Sarkar, Hajrah; (2022) Understanding disease mechanisms and developing new therapies for RDH12-related retinopathies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Retinol dehydrogenase 12 (RDH12) is expressed in photoreceptor inner segments and catalyses the reduction of all-trans retinal (atRAL) to all-trans retinol as part of the visual cycle. Mutations in RDH12 are primarily associated with Leber congenital amaurosis (LCA), a severe early-onset retinal dystrophy causing childhood blindness, and in rare cases autosomal dominant retinitis pigmentosa (RP), causing a late onset milder phenotype. There are currently no treatments for RDH12-related retinopathies. In this thesis, several models were generated to study the disease mechanisms and test new therapeutics. Recombinant expression and purification of RDH12 was attempted to study protein structure, however difficulties in purification were encountered, with protein aggregation and low yields. CRISPR/Cas9 gene editing was used to generate a rdh12 zebrafish mutant model. rdh12 fish displayed a late onset rod-predominant degeneration, with defects in rhodopsin trafficking. Early indicators of stress were detected in the adult retina, with reduced expression of autophagy and oxidative stress markers and increased phagosome size. HEK-293 stable cell lines expressing wildtype (WT) and mutant RDH12 were generated. WT RDH12 protected cells from atRAL-induced toxicity. 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. Zebrafish and cell line models revealed a number of disrupted pathways, representing potential therapeutic targets. A number of drugs were screened and pregabalin, a retinal scavenger, was found to reduce atRAL induced ER stress in RDH12 mutant cells, representing a new class of potential drugs that can be targeted for RDH12-retinopathies. Induced pluripotent stem cell lines were generated from two patients, to be used for differentiation to retinal organoids facilitating further investigation of disease mechanisms and drug screening. The models created in this project provide a valuable resource for further study of RDH12-retinopathies and development of novel therapeutics.

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