Photoreceptors are sensitive neuronal cells with great metabolic demands, as they are responsible for carrying out visual phototransduction, a complex and multistep process that requires the exquisite coordination of a large number of signalling protein components. Therefore, the viability of photoreceptors relies on mechanisms that ensure a well-balanced and functional proteome that maintains the protein homeostasis, or proteostasis, of the cell. This review explores how the different isoforms of Hsp90, including the cytosolic Hsp90α/β, the mitochondrial TRAP1, and the ER-specific GRP94, are involved in the different proteostatic mechanisms of photoreceptors, and elaborates on Hsp90 function when retinal homeostasis is disturbed. In addition, several studies have shown that chemical manipulation of Hsp90 has significant consequences, both in healthy and degenerating retinae, and this can be partially attributed to the fact that Hsp90 interacts with important photoreceptor-associated client proteins. Here, the interaction of Hsp90 with the retina-specific client proteins PDE6 and GRK1 will be further discussed, providing additional insights for the role of Hsp90 in retinal disease.

Bialleleic pathogenic variants in LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report the use of gene editing to generate isogenic LCA5 knock-out (LCA5 KO) induced pluripotent stem cells (iPSC) and their differentiation to retinal organoids. The molecular and cellular phenotype of the LCA5 KO retinal organoids was studied in detail and compared to isogenic controls as well as patient-derived retinal organoids. The absence of LCA5 was confirmed in retinal organoids by immunohistochemistry and western blotting. There were no major changes in retinal organoid differentiation or ciliation, however, the localisation of CEP290 and IFT88 was significantly altered in LCA5 KO and patient photoreceptor cilia with extension along the axoneme. The LCA5-deficient organoids also had shorter outer segments and rhodopsin was mislocalised to the outer nuclear layer. We also identified transcriptomic and proteomic changes associated with the loss of LCA5. Importantly, treatment with the small molecules eupatilin, fasudil or a combination of both drugs reduced CEP290 and IFT88 accumulation along the cilia. The treatments also improved rhodopsin traffic to the outer segment and reduced mislocalisation of rhodopsin in the outer nuclear layer (ONL). The improvements in cilia-associated protein localisation and traffic were accompanied by significant changes in the transcriptome towards control gene expression levels in many of the differentially expressed genes. In summary, iPSC-derived retinal organoids are a powerful model for investigating the molecular and cellular changes associated with loss of LCA5 function and highlight the therapeutic potential of small molecules to treat retinal ciliopathies.

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.

Retinitis Pigmentosa (RP) is an inherited retinal dystrophy characterised by the progressive loss of rod photoreceptors. Sector RP is a form of RP where degeneration originates in the inferior retina, mainly influenced by light exposure. Over 200 RHO variants are pathogenic and associated with autosomal dominant RP. RHO ^M39R is one of the most common RHO variants linked to sector RP in the UK. A knock-in (KI) mouse model expressing Rho ^M39R was generated and characterised to investigate the mechanisms of degeneration associated with this variant and explore novel therapeutic strategies for rhodopsin sector RP. Under cyclic ambient light, Rho ^M39R/+ KI mice exhibited impaired retinal function by ERG, with some defects in OS ultrastructure, but retained normal outer nuclear layer (ONL) thickness. Repeated exposure to bright light led to photoreceptor loss. In contrast, Rho ^M39R/M39R KI mice in cyclic ambient light displayed severe retinal dysfunction, ONL thinning, and grossly abnormal OS ultrastructure. In homozygous mice, a single bright light exposure significantly reduced ONL thickness within 48 h. The rescue of these models was achieved through reduced light exposure and pharmacological intervention. Rearing in dim red light (red cage condition) restored ERG responses in Rho ^M39R/+ KI mice and improved ONL thickness in Rho ^M39R/M39R KI mice. Transcriptomic analysis in Rho ^M39R/M39R KI mice revealed upregulation of Sphingosine 1-P Receptor ( S1pr ) transcripts. Treatment with the S1PR agonist Fingolimod (FTY720) before bright light exposure significantly reduced degeneration, demonstrating a protective effect in both heterozygous and homozygous models and suggesting potential as a therapeutic approach for sector RP patients.

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 both 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 susceptibility to morphological and functional changes related to phototransduction. These results demonstrate that disruption of Müller glia signaling contributes to a loss of retinal integrity, providing novel insights into USH1C pathogenesis and offering targets for therapeutic interventions.

Retinitis Pigmentosa (RP) is an inherited retinal dystrophy characterized by the progressive loss of rod photoreceptors. Sector RP is a form of RP, where degeneration originates in the inferior retina, mainly influenced by light exposure. Over 200 RHO variants are pathogenic and associated with autosomal dominant RP. RHO ^M39R is one of the most common RHO variants linked to sector RP in the UK. A knock-in (KI) mouse model expressing Rho ^M39R was generated and characterized to investigate the mechanisms of degeneration associated with this variant and explore novel therapeutic strategies for rhodopsin sector RP. Under ambient light, Rho ^M39R/+ KI mice exhibited impaired retinal function by ERG, with some defects in OS ultrastructure, but retained normal outer nuclear layer (ONL) thickness. Repeated exposure to bright light led to photoreceptor loss. In contrast, Rho ^M39R/M39R KI mice in ambient light displayed severe retinal dysfunction, ONL thinning, and grossly abnormal OS ultra structure. In homozygous mice, a single bright light exposure significantly reduced ONL thickness within 48 h. The rescue of these models was achieved through reduced light exposure and pharmacological intervention. Rearing in dim red light (red cage condition) restored ERG responses in Rho ^M39R/+ KI mice and improved ONL thickness in Rho ^M39R/M39R KI mice. Transcriptomic analysis in Rho ^M39R/M39R KI mice revealed upregulation of Sphingosine 1-P Receptor (S1PR) transcripts. Treatment with the S1PR agonist Fingolimod (FTY720) before bright light exposure significantly reduced degeneration, demonstrating a protective effect in both heterozygous and homozygous models and suggesting potential a therapeutic approach for sector RP patients.