The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes

Background The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. Results Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. Conclusions We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.

Pathophysiological features of the visual cycle, cascade and metabolic pathways in retinitis pigmentosa

This literature review offers a detailed description of the genes and proteins involved in pathophysiological processes in isolated retinitis pigmentosa (RP). To date, 84 genes and 7 candidate genes have been described for non-syndromic RP. Each of these genes encodes a protein that plays a role in vital processes in the retina and / or retinal pigment epithelium, including the cascade of phototransduction (transmission of the visual signal), the visual cycle, ciliary transport, the environment of photoreceptor cilia and the interphotoreceptor matrix. The identification and study of pathophysiological pathways affected in non-syndromic RP is important for understanding the main pathogenic ways and developing approaches to target treatment.

Molecular structures of the eukaryotic retinal importer ABCA4

The ATP-binding cassette (ABC) transporter family contains thousands of members with diverse functions. Movement of the substrate, powered by ATP hydrolysis, can be outward (export) or inward (import). ABCA4 is a eukaryotic importer transporting retinal to the cytosol to enter the visual cycle. It also removes toxic retinoids from the disc lumen to the cytosol. Mutations in ABCA4 cause impaired vision or blindness. Despite decades of clinical, biochemical, and animal model studies, the molecular mechanism of ABCA4 is unknown. Here we report the structures of human ABCA4 in two conformations. In the absence of ATP, ABCA4 adopts an outward-facing conformation, poised to recruit substrate. The presence of ATP induces large conformational changes that could lead to substrate release. These structures provide a molecular basis to understand many disease-causing mutations and a rational guide for new experiments to uncover how ABCA4 recruits, flips, and releases retinoids.

Function of mammalian M-cones depends on the level of CRALBP in Müller cells

Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the Müller cell–driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in Müller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by ∼50% in both the RPE and retina of Rlbp1+/− mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/− mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/− mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the Müller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1−/− mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation.