ABSTRACT
Corneal dystrophies are phenotypically and genetically heterogeneous, often resulting in visual impairment caused by corneal opacification. We investigated the genetic cause of an autosomal dominant corneal stromal dystrophy in a pedigree with eight affected individuals in three generations. Affected individuals had diffuse central stromal opacity, with reduced visual acuity in older family members. Histopathology of affected cornea tissue removed during surgery revealed mild stromal textural alterations with alcianophilic deposits. Whole genome sequence data were generated for four affected individuals. No rare variants (MAF < 0.001) were identified in established corneal dystrophy genes. However, a novel heterozygous missense variant in exon 4 of SPARCL1, NM_004684: c.334G > A; p.(Glu112Lys), which is predicted to be damaging, segregated with disease. SPARC-like protein 1 (SPARCL1) is a secreted matricellular protein involved in cell migration, cell adhesion, tissue repair, and remodelling. Interestingly, SPARCL1 has been shown to regulate decorin. Heterozygous variants in DCN, encoding decorin, cause autosomal dominant congenital stromal corneal dystrophy, suggesting a common pathogenic pathway. Therefore, we performed immunohistochemistry to compare SPARCL1 and decorin localisation in corneal tissue from an affected family member and an unaffected control. Strikingly, the level of decorin was significantly decreased in the corneal stroma of the affected tissue, and SPARCL1 appeared to be retained in the epithelium. In summary, we describe a novel autosomal dominant corneal stromal dystrophy associated with a missense variant in SPARCL1, extending the phenotypic and genetic heterogeneity of inherited corneal disease.
ABSTRACT
Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.
Subject(s)
Fuchs' Endothelial Dystrophy , Transcription Factor 4 , Trinucleotide Repeat Expansion , Humans , Male , Alternative Splicing/genetics , Endothelial Cells/metabolism , Endothelium, Corneal/metabolism , Endothelium, Corneal/pathology , Fuchs' Endothelial Dystrophy/genetics , Transcription Factor 4/genetics , Transcription Factor 4/metabolism , Transcriptome/genetics , Trinucleotide Repeat Expansion/genetics , FemaleABSTRACT
Animals with enhanced dim-light sensitivity are at higher risk of light-induced retinal degeneration when exposed to bright light conditions.1,2,3,4 This trade-off is mediated by the rod photoreceptor sensory protein, rhodopsin (RHO), and its toxic vitamin A chromophore by-product, all-trans retinal.5,6,7,8 Rod arrestin (Arr-1) binds to RHO and promotes sequestration of excess all-trans retinal,9,10 which has recently been suggested as a protective mechanism against photoreceptor cell death.2,11 We investigated Arr-1 evolution in animals at high risk of retinal damage due to periodic bright-light exposure of rod-dominated retinas. Here, we find the convergent evolution of enhanced Arr-1/RHO all-trans-retinal sequestration in owls and deep-diving whales. Statistical analyses reveal a parallel acceleration of Arr-1 evolutionary rates in these lineages, which is associated with the introduction of a rare Arr-1 mutation (Q69R) into the RHO-Arr-1 binding interface. Using in vitro assays, we find that this single mutation significantly enhances RHO-all-trans-retinal sequestration by â¼30%. This functional convergence across 300 million years of evolutionary divergence suggests that Arr-1 and RHO may play an underappreciated role in the photoprotection of the eye, with potentially vast clinical significance.
Subject(s)
Retinal Degeneration , Strigiformes , Animals , Strigiformes/metabolism , Retinaldehyde/metabolism , Whales , Retinal Rod Photoreceptor Cells , Retina/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Rhodopsin/metabolismABSTRACT
Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS â¼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV-violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.
Subject(s)
Color Vision , Opsins , Animals , Fresh Water , Opsins/genetics , Opsins/metabolism , Phylogeny , Retinal Cone Photoreceptor Cells/metabolism , Rod Opsins/genetics , Snakes/genetics , Snakes/metabolismABSTRACT
Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field.
Subject(s)
Fuchs' Endothelial Dystrophy/genetics , Transcription Factor 4/genetics , Trinucleotide Repeat Expansion/genetics , Fuchs' Endothelial Dystrophy/pathology , Gene Expression Regulation , Genetic Predisposition to Disease , Genotype , Humans , Polymorphism, GeneticABSTRACT
Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS ∼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV–violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.
ABSTRACT
Most vertebrates use a combination of rod and cone photoreceptors to enable vision in conditions ranging from starlight to direct sunlight. Nocturnal geckos, however, have simplex retinas that contain only rods in terms of morphology and physiology, but these rods are thought to be derived from cones through an evolutionary process known as photoreceptor transmutation. To investigate this, we generated eye transcriptomes and analyzed patterns of phototransduction gene evolution in geckos in comparison to other reptiles. We confirm that geckos have lost several major components of the rod phototransduction pathway, including rod opsin (RH1), which we identified as a pseudogene in multiple genomes. We also identified a partial rod transducin transcript, but found no evidence of the protein in retinal sections. However, we find that geckos express several complete rod phototransduction transcripts in the eye, which may contribute to the rod-like physiology of nocturnal gecko photoreceptors. Finally, we found surprising evidence that even though photoreceptor transmutation evolved independently in geckos and snakes, they have experienced parallel shifts in selective constraint on phototransduction genes. These results implicate adaptive change in the underlying molecular machinery of visual transduction, in addition to the convergent changes in cellular morphology, during photoreceptor transmutation.
Subject(s)
Biological Evolution , Lizards/physiology , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/physiology , Snakes/physiology , Animals , Light , Opsins/genetics , Phylogeny , Pseudogenes , Selection, Genetic , Species Specificity , Transcriptome , Vision, OcularABSTRACT
G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin-a GPCR activated by light-for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease.
Subject(s)
Mutation, Missense , Retinitis Pigmentosa/genetics , Rhodopsin/metabolism , Signal Transduction , Cell Line, Tumor , Genes, Mating Type, Fungal/genetics , Humans , Retinitis Pigmentosa/pathology , Rhodopsin/chemistry , Rhodopsin/genetics , Saccharomyces cerevisiaeABSTRACT
Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.
Subject(s)
Evolution, Molecular , Genome , Molecular Sequence Annotation , Predatory Behavior , Snakes/genetics , Adaptation, Physiological , Animals , Female , Photoreceptor Cells, Vertebrate , Receptors, Odorant/genetics , Reptiles/classification , Reptiles/genetics , Retinal Pigments/genetics , Selection, Genetic , Snakes/classification , Snakes/physiology , Venoms/genetics , Voltage-Gated Sodium Channels/geneticsABSTRACT
High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates-properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.
Subject(s)
Altitude , Rhodopsin/chemistry , Animals , Bayes Theorem , Biological Evolution , Bolivia , Catfishes , Cold Shock Proteins and Peptides/chemistry , Cold Temperature , Crystallography, X-Ray , Ecuador , Evolution, Molecular , Geography , HEK293 Cells , Humans , Kinetics , Mutation , Peru , PhylogenyABSTRACT
Colubridae is the largest and most diverse family of snakes, with visual systems that reflect this diversity, encompassing a variety of retinal photoreceptor organizations. The transmutation theory proposed by Walls postulates that photoreceptors could evolutionarily transition between cell types in squamates, but few studies have tested this theory. Recently, evidence for transmutation and rod-like machinery in an all-cone retina has been identified in a diurnal garter snake (Thamnophis), and it appears that the rhodopsin gene at least may be widespread among colubrid snakes. However, functional evidence supporting transmutation beyond the existence of the rhodopsin gene remains rare. We examined the all-cone retina of another colubrid, Pituophis melanoleucus, thought to be more secretive/burrowing than Thamnophis We found that P. melanoleucus expresses two cone opsins (SWS1, LWS) and rhodopsin (RH1) within the eye. Immunohistochemistry localized rhodopsin to the outer segment of photoreceptors in the all-cone retina of the snake and all opsin genes produced functional visual pigments when expressed in vitro Consistent with other studies, we found that P. melanoleucus rhodopsin is extremely blue-shifted. Surprisingly, P. melanoleucus rhodopsin reacted with hydroxylamine, a typical cone opsin characteristic. These results support the idea that the rhodopsin-containing photoreceptors of P. melanoleucus are the products of evolutionary transmutation from rod ancestors, and suggest that this phenomenon may be widespread in colubrid snakes. We hypothesize that transmutation may be an adaptation for diurnal, brighter-light vision, which could result in increased spectral sensitivity and chromatic discrimination with the potential for colour vision.
Subject(s)
Adaptation, Biological , Colubridae/physiology , Reptilian Proteins/metabolism , Retinal Cone Photoreceptor Cells/physiology , Rhodopsin/metabolism , Animals , Photoperiod , Sequence Analysis, DNAABSTRACT
Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments.
Subject(s)
Models, Molecular , Retina/metabolism , Retinitis Pigmentosa/metabolism , Rhodopsin/metabolism , Amino Acid Substitution , Animals , Cattle , Genetic Predisposition to Disease , HEK293 Cells , Hot Temperature/adverse effects , Humans , Light , Mutagenesis, Site-Directed , Mutation , Phylogeny , Protein Interaction Domains and Motifs/radiation effects , Protein Stability/radiation effects , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Retina/pathology , Retina/radiation effects , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/pathology , Rhodopsin/chemistry , Rhodopsin/genetics , Schiff Bases/chemistry , Solubility/radiation effects , SpectrophotometryABSTRACT
Lake Baikal is the deepest and one of the most ancient lakes in the world. Its unique ecology has resulted in the colonization of a diversity of depth habitats by a unique fauna that includes a group of teleost fish of the sub-order Cottoidei. This relatively recent radiation of cottoid fishes shows a gradual blue-shift in the wavelength of the absorption maximum of their visual pigments with increasing habitat depth. Here we combine homology modeling and quantum chemical calculations with experimental in vitro measurements of rhodopsins to investigate dim-light adaptation. The calculations, which were able to reproduce the trend of observed absorption maxima in both A1 and A2 rhodopsins, reveal a Barlow-type relationship between the absorption maxima and the thermal isomerization rate suggesting a link between the observed blue-shift and a thermal noise decrease. A Nakanishi point-charge analysis of the electrostatic effects of non-conserved and conserved amino acid residues surrounding the rhodopsin chromophore identified both close and distant sites affecting simultaneously spectral tuning and visual sensitivity. We propose that natural variation at these sites modulate both the thermal noise and spectral shifting in Baikal cottoid visual pigments resulting in adaptations that enable vision in deep water light environments.
Subject(s)
Fishes , Lakes , Light , Rhodopsin , Amino Acids , Animals , Models, Molecular , Molecular Conformation , Retinal Cone Photoreceptor Cells/metabolism , Rhodopsin/chemistry , Rhodopsin/metabolism , Structure-Activity RelationshipABSTRACT
Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the "transmutation" theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single "cones." Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.
Subject(s)
Biological Evolution , Circadian Rhythm , Colubridae/physiology , Retinal Cone Photoreceptor Cells/cytology , Animals , Immunohistochemistry , Mice , Models, Biological , Molecular Sequence Data , Retinal Cone Photoreceptor Cells/ultrastructure , Retinal Pigments/metabolism , Retinal Rod Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/ultrastructure , Rhodopsin/metabolism , Transducin/metabolismABSTRACT
Most soluble lysosomal hydrolases are sorted in the trans-Golgi network (TGN) and delivered to the lysosomes by the mannose 6-phosphate receptor (M6PR). However, the non-enzymic sphingolipid activator protein (SAP), prosaposin, as well as certain soluble lysosomal hydrolases, is sorted and trafficked to the lysosomes by sortilin. Based on previous results demonstrating that prosaposin requires sphingomyelin to be targeted to the lysosomes, we hypothesized that sortilin and its ligands are found in detergent-resistant membranes (DRMs). To test this hypothesis we have analyzed DRM fractions and demonstrated the presence of sortilin and its ligand, prosaposin. Our results showed that both the M6PR and its cargo, cathepsin B, were also present in DRMs. Cathepsin H has previously been demonstrated to interact with sortilin, while cathepsin D interacts with both sortilin and the M6PR. Both of these soluble lysosomal proteins were also found in DRM fractions. Using sortilin shRNA we have showed that prosaposin is localized to DRM fractions only in the presence of sortilin. These observations suggest that in addition to interacting with the same adaptor proteins, such as GGAs, AP-1 and retromer, both sortilin and the M6PR localize to similar membrane platforms, and that prosaposin must interact with sortilin to be recruited to DRMs.