Rhodopsin-regulated Insulin Receptor Signaling Pathway in Rod Photoreceptor Neurons

The retina is an integral part of the central nervous system and retinal cells are known to express insulin receptors (IR), although their function is not known. This article describes recent studies that link the photoactivation of rhodopsin to tyrosine phosphorylation of the IR and subsequent activation of phosphoinositide 3-kinase, a neuron survival factor. Our studies suggest that the physiological role of this process is to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis. We focus mainly on our recently identified regulation of the IR pathway through the G-protein-coupled receptor rhodopsin. Various mutant and knockout proteins of phototransduction cascade have been used to study the light-induced activation of the retinal IR. Our studies suggest that rhodopsin may have additional previously uncharacterized signaling functions in photoreceptors.     

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.     

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.     

Suppression of constant-light-induced blindness but not retinal degeneration by inhibition of the rhodopsin degradation pathway

BACKGROUND: Continuous exposure to light, even at relatively low intensities, leads to retinal damage and blindness in wild-type animals. However, the molecular mechanisms underlying constant-light-induced blindness are poorly understood. It has been presumed that the visual impairment resulting from long-term, continuous exposure to ambient light is a secondary consequence of the effects of light on retinal morphology, but this has not been addressed. RESULTS: To characterize the mechanism underlying light-induced blindness, we applied a molecular genetic approach using the fruit fly, Drosophila melanogaster. We found that the temporal loss of the photoresponse was paralleled by a gradual decline in the concentration of rhodopsin. The decline in rhodopsin and the visual response were suppressed by a C-terminal truncation of rhodopsin, by mutations in arrestin, and by elimination of a lysosomal protein, Sunglasses. Conversely, the visual impairment was greatly enhanced by mutation of the rhodopsin phosphatase, rdgC. Surprisingly, the mutations that suppressed light-induced blindness did not reduce the severity of the retinal degeneration resulting from constant light. Moreover, mutations known to suppress retinal degeneration did not ameliorate the light-induced blindness. CONCLUSIONS: These data demonstrate that the constant light-induced blindness and retinal degeneration result from defects in distinct molecular pathways. Our results support a model in which visual impairment caused by continuous illumination occurs through an arrestin-dependent pathway that promotes degradation of rhodopsin.     

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.     

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.     

Enhanced Retinal Insulin Receptor-activated Neuroprotective Survival Signal in Mice Lacking the Protein-tyrosine Phosphatase-1B Gene

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated in the negative regulation of insulin signaling. We previously demonstrated that light-induced tyrosine phosphorylation of the retinal insulin receptor (IR) results in the activation of phosphoinositide 3-kinase/Akt survival pathway in rod photoreceptor cells. The molecular mechanism behind light-induced activation of IR is not known. We investigated the in vivo mechanism of IR activation and found that PTP1B activity in dark-adapted retinas was significantly higher than in light-adapted retinas. We made a novel finding in this study that the light-dependent regulation of PTP1B activity is signaled through photobleaching of rhodopsin. Conditional deletion of PTP1B in rod photoreceptors by the Cre-loxP system resulted in enhanced IR signaling. Further PTP1B activity negatively regulated the neuroprotective survival signaling in the retina. One of the challenging questions in the retina research is how mutations in human rhodopsin gene slowly disable and eventually disrupt photoreceptor functions. Our studies suggest that a defect in the photobleaching of rhodopsin and mutation in rhodopsin gene enhances the activity of PTP1B, and this activated activity could down-regulate the IR survival signaling. Our studies suggest that PTP1B antagonists could be potential therapeutic agents to treat stress-induced photoreceptor degenerations and provide further evidence that rhodopsin photoexcitation may trigger signaling events alternative to the classic phototransduction.     

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 diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell‐autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild‐type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild‐type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival‐promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival‐promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival‐promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 475–490, 1999     

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.     

Engineering of an artificial light-modulated potassium channel

Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.     

Rhodopsin activation causes retinal degeneration in Drosophila rdgC mutant

Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.     

Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors

Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.     

Regulation of phototransduction responsiveness and retinal degeneration by a phospholipase D-generated signaling lipid

Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld(null) flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.     

Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration

4-Hydroxynonenal (4-HNE) is a reactive aldehyde species generated endogenously from the nonenzymatic oxidation of n-6 polyunsaturated fatty acids under physiological conditions. We have reported that intense white light exposure increases 4-HNE-protein modification in the retina prior to the onset of photoreceptor cell apoptosis. To understand the molecular mechanism(s) underlying the retinal degeneration induced by photooxidative stress, we identified 4-HNE-modified retinal proteins using a proteomic approach. Albino rats were exposed to 5 k lx white fluorescent light for 3 h and retinas were removed 24 h later and pooled. By Western dot blot analysis, the total intensity of 4-HNE-modified proteins was increased 1.5-fold following the exposure compared to dim light controls. In two independent sets of two-dimensional gel electrophoresis/Western blots followed by peptide mass fingerprinting (PMF), nine proteins including voltage-dependent anion channel, enolase 1, aldolase C, crystallins A and βB3, heterogeneous nuclear ribonucleoprotein A2/B1, albumin, and glutamine synthetase were identified. We observed that 4-HNE modifications of retinal proteins are specific to a particular set of proteins rather than random events on abundant proteins. By immunohistochemistry, localization of 3 identified proteins overlapped with immunoreactivity of 4-HNE-modified proteins in light-exposed retinas. Intense light exposure increases 4-HNE-protein modifications on specific retinal proteins in several functional categories including energy metabolism, glycolysis, chaperone, phototransduction, and RNA processing. Together with previous reports that 4-HNE modification changes protein activities, these results suggest a close association of 4-HNE-protein modifications with the initiation of light-induced retinal degeneration.     

Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration

4-Hydroxynonenal (4-HNE) is a reactive aldehyde species generated endogenously from the nonenzymatic oxidation of n-6 polyunsaturated fatty acids under physiological conditions. We have reported that intense white light exposure increases 4-HNE-protein modification in the retina prior to the onset of photoreceptor cell apoptosis. To understand the molecular mechanism(s) underlying the retinal degeneration induced by photooxidative stress, we identified 4-HNE-modified retinal proteins using a proteomic approach. Albino rats were exposed to 5 k lx white fluorescent light for 3 h and retinas were removed 24 h later and pooled. By Western dot blot analysis, the total intensity of 4-HNE-modified proteins was increased 1.5-fold following the exposure compared to dim light controls. In two independent sets of two-dimensional gel electrophoresis/Western blots followed by peptide mass fingerprinting (PMF), nine proteins including voltage-dependent anion channel, enolase 1α, aldolase C, crystallins αA and βB3, heterogeneous nuclear ribonucleoprotein A2/B1, albumin, and glutamine synthetase were identified. We observed that 4-HNE modifications of retinal proteins are specific to a particular set of proteins rather than random events on abundant proteins. By immunohistochemistry, localization of 3 identified proteins overlapped with immunoreactivity of 4-HNE-modified proteins in light-exposed retinas. Intense light exposure increases 4-HNE-protein modifications on specific retinal proteins in several functional categories including energy metabolism, glycolysis, chaperone, phototransduction, and RNA processing. Together with previous reports that 4-HNE modification changes protein activities, these results suggest a close association of 4-HNE-protein modifications with the initiation of light-induced retinal degeneration.     

Drosophila mosaic screen identifies diehard mutants as norpA P24 suppressors

It is well-known that malfunctioning of the phototransduction mechanism stimulates the cell death machinery, resulting in retinal degeneration both in vertebrates and invertebrates. Genetic screens have often failed to identify modifiers underlying such degenerative syndromes because of the lethality associated with their fundamental function during development. A norpA ^P24 suppressor screen was successful performed with mosaic flies generated by the yeast site-specific recombination FLP-FRT system. Three independent diehard mutants, identified by mutagenizing the 2L FRT chromosome in the norpA ^P24 background, include both essential and non-essential mutations. Their suppressive effects on the norpA ^P24 -triggered retinal degeneration depend on the intensity and duration of light based on a deep pseudopupil analysis and histology. These results suggest that the suppressor screen using mosaic flies provides a valuable tool for recovering both essential and non-essential genes within the fundamental genetic pathways. Further studies of diehard mutants will provide insights into the disease mechanism that underlies the retinal degeneration of phototransduction mutants.     

Unknown mechanisms of the GPCR-signaling cascade in vertebrate photoreceptors

Among the GPCR-signaling cascades, phototransduction in vertebrate retinal photoreceptors has been characterized in unprecedented details. It is believed that basic mechanisms of phototransduction and adaptation are reliably and completely established, and phototransduction may serve as a benchmark for understanding other G-protein-coupled systems. In this review, we compare present scheme of phototransduction with other GPCR-cascades in order to reveal their similarities and specific features. We show, based mainly on our physiological and biophysical data, that the existing scheme misses a few important regulations whose molecular basis is unknown. There exists a fast and efficient mechanism that accelerates the turn-off of the activated G-protein (transducin) during light adaptation. A few slowly acting processes result in a long-lasting modification of the cascade's components and regulate the speed of rhodopsin and transducin quenching. Similarly to other GPCR-cascades, one may suggest that there are multiple signalling pathways that start from photoactivated rhodopsin and rely on different secondary messengers (e.g. cAMP vs. cGMP). We also show that rhodopsin in retinal rods may form areas of paracristalline organization, and that the oligomerization might be a mechanism for controlling the amplification of the signalling cascade. The missing mechanisms are by no means minor, and could ensure sensitivity regulation within two orders of magnitude range.     

An Unexpected Diversity of Photoreceptor Classes in the Longfin Squid,Doryteuthis pealeii

Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.