T. Michael Redmond

Carotenoid cleavage dioxygenases (CCDs) are a superfamily of non-heme iron-containing enzymes ubiquitous across all kingdoms of life. Previously, we identified a new subfamily of CCDs termed beta-carotene oxygenase-like (BCOL) proteins, a chordate expansion of an ancestral sister clade of metazoan CCDs, which are widely distributed in marine invertebrates, including mollusks and lancelets. Here we present the three-dimensional structure of a representative BCOL protein from the lancelet Branchiostoma fforidae (BCOLg) at 2.4 Å resolution and its functional properties. BCOLg symmetrically cleaves β-carotene into all- trans retinal. RNAseq of wild-caught animals detected six of the seven annotated BCOL transcripts in Branchiostoma gut tissue. We expressed and crystallized recombinant BCOLg. BCOLg is heavily glycosylated, with four N-linked glycosylation sites, and adopts the seven-bladed propeller fold characteristic of the superfamily, with one disulfide bond contributing to structural stability. BCOLg and several other BCOL proteins contain Asp substitutions at two of three Glu positions found in other CCDs, stabilizing three of four His residues involved in Fe ²⁺ coordination. Notably, BCOLg lacks the canonical -PDPCK-motif characteristic of other metazoan CCDs. Summary: Reporting the structure of lancelet beta-carotene oxygenase-like BCOLg protein at 2.4 Å, an unusual carotenoid cleavage dioxygenase (CCD) superfamily member. BCOLg is a heavily glycosylated protein with a distinctive iron-coordination environment and lacking the canonical metazoan -PDPCK-motif.

Non-coding RNAs play critical roles in regulating gene expression and diverse biological processes. While the roles of microRNAs (miRNAs/miRs) are well known, the roles of long non-coding RNAs (lncRNAs; >200 nucleotides, with little or no protein-coding potential) remain less defined. In this study, we investigated the function of lncRNAs during the differentiation of human retinal pigment epithelial (ARPE-19) cells, identifying LINC00276 as being consistently and robustly upregulated during RPE differentiation. Silencing LINC00276-202 (ENST00000418420.1) using antisense GapmeRs significantly reduced the expression of RPE differentiation-associated genes, including RPE65 , MITF , TRPM1 , and TRPM3 , whereas LINC00276-202 overexpression restored their expression levels. In silico analysis identified a shared miR-204-5p/miR-211-5p-responsive region within LINC00276-202 . Both miRNAs produced modest increases in LINC00276-202 -associated reporter activity, whereas deletion of the shared seed-recognition region attenuated this response. Importantly, despite increased accumulation of the seed-deletion transcript, the mutant construct failed to induce RPE65 expression to the extent observed with WT LINC00276-202 , indicating that sequence integrity contributes to its biological activity. RNA pull-down followed by mass spectrometry identified PURB, SART3, ANXA6, and hnRNPL as potential protein binding partners. Of these, hnRNPL was validated as a LINC00276-202 -interacting protein by RNA immunoprecipitation and immunoblotting. LINC00276-202 modulated hnRNPL protein levels, and hnRNPL contributed to LINC00276-202 -mediated regulation of β-catenin protein abundance and TCF/LEF transcriptional activity. Together, these findings identify LINC00276-202 as a novel regulator of RPE differentiation involving a miR-204/miR-211-responsive region, hnRNPL interaction, and β-catenin-associated signaling, with potential relevance to retinal degenerative disease.

The visual cycle is an important pathway in the retinal pigment epithelium (RPE) which regenerates 11-cis retinal chromophore for the retinal photoreceptors. The central enzyme in the visual cycle is RPE65 retinol isomerase. Expression of RPE65 mRNA and protein levels are significantly lower in RPE cell culture models when compared to native RPE. This limits the use of these models to study the visual cycle. To determine the main drivers of RPE65 regulation we compared the transcriptional profiles of native and cell culture models of RPE with various levels of RPE65 expression. We also compared the levels of RPE65 expression between ARPE-19 cells grown in media supplemented with 1 mM pyruvate (PYR) or 10 mM nicotinamide (NAM). In addition, we performed experiments directed at transcriptional and translational regulation of RPE65. We show that RPE65 mRNA and protein expression is significantly higher in NAM media grown cells than PYR cells. Transfection of cells with a variety of different vectors containing RPE65 ORFs with different promoters, codon optimization, IRES, 3’ UTRs, suggest that translational effects are less important than transcriptional status. Importantly, we found that feeding with rod outer segments (ROS) decreases RPE65 expression in NAM grown cells, suggesting that certain primary functions of the RPE (here, visual cycle and phagocytosis) are not positively linked. Analysis of differentially regulated microRNAs (miRs) provides a basis for this downregulation. It appears that the regulation of RPE65 expression in ARPE-19 cells, in particular, is multifactorial, involving primarily metabolic and transcriptional status of the cells, with translation of RPE65 mRNA playing a smaller role.

Recessive RPE65-related retinopathy is an inherited retinal disease (IRD) that is a well-established target for gene therapy. Dominant RPE65-related retinopathy, however, due to Irish founder variant p.(D477G), is extremely rare. Here, we report the discovery, replication and characterization of a novel dominant retinopathy caused by RPE65 variant p.(E519K), identified in 83 individuals of European ancestry across IRD registries (Belgian discovery cohort, n=2,873; replication cohort, n=18,796). Long-read sequencing-based haplotyping revealed a shared region of 464 kb, supporting a founder effect. Genotype-phenotype data support dominant inheritance and phenotypic variability respectively, characterized by late-onset macular dystrophy with two main subtypes, a pathognomonic mottled subtype and a pattern dystrophy subtype. Functional studies showed that the p.(E519K) variant affects RPE65 enzymatic activity, correlating with lower protein expression. Protein modelling and cellular thermal shift assays further supported a destabilizing effect on protein structure. Overall, our work provides strong genetic, clinical, molecular and functional evidence for a novel dominant RPE65 retinopathy in multiple families in Europe and North America due to a Belgian founder variant. This discovery reduces the diagnostic gap in dominant IRD, particularly in individuals of European ancestry. Finally, it lays the foundation for developing therapeutic strategies targeting dominant RPE65 retinopathy.

Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous disorders characterised by progressive vision loss. Over 270 causative genes have been identified and variants within the same gene can give rise to clinically distinct disorders. Human induced pluripotent stem cells (iPSCs) have revolutionised disease modelling, by allowing pathophysiological and therapeutic studies in the patient and tissue context. The IRD gene RLBP1 encodes CRALBP, an actor of the rod and cone visual cycles in the retinal pigment epithelium (RPE) and Müller cells, respectively. Variants in RLBP1 lead to three clinical subtypes: Bothnia dystrophy, Retinitis punctata albescens and Newfoundland rod-cone dystrophy. We modelled RLBP1 -IRD subtypes by patient-specific iPSC-derived RPE and identified pertinent therapeutic read-outs. We developed an AAV2/5-mediated gene replacement strategy and provided a proof-of-concept in the ex vivo human models that was validated in an in vivo Rlbp1 ^−/− murine model. Most importantly, we identified a previously unsuspected smaller CRALBP isoform that is naturally and differentially expressed in both human and murine retina. The new isoform arises from an alternative methionine initiation site and plays a role in the visual cycle. This work provides novel insights into CRALBP expression and RLBP1 -associated pathophysiology and raises important considerations for successful gene supplementation therapy.

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.

The visual cycle is a complex biological process that involves the sequential action of proteins in the retinal pigment epithelial (RPE) cells and photoreceptors to modify and shuttle visual retinoids. A majority of the visual cycle proteins are membrane proteins, either integral or peripheral membrane proteins. Despite significant progress in understanding their physiological function, very limited structural information is available for the visual cycle proteins. Moreover, the mechanism of membrane interaction is not yet clear in all cases. Here, we demonstrate the presence of an amphipathic helix in selected RPE visual cycle proteins, using in silico tools, and highlight their role in membrane association and function.

RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107–125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107–125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107–125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15^R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15^R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15^R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9-cis retinal improved ERG amplitudes in Pcdh15^R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.