161A mutations in this ethnically-mixed population and to assess the phenotype of positive cases. Out of 273 unrelated patients, only 3 subjects had defects in FAM161A. A fourth positive patient, the sister of one of these index cases, was also identified following pedigree analysis. They were all homozygous for the p.T452Sfx3 mutation, which was previously reported as a founder DNA variant in the Israeli and Palestinian populations. Analysis of cultured lymphoblasts from patients revealed that mutant FAM161A transcripts were actively degraded by nonsense-mediated mRNA decay. Electroretinographic testing showed 30 Hz cone flicker responses in the range of 0.10 to 0.60 microvolts in all cases at their first visit (age 12 to 23) (lower norm = 50 μV) and of 0.06 to 0.32 microvolts at their most recent examination (age 27 to 43), revealing an early-onset of this progressive disease. Our data indicate that mutations in FAM161A are responsible for 1% of recessive RP cases in North America, similar to the prevalence detected in Germany and unlike the data from Israel and the Palestinian territories. We also show that, at the molecular level, the disease is likely caused by FAM161A protein deficiency.

Oxygen-induced retinopathy in mice with retinal photoreceptor cell degeneration

AimsIt is reported that retinal neovascularization seems to rarely co-exist with retinitis pigmentosa in patients and in some mouse models; however, it is not widely acknowledged as a universal phenomenon in all strains of all animal species. We aimed to further explore this phenomenon with an oxygen-induced retinopathy model in mice with retinal photoreceptor cell degeneration. Main methodsOxygen-induced retinopathy of colored and albino mice with rapid retinal degeneration were compared to homologous wild-type mice. The retinas were analyzed using high-molecular-weight FITC-dextran stained flat-mount preparation, hematoxylin and eosin (H&E) stained cross-sections, an immunohistochemical test for vascular endothelial growth factor (VEGF) distribution and Western blotting for VEGF expression after exposure to hyperoxia between postnatal days 17 (P17) and 21. Key findingsLeakage and areas of non-perfusion of the retinal blood vessels were alleviated in the retinal degeneration mice. The number of preretinal vascular endothelial cell nuclei in the retinal degeneration mice was smaller than that in the homologous wild-type mice after exposure to hyperoxia (P<0.01). The degree of oxygen-induced retinopathy was positively correlated with the VEGF expression level. However, the VEGF expression level was lower in the retinal degeneration mice. SignificanceProliferative retinopathy occurred in mice with rapid retinal degeneration, but retinal photoreceptor cell degeneration could partially restrain the retinal neovascularization in this rapid retinal degeneration mouse model.

A new ocular phenotype associated with an unexpected but known systemic disorder and mutation: novel use of genomic diagnostics and exome sequencing

We identified a 28-year-old cognitively normal patient with severe visual loss, absent electrical signals from the photoreceptors by electroretinogram (ERG) and nystagmus due to Leber congenital amaurosis (LCA), associated with...

Microglial response to light‐induced photoreceptor degeneration in the mouse retina

The microglial response elicited by degeneration of retinal photoreceptor cells was characterized in BALB/c mice exposed to bright light for 7 hours and then kept in complete darkness for survival times ranging from 0 hours to 10 days. Photodegeneration resulted in extensive cell death in the retina, mainly in the outer nuclear layer (ONL), where the photoreceptor nuclei are located. Specific immunolabeling of microglial cells with anti-CD11b, anti-CD45, anti-F4/80, anti-SRA, and anti-CD68 antibodies revealed that microglial cells were activated in light-exposed retinas. They migrated to the ONL, changed their morphology, becoming rounded cells with short and thick processes, and, finally, showed immunophenotypic changes. Specifically, retinal microglia began to strongly express antigens recognized by anti-CD11b, anti-CD45, and anti-F4/80, coincident with cell degeneration. In contrast, upregulation of the antigen recognized by anti-SRA was not detected by immunocytochemistry until 6 hours after light exposure. Differences were also observed at 10 days after light exposure: CD11b, CD45, and F4/80 continued to be strongly expressed in retinal microglia, whereas the expression of CD68 and SRA had decreased to near-normal values. Therefore, microglia did not return to their original state after photodegeneration and continued to show a degree of activation. The accumulation of activated microglial cells in affected regions simultaneously with photoreceptor degeneration suggests that they play some role in photodegeneration.

A new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent, cobalt chloride

Retinal photoreceptor cell degeneration was induced by cobalt chloride, a chemical hypoxia-mimicking agent in rodents. Time course and dose–response of photoreceptor cell degeneration in mouse retina after intravitreal injection of cobalt chloride were examined by conventional histological analysis by hematoxylin and eosin staining and in situ terminal dUTP-biotin nick end labeling of DNA fragments (TUNEL) method with the use of paraffin-embedded sections. The dose–response of photoreceptor cell degeneration in rat retina was also examined. Photoreceptor cells progressively degenerated with time and under dose–response relationship. The suitable dose of cobalt chloride for the selective photoreceptor cell degeneration in mice is 10–12 nmol intravitreal injection at the volume of 2 μl. The retinal morphology of the mice 2 weeks after the 10–12 nmol intravitreal injection was similar to that of retinal degeneration in the mutant rd mouse. Retinal damage of total retinal layers was induced by an excessive dose of cobalt chloride. The progression of retinal damage after cobalt chloride injection, measured morphologically, was completed at 1 week. However, nuclear DNA fragmentation, mainly detected at outer nuclear layer by TUNEL, peaked at 48 h after 12 nmol cobalt chloride injection. Thus, the selective photoreceptor cell degeneration induced by cobalt chloride follows DNA fragmentation at outer nuclear layer. The photoreceptor cell degeneration is established optionally by cobalt chloride without use of the retinal degeneration mutant animals. Thus, we have described the development of a new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent.

Cancer-related diseases of the eye: the role of calcium and calcium-binding proteins

The eye provides unique opportunities to study complex biochemical pathways and to describe how components of these pathways contribute to the molecular basis of disease. In this article, the role of calcium-binding proteins in cancer-related diseases of the eye is reviewed. First, paraneoplastic syndromes, or so-called remote effects of cancer, arise from damage to tissues distant from any tumor or its metastases. Many of these syndromes are believed to be immune-mediated. Cancer-associated retinopathy (CAR), a blinding disease due to the degeneration of retinal photoreceptor cells, is one of the best characterized of the paraneoplastic syndromes. The CAR autoantigen has been identified as recoverin, a calcium-binding protein of the EF-hand superfamily. Its features as a calcium-binding protein, along with its function in photoreceptor cells and its role as the CAR autoantigen, are discussed. Next, unlike visual symptoms instigated by a distant tumor, ocular melanoma is the primary malignancy originating in the eye. ALG-2 encodes a pro-apoptotic calcium-binding protein that is down-regulated in ocular melanoma, thus providing these tumor cells with a selective advantage. In addition to background discussion of ALG-2, data describing the expression, cellular localization, and dimerization characteristics of ALG-2 in melanoma cells are presented. Biochemical studies of ALG-2 and its interactions with its target Alix/AIP1 also are presented. Finally, the function of ALG-2 in calcium-induced cell death is discussed. Additional calcium-binding proteins in retina and in ocular tumors are described in relation to different disease entities. Such proteins and their expression in the eye provide valuable examples bridging studies of protein chemistry, cellular function, and human disease.

Clinicopathological features of gastric cancer cases and aberrantly expressed recoverin.

Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin, a retina-specific calcium binding protein, and other retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. Recently we reported that aberrant expression of recoverin was identified in more than 50% of tumor cells and their cell lines from several kinds of cancers, including gastric cancer, lung carcinoma, and other cancers. To elucidate the clinicopathological significance of aberrantly expressed recoverin in cancer cells, we performed immunocytochemical analysis using a monoclonal antibody against human recoverin. Within 18 patients with different clinical stages (I-IV) of gastric cancer, the aberrant expression of recoverin in tumor cells was recognized in 6 out of 18 patients (2 out of 2 stage IA, 1 out of 1 stage IB, 2 out of 3 stage II, 0 out of 7 stage IIIA, 0 out of 1 stage IIIB, and 1 out 4 stage IV). The present data are consistent in part with the previous observations that recoverin-expressing cancer cells induced tumor immunity and provide a favorable prognosis for primary cancer in CAR patients.

An anti-angiogenic state in mice and humans with retinal photoreceptor cell degeneration.

Abnormal angiogenesis accompanies many pathological conditions including cancer, inflammation, and eye diseases. Proliferative retinopathy because of retinal neovascularization is a leading cause of blindness in developed countries. Another major cause of irreversible vision loss is retinitis pigmentosa, a group of diseases characterized by progressive photoreceptor cell degeneration. Interestingly, anecdotal evidence has long suggested that proliferative diabetic retinopathy is rarely associated clinically with retinitis pigmentosa. Here we show that neonatal mice with classic inherited retinal degeneration (Pdeb(rd1)/Pdeb(rd1)) fail to mount reactive retinal neovascularization in a mouse model of oxygen-induced proliferative retinopathy. We also present a comparable human paradigm: spontaneous regression of retinal neovascularization associated with long-standing diabetes mellitus occurs when retinitis pigmentosa becomes clinically evident. Both mouse and human data indicate that reactive retinal neovascularization either fails to develop or regresses when the number of photoreceptor cells is markedly reduced. Our findings support the hypothesis that a functional mechanism underlying this anti-angiogenic state is failure of the predicted up-regulation of vascular endothelial growth factor, although other growth factors may also be involved. Preventive and therapeutic strategies against both proliferative and degenerative retinopathies may emerge from this work.

Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.

Early disappearance of alpha-transducin in light-induced photoreceptor degeneration in albino rats

Progressive degeneration of retinal photoreceptor cells occurs in albino rats when exposed to continuous lighting. Three proteins involved in the phototransduction cascade were immunodetected in these cells after various durations of continuous illumination. We found that S-antigen (arrestin) and rhodopsin immunoreactivities persisted for 1-2 months during the degenerative process, whilst immunoreactivity of the alpha subunit of transducin totally disappeared between day 2 and day 4 of continuous light exposure. This suggests that continuous illumination could impair alpha-transducin synthesis, a possible causal factor of photoreceptor damage.

The gene for the γ-subunit of retinal cGMP-phosphodiesterase is on mouse chromosome 11

Mice carrying the rd mutation are affected with an autosomal recessive disease characterized by the total degeneration of retinal photoreceptor cells, which begins at postnatal day 8 and reaches completion by 3 wk of life. Biochemical studies have led to the proposal that a lesion in cGMP metabolism may be the cause of the rd photoreceptor degeneration, since cGMP reaches abnormally high levels in the rd retina a few days before the morphological pathology starts. The abnormal cGMP level is due to deficient cGMP-phosphodiesterase (cGMP-PDE) activity. A cDNA for the gamma-subunit of mouse cGMP-PDE has recently been cloned and characterized. We have mapped this cDNA to mouse chromosome 11 using a panel comprised of 19 hamster-mouse somatic cell hybrids by Southern blot hybridization. Our results suggest that the structural gene for the gamma-subunit of cGMP-PDE from mouse retina, which we have designated 'Pdeg', is not the primary defect in rd disease, as the locus of this genetic defect is on mouse chromosome 5.

Photoreceptor degeneration after exposure of rats to incandescent illumination.

The retina of the albino rat undergoes degenerative changes when exposed to low intensity incandescent light. The retinal degeneration is limited specifically to the photoreceptor cells, and the pigment epithelium is unaffected. Early changes in the receptors included fragmentation of the inner and outer segments and pyknosis of the receptor cell nuclei. Phagocytic cells invaded and occupied the central retinal area of degeneration, between the receptor layer and the pigment epithelium, in the 4 and 5 day exposure periods. They were absent centrally after 14 and 30 days of exposure, but were present at these time periods in the peripheral retina, where photoreceptor destruction was still in progress. The destruction of photoreceptor cells, including the receptor and outer nuclear layers of the retina, by incandescent light progressed at a slightly reduced rate as compared to that after exposure to fluorescent light of the same intensity. These experiments indicate that exposure to either low intensity incandescent or fluorescent light will cause a selective degeneration of retinal photoreceptor cells, and therefore provide an easily reproducible model for the study of retinal structure and function in the absence of the receptors.