The RLBP1 gene encodes the 36 kDa cellular retinaldehyde binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod-cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Müller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.
Citation: Schlegel DK, Glasauer SMK, Mateos JM, Barmettler G, Ziegler U, Neuhauss SCF. A new zebrafish model for CACNA2D4-dysfunction. Invest Ophthalmol Vis Sci. 2019;60:5124-5135. https://doi.org/10.1167/ iovs.19-26759 PURPOSE. Mutations in CACNA2D4, encoding the a 2 d 4 subunit of retinal voltage-gated calcium channels (Ca v ), cause a rare type of retinal dysfunction in human, mainly affecting cone vision. Here, we investigate the role of CACNA2D4 in targeting of Ca v , its influence on conemediated signal transmission, and the cellular and subcellular changes upon loss of a 2 d 4 by exploiting the advantages of the cone-dominant zebrafish as model system. METHODS.We identified two zebrafish CACNA2D4 paralogs (cacna2d4a and cacna2d4b), analyzed their expression by RNA in situ hybridization and introduced truncating frameshift mutations through CRISPR/Cas9-mediated mutagenesis. We analyzed retinal function and morphology of the single and double mutant lines by electroretinography, immunohistochemistry, light-and electron microscopy. RESULTS. Knockout of cacna2d4b reduces the expression of Cacna1fa, the pore-forming subunit of retinal Ca v 1.4, whereas loss of cacna2d4a did not. Only knockout of both paralogs impaired cone-mediated ERG b-wave amplitude. The number of ''floating'' ribbons is increased in double-KO, while retinal morphology and expression of postsynaptic mGluR6b remain largely unaffected. Both Cacna1fa and Ribeyeb show ectopic punctate expression in cacna2d4b-KO and double-KO photoreceptors.CONCLUSIONS. We find that increasing the expression of Ca v at the synaptic membrane is an evolutionarily conserved function of Cacna2d4b. Yet, since both paralogs participate in cone synaptic transmission, we propose partial subfunctionalization in zebrafish. Similar to human patients, our double KO zebrafish model shows mild cone dysfunction, which was not associated with signs of retinal degeneration. Therefore, cacna2d4-KO zebrafish is a suitable model to study the pathophysiological mechanisms underlying CACNA2D4 dysfunction in human.Keywords: calcium channel, cone photoreceptor, zebrafish, disease model Recent studies on two different Cacna2d4-KO mice suggest a role for Cacna2d4 in structural organization and function of rod 23,24 and cone synapses. 24 Interestingly, in all three studies, Cacna2d4 mutation affects rods more severely than cones, and
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