Loss of ABCA4 from photoreceptor discs is associated with glial transcriptomic changes in retinal organoids

Single-cell analysis reveals impaired Müller glia-mediated intercellular communication and photoreceptor pathology in USH1C retinal organoids

Usher syndrome type 1, caused by pathogenic variants in the USH1C gene, leads to congenital deafness and progressive retinal degeneration resulting in vision loss. While auditory deficits can be compensated by cochlea implants and hearing aids, no treatment exists to prevent retinal degeneration. Here, we generated retinal organoids from induced pluripotent stem cells of two USH1C patients to elucidate the cellular and molecular mechanisms driving ocular pathogenesis. Single-cell RNA sequencing of healthy and USH1C retinal organoids identified differential expression of genes related to phototransduction in photoreceptors, as well as alterations in cell adhesion and canonical Wnt signaling in Müller glia cells. Analysis of intercellular communication revealed an overall reduced signaling efficiency, particularly affecting Müller glia-mediated retinal adhesion processes. Morphological characterization of organoids confirmed transcriptome changes by showing degeneration of the outer limiting membrane and loss of adherens junction architecture. Moreover, photoreceptors revealed increased levels of apoptosis, as well as morphological and functional changes related to phototransduction. Our results demonstrate that disruption of Müller glia signaling contributes to an overall loss of retinal integrity, providing novel insights into USH1C pathogenesis and offering targets for therapeutic interventions.

Single-cell spatial transcriptomics reveals rewiring of RPE and Müller glia signaling toward photoreceptors following outer segment disruption in Prph2C213Y mice.

PRPH2 mutations cause inherited retinal dystrophies (IRDs), but how photoreceptor outer segment (OS) disruption reshapes the surrounding retina remains unclear. Using a heterozygous Prph2C213Y/+ mouse model generated by CRISPR/Cas9, we characterized age-related retinal pathology and responses of retinal pigment epithelium (RPE) and Müller glia. Independent age- and sex-matched cohorts were examined at 1, 3, and 6 months by electroretinography, optical coherence tomography, and fundus autofluorescence. Mutant mice showed rod dysfunction from 1 month, RPE dysfunction from 3 months, and cone dysfunction by 6 months, accompanied by progressive outer retinal thinning and hyperautofluorescent deposits. Histological and ultrastructural analyses revealed OS disorganization, shortened RPE microvilli, RPE monolayer remodeling, increased RPE autofluorescence, and reactive Müller gliosis. Single-cell spatial transcriptomics of wild-type and mutant retinas at 6 months resolved nine cell populations and identified RPE cells and Müller glia as prominently perturbed non-photoreceptor populations. RPE cells showed an epithelial-mesenchymal transition-related remodeling state linked to a candidate Nfib-Fstl1 module, whereas Müller glia showed activation of activator protein 1 (AP-1) regulons, including Fos, Fosl2, and Junb, with predicted targets Osmr, A2m, and Stat3. Cell-cell communication analyses indicated coordinated changes in neuroprotective, inflammatory, and matrix-related signaling from RPE cells and Müller glia toward photoreceptors. These findings indicate that PRPH2-associated retinal dystrophy is a multicellular process in which OS disruption drives coordinated RPE and Müller glial remodeling with potentially protective or pro-degenerative effects, and nominate the RPE Nfib-Fstl1 program, Müller glial AP-1 responses with predicted STAT3 involvement, and support-cell-derived growth factor signaling as candidate mutation-independent therapeutic targets.

Retinal degeneration in a mouse model of CRB1 disease rescued by the photoreceptor-specific CRB1-B isoform

Many genes involved in inherited diseases produce alternate mRNA isoforms that remain poorly characterized. Functional assessment of these isoforms could therefore unlock new insights into disease pathobiology or treatment. Here we investigated the function of the newly discovered “B” isoform of CRB1, a gene implicated in inherited retinal degenerations. CRB1-B is the most abundant retinal isoform, and differs from the canonical CRB1-A isoform in several important ways. Crucially, CRB1-B is the only isoform expressed by photoreceptors – the cells vulnerable to degeneration. Using a mouse model of CRB1-associated degeneration with degraded visual acuity, we find that restoring CRB1-B to photoreceptors prevents degeneration and completely rescues visual function, despite the absence of other isoforms. The therapeutic mechanism involves preservation of adherens junctions between photoreceptors and supporting glia: We establish that progressive loss of these junctions is a key pathobiological mechanism underlying photoreceptor death, and that junction loss is prevented by restoring photoreceptor expression of CRB1-B. Our findings nominate CRB1-B replacement as a promising gene therapy strategy. More broadly, they suggest that exploring disease gene isoform diversity offers an untapped opportunity to devise novel therapeutics.

Identification of Gene Expression Changes Associated with the Progression of Retinal Degeneration in the rd1 Mouse

APTX deficiency is associated with altered neuronal differentiation and alternative splicing in patient-derived neurons.

It is demonstrated that AOA1-derived neurons exhibit neurite morphology and maturation defects correlated with the accumulation of DNA single-strand break (SSB) signals, and DNA-damage and PAR signals showed greater DNA-damage and PAR signals together with lower protein-normalized NAD(H) and ATP after genotoxic exposure.

CDHR1-associated retinal degeneration: Clinical phenotypes and therapeutic approaches.

CDHR1 is a recently identified cause of autosomal recessive retinal degeneration manifesting as three distinct clinical phenotypes: macular dystrophy, cone-rod dystrophy or retinitis pigmentosa. In this review, we summarise the discovery and characterisation of CDHR1, clinical phenotypes, natural history and therapeutic approaches including gene supplementation and CRISPR gene editing. CDHR1 is a non-classical cadherin that is highly expressed in cone and rod photoreceptors and is essential for the higher-order organisation of the functionally critical outer segments. Promising pre-clinical data show that AAV gene supplementation therapy delivered by subretinal injection can lead to long-term morphological, structural, functional and behavioural improvements in the Cdhr1 knockout mouse model. Notably, CDHR1 supplementation restored full-length photoreceptor outer segments that are usually shortened and disorganised in disease models and prolonged photoreceptor survival - key mechanisms for the functional and behavioural rescue effects that were observed. The disease is likely to be underdiagnosed because CDHR1-associated macular dystrophy - likely to be the most common disease phenotype - is most often caused by a 'silent' nucleotide substitution that has previously been overlooked by genetic testing. Since the macular dystrophy phenotype has phenotypic similarities to advanced dry age-related macular degeneration (AMD), misdiagnoses are common. CDHR1-associated macular dystrophy also shares phenotypic features with a variety of monogenic masquerades such as ABCA4, PRPH2 and GUCY2D-associated macular dystrophies; we present a flowchart to guide the clinical distinction of these disorders which is now critical for patients as their treatments diverge. AAV gene therapy may be beneficial across the CDHR1 disease spectrum - including those with hypomorphic variants associated with macular dystrophy or retinitis pigmentosa. The coding sequence fits into AAV and with the macular dystrophy phenotype, there is a large time window for potential intervention and a large treatable population. Hence clinical trials are anticipated. It is therefore important that patients with CDHR1-associated retinal degeneration are accurately phenotyped and genetically confirmed to support the application of CDHR1 gene therapy.