Constitutive opsin signaling: night blindness or retinal degeneration?

A subset of genetic mutations in photoreceptor-specific genes results in abnormally prolonged activation of transducin-mediated photosignaling in rod cells. In humans and animal models, these mutations cause visual dysfunctions ranging from a mild stationary night blindness to severe, early-onset retinal degeneration. There are mechanistic differences between mutations causing night blindness and those causing retinal degeneration. Here, we hypothesize that mutations causing continuous activation of the visual cascade as the result, for example, of the inability of the photoreceptor to regenerate rhodopsin, lead to retinal degeneration; those mutations that can terminate signaling, even if only partially and intermittently, slow the rate of degeneration sufficiently to give rise to stationary night blindness. Furthermore, we hypothesize that a prolonged decrease in intracellular calcium concentration resulting from persistent activation is responsible for triggering apoptotic rod-cell death.     

Unfolding retinal dystrophies: a role for molecular chaperones?

Inherited retinal dystrophy is a major cause of blindness worldwide. Recent molecular studies have suggested that protein folding and molecular chaperones might play a major role in the pathogenesis of these degenerations. Incorrect protein folding could be a common consequence of causative mutations in retinal degeneration disease genes, particularly mutations in the visual pigment rhodopsin. Furthermore, several retinal degeneration disease genes have recently been identified as putative facilitators of correct protein folding, molecular chaperones, on the basis of sequence homology. We also consider whether manipulation of chaperone levels or chaperone function might offer potential novel therapies for retinal degeneration.     

A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila

Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. Here, we uncover a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism. This process involves the formation of membrane complexes of phosphorylated, activated rhodopsin and its inhibitory protein arrestin, and subsequent clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment. Together, these data define the proapoptotic molecules in Drosophila photoreceptors and indicate a novel signaling pathway for light-activated rhodopsin molecules in control of photoreceptor viability.     

Identification of novel rhodopsin mutations associated with retinitis pigmentosa by GC-clamped denaturing gradient gel electrophoresis.

Retinitis pigmentosa (RP) is a group of disorders characterized by progressive degeneration of the outer retina, resulting in night blindness, visual field loss, an abnormal electroretinogram, and characteristic retinal pigmentary changes. An important step in the understanding of RP has been the recognition that some cases of autosomal dominant RP (ADRP) are caused by mutations in the rhodopsin gene. Multiple different point mutations within the coding sequence of the rhodopsin gene have been associated with ADRP. We have developed a GC-clamped denaturing-gradient-gel electrophoresis (DGGE) assay for the coding region of the rhodopsin gene and have used this assay to screen ADRP patients for mutations. The assay consists of amplifying with PCR the five exons of the rhodopsin gene and then analyzing each PCR product by DGGE. We have used this assay to detect three previously unreported rhodopsin base substitutions associated with ADRP. The use of this assay to identify ADRP patients who have various rhodopsin mutations has allowed us to begin studies seeking to correlate molecular genotype with clinical phenotype. Furthermore, GC-clamped DGGE has allowed us to identify families with ADRP not caused by a rhodopsin mutation. Such families will be important in the search for other genes involved in ADRP.     

Light adaptation through phosphoinositide-regulated translocation of Drosophila visual arrestin

Photoreceptor cells adapt to bright or continuous light, although the molecular mechanisms underlying this phenomenon are incompletely understood. Here, we report a mechanism of light adaptation in Drosophila, which is regulated by phosphoinositides (PIs). We found that light-dependent translocation of arrestin was defective in mutants that disrupt PI metabolism or trafficking. Arrestin bound to PIP(3) in vitro, and mutation of this site delayed arrestin shuttling and resulted in defects in the termination of the light response, which is normally accelerated by prior exposure to light. Disruption of the arrestin/PI interaction also suppressed retinal degeneration caused by excessive endocytosis of rhodopsin/arrestin complexes. These findings indicate that light-dependent trafficking of arrestin is regulated by direct interaction with PIs and is required for light adaptation. Since phospholipase C activity is required for activation of Drosophila phototransduction, these data point to a dual role of PIs in phototransduction.     

Unusual thermal and conformational properties of the rhodopsin congenital night blindness mutant Thr-94 --> Ile

Naturally occurring point mutations in the opsin gene cause the retinal diseases retinitis pigmentosa and congenital night blindness. Although these diseases involve similar mutations in very close locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal degeneration. We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site. T94I mutant rhodopsin folded properly and was able to bind 11-cis-retinal to form chromophore, but it showed a blue-shifted visible band at 478 nm and reduced molar extinction coefficient. Furthermore, T94I showed dramatically reduced thermal stability, extremely long lived metarhodopsin II intermediate, and highly increased reactivity toward hydroxylamine in the dark, when compared with wild type rhodopsin. The results are consistent with the location of Thr-94 in close proximity to Glu-113 counterion in the vicinity of the Schiff base linkage and suggest a role for this residue in maintaining the correct dark inactive conformation of the receptor. The reported results, together with previously published data on the other two known congenital night blindness mutants, suggest that the molecular mechanism underlying this disease may not be structural misfolding, as proposed for retinitis pigmentosa mutants, but abnormal functioning of the receptor by decreased thermal stability and/or constitutive activity.     

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp(14) with an amino acid substitution juxtaposed to the TRP domain. The trp(14) flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp(Delta)(1272), had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na(+)/Ca(2+) exchanger, CalX, indicating that the cell death was due primarily to deficient Ca(2+) entry rather than disruption of the TRP-anchoring function.     

视紫红质及RPE65蛋白在光致视网膜变性疾病中的作用机制

视紫红质是感光细胞中的一种视色素,在光线的接收和视觉电位的产生方面具有重要的生理作用,由视紫红质介导的过度光信号传导是光性视网膜变性的主要原因。近年的研究表明,视网膜色素上皮细胞中的RPE65蛋白作为影响视紫红质再生的关键因素,与视网膜光损伤的易感性密切相关。就视紫红质和RPE65蛋白在光致视网膜变性中的作用机理作一探讨。     

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.     

Systemic administration of the iron chelator deferiprone protects against light-induced photoreceptor degeneration in the mouse retina

Oxidative stress plays a key role in a light-damage (LD) model of retinal degeneration as well as in age-related macular degeneration (AMD). Since iron can promote oxidative stress, the iron chelator deferiprone (DFP) was tested for protection against light-induced retinal degeneration. To accomplish this, A/J mice were treated with or without oral DFP and then were placed in constant bright white fluorescent light (10,000 lx) for 20 h. Retinas were evaluated at several time points after light exposure. Photoreceptor apoptosis was assessed using the TUNEL assay. Retinal degeneration was assessed by histology 10 days after exposure to damaging white light. Two genes upregulated by oxidative stress, heme oxygenase 1 (Hmox1) and ceruloplasmin (Cp), as well as complement component 3 (C3) were quantified by RT-qPCR. Cryosections were immunolabeled for an oxidative stress marker (nitrotyrosine), a microglial marker (Iba1), as well as both heavy (H) and light (L) ferritin. Light exposure resulted in substantial photoreceptor-specific cell death. Dosing with DFP protected photoreceptors, decreasing the numbers of TUNEL-positive photoreceptors and increasing the number of surviving photoreceptors. The retinal mRNA levels of oxidative stress-related genes and C3 were upregulated following light exposure and diminished by DFP treatment. Immunostaining for nitrotyrosine indicated that DFP reduced the nitrative stress caused by light exposure. Robust H/L-ferritin-containing microglial activation and migration to the outer retina occurred after light exposure and DFP treatment reduced microglial invasion. DFP is protective against light-induced retinal degeneration and has the potential to diminish oxidative stress in the retina.     

A lysosomal tetraspanin associated with retinal degeneration identified via a genome-wide screen

The Drosophila visual system has provided a model to study phototransduction and retinal degeneration. To identify new candidate proteins that contribute to these processes, we conducted a genome-wide screen for genes expressed predominately in the eye, using DNA microarrays. This screen appeared to be comprehensive as it led to the identification of all 22 eye-enriched genes previously shown to function in phototransduction or implicated in retinal degeneration. In addition, we identified 93 eye-enriched genes whose roles have not been previously defined. One of the eye-enriched genes encoded a member of a large family of transmembrane proteins, referred to as tetraspanins. We created a null mutation in the eye-enriched tetraspanin, Sunglasses (Sun), which resulted in light-induced retinal degeneration. We found that the Sun protein was distributed primarily in lysosomes, and functioned in a long-known but poorly understood phenomenon of light-induced degradation of rhodopsin. We propose that lysosomal tetraspanins in mammalian cells may also function in the downregulation of rhodopsin and other G-protein-coupled receptors, in response to intense or prolonged agonist stimulation.     

New retinal light damage QTL in mice with the light-sensitive RPE65 LEU variant

The purpose of this study was to determine the QTL that influence acute, light-induced retinal degeneration differences between the BALB/cByJ and 129S1/SvImJ mouse strains. Five- to 6-week-old F₂ progeny of an intercross between the two strains were exposed to 15,000 LUX of white light for 1 h after their pupils were dilated, placed in the dark for 16 h, and kept for 10–12 days in dim cyclic light before retinal rhodopsin was measured spectrophotometrically. This was used as the quantitative trait for retinal degeneration. Neither gender nor pigmentation had a significant influence on the amount of rhodopsin after light exposure in the F₂ progeny. For genetic study, DNAs of the 27–36 F₂ progeny with the highest and 27–36 F₂ with the lowest levels of rhodopsin after light exposure were genotyped with 71 dinucleotide repeat markers spanning the genome. Any marker with a 95% probability of being associated with phenotype was tested in all 289 F₂ progeny. Data were analyzed with Map Manager QTX. Significant QTL were found on mouse Chrs 1 and 4, and suggestive QTL on Chrs 6 and 2. The four QTL together equal an estimated 78% of the total genetic effect, and each of the QTL represents a gene with BALB/c susceptible alleles. The Chr 6 QTL is in the same region as a highly significant age-related retinal degeneration QTL found previously. Identification of these QTL is a first step toward identifying the modifier genes/alleles they represent, and identification of the modifiers may provide important information for human retinal diseases that are accelerated by light exposure.     

Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration

Mutations in the human transmembrane protein CRB1 are associated with severe forms of retinal dystrophy, retinitis pigmentosa 12 (RP12), and Leber's congenital amaurosis (LCA). The Drosophila homolog, crumbs, is required for polarity and adhesion in embryonic epithelia and for correct formation of adherens junctions and proper morphogenesis of photoreceptor cells. Here, we show that mutations in Drosophila crumbs result in progressive, light-induced retinal degeneration. Degeneration is prevented by expression of p35, an inhibitor of apoptosis, or by reduction of rhodopsin levels through a vitamin A-deficient diet. In the dark, rhabdomeres survive but exhibit morphogenetic defects. We demonstrate that it is the extracellular portion of the Crumbs protein that is essential to suppress light-induced programmed cell death, while proper morphogenesis depends on the intracellular part. We conclude that human and Drosophila Crumbs proteins are functionally conserved to prevent light-dependent photoreceptor degeneration. This experimental system is now ideally suited to study the genetic and molecular basis of RP12- and LCA-related retinal degeneration.     

Suppressing autophagy protects photoreceptor cells from light-induced injury

Autophagy, a conserved cellular self-degradation process, not only serves to protect cells at critical times during development and nutrient stress, but also contributes to cell death. Photoreceptor cells are unique neurons which when directly exposed to the light, transduces light stimuli into visual signal. However, intense light exposure can be cytotoxic to the retina. So far, the precise mechanism underlying retina light injury remains unknown, and the effective therapy is still unavailable. Here, we found that visible light exposure activated the mitogen-activated protein kinases (MAPK) pathway and led to remarkable autophagy in photoreceptor cells (661W cells). Directly blocking autophagy with 3MA or LY294002 markedly attenuated light-induced death in 661W cells. Among the activated downstream factors of MAPK pathway, ERK, not JNK or p-38, played a critical role in light-induced death mechanism. Inhibiting the activation of ERK with its specific inhibitor PD98059 significantly suppressed light-induced autophagy and protected 661W cells from light injury. These results indicate that autophagy is an essential event in light-induced photoreceptor death and that directly blocking autophagy or suppressing autophagy by inhibiting the ERK pathway could effectively attenuates light-induced damage. These observations may have a potential application in the treatment of retinal light injury.     

Accumulation of Rhodopsin in Late Endosomes Triggers Photoreceptor Cell Degeneration

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.     

无脉络膜症的发病机制及诊疗进展

无脉络膜(CHM)是一种X染色体连锁的遗传性进行性视网膜色素上皮,光感受细胞和脉络膜血管逐渐退化,最终致盲的疾病。该病是由于位于Xq21上的REP-1缺失突变导致失活,导致CHM基因不能表达,从而出现脉络膜血管层发育障碍,进行性视网膜色素上皮和脉络膜营养不良,变性及进行性脉络膜萎缩消失。男性患者一般在十几岁至二十几岁时开始出现夜盲,周边视野逐渐丧失,形成管状视野,严重者仅剩5-10度的中央视野,最终失明。女性携带者大多无症状。     

Increased Light Exposure Alleviates One Form of Photoreceptor Degeneration Marked by Elevated Calcium in the Dark

Background

In one group of gene mutations that cause photoreceptor degeneration in human patients, guanylyl cyclase is overactive in the dark. The ensuing excess opening of cGMP-gated cation channels causes intracellular calcium to rise to toxic levels. The Y99C mutation in guanylate cyclase-activating protein 1 (GCAP1) has been shown to act this way. We determined whether prolonged light exposure, which lowers cGMP levels through activation of phototransduction, might protect photoreceptors in a line of transgenic mice carrying the GCAP1-Y99C.

Methodology/Principal Findings

We reared cohorts of GCAP1-Y99C transgenic mice under standard cyclic, constant dark and constant light conditions. Mouse eyes were analyzed by histology and by immunofluorescence for GFAP upregulation, a non-specific marker for photoreceptor degeneration. Full-field electroretinograms (ERGs) were recorded to assess retinal function. Consistent with our hypothesis, constant darkness accelerated disease, while continuous lighting arrested photoreceptor degeneration.

Conclusions/Significance

In contrast to most forms of retinal degeneration, which are exacerbated by increased exposure to ambient light, a subset with mutations that cause overly active guanylyl cyclase and high intracellular calcium benefitted from prolonged light exposure. These findings may have therapeutic implications for patients with these types of genetic defects.     

Light-induced calcium release in isolated intact cattle rod outer segments upon photoexcitation of rhodopsin.

By applying flash-spectrophotometry with the calcium-indicating dye arsenazo III rapid light-triggered calcium release in various cattle rod outer segment preparations was studied. It is shown that light-induced calcium signals can be unambiguously discriminated from underlying absorption changes due to photolysis of rhodopsin and apparent absorption changes resulting from lightscattering transients. The following results have been obtained: 1. Calcium-induced arsenazo III responses can be quantitatively and kinetically resolved within the time domain of the visual transduction process. 2. Photoexcitation of rhodopsin results in calcium release from intradiscal binding sites. 3. Calcium released does not appear in the cytoplasmic space unless the disc membrane is made permeable to calcium ions by an ionophore. 4. The shortest observed half-rise time of calcium release (300 ms) is possibly limited by the ionophore. 5. The stoichiometric ratio of calcium released/rhodopsin bleached is 0.5 at a free calcium concentration of 2 microM. The amount of calcium released is proportional to the precentage of rhodopsin bleaching (from 1--10%). 6. Upon disruption of the disc stack by lysis of intact rod outer segments the light-induced calcium release is greatly altered. The results are discussed in relation to previous reports on a light-induced calcium release from retinal discs and in terms of the proposed role of calcium as an intracellular transmitter in vertebrate photoreceptors.     

Development and Regulation of Rhodopsin Kinase in Rat Pineal and Retina

Rhodopsin kinase, once thought to be a retinal enzyme, was recently found at high levels in the pineal gland. In the present study the developmental pattern and the regulation by environmental lighting of this enzyme in both tissues was studied in the rat. Enzyme activity was present in the neonatal pineal gland several days earlier than in the retina, and increased gradually up to 20 days of age and remained at that level thereafter; the retinal enzyme appeared to increase until day 60. Pineal and retinal rhodopsin kinase activities showed a 25% increase in in the middle of the dark and the beginning of the light period, respectively. Exposure to constant light caused a 50% decrease in rhodopsin kinase levels in both tissues. However, only pineal rhodopsin kinase activity declined followed bilateral superior cervical ganglionectomy. This indicates pineal rhodopsin kinase activity is similar to other pineal enzymes in that it is controlled by light acting through the sympathetic nervous system. In contrast, the light-induced decrease in retinal rhodopsin kinase may be due to the direct destructive effect of light on the retina. The finding of neural control of pineal rhodopsin kinase in the pineal gland of adult rats is consistent with a function of the enzyme in the neural regulation of pineal function.