Disturbed retinoid metabolism upon loss of rlbp1a impairs cone function and leads to subretinal lipid deposits and photoreceptor degeneration in the zebrafish retina

  1. Domino K Schlegel
  2. Srinivasagan Ramkumar
  3. Johannes von Lintig
  4. Stephan CF Neuhauss

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    Evaluation Summary:

    In mammals the cellular retinaldehyde binding protein, CRALBP, is expressed in the pigment epithelium (RPE) and in Müller glial cells (MGCs) in the retina. Zebrafish has two copies of the gene, each expressed in one of the cell types. By knocking out each gene with CRISPR/Cas9, the authors could show that it is the copy expressed in the RPE that is essential for turnover of retinal and for cone function. Thus, the zebrafish gene duplication suggests that the RPE role of CRALBP is the important one also in humans, implying the RPE as target for future gene therapy in humans with mutations in CRALBP.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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Abstract

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.

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  1. Version published to 10.7554/elife.71473 on eLife
  2. eLife

    Evaluation Summary:

    In mammals the cellular retinaldehyde binding protein, CRALBP, is expressed in the pigment epithelium (RPE) and in Müller glial cells (MGCs) in the retina. Zebrafish has two copies of the gene, each expressed in one of the cell types. By knocking out each gene with CRISPR/Cas9, the authors could show that it is the copy expressed in the RPE that is essential for turnover of retinal and for cone function. Thus, the zebrafish gene duplication suggests that the RPE role of CRALBP is the important one also in humans, implying the RPE as target for future gene therapy in humans with mutations in CRALBP.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Schlegel et al. dug into the function of the CRALBP protein in relation to a series of autosomal recessive retinal diseases. They used zebrafish as a model since they hold two duplicated genes (rlbp1a and rlbp1b paralogs) which are orthologs for the human RLBP1. Interestingly, a subfunctionalisation has happened in zebrafish that led each one of the paralogs to be expressed exclusively in either the RPE (rlbp1a) or the Müller glial cells (rlbp1b). This circumstance is very favourable for the study of their function in the visual cycle, independently and together, by knocking them out using goal directed mutagenesis. To do so, Neuhauss lab incorporated to his lab the state-of-art technique CRIPSR-Cas to specifically KO each of the two genes of interest.

    Subsequently, to analyse the results, they confirmed the absence of the targeted proteins by immunohistochemistry and afterwards analysed the retinal function by electroretinogram and the presence of different metabolites by HPLC.
    The results and the conclusions extracted from them are totally sound and the discussion is well-structured touches each single point.

    The goal of the study was to shed light on the aetiology of the diseases known to be by associated with mutations in the gene RLBP1 and the authors succeeded by generating a reliable model recapitulating the main features common to these autosomal recessive retinal diseases, including an essential factor as aging is. The generation of a new model opens doors in the respective field by facilitating the study of the related phenotype-related alterations, something important in this case due to the incidence of the blinding diseases caused by RLBP1 mutations.

    As a major strength I see the continuity of the reliable, rigorous and meticulous work of Neuhauss lab. It is a pleasure to see regular publications, evolving together with the advances in technology. In this case, incorporating the technique awarded in the last Nobel prize in Chemistry. In addition, the generation of a new model to study RLBP1-KO-related diseases adds a new tool to investigate the molecular mechanisms triggering these diseases.

    As a weakness I see the use of a CRALBP1 serum to validate the KOs, instead of using specific antibodies. I understand the difficulty of generating antibodies but perhaps the use of riboprobes could be a more adequate way.

  4. eLife

    Reviewer #2 (Public Review):

    Using CRISPR/Cas9 mutagenesis the authors generated two homozygous knockout fish lines deleting separately rlbp1a in RPE and rlbp1b in MGC and investigated the pathological effects of the protein deficiencies on visual function. It was found that deletion of rlbp1a results in reduced chromophore levels and suppressed cone responses to light. This mutant also accumulated lipid droplets containing 11-cis and all-trans-retinyl esters which are similar to subretinal lesions in patients with RLBP1 mutations. During aging rlbp1a mutant fish develops cone and rod dystrophy and retinal thinning. In contrast, rlbp1b deletion did not result in retina degeneration or dysfunction. The double mutant demonstrated the same phenotype as a single rlbp1a mutant. This means that CRALBP1a is an essential protein which plays an important role in generation of 11-cis-retinal by accelerating the retinol isomerase reaction catalyzed by RPE65 and, probably, assisting to RGR photoisomerase in RPE cells. It may also assist to yet unknown retinyl ester hydrolase which hydrolyzes 11-cis-retinyl esters in retinosomes to produce 11-cis-retinol. The results of this work show that MGC produced CRALBP1b does not participate in visual cycle in zebrafish. Most likely, mammalian CRALBP expressed in MGC also plays only minor role in visual function. It means that Muller cells contribution to chromophore regeneration previously postulated by other authors maybe overestimated. The role of rlbp1b remains to be investigated in future.

    The manuscript is written clearly and carefully. Both the data and the conclusion of the manuscript are solid and certainly worthy of publication.

  5. eLife

    Reviewer #3 (Public Review):

    Schlegel et al take advantage of the fact that in teleost fish there has been a duplication of the RLBP1 gene, with rlbp1a expressed in the retinal pigment epithelium (RPE) and rlbp1b expressed in Müller glial cells (MGCs). Selective knockout of these paralogs in zebrafish has enabled the authors to separately examine the roles of the expressed cellular retinaldehyde binding protein, CRALBP, in these two classes of cell. They have thereby been able (in a cone-dominant species) to disentangle the contributions of CRALBP within the two retinoid re-cycling pathways: the canonical RPE cycle and the more recently discovered intra-retinal cycle.

    Importantly, the authors show that their zebrafish rlbp1a KO model exhibits features found in certain human blinding diseases caused by mutations in the RLBP1 gene (e.g. Bothnia dystrophy, fundus albipunctatus, etc.). Most notable is the occurrence of sub-retinal lipid droplets (that appear to contain the accumulation of retinyl esters), together with the occurrence of age-related retinal degeneration.

    The experiments have been well-designed and for the most part well-executed, and the results and interpretation generally appear very solid. Nevertheless, there are a few areas where clarification or additional data are called for.

    1. One area where additional explanation is needed concerns the measurement and interpretation of 11-cis retinaldehyde (11cisRAL) levels. The text refers to "11cisRAL" as the aldehyde that binds to CRALBP. However, it is unclear whether the measurements of "11cisRAL" refer to the total of 11-cis isomer covalently bound as visual pigment (in cones and rods) in addition to the non-covalently bound 11cisRAL. I am not a chemist, and am unable to determine what was extracted. It will be important for the paper to make absolutely clear what was measured, and to interpret the measurements appropriately, according to whether or not covalently bound retinaldehyde was included. If the measurements did include rhodopsin and cone opsins, then I would urge that a term distinct from "11cisRAL" be employed throughout the paper.

    2. Another area that would benefit from additional explanation is the considerable degree of variability in the measurements of retinoid content in WT adult animals in Figure 2. Between panels A, C and E, the mean WT levels differ markedly: for 11cisRAL (312, 177 and 280); for 11cisRE (12.7, 86.1 and 7.2); and for atRE (168, 460 and 187). Are these WT values significantly different from each other? Would it be better to use the WT grand means in the tests of significance for the knockouts?

    3. Before the authors can state that "We observed no pronounced changes of cone responses in Cralbpb-deficient larval eyes" they need to illustrate rlbp1b KO responses in Figure 4. At present, the double KO plots in panel B2 ("BL", and "BL+reDA") appear to show considerably greater attenuation than for the rlbp1a KO plots in panel A2, strongly suggesting an effect of Cralbpb. So I can't see how the authors can make the above statement unless they present the data.

    Subject to clarification of the points above, this paper represents an important contribution to knowledge of the cellular mechanisms of retinoid recycling for cone photoreceptors.

  6. Version published to 10.1101/2021.06.18.448986v1 on bioRxiv