Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Sep 12;18(9):1793-1810.
doi: 10.1016/j.stemcr.2023.07.001. Epub 2023 Aug 3.

CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids

Thilo M Buck  1 , Peter M J Quinn  1 , Lucie P Pellissier  2 , Aat A Mulder  3 , Aldo Jongejan  4 , Xuefei Lu  1 , Nanda Boon  1 , Daniëlle Koot  1 , Hind Almushattat  1 , Christiaan H Arendzen  5 , Rogier M Vos  2 , Edward J Bradley  6 , Christian Freund  5 , Harald M M Mikkers  7 , Camiel J F Boon  8 , Perry D Moerland  4 , Frank Baas  9 , Abraham J Koster  3 , Jacques Neefjes  3 , Ilana Berlin  3 , Carolina R Jost  3 , Jan Wijnholds  10
Affiliations

CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids

Thilo M Buck et al. Stem Cell Reports. .

Abstract

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

Keywords: CRB1; NOTCH1; RAB11A; VPS35; WDFY1; autophagy; cell polarity; endolysosomal system; organoids; retina; retromer.

PubMed Disclaimer

Conflict of interest statement

Conflict of interests The authors declare no competing interests. The LUMC is holder of patent number PCT/NL2014/050549, which describes the potential clinical use of CRB2; J.W. and L.P.P. are listed as co-inventor of this patent, and J.W. is an employee of the LUMC.

Figures

None
Graphical abstract
Figure 1
Figure 1
mRNA transcript levels are marginally different in Crb1KOCrb2ΔRPC against Crb1KO retina (A and B) Retinal morphology on plastic sections of Crb1KO and Crb1KOCrb2ΔRPC mice on a 100% C57/B6 genetic background at embryonic day 15.5 (E15.5). (A) Crb1KO retinas appeared to be unaffected, while (B) Crb1KOCrb2ΔRPC retinas had protrusions of neuroblast nuclei in the subretinal space (inset). (C) Protrusions per retinal section in a 50% and 100% C57/B6 genetic background at E13.5. (D–J) 100% C57/B6 genetic background. (D and E) Immunofluorescence labeling of pHH3+ nuclei (late G2 cell cycle and mitosis maker) on postnatal day P1 in (D) Crb1KO and (E) and Crb1KOCrb2ΔRPC retina. (F) Quantification of pHH3+ cells on E15.5, E17.5, P1, and P5. ∗p < 0.05. (G–J) All changed genes are indicated in blue circles (p < 0.01; log2(Fold Change) > 1.5). (G–I) Crb1KOCrb2ΔRPC against Crb1KO retina transcripts (run 1) on E15.5, E17.5, and P1. (J) Crb1KOCrb2ΔRPC compared with WT retina transcripts on E15.5 (run 2). (K) Potential WDFY1 involvement in CRB1 protein trafficking (modified from Aguilar-Aragon et al., 2020). Scale bars, 20 μm. WT, wild type; TGN, trans-Golgi network; MVB, multivesicular body. See also Figures S1 and S2.
Figure 2
Figure 2
Apical NOTCH1 is lost in CRB1 patient retinal organoids (A) CTRL1 (LUMC004iCTRL10) RPE expresses NOTCH1 on DD180 (BF, bright field; to indicate pigments in the RPE). (B–F) CTRL1 (B), CTRL2 (LUMC044iCTRL44) (C), patient P116 (LUMC0116iCRB09) (D), patient P117 (LUMC0117iCRB01) (E), and patient P128 (LUMC0128iCRB01) (F). (A–F) NOTCH1 is expressed specifically in DD180 Müller glial apical villi (magenta, NOTCH1; yellow, CD44; blue, recoverin; gray, DAPI). (G–J) PLA of NOTCH1-ECD and CRB1-ECD (green signal) shows interaction at CTRL OLM but reduced interaction at patient OLM with increased localization in patient ONL. Scale bars, 25 μm.
Figure 3
Figure 3
High-resolution TEM imaging of retinal organoids on DD180 Shown are electron-dense OLM (red arrows), electron-dense degradative compartments/vacuoles (arrowheads), and nuclei above the OLM (asterisks). (A and B) Gene-corrected lines (ISO-03 P116, iso3LUMC0116iCRB09; ISO-02-P128, iso2LUMC0128iCRB01). (C and D) CTRL lines (CTRL2, LUMC0044iCTRL44; CTLR3, LUMC080iCTRL12). (E–G) Patient lines P116, P128, and P117 (LUMC0116iCRB09, LUMC0128iCRB01, and LUMC0117iCRB01, respectively). Scale bars, 5 μm. Related to Figure S3.
Figure 4
Figure 4
The immunofluorescence signal of CRB1 labeling is reduced in CRB1 patient retinal organoids at the OLM (A–H) Triple staining of CRB1-ICD (AK2 antibody, magenta), phalloidin (filamentous actin [F-actin], green), and ARL8A/B (yellow). (I and J) CRB1 fluorescence signal measured by puncta in the OLM and ONL. (K and L) CRB1 fluorescence signal measured by puncta in the OLM and ONL. Scale bars, 25 μm. Each data point in the graphs represents individual organoids, of which an average was taken of 3 representative images. The SEM is derived from these averages. The numbers of individual organoids per condition (CTRL1, CTRL2, CTRL3, ISO-03 P116, ISO-02 P128, P116, P117, and P128) in (I) and (J) are 9, 9, 9, 10, 10, 12, 9, and 13, respectively, and for (K) and (L) 7, 8, 3, 8, 8, 12, 6, and 8, respectively (from at least two independent organoid batches). Statistical analysis: ∗p < 0.05, ∗∗∗p < 0.001. Related to Figures S4 and S5.
Figure 5
Figure 5
More degradative vesicles/compartments are present in CRB1 patient retinal organoids (A–F) Immunofluorescence triple staining of CRB1-ICD (AK2 antibody, magenta), p62 (green), and LC3B (yellow). (G and H) LC3B localized more in the ONL and OLM layers in CRB1 RP retinal organoids. (I–K) Western blots of individual organoid lysates (plus symbol, isogenic 2 line (iso02-128iCRB01); minus symbol, patient 3 (line LUMC0128CRB01) stained for LC3B (LC3-I and LC3-II; 19/17 kD; recoverin for photoreceptors (26 kD), and GAPDH (housekeeping CTRL, 37 kD). Autophagic flux was decreased in CRB1 patient retinal organoids. Scale bars, 25 μm. Each data point in the graphs in (J) and (K) represents individual organoids, of which an average was taken. The SEM is derived from these averages. The numbers of individual organoids in (G) and (H) per condition (CTRL1, CTRL2, CTRL3, ISO-03 P116, ISO-02 P128, P116, P117, and P128) are 7, 6, 9, 8, 9, 10, 11, and 10, respectively (from at least two independent organoid batches). Statistical analysis: ∗p < 0.05, ∗∗∗p < 0.001. Related to Figures S5, S6, and S8.
Figure 6
Figure 6
Dysregulation of the endolysosomal system in CRB1 patient organoids (A–L) Immunofluorescence stainings, with the antibodies indicated on the right sides of the panels, on isogenic CTRL organoid lines 1 and 2 (A, C, E, G, I, and K) related to patient organoid lines 1 (LUMC0116iCRB09) and 3 (LUMC0128iCRB01) and lines 1 and 3 (B, D, F, H, and J–L). (A–D) Recycling endosomes (RAB11A, magenta) and phalloidin (F-actin, OLM, green). The OLM in retinal organoids is stained by F-actin. (E–H) Late endosomes (RAB7, magenta), retromer complex (VPS35, green), and endolysosomes (ARL8A/B, yellow). (I–L) Late endosomes (RAB7, magenta) and EEs (EEA1, green). (M–P) Quantification of the fluorescence signal of EEs (EEA1), late endosomes (RAB7), retromer (VSP35), and recycling endosomes (RAB11A) in the OLM. (M) RAB7 in the OLM. (N) EEA1 in the OLM. (O) VPS35 in the OLM. (P) RAB11A in the OLM. Scale bars, 10 μm. Each data point in the graphs represents individual organoids, of which an average was taken of 3 representative images. The SEM is derived from these averages. The numbers of individual organoids per condition (ISO-03 P116, ISO-02 P128, P116, and P117) for (M) and (O) are 8, 9, 16, and 12, respectively; for (N) 6, 7, 5, and 6, respectively; and for (P) 9, 9, 8, and 8, respectively (from at least two independent organoid batches). Statistical analysis: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Related to Figure S7.
Figure 7
Figure 7
Increased levels of WDFY1 in EE s of patient CRB1 patient retinal organoidsAll organoids are shown at DD180 (A–F) Immunofluorescence labeling of WDFY1 (magenta), EEA1 (green), and Cathepsin D (yellow). The overlay is shown in white (A‴–F‴). (G and H) Semiquantification of the fluorescence signal of WDFY1. (G) WDFY1 particle area/total OLM area in percent. (H) WDFY1 particle area/total ONL area in percent. Scale bars, 10 μm. Each data point in the graph represents individual organoids, of which an average was taken of 3 representative images. The SEM is derived from these averages. The numbers of individual organoids per condition (CTRL1, CTRL2, CTRL3, ISO-03 P116, ISO-02 P128, P116, P117, and P128) in (G) and (H) are 8, 9, 9, 10, 8, 10, 11, and 14, respectively (from at least two independent organoid batches). Statistical analysis: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
See this image and copyright information in PMC

References

    1. Aguilar-Aragon M., Fletcher G., Thompson B.J. The cytoskeletal motor proteins Dynein and MyoV direct apical transport of Crumbs. Dev. Biol. 2020;459:126–137. doi: 10.1016/j.ydbio.2019.12.009. - DOI - PMC - PubMed
    1. Alves C.H., Sanz A.S., Park B., Pellissier L.P., Tanimoto N., Beck S.C., Huber G., Murtaza M., Richard F., Sridevi Gurubaran I., et al. Loss of CRB2 in the mouse retina mimics human retinitis pigmentosa due to mutations in the CRB1 gene. Hum. Mol. Genet. 2013;22:35–50. doi: 10.1093/hmg/dds398. - DOI - PubMed
    1. Alves C.H., Bossers K., Vos R.M., Essing A.H.W., Swagemakers S., van der Spek P.J., Verhaagen J., Wijnholds J. Microarray and morphological analysis of early postnatal CRB2 mutant retinas on a pure C57BL/6J genetic background. PLoS One. 2013;8:e82532. doi: 10.1371/journal.pone.0082532. - DOI - PMC - PubMed
    1. Boon N., Lu X., Andriessen C.A., Moustakas I., Buck T.M., Freund C., Arendzen C.H., Böhringer S., Mei H., Wijnholds J. AAV-mediated gene augmentation therapy of CRB1 patient-derived retinal organoids restores the histological and transcriptional retinal phenotype. Stem Cell Rep. 2023;18:1123–1137. doi: 10.1016/j.stemcr.2023.03.014. - DOI - PMC - PubMed
    1. Boon N., Wijnholds J., Pellissier L.P. Research models and gene augmentation therapy for CRB1 retinal dystrophies. Front. Neurosci. 2020;14:860. doi: 10.3389/fnins.2020. 00860. - DOI - PMC - PubMed

Publication types