A Proximity Complementation Assay to Identify Small Molecules That Enhance the Traffic of ABCA4 Misfolding Variants.

ABCA4-related retinopathy is the most common inherited Mendelian eye disorder worldwide, caused by biallelic variants in the ATP-binding cassette transporter ABCA4. To date, over 2200 ABCA4 variants have been identified, including missense, nonsense, indels, splice site and deep intronic defects. Notably, more than 60% are missense variants that can lead to protein misfolding, mistrafficking and degradation. Currently no approved therapies target ABCA4. In this study, we demonstrate that ABCA4 misfolding variants are temperature-sensitive and reduced temperature growth (30 °C) improves their traffic to the plasma membrane, suggesting the folding of these variants could be rescuable. Consequently, an in vitro platform was developed for the rapid and robust detection of ABCA4 traffic to the plasma membrane in transiently transfected cells. The system was used to assess selected candidate small molecules that were reported to improve the folding or traffic of other ABC transporters. Two candidates, 4-PBA and AICAR, were identified and validated for their ability to enhance both wild-type ABCA4 and variant trafficking to the cell surface in cell culture. We envision that this platform could serve as a primary screen for more sophisticated in vitro testing, enabling the discovery of breakthrough agents to rescue ABCA4 protein defects and mitigate ABCA4-related retinopathy.

Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies.

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.

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision.

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.

Biallelic variants in coenzyme Q10 biosynthesis pathway genes cause a retinitis pigmentosa phenotype.

The aim of this study was to investigate coenzyme Q10 (CoQ10) biosynthesis pathway defects in inherited retinal dystrophy. Individuals affected by inherited retinal dystrophy (IRD) underwent exome or genome sequencing for molecular diagnosis of their condition. Following negative IRD gene panel analysis, patients carrying biallelic variants in CoQ10 biosynthesis pathway genes were identified. Clinical data were collected from the medical records. Haplotypes harbouring the same missense variant were characterised from family genome sequencing (GS) data and direct Sanger sequencing. Candidate splice variants were characterised using Oxford Nanopore Technologies single molecule sequencing. The CoQ10 status of the human plasma was determined in some of the study patients. 13 individuals from 12 unrelated families harboured candidate pathogenic genotypes in the genes: PDSS1, COQ2, COQ4 and COQ5. The PDSS1 variant c.589 A > G was identified in three affected individuals from three unrelated families on a possible ancestral haplotype. Three variants (PDSS1 c.468-25 A > G, PDSS1 c.722-2 A > G, COQ5 c.682-7 T > G) were shown to lead to cryptic splicing. 6 affected individuals were diagnosed with non-syndromic retinitis pigmentosa and 7 had additional clinical findings. This study provides evidence of CoQ10 biosynthesis pathway gene defects leading to non-syndromic retinitis pigmentosa in some cases. Intronic variants outside of the canonical splice-sites represent an important cause of disease. RT-PCR nanopore sequencing is effective in characterising these splice defects.

A system of coordinated autonomous robots for Lagrangian studies of microbes in the oceanic deep chlorophyll maximum.

The deep chlorophyll maximum (DCM) layer is an ecologically important feature of the open ocean. The DCM cannot be observed using aerial or satellite remote sensing; thus, in situ observations are essential. Further, understanding the responses of microbes to the environmental processes driving their metabolism and interactions requires observing in a reference frame that moves with a plankton population drifting in ocean currents, i.e., Lagrangian. Here, we report the development and application of a system of coordinated robots for studying planktonic biological communities drifting within the ocean. The presented Lagrangian system uses three coordinated autonomous robotic platforms. The focal platform consists of an autonomous underwater vehicle (AUV) fitted with a robotic water sampler. This platform localizes and drifts within a DCM community, periodically acquiring samples while continuously monitoring the local environment. The second platform is an AUV equipped with environmental sensing and acoustic tracking capabilities. This platform characterizes environmental conditions by tracking the focal platform and vertically profiling in its vicinity. The third platform is an autonomous surface vehicle equipped with satellite communications and subsea acoustic tracking capabilities. While also acoustically tracking the focal platform, this vehicle serves as a communication relay that connects the subsea robot to human operators, thereby providing situational awareness and enabling intervention if needed. Deployed in the North Pacific Ocean within the core of a cyclonic eddy, this coordinated system autonomously captured fundamental characteristics of the in situ DCM microbial community in a manner not possible previously.

New variants and in silico analyses in GRK1 associated Oguchi disease.

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.

AAV-mediated ERdj5 overexpression protects against P23H rhodopsin toxicity.

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.

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The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy.

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.

The integrity and organization of the human AIPL1 functional domains is critical for its role as a HSP90-dependent co-chaperone for rod PDE6.

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.

The role of the ER stress-response protein PERK in rhodopsin retinitis pigmentosa.

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.

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

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.

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration.

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.

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

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Investigation of Aberrant Splicing Induced by AIPL1 Variations as a Cause of Leber Congenital Amaurosis.

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.

Encephalic photoreception and phototactic response in the troglobiont Somalian blind cavefish Phreatichthys andruzzii.

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 λ(max) around 500 nm when reconstituted with an A(1) 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 A(2) absorbance spectra corrected for skin/skull absorbance indicates that the A(2) versions of either or both of these pigments could explain the observed behavioural spectral sensitivity.