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The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis, and plausible pathogenic variants and clinical phenotype evaluated by the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. Previously unreported, likely pathogenic, non-coding variants in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10 and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5(-/-) and P23H(+/-):Erdj5(-/-) mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin. Funding Agencies|Wellcome TrustWellcome Trust [092621, 205041, 099173]; Retina UK [GR576]; Fight for Sight; Foundation Fighting Blindness USA; UCL and Moorfields Eye Hospital NIHR Biomedical Research Centre

Biallelic mutations in G‐Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in‐depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients’ genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure‐based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease‐causing variants may impede protein function in‐silico., In this study, Poulter et al. expand the number of mutations in Rhodopsin Kinase (GRK1), associated with Oguchi disease, from 13 to 21. The authors compare disease associated mutations with likely nonpathogenic variants in a range of bioinformatic prediction software. In silico analyses of the mutations, using a homology model, suggest mutations result in one of three potential mechanisms of disease: loss of protein, loss of kinase function or a failure of prenylation leading to mislocalisation of the protein.

Inherited mutations in the rod visual pigment, rhodopsin, cause the degenerative blinding condition, retinitis pigmentosa (RP). Over 150 different mutations in rhodopsin have been identified and, collectively, they are the most common cause of autosomal dominant RP (adRP). Mutations in rhodopsin are also associated with dominant congenital stationary night blindness (adCSNB) and, less frequently, recessive RP (arRP). Recessive RP is usually associated with loss of rhodopsin function, whereas the dominant conditions are a consequence of gain of function and/or dominant negative activity. The in-depth characterisation of many rhodopsin mutations has revealed that there are distinct consequences on the protein structure and function associated with different mutations. Here we categorise rhodopsin mutations into seven discrete classes; with defects ranging from misfolding and disruption of proteostasis, through mislocalisation and disrupted intracellular traffic to instability and altered function. Rhodopsin adRP offers a unique paradigm to understand how disturbances in photoreceptor homeostasis can lead to neuronal cell death. Furthermore, a wide range of therapies have been tested in rhodopsin RP, from gene therapy and gene editing to pharmacological interventions. The understanding of the disease mechanisms associated with rhodopsin RP and the development of targeted therapies offer the potential of treatment for this currently untreatable neurodegeneration.

PurposeBiallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify pathogenic GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity.MethodsPatients’ genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Pathogenic variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools.ResultsWe identified eleven previously unpublished cases with biallelic pathogenic GRK1 variants, including seven novel variants, and reviewed all GRK1 pathogenic variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. Additionally, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate pathogenic from non-pathogenic variants.ConclusionWe identified new GRK1 pathogenic variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function, giving new insights into the mechanisms of pathogenicity. All pathogenic GRK1 variants described to date have been collated into a Leiden Open Variation Database (http://dna2.leeds.ac.uk/GRK1_LOVD/genes/GRK1).

Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of dominant retinitis pigmentosa (RP), a type of inherited blindness caused by the dysfunction and death of photoreceptor cells. The P23H mutation, the most frequent single cause of RP in the USA, causes rhodopsin misfolding and induction of the unfolded protein response (UPR), an adaptive ER stress response and signalling network that aims to enhance the folding and degradation of misfolded proteins to restore proteostasis. Prolonged UPR activation, and in particular the PERK branch, can reduce protein synthesis and initiate cell death through induction of pro-apoptotic pathways. Here, we investigated the effect of pharmacological PERK inhibition on retinal disease process in the P23H-1 transgenic rat model of retinal degeneration. PERK inhibition with GSK2606414A led to an inhibition of eIF2α phosphorylation, which correlated with reduced ERG function and decreased photoreceptor survival at both high and low doses of PERK inhibitor. Additionally, PERK inhibition increased the incidence of inclusion formation in cultured cells overexpressing P23H rod opsin, and increased rhodopsin aggregation in the P23H-1 rat retina, suggesting enhanced P23H misfolding and aggregation. In contrast, treatment of P23H-1 rats with an inhibitor of eIF2α phosphatase, salubrinal, led to improved photoreceptor survival. Collectively, these data suggest the activation of PERK is part of a protective response to mutant rhodopsin that ultimately limits photoreceptor cell death.

Biallelic mutations in the photoreceptor-expressed aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) are associated with autosomal recessive Leber congenital amaurosis (LCA), the most severe form of inherited retinopathy in early childhood. AIPL1 functions as a photoreceptor-specific co-chaperone that interacts with the molecular chaperone HSP90 to facilitate the stable assembly of the retinal cyclic GMP (cGMP) phosphodiesterase (PDE6) holoenzyme. In this study, we characterized the functional deficits of AIPL1 variations, some of which induce aberrant pre-mRNA AIPL1 splicing leading to the production of alternative AIPL1 isoforms. We investigated the ability of the AIPL1 variants to mediate an interaction with HSP90 and modulate the rod cGMP PDE6 stability and activity. Our data revealed that both the FK506 binding protein (FKBP)-like domain and the tetratricopeptide repeat (TPR) domain of AIPL1 are required for interaction with HSP90. We further demonstrate that AIPL1 significantly modulates the catalytic activity of heterologously expressed rod PDE6. Although the N-terminal FKBP-like domain of AIPL1 binds the farnesylated PDE6α subunit through direct interaction with the farnesyl moiety, mutations compromising the integrity of the C-terminal TPR domain of AIPL1 also failed to modulate PDE6 activity efficiently. These AIPL1 variants moreover failed to promote the HSP90-dependent stabilization of the PDE6α subunit in the cytosol. In summary, we have successfully validated the disease-causing status of the AIPL1 variations in vitro. Our findings provide insight into the mechanism underlying the co-chaperone role of AIPL1 and will be critical for ensuring an early and effective diagnosis of AIPL1 LCA patients.

Neil Jacobstein James Bellingham Guang-Zhong Yang The human body evolved with a very specialized and narrow set of system parameters, such as atmospheric pressure at sea level of 101.3 kPa, gravity of 9.807 m/s2, internal temperature of 37°C, a blood pH of 7.4, radiation exposure of 620 mrem (milli–roentgen-equivalent-man)/year, and an atmospheric mix of 78% nitrogen, 21% oxygen, 0.9% argon, and 0.039% carbon dioxide. We operate in environments where the electromagnetic spectrum allows imaging and communications at great ranges and high bandwidths. Our comfort and safety parameters mean that humans operating in environments with significantly different parameters, such as those commonly found in space or marine environments, require extensive and expensive redundant life support systems found on submarines and spacecraft designed specifically for humans. In contrast, robots are already able to operate cost-effectively and with high precision in extremely harsh marine environments and in space. For example, small Arctic robots can image …

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic ‘gain of function’, such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

Photoreception by vertebrates enables both image-forming vision and non-image-forming responses such as circadian photoentrainment. Over the recent years, distinct non-rod non-cone photopigments have been found to support circadian photoreception in diverse species. By allowing specialization to this sensory task a selective advantage is implied, but the nature of that specialization remains elusive. We have used the presence of distinct rod opsin genes specialized to either image-forming (retinal rod opsin) or non-image-forming (pineal exo-rod opsin) photoreception in ray-finned fish (Actinopterygii) to gain a unique insight into this problem. A comparison of biochemical features for these paralogous opsins in two model teleosts, Fugu pufferfish (Takifugu rubripes) and zebrafish (Danio rerio), reveals striking differences. While spectral sensitivity is largely unaltered by specialization to the pineal environment, in other aspects exo-rod opsins exhibit a behavior that is quite distinct from the cardinal features of the rod opsin family. While they display a similar thermal stability, they show a greater than tenfold reduction in the lifetime of the signaling active Meta II photoproduct. We show that these features reflect structural changes in retinal association domains of helices 3 and 5 but, interestingly, not at either of the two residues known to define these characteristics in cone opsins. Our findings suggest that the requirements of non-image-forming photoreception have lead exo-rod opsin to adopt a characteristic that seemingly favors efficient bleach recovery but not at the expense of absolute sensitivity. Electronic supplementary material The online version of this article (doi:10.1007/s00018-011-0665-y) contains supplementary material, which is available to authorized users.

A small number of mammalian retinal ganglion cells act as photoreceptors for regulating certain non-image forming photoresponses. These intrinsically photosensitive retinal ganglion cells express the putative photopigment melanopsin. Ablation of the melanopsin gene renders these cells insensitive to light; however, the precise role of melanopsin in supporting cellular photosensitivity is unconfirmed. Here we show that heterologous expression of human melanopsin in a mouse paraneuronal cell line (Neuro-2a) is sufficient to render these cells photoreceptive. Under such conditions, melanopsin acts as a sensory photopigment, coupled to a native ion channel via a G-protein signalling cascade, to drive physiological light detection. The melanopsin photoresponse relies on the presence of cis-isoforms of retinaldehyde and is selectively sensitive to short-wavelength light. We also present evidence to show that melanopsin functions as a bistable pigment in this system, having an intrinsic photoisomerase regeneration function that is chromatically shifted to longer wavelengths.

We have cloned and characterised the expression of a new opsin gene, neuropsin (Opn5), in mice and humans. Neuropsin comprises seven exons on mouse chromosome 17. Its deduced protein sequence suggests a polypeptide of 377 amino acids in mice (354 in humans), with many structural features common to all opsins, including a lysine in the seventh transmembrane domain required to form a Schiff base link with retinaldehyde. Neuropsin shares 25-30% amino acid identity with all known opsins, making it the founding member of a new opsin family. It is expressed in the eye, brain, testis and spinal cord.

We have determined that the human SEPW1 (selenoprotein W) gene maps to chromosome 19q13.3, spans approximately 6.3 kb and comprises six exons, in contrast to the previously published five exons. The gene lacks canonical TATA and CAAT boxes, but has numerous Sp1 consensus binding sites upstream of multiple transcription start sites. SEPW1 is expressed in all of the 22 tissues assayed, and shows highest expression in skeletal muscle and heart. Additionally, we have also identified a retroprocessed SEPW1 pseudogene, SEPW1P, which maps to chromosome 1p34-35.

PURPOSE: Biallelic mutations in AIPL1 cause Leber congenital amaurosis (LCA), a devastating retinal degeneration characterized by the loss or severe impairment of vision within the first few years of life. AIPL1 is highly polymorphic with more than 50 mutations and many more polymorphisms of uncertain pathogenicity identified. As such, it can be difficult to assign disease association of AIPL1 variations. In this study, we investigate suspected disease-associated AIPL1 variations, including nonsynonymous missense and intronic variants to validate their pathogenicity. METHODS: AIPL1 minigenes harboring missense and intronic variations were constructed by amplification of genomic fragments of the human AIPL1 gene. In vitro splice assays were performed to identify the resultant AIPL1 transcripts. RESULTS: We show that all nine of the suspected disease-associated AIPL1 variations investigated induced aberrant pre-mRNA splicing of the AIPL1 gene, and our study is the first to show that AIPL1 missense mutations alter AIPL1 splicing. We reveal that the presumed rare benign variant c.784G>A [p.(G262S)] alters in vitro AIPL1 splicing, thereby validating the disease-association and clarifying the underlying disease mechanism. We also reveal that in-phase exon skipping occurs normally at a low frequency in the retina, but arises abundantly as a consequence of specific AIPL1 variations, suggesting a tolerance threshold for the expression of these alternative transcripts in the retina normally, which is exceeded in LCA. CONCLUSIONS: Our data confirm the disease-association of the AIPL1 variations investigated and reveal for the first time that aberrant splicing of AIPL1 is an underlying mechanism of disease in LCA.

In previous studies, fully functional rod and long-wavelength-sensitive (LWS) cone photopigments have been isolated from the eye of the subterranean blind mole rat (Spalax ehrenbergi superspecies). Spalax possesses subcutaneous atrophied eyes and lacks any ability to respond to visual images. By contrast this animal retains the ability to entrain circadian rhythms of locomotor behaviour to environmental light cues. As this is the only known function of the eye, the rod and LWS photopigments are thought to mediate this response. Most mammals are dichromats possessing, in addition to a single rod photopigment, two classes of cone photopigment, LWS and ultraviolet-sensitive/violet-sensitive (UVS/VS) with differing spectral sensitivities which mediate colour vision. In this paper we explore whether Spalax is a dichromat and has the potential to use colour discrimination for photoentrainment. Using immunocytochemistry and molecular approaches we demonstrate that Spalax is a LWS monochromat. Spalax lacks a functional UVS/VS cone photopigment due to the accumulation of several deleterious mutational changes that have rendered the gene nonfunctional. Using phylogenetic analysis we show that the loss of this class of photoreceptor is likely to have arisen from the visual ecology of this species, and is not an artefact of having an ancestor which lacked a functional UVS/VS cone photopigment. We conclude that colour discrimination is not a prerequisite for photoentrainment in this species.

Research over the past decade has provided overwhelming evidence that photoreception in the vertebrate eye is not confined to the rod and cone photoreceptors. It appears that photoreceptor cells within the inner retina provide irradiance information to a wide variety of different photosensory tasks including photoentrainment, pupillary constriction and masking behaviour. Action spectra in mice lacking all rod and cone photoreceptors ( rd/rd cl) have demonstrated the existence of a previously uncharacterised, opsin/vitamin-A-based photopigment with peak sensitivity at 479 nm (opsin photopigment/OP(479)). The review addresses the question: has the gene encoding OP(479) already been isolated, and if not, what type of gene should we be seeking and where in the eye might this gene be expressed? On the basis of available data, the gene that encodes OP(479) remains unidentified, and two broad possibilities exist. On the assumption that OP(479) will be like all of the other vertebrate photopigments (ocular and extraocular) and share a close phylogenetic relationship based upon amino acid identity and a conserved genomic structure, then the gene encoding OP(479) has yet to be isolated. Alternatively, there may have been a separate line of photopigment evolution in the vertebrates that has given rise to the melanopsin family. If true then the mammalian melanopsin gene may encode OP(479). Only when melanopsin and other candidates for OP(479) have been functionally expressed, and shown to encode a photopigment that matches the action spectrum of OP(479), can firm conclusions about the identity of the non-rod, non-cone ocular photoreceptor of mammals be made.

A member of a new photopigment family first isolated from teleost fish, vertebrate ancient (VA) opsin, has recently been shown to form a functional photopigment and to be expressed within a subset of horizontal and amacrine cells of the inner retina. These sites of expression (and structural features) of VA opsin suggest that this photopigment might mediate non-image-forming light-detection tasks. We attempted to gain support for this hypothesis by examining the expression of VA opsin within the central nervous system (CNS) (pineal and deep brain) of the Atlantic salmon Salmo salar. In addition, we examined the sites of rod-opsin, cone-opsin and α -transducin expression within the salmon CNS to provide a more complete description of the extraretinal photoreceptors of a teleost vertebrate. We show that multiple populations of cells within the salmon CNS appear to contain photoreceptors: VA opsin was strongly expressed in the pineal organ and in bilateral columns of subependymal cells in the epithalamus; anti-cone-opsin antibodies labelled cells within the pineal and numerous cells in the anterior hypothalamus (suprachiasmatic nucleus, nucleus preopticus magnocellularis, nucleus preopticus parvocellularis); anti-rod-opsin antibodies labelled cells within the pineal but no other areas within the central brain; and anti- α -transducin antibodies labelled cells within the pineal and the ventral telencephalon. Collectively, our results suggest that VA opsin is a photopigment specialised for irradiance detection tasks within the eye, pineal and central brain, and that the salmon has multiple and varied populations of photoreceptors within the CNS. We review the significance of these findings within the broad context of vertebrate extraretinal photoreception.

Doyne honeycomb retinal dystrophy (DHRD) is a late-onset autosomal dominant disorder that causes degeneration of the retina and can lead to blindness. We have previously assigned DHRD to a 5-cM region of chromosome 2p16 between marker loci D2S2739 and D2S378. Using sequence-tagged sites (STSs), expressed sequence tags (ESTs) and polymorphic markers within the DHRD region, we have identified 18 yeast artificial chromosomes (YACs) encompassing the DHRD locus, spanning approximately 3 Mb. The YAC contig was constructed by STS content mapping of these YACs and incorporates 13 STSs, including four genes and six polymorphic marker loci. We also report the genetic mapping of two families with a dominant drusen phenotype to the DHRD locus, and genetic refinement of the disease locus to a critical interval flanked by microsatellite marker loci D2S2352 and D2S2251, a distance of approximately 700 kb. These studies exclude a number of candidate genes and provide a resource for construction of a transcriptional map of the region, as a prerequisite to identification of the DHRD disease-causing gene and genes for other diseases mapping in the region, such as Malattia leventinese and Carney complex.

In this study, we report the identification and characterisation of a novel carbonic anhydrase related-protein. We have determined that the full length coding sequence of an anonymous expressed sequenced tag, D19S799E, encodes a novel carbonic anhydrase related-protein (CARP-2) that is 328 amino acids in length. This peptide exhibits between 23.1-28.8% amino acid identity with the seven active human carbonic anhydrase (CA) isozymes. Four substitutions of key amino acids in the catalytic domain of CAs (equivalent to His94Arg, His96Leu, His119Gln, and Thr199Ser) are likely to render CARP-2 inactive as a carbonic anhydrase. Northern blot analysis of 23 human tissues indicates that CARP2 is expressed abundantly in the brain with moderate expression also present in spinal cord and thyroid. D19S799E (and thus CARP2) has previously been localised close to the polymorphic marker D19S412 and the genes DBP and FUT1/FUT2 on 19q13. 3.

A study of the sequences of the rhodopsin-encoding genes (Rh) in eight fish species from two of the major subdivisions of the teleosts reveals that no introns are present in the coding region. This contrasts with the opsin-encoding genes of all other vertebrates where either four or five introns are invariably found. Phylogenetic analysis shows that this intronless teleost Rh is homologous to the intron-containing Rh of amphibia, birds and mammals. Possible mechanisms for intron loss are discussed, including replacement by homologous conversion of Rh with a processed cDNA.

SUMMARY Many physiological and behavioural responses to changes in environmental lighting conditions are mediated by extraocular photoreceptors. Here we investigate encephalic photoreception in Phreatichthys andruzzii, a typical cave-dwelling fish showing an extreme phenotype with complete anophthalmy and a reduction in size of associated brain structures. We firstly identified two P. andruzzii photopigments, orthologues of rod opsin and exo-rod opsin. In vitro, both opsins serve as light-absorbing photopigments with λmax around 500 nm when reconstituted with an A1 chromophore. When corrected for the summed absorption from the skin and skull, the spectral sensitivity profiles shifted to longer wavelengths (rod opsin: 521 nm; exo-rod opsin: 520 nm). We next explored the involvement of both opsins in the negative phototaxis reported for this species. A comparison of the spectral sensitivity of the photophobic response with the putative A2 absorbance spectra corrected for skin/skull absorbance indicates that the A2 versions of either or both of these pigments could explain the observed behavioural spectral sensitivity.

Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouseOpn4locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigmentin vitro. Quantitative PCR revealed thatOpn4Sis 40 times more abundant thanOpn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

Until recently, all ocular photoreception was attributed to the rods and cones of the retina. However, studies on mice lacking rod and cone photoreceptors (rd/rd cl), has shown that these mice can still use their eyes to detect light to regulate their circadian rhythms, suppress pineal melatonin, modify locomotor activity and modulate pupil size. In addition, action spectra for some of these responses have characterized a novel opsin/vitamin A-based photopigment with a lambda(max) approximately 480 nm. Electrophysiological studies have shown that a subset of retinal ganglion cells are intrinsically photosensitive, and melanopsin has been proposed as the photopigment mediating these responses to light. In contrast to mammals, an inner retinal photopigment gene has been identified in teleost fish. Vertebrate ancient (VA) opsin forms a photopigment with a lambda(max) between 460-500 nm, and is expressed in a sub-set of retinal horizontal cells, and cells in the amacrine and ganglion cell layers. Electrophysiological analysis suggests that VA opsin horizontal cells are intrinsically photosensitive and encode irradiance information. In contrast to mammals, however, the function of these novel ocular photoreceptors remains unknown. We compare non-rod, non-cone ocular photoreceptors in mammals and fish, and examine the criteria used to place candidate photopigment molecules into a functional context.

3 páginas, 2 figuras, 1 tabla.-- et al., Retinal photoreceptor dystrophies (RD) are a highly heterogeneous group of genetic disorders of the retina, representing the most frequently inherited form of visual handicap, affecting approximately 1.5 million people world wide. To date, more than 40 genetic loci have been implicated in RD. One of them, the CORD2 locus, for an autosomal dominant form of cone-rod dystrophy (CRD), maps to chromosome 19q and has previously been reported in a single large family of British origin. We now report a new family with severe early onset CRD, phenotypically very similar to the British family, which also maps to 19q, but is of Greek origin. Haplotype data of the Greek family showed no recombination between and including markers D19S219 and D19S246 and linkage analysis gave a lod score of 2.7 (at theta=0) with marker D19S412, confirming the data obtained in the British family., M Papaioannou is a fellow of the National Greek Scholarship Foundation. Drs D Bessant and A Payne are funded by the Medical Research Council of the UK (grant No G9301094).

In mammals, the melanopsin gene (Opn4) encodes a sensory photopigment that underpins newly discovered inner retinal photoreceptors. Since its first discovery in Xenopus laevis and subsequent description in humans and mice, melanopsin genes have been described in all vertebrate classes. Until now, all of these sequences have been considered representatives of a single orthologous gene (albeit with duplications in the teleost fish). Here, we describe the discovery and functional characterisation of a new melanopsin gene in fish, bird, and amphibian genomes, demonstrating that, in fact, the vertebrates have evolved two quite separate melanopsins. On the basis of sequence similarity, chromosomal localisation, and phylogeny, we identify our new melanopsins as the true orthologs of the melanopsin gene previously described in mammals and term this grouping Opn4m. By contrast, the previously published melanopsin genes in nonmammalian vertebrates represent a separate branch of the melanopsin family which we term Opn4x. RT-PCR analysis in chicken, zebrafish, and Xenopus identifies expression of both Opn4m and Opn4x genes in tissues known to be photosensitive (eye, brain, and skin). In the day-14 chicken eye, Opn4m mRNA is found in a subset of cells in the outer nuclear, inner nuclear, and ganglion cell layers, the vast majority of which also express Opn4x. Importantly, we show that a representative of the new melanopsins (chicken Opn4m) encodes a photosensory pigment capable of activating G protein signalling cascades in a light- and retinaldehyde-dependent manner under heterologous expression in Neuro-2a cells. A comprehensive in silico analysis of vertebrate genomes indicates that while most vertebrate species have both Opn4m and Opn4x genes, the latter is absent from eutherian and, possibly, marsupial mammals, lost in the course of their evolution as a result of chromosomal reorganisation. Thus, our findings show for the first time that nonmammalian vertebrates retain two quite separate melanopsin genes, while mammals have just one. These data raise important questions regarding the functional differences between Opn4x and Opn4m pigments, the associated adaptive advantages for most vertebrate species in retaining both melanopsins, and the implications for mammalian biology of lacking Opn4x.

Research over the past decade has provided overwhelming evidence that photoreception in the vertebrate eye is not confined to the rods and cones. The discovery of non-rod, non-cone ocular photoreceptors in mammals and fish arose from quite different lines of investigation. In transgenic mice entirely lacking functional rod and cone photoreceptors a range of responses to light, including the regulation of the circadian system and a pupillary light reflex, are preserved. Electrophysiological and imaging approaches were then able to characterise a coupled plexus of directly light sensitive ganglion cells. Most recently action spectroscopy has shown that a novel ‘blue-light’ sensitive photopigment based upon opsin/vitamin A (OP480) mediates these responses to light. Several candidate genes have emerged for OP480, with melanopsin being by far the strongest. A definitive link, however, between this gene and OP480 has still to be established. In contrast to the mammals, the discovery of inner retinal photoreceptors (IRPs) in fish started with the discovery of a new gene family (VA opsin). The teleost VA opsins form functional photopigments and are expressed in several different types of inner retinal neuron, including retinal horizontal cells. Recent studies have investigated the electrical properties of these photosensitive neurones, but their light-sensing role remains a matter of speculation. Thus the study of IRP is developing along quite separate lines. In the mammals the research is directed towards a molecular identification of the photopigment (OP480) and its cascade, whilst in fish the major effort is directed towards identifying a role for these novel photoreceptors using physiological approaches. The discovery of IRPs in the vertebrates tells us that despite 150 years of research, we still have much to learn about how the eye processes light.

Until recently, all ocular photoreception was attributed to the rods and cones of the retina. However, studies on mice lacking rod and cone photoreceptors (rd/rd cl), has shown that these mice can still use their eyes to detect light to regulate their circadian rhythms, suppress pineal melatonin, modify locomotor activity and modulate pupil size. In addition, action spectra for some of these responses have characterized a novel opsin/vitamin A-based photopigment with a lambda(max) approximately 480 nm. Electrophysiological studies have shown that a subset of retinal ganglion cells are intrinsically photosensitive, and melanopsin has been proposed as the photopigment mediating these responses to light. In contrast to mammals, an inner retinal photopigment gene has been identified in teleost fish. Vertebrate ancient (VA) opsin forms a photopigment with a lambda(max) between 460-500 nm, and is expressed in a sub-set of retinal horizontal cells, and cells in the amacrine and ganglion cell layers. Electrophysiological analysis suggests that VA opsin horizontal cells are intrinsically photosensitive and encode irradiance information. In contrast to mammals, however, the function of these novel ocular photoreceptors remains unknown. We compare non-rod, non-cone ocular photoreceptors in mammals and fish, and examine the criteria used to place candidate photopigment molecules into a functional context.

In Teleost fish examined to date the ocular rod opsin gene, rho, is intronless, unlike the rod opsin genes of other vertebrate classes which possess a five exon/four intron structure. We have examined in silico the structure of rho (which is expressed uniquely in the retina) and the closely related extraretinal rod-like opsin (exo-rhodopsin) gene, errlo (which is expressed uniquely in the pineal), in the puffer-fish, Fugu rubripes (Takifugu rubripes). Whilst the ocular rho is intronless in common with other Teleosts, the pineal errlo has the five exon/four intron structure common to the rod opsin gene of other vertebraes. A comparison of the sequence surrounding the errlo and rho loci indicates that the errlo locus is syntenic with RHO, the human rod opsin gene, rather than rho. We suggest that the intronless rho may have arisen through an ancient retrotransposition of a mature mRNA originating from errlo. This duplication event has occurred early in the evolution of the Actinopterygii (ray-finned fish) since the rho of the primitive Actinopterygians such as sturgeon, bowfin, and gar is also intronless. Since it appears that the intron containing errlo is the ancestral opsin gene that gave rise to the intronless rho in the Teleostei, errlo is therefore the true orthologue of the rod opsin gene in other vertebrate classes. We suggest that loss of expression of errlo in the retina could be related to the metabolic and physiological advantages, such as a reduction in splicing events during RNA processing, that may be conferred through possession of an additional, intronless rod opsin gene in the form of rho.

Background: Although photoreception is best understood in rods and cones, it is increasingly clear that these are not the only photoreceptive cells of the vertebrate retina. While considerable attention has been paid to the role of melanopsin in the generation of intrinsic light sensitivity in the retinal ganglion cells of mammals, nothing is known about the photoreceptive capacity of the horizontal cells of the fish retina in which both VA opsin and melanopsin are expressed. As yet, there has been little more than speculation as to the physiological function of these opsins within local retinal circuit neurons. Results: VA opsin and melanopsin have been isolated and localized within the well-characterized cyprinid retina of the roach ( Rutilus rutilus ). Parallel electrophysiological studies identified a novel subtype of horizontal cell (HC-RSD) characterized by a depolarizing response that fits an opsin photopigment with a λ max of 477 nm. The HC-RSD cells mediate responses to light that are characterized by long integration times, well beyond those observed for rods and cones. Significantly, HC-RSD responses persist when the conventional photoreceptor inputs are saturated by background light. Conclusions: The syncytium of coupled horizontal cells has long been considered to provide a signal of overall retinal irradiance. Our data suggest that this light information is, at least in part, derived from a population of intrinsically photosensitive VA opsin and/or melanopsin horizontal cells.

Isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24-h light/dark cycle. The mechanism underlying this cellular photosensitivity is unknown. We report the identification of a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks.

We have compared the onsets of expression of the classical visual opsins with those of the non-rod, non-cone opsins in foetal and post-natal eye tissue from mice and humans. Mouse Rgr-opsin, peropsin, encephalopsin and melanopsin are all expressed in foetal development by E11.5, unlike the murine rod and cone opsins that exhibit post-natal expression, e.g. P1 for ultraviolet cone opsin and P5 for rod opsin. Human non-rod, non-cone opsins are also all expressed early, by 8.6 weeks post-conception. The implications of these observations are discussed with regard to the possible functions of these opsins at early stages of ocular development.

Background The vertebrate opsins are proteins which utilise a retinaldehyde chromophore in their photosensory or photoisomerase roles in the visual/irradiance detection cycle. The majority of the opsins, such as rod and cone opsins, have a very highly conserved gene structure suggesting a common lineage. Exceptions to this are RGR-opsin and melanopsin, whose genes have very different intron insertion positions. The gene structure of another opsin, peropsin (retinal pigment epithelium-derived rhodopsin homologue, RRH) is unknown. Results By in silico analysis of the GenBank database we have determined that the human RRH comprises 7 exons spanning approximately 16.5 kb and is localised to chromosome 4q25 in the following gene sequence: cen-EGF-RRH-IF-qter – a position that excludes this gene as a candidate for the RP29 autosomal recessive retinitis pigmentosa locus. A comparison of opsin gene structures reveals that RRH and RGR share two common intron (introns 1 and 4) insertion positions which may reflect a shared ancestral gene. Conclusion The opsins comprise a diverse group of genes which appear to have arisen from three different lineages. These lineages comprise the "classical opsin superfamily" which includes the rod and cone opsins, pinopsin, VA-opsin, parapinopsin and encephalopsin; the RRH and RGR group; and the melanopsin line. A common lineage for RRH and RGR, together with their sites of expression in the RPE, indicates that peropsin may act as a retinal isomerase.

Photoreception is best understood in retinal rods and cones, but it is not confined to these cells. In non-mammals, intrinsically photosensitive cells have been identified within several structures including the pineal, hypothalamus and skin. More recently novel light sensitive cells have been identified in the inner/basal retina of both teleosts and rodents. Melanopsin has been proposed as the photopigment mediating many of these non-rod, non-cone responses to light. However, much about the melanopsin gene family remains to be clarified including their potential role as photopigments, and taxonomic distribution. We have isolated the first orthologue of melanopsin from a teleost fish and show expression of this gene in a sub-set of retinal horizontal cells (type B). Zebrafish melanopsin, and orthologues of this gene, differ markedly from the vertebrate photopigment opsins. The putative counterion is not a glutamate but a tyrosine, the putative G-protein binding domain in the third cytoplasmic loop is not conserved, and they show low levels of amino acid identity (approximately 27%) to both the known photopigment opsins and to other members of the melanopsin family. Mouse melanopsin is only 58% identical to Xenopus, and 68% identical to zebrafish. By contrast, the photosensory opsin families show approximately 75% conservation. On the basis of their structure, genomic organisation, discrete evolutionary lineage, and their co-expression with other opsins, the melanopins are not obvious photosensory opsins. They might represent a separate branch of photopigment evolution in the vertebrates or they may have a non-direct photosensory function, perhaps as a photoisomerase, in non-rod, non-cone light detection.

As part of an ongoing search to identify novel mammalian photopigments that may mediate nonvisual tasks such as circadian entrainment and acute suppression of pineal melatonin levels, a number of recently cloned nonvisual opsin sequences were used to search dbEST. panopsin (OPN3) was one of the clones identified using this approach. Expression analysis detects two transcripts of approximately 2.1 and 2.5 kb, in a wide range of tissues including brain, liver, and retina, which encode a predicted protein of 403 amino acids. The gene was localized to the region of chromosome 1q43 also encompassing the kynurenine monooxygenase (KMO) and choroideremia-like Rab escort protein 2 (CHML) genes. KMO and panopsin overlap at their 3' ends but are transcribed in opposite directions. CHML, an intronless gene, lies in intron 1 of panopsin.

We report the isolation and characterisation of a novel opsin cDNA from the retina and pineal of the common carp (Cyprinus carpio L.). When a comparison of the amino acid sequences of salmon vertebrate ancient opsin (sVA) and the novel carp opsin are made, and the carboxyl terminus is omitted, the level of identity between these two opsins is 81% and represents the second example of the VA opsin family. We have therefore termed this C. carpio opsin as carp VA opsin (cVA opsin). We show that members of the VA opsin family may exist in two variants or isoforms based upon the length of the carboxyl terminus and propose that the mechanism of production of the short VA opsin isoform is alternative splicing of intron 4 of the VA opsin gene. The VA opsin gene consists of five exons, with intron 2 significantly shifted in a 3′ direction relative to the corresponding intron in rod and cone opsins. The position (or lack) of intron 2 appears to be a diagnostic feature which separates the image forming rod and cone opsin families from the more recently discovered non-visual opsin families (pin-opsins (P), vertebrate ancient (VA), parapinopsin (PP)). Finally, we suggest that lamprey P opsin should be reassigned to the VA opsin family based upon its level of amino acid identity, genomic structure with respect to the position of intron 2 and nucleotide phylogeny.

We have isolated a novel opsin from the pineal complex of Atlantic salmon (Salmo salar) and from the brain of the puffer fish (Fugu rubripes). These extra-retinal opsins share approximately 74% identity at the nucleotide and amino acid level with rod-opsins from the retina of these species. By PCR, we have determined that the novel rod-like opsin is not expressed in the salmon retina, and the retinal rod-opsin is not expressed in the salmon pineal. Phylogenetic analysis suggests that the rod-like opsins arose from a gene duplication event approximately 205 million years ago, a time of considerable adaptive radiation of the bony fish. In view of the large differences in the coding sequences of the pineal/brain rod-like opsins, their extra-retinal sites of expression, and phylogenetic position we have termed these novel opsins ‘extra-retinal rod-like opsins’ (ERrod-like opsins). We speculate that the differences between retinal rod-opsins and ERrod-like opsins have arisen from their differing photosensory roles and/or genetic drift after the gene duplication event in the Triassic.

We report the identification and characterisation of a novel human orphan G-protein-coupled receptor (GPR) which maps to chromosome 2p16. We have determined the full-length coding sequence and genomic structure of a gene corresponding to the anonymous expressed sequenced tag, WI-31133. This gene encodes a novel protein that is 540 amino acids in length. Protein sequence analysis predicts the presence of seven transmembrane domains, a characteristic feature of GPRs. In situ hybridisation to human retina and Northern blot analysis of human retinal pigment epithelium (RPE) showed localisation of this transcript to the RPE and cells surrounding retinal arterioles. In contrast, the transcript was localised to the photoreceptor inner segments and the outer plexiform layer in mouse sections. Northern blot analysis demonstrated a 7 kb transcript highly expressed in the brain. No mutations were identified during a screen of patients suffering from Doyne's honeycomb retinal dystrophy (DHRD), an inherited retinal degeneration which maps to chromosome 2p16.

The cephalopod molluscs are a group of invertebrates that occupy a wide range of oceanic photic environments. They are an ideal group of animals, therefore, in which to study the evolution of rhodopsin. The cDNA sequence of the rhodopsin gene of the cuttlefish Sepia officinalis (L.) (Sub-class Coleoidea, Order Sepiida) is presented, together with an analysis of the structure of the gene. A proline-rich C terminus is present; this structure is characteristic of cephalopod rhodopsins. In common with all invertebrate opsins studied so far, the equivalent site to the counterion in vertebrate opsins is occupied by an aromatic amino acid. An intron is present that splits codon 107, in contrast to the intronless rhodopsin gene in two species of myopsid squid. A spectral tuning model involving substitutions at only three amino acid sites is proposed for the spectral shifts between the rhodopsins of Sepia officinalis, three species of squid and Paroctopus defleini.

The cDNA sequence of the ultraviolet-sensitive opsin in the honey-bee, Apis mellifera, with associated 5' and 3' untranslated regions, is presented. The analysis of genomic structure reveals seven introns in the coding region of the gene, with six at novel positions for an insect opsin gene. The equivalent site to the counterion in vertebrate opsins is occupied by a Tyr residue. This contrasts with the presence of Phe at this site in the ultraviolet-sensitive opsins of Drosophila sps. A comparison of the amino acid sequence within the seven alpha-helical transmembrane regions of insect ultraviolet/blue-sensitive opsins identifies substitution at five sites that involve either replacement of a polar with a non-polar residue, or a change in charge. Such changes are known to result in spectral shifts in vertebrate pigments. Phylogenetic analysis indicates that the ultraviolet-sensitive pigments represent an ancient class of insect opsins.

Choroidoretinal dystrophies are incurable and essentially untreatable, representing the most common cause of genetic visual loss in childhood (1). They are a clinically and genetically heterogeneous group of disorders. Dystrophies which primarily affect rod function such as retinitis pigmentosa have been extensively studied and a number of genes have been implicated in the disease pathogenesis (2). In contrast dystrophies which primarily affect cone photoreceptors have been less well studied and examples of this group of diseases includes cone dystrophies and cone-rod dystrophies. Cone dystrophies are characterised by photophobia, loss of visual acuity and colour vision defects associated with reduced cone photoreceptor ERG responses. Abnormal pigmentation with atrophy is often seen at the macula. Cone-rod dystrophies are distinct from cone dystrophies in that abnormalities of cone dysfunction is seen with progressive peripheral retinal disease. Diminished visual acuity and loss of colour discrimination is followed by nyctalopia, progressive peripheral visual field deficit and decreasing rod photoreceptor ERG amplitudes from an early age. Advancing chorioretinal atrophy of the central and peripheral retina is characteristic (3). Autosomal dominant, recessive and X-linked patterns of inheritance for cone dystrophies and cone-rod dystrophy (CRD) have been described and studies have implicated a number of loci for the disease-causing genes. Loci associated with cone dystrophies include a balanced translocation on chromosome 6q which was reported in a patient with mental retardation and cone dystrophy (4), an X-linked cone dystrophy mapping to Xp21.1–p11.3 (5) and two independent studies have used linkage analysis to identify an autosomal dominant cone dystrophy locus on chromosome 17p (6–7). Loci implicated in CRD include two case reports that have suggested localisation of CRD genes on chromosome 18q (8) and 17q (9), an autosomal dominant form of CRD mapping to chromosome 19q (10) and a transverse mutation in the peripherin/RDS gene (Asn244His) has been found in one Japanese CRD family (11).