Loss of REP1 impacts choroidal melanogenesis and vasculogenesis in choroideremia.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, however, the involvement of the choroid in disease progression is not fully understood. CHM is caused by mutations in the CHM gene, encoding the ubiquitously expressed Rab escort protein 1 (REP1). REP1 plays an important role in intracellular trafficking of vesicles, including melanosomes. In this study, we examined the ultrastructure of the choroid in chmru848 fish and Chmnull/WT mouse models using transmission electron and confocal microscopy. Significant pigmentary disruptions were observed, with lack of melanosomes in the choroid of chmru848 fish from 4 days post fertilisation (4dpf), and a reduction in choroidal blood vessel diameter and interstitial pillars suggesting a defect in vasculogenesis. Total melanin and expression of melanogenesis genes tyr, tryp1a, mitf, dct and pmel were also reduced from 4dpf. In Chmnull/WT mice, choroidal melanosomes were significantly smaller at 1 month, with reduced eumelanin at 1 year. The choroid in CHM patients were also examined using spectral domain optical coherence tomography (SD-OCT) and OCT-angiography (OCT-A) and the area of preserved choriocapillaris (CC) was found to be smaller than that of overlying photoreceptors, suggesting that the choroid is degenerating at a faster rate. Histopathology of an enucleated eye from a 74-year-old CHM male patient revealed isolated areas of RPE but no associated underlying CC. Pigmentary disruptions in CHM animal models reveal an important role for REP1 in melanogenesis, and drugs that improve melanin production represent a potential novel therapeutic avenue.

Oxidative and Endoplasmic Reticulum Stress Represent Novel Therapeutic Targets for Choroideremia.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy, affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, with no approved therapy. CHM is caused by mutations in the CHM gene, which encodes the ubiquitously expressed Rab escort protein 1 (REP1). REP1 is involved in prenylation, a post-translational modification of Rab proteins, and plays an essential role in intracellular trafficking. In this study, we examined oxidative and endoplasmic reticulum (ER) stress pathways in chmru848 zebrafish and CHMY42X patient fibroblasts, and screened a number of neuroprotectants for their ability to reduce stress. The expression of the oxidative stress markers txn, cat and sod3a, and the ER stress markers bip, atf4 and atf6, were dysregulated in chmru848 fish. The expression of SOD2 was also reduced in CHMY42X fibroblasts, along with reduced BIP and increased CHOP expression. The lack of REP1 is associated with defects in vesicular trafficking, photoreceptor outer segment phagocytosis and melanosome transport, leading to increased levels of stress within the retina and RPE. Drugs targeting oxidative and ER stress pathways represent novel therapeutic avenues.

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs.

Congenital aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough-inducing drugs (TRIDs) have the ability to weaken the recognition of in-frame premature termination codons (PTCs), permitting full-length protein to be translated. We established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from two aniridia patients with prevalent PAX6 nonsense mutations. Both in vitro models show reduced PAX6 protein levels, mimicking the disease. The repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP) and the positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESCs. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia.

Choroideremia: molecular mechanisms and therapies.

Choroideremia (CHM) is a monogenic X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE), and choroid; it is caused by mutations involving the CHM gene. CHM is characterized by night blindness in early childhood, progressing to peripheral visual field loss and eventually to complete blindness from middle age. CHM encodes the ubiquitously expressed Rab escort protein 1 (REP1), which is responsible for prenylation of Rab proteins and is essential for intracellular trafficking of vesicles. In this review we explore the role of REP1 in the retina and its newly discovered systemic manifestations, and discuss the therapeutic strategies for tackling this disease, including the outcomes from recent clinical trials.

Identification of 4 novel human ocular coloboma genes ANK3, BMPR1B, PDGFRA, and CDH4 through evolutionary conserved vertebrate gene analysis.

PURPOSE: Ocular coloboma arises from genetic or environmental perturbations that inhibit optic fissure (OF) fusion during early eye development. Despite high genetic heterogeneity, 70% to 85% of patients remain molecularly undiagnosed. In this study, we have identified new potential causative genes using cross-species comparative meta-analysis. METHODS: Evolutionarily conserved differentially expressed genes were identified through in silico analysis, with in situ hybridization, gene knockdown, and rescue performed to confirm spatiotemporal gene expression and phenotype. Interrogation of the 100,000 Genomes Project for putative pathogenic variants was performed. RESULTS: Nine conserved differentially expressed genes between zebrafish and mouse were identified. Expression of zebrafish ank3a, bmpr1ba/b, cdh4, and pdgfaa was localized to the OF, periocular mesenchyme cells, or ciliary marginal zone, regions traversed by the OF. Knockdown of ank3, bmpr1b, and pdgfaa revealed a coloboma and/or microphthalmia phenotype. Novel pathogenic variants in ANK3, BMPR1B, PDGFRA, and CDH4 were identified in 8 unrelated coloboma families. We showed BMPR1B rescued the knockdown phenotype but variant messenger RNAs failed, providing evidence of pathogenicity. CONCLUSION: We show the utility of cross-species meta-analysis to identify several novel coloboma disease-causing genes. There is a potential to increase the diagnostic yield for new and unsolved patients while adding to our understanding of the genetic basis of OF morphogenesis.

Involvement of Oxidative and Endoplasmic Reticulum Stress in RDH12-Related Retinopathies.

Retinol dehydrogenase 12 (RDH12) is expressed in photoreceptor inner segments and catalyses the reduction of all-trans retinal (atRAL) to all-trans retinol (atROL), as part of the visual cycle. Mutations in RDH12 are primarily associated with autosomal recessive Leber congenital amaurosis. To further our understanding of the disease mechanisms, HEK-293 cell lines expressing wildtype (WT) and mutant RDH12 were created. The WT cells afforded protection from atRAL-induced toxicity and oxidative stress. Mutant RDH12 cells displayed reduced protein expression and activity, with an inability to protect cells from atRAL toxicity, inducing oxidative and endoplasmic reticulum (ER) stress, with upregulation of sXBP1, CHOP, and ATF4. Pregabalin, a retinal scavenger, attenuated atRAL-induced ER stress in the mutant RDH12 cell lines. A zebrafish rdh12 mutant model (rdh12u533 c.17_23del; p.(Val6AlafsTer5)) was generated through CRISPR-Cas9 gene editing. Mutant fish showed disrupted phagocytosis through transmission electron microscopy, with increased phagosome size at 12 months post-fertilisation. Rhodopsin mislocalisation and reduced expression of atg12 and sod2 indicated early signs of a rod-predominant degeneration. A lack of functional RDH12 results in ER and oxidative stress representing key pathways to be targeted for potential therapeutics.

Generation of two human iPSC lines from patients with autosomal dominant retinitis pigmentosa (UCLi014-A) and autosomal recessive Leber congenital amaurosis (UCLi015-A), associated with RDH12 variants.

Induced pluripotent stem cell (iPSC) lines were generated from two patients with RDH12 variants. UCLi014-A is from a patient with heterozygous frameshift mutation c.759del p.(Phe254Leufs*24), associated with autosomal dominant retinitis pigmentosa. UCLi015-A is from a patient with homozygous missense mutation c.619A > G p.(Asn207Asp), associated with Leber congenital amaurosis. Fibroblasts were derived from skin biopsies and reprogrammed using integration free episomal reprogramming plasmids. The iPSC lines expressed pluripotency markers, exhibited differentiation potential in vitro and displayed normal karyotypes. These cell lines will act as a tool for disease modelling, enabling comparison of disease mechanisms, identification of therapeutic targets and drug screening.

Generation of human iPSC line (UCLi013-A) from a patient with microphthalmia and aniridia, carrying a heterozygous missense mutation c.372C>A p.(Asn124Lys) in PAX6.

A human induced pluripotent stem cell (hiPSC) line (UCLi013-A) was generated from fibroblast cells of a 34-year-old donor with multiple ocular conditions including severe microphthalmia and aniridia. The patient had a heterozygous missense mutation in PAX6 c.372C>A, p.(Asn124Lys), validated in the fibroblasts through Sanger sequencing. Fibroblasts derived from a skin biopsy were reprogrammed using integration free episomal reprogramming. The established iPSC line was found to express pluripotency markers, exhibit differentiation potential in vitro and display a normal karyotype. This cell line will act as a tool for disease modelling of microphthalmia and aniridia, identification of therapeutic targets and drug screening.

Generation of two human control iPS cell lines (UCLi016-A and UCLi017-A) from healthy donors with no known ocular conditions.

Two human induced pluripotent stem cell (hiPSC) lines (UCLi016-A and UCLi017-A) were generated from fibroblast cells of 23- and 34-year-old healthy male donors with no known ocular conditions. Fibroblast cells were derived from skin biopsies and reprogrammed using integration free episomal reprogramming. The established iPSC lines were found to express pluripotency markers, exhibit differentiation potential in vitro and display a normal karyotype. These cell lines will act as a control lines for researchers studying ocular diseases.

Novel Heterozygous Deletion in Retinol Dehydrogenase 12 (RDH12) Causes Familial Autosomal Dominant Retinitis Pigmentosa.

Mutations in the retinol dehydrogenase 12 (RDH12) gene are primarily associated with Leber congenital amaurosis (LCA) type 13, a severe early onset autosomal recessive retinal dystrophy. Only one family with a heterozygous variant, associated with mild retinitis pigmentosa (RP), has been reported. We report a novel heterozygous variant [(c.759del; p.(Phe254Leufs∗24)], resulting in a frameshift and premature termination identified in two unrelated individuals with familial autosomal dominant RP. Both heterozygous variants are associated with a late onset RP phenotype, suggesting a possible genotype-phenotype correlation.

Retinol dehydrogenase 12 (RDH12): Role in vision, retinal disease and future perspectives.

Retinol dehydrogenase 12 (RDH12) is an NADPH-dependent retinal reductase, which is expressed in the inner segments of the photoreceptors. It functions as part of the visual cycle, which is a series of enzymatic reactions required for the regeneration of the visual pigment, and has also been implicated in detoxification of lipid peroxidation products. Mutations in RDH12 have been linked to Leber congenital amaurosis (LCA) and autosomal dominant retinitis pigmentosa. A number of in-vitro studies have shown that mutations in RDH12 result in little or no enzyme activity. Knockout mouse models however do not recapitulate the severe phenotype observed in patients, resulting in a limited understanding of the disease mechanisms. With gene replacement and small molecule drugs emerging for inherited retinal dystrophies, herein we provide a review of RDH12 structure, its role in vision and the current understanding of disease mechanisms linked to clinical phenotype to support therapeutic development.

Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics.

Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense-mediated mRNA decay (NMD) is the cell's natural surveillance mechanism that detects and destroys PTC-containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using Quantitative reverse transcription PCR (RT-qPCR), we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation [c.715 C>T; p.(R239*)]. These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their induced pluripotent stem cell-derived retinal pigment epithelium. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wild-type levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated.

Dynamic Equilibrium of the Aurora†A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy.

The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small-molecule inhibitors. By using single-molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora A kinase is in dynamic equilibrium between a DFG-in-like active T-loop conformation and a DFG-out-like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora A activating protein TPX2 shifts the equilibrium towards an active T-loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T-loop. We show that Aurora A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. Our approach will enable conformation-specific effects to be integrated into inhibitor discovery across the kinome, and we outline some immediate consequences for structure-based drug discovery.

Unravelling the role of SNM1 in the DNA repair system of Trypanosoma brucei.

All living cells are subject to agents that promote DNA damage. A particularly lethal lesion are interstrand cross-links (ICL), a property exploited by several anti-cancer chemotherapies. In yeast and humans, an enzyme that plays a key role in repairing such damage are the PSO2/SNM1 nucleases. Here, we report that Trypanosoma brucei, the causative agent of African trypanosomiasis, possesses a bona fide member of this family (called TbSNM1) with expression of the parasite enzyme able to suppress the sensitivity yeast pso2Δ mutants display towards mechlorethamine, an ICL-inducing compound. By disrupting the Tbsnm1 gene, we demonstrate that TbSNM1 activity is non-essential to the medically relevant T. brucei life cycle stage. However, trypanosomes lacking this enzyme are more susceptible to bi- and tri-functional DNA alkylating agents with this phenotype readily complemented by ectopic expression of Tbsnm1. Genetically modified variants of the null mutant line were subsequently used to establish the anti-parasitic mechanism of action of nitrobenzylphosphoramide mustard and aziridinyl nitrobenzamide prodrugs, compounds previously shown to possess potent trypanocidal properties while exhibiting limited toxicity to mammalian cells. This established that these agents, following activation by a parasite specific type I nitroreductase, produce metabolites that promote formation of ICLs leading to inhibition of trypanosomal growth.