Abstract
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.
Highlights
Leber congenital amaurosis (LCA) is a severe early onset autosomal recessive retinal dystrophy, and mutations in the retinol dehydrogenase 12 (RDH12) gene (OMIM: 608830) account for approximately 10% of all cases [1]
RDH12 is expressed in photoreceptor inner segments and is responsible for the reduction of all-trans retinal to all-trans retinol, as part of the visual cycle [5]. atRAL is primarily reduced to atROL in photoreceptor outer segments (POSs) by RDH8; in periods of intense illumination, atRAL leaks to the inner segments where it is reduced by RDH12 [6]
We have identified a number of pathways that appear to be disrupted, including oxidative stress, endoplasmic reticulum (ER) stress, and autophagy, representing potential future therapeutic targets
Summary
Leber congenital amaurosis (LCA) is a severe early onset autosomal recessive retinal dystrophy, and mutations in the retinol dehydrogenase 12 (RDH12) gene (OMIM: 608830) account for approximately 10% of all cases [1]. Treatment of ARPE-19 cells with atRAL resulted in a significant increase in intracellular ROS and upregulation of NRF2, HO-1, and γ-GCSh. Nrf (nuclear factor E2-related factor 2) is a transcription factor that regulates the cells’ response to oxidative stress through the expression of antioxidant and detoxifying genes. Activation of ATF6 results in its translocation to the Golgi, where it is cleaved and translocated back to the nucleus, where it induces expression of target genes, including chaperones The role of these pathways is to alleviate ER stress and restore ER homeostasis; when ER stress becomes overwhelmingly high, ATF4 activates C/EBP homologous protein (CHOP), which in turn triggers apoptosis [19,20]. We have identified a number of pathways that appear to be disrupted, including oxidative stress, ER stress, and autophagy, representing potential future therapeutic targets
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