Eupatilin improves cilia defects in human CEP290 ciliopathy models

Abstract

The photoreceptor outer segment is a highly specialized primary cilium essential for phototransduction and vision. Biallelic pathogenic variants in the cilia-associated gene CEP290 cause non-syndromic Leber congenital amaurosis 10 (LCA10) and syndromic diseases, where the retina is also affected. While RNA antisense oligonucleotides and gene editing are potential treatment options for the common deep intronic variant c.2991+1655A>G in CEP290 , there is a need for variant-independent approaches that could be applied to a broader spectrum of ciliopathies. Here, we generated several distinct human models of CEP290 -related retinal disease and investigated the effects of the flavonoid eupatilin as a potential treatment. Eupatilin improved cilium formation and length in CEP290 LCA10 patient-derived fibroblasts, in gene-edited CEP290 knockout (CEP290 KO) RPE1 cells, and in both CEP290 LCA10 and CEP290 KO iPSCs-derived retinal organoids. Furthermore, eupatilin reduced rhodopsin retention in the outer nuclear layer of CEP290 LCA10 retinal organoids. Eupatilin altered gene transcription in retinal organoids, by modulating the expression of rhodopsin, and by targeting cilia and synaptic plasticity pathways. This work sheds light into the mechanism of action of eupatilin, and supports its potential as a variant-independent approach for CEP290 -associated ciliopathies.

Figure 1.. Eupatilin increases cilia incidence and length in CEP290 LCA10 patient fibroblasts.

A, Representative images of control (CEP290 WT) and CEP290 LCA10 fibroblasts treated with eupatilin (20 μM) or vehicle, stained for pericentrin (PCN, green, basal body) and ARL13b (magenta, axoneme). Scale bar: 20 μm. Inset shows cilia at higher magnification. Scale bar 5 μm. B-C, Cilia incidence and length were scored using the elaboration of an ARL13B positive axoneme from a PCN positive basal body. Number of cilia counted in B: WT vehicle (−) n=89, Eupatilin (+) n=147; CEP290 LCA10 vehicle (−) n=99, Eupatilin (+) n=87 (B). Number of cilia counted in C: WT vehicle (−) n=271, Eupatilin (+) n=231; CEP290 LCA10 vehicle (−) n=214, Eupatilin (+) n=345. Mean ± standard deviation (SD), ^** P<0.01, ^*** P<0.005, ^**** p<0.0001 one-way ANOVA test.

Figure 2.. Eupatilin improves cilia incidence and length in CEP290 KO RPE1 cells.

A, Sanger sequence showing CRISPR/Cas9-induced deletion of 1 bp in CEP290 (black box) and predicted effect on amino acid sequence. Stop codon is highlighted in exon 6 (red box). B, qPCR analyses of CEP290 transcripts shows reduced levels in CEP290 KO RPE1 cells. Primers were designed between exon 13 and 14, n=4. C, Western blot of CEP290 protein shows reduced level in CEP290 KO cells, β-tubulin and vinculin were used as reference proteins. D, Quantification of western blot, showing reduced CEP290 expression. n=3 independent western blots. E, Representative images of CEP290 WT (control) and CEP290 KO RPE1 cells treated with vehicle or eupatilin (20 μM) with cilia stained for PCN (green) and ARL13b (magenta). Scale bar: 40 μm. Inset shows cilium at higher magnification. Scale bar: 2 μm. Graphical representation of cilia incidence (F), and cilia length (G), in WT and CEP290 KO RPE1 cells treated with 20 μM eupatilin. F-G, 24–28 independent fields of view, from 3 separate experiment repeats were scored, and 207–630 cilia were measured. H-I, The effect of eupatilin treatment for 24 hours at the indicated doses on cilia incidence and length was measured. H, 8 independent images, from 2 separate experiment repeats were scored, and 19–92 cilia were measured (I). Median ± min and max, ^** p<0.01, ^*** p<0.005, ^**** p<0.0001 ordinary one-way ANOVA with Tukey’s post-hoc test.

Figure 3.. Eupatilin treatment restores ciliation and cilia length in iPSC-derived CEP290 KO and CEP290 LCA10 retinal organoids.

A, Bright field images of CEP290 retinal organoids at differentiation day 70. Scale bar: 500 μm. B, RCVRN (green) and CRX (magenta) staining of day 120 retinal organoids. Scale bar: 40 μm C, Eupatilin treatment of retinal organoids from days 90 to 120. Cilia were stained with ARL13B (green) and PCN (magenta). Scale bar: 40 μm. Insets show a higher magnification of the photoreceptor cilia. Scale bar: 1 μm D, Quantification of photoreceptor ciliation and photoreceptor cilium length (E) in CEP290 retinal organoids at day 120 after eupatilin treatments. CEP290 WT Vehicle n=4 ROs (141 cilia), CEP290 WT EUP n=4 ROs (135 cilia), CEP290 KO Vehicle n=3 (148 cilia), CEP290 KO EUP n=2 (88 cilia), CEP290 LCA10 Vehicle n=4 (114 cilia), CEP290 LCA10 EUP n=4 (176 cilia). * p<0.05 ^** p<0.01, ^*** P<0.001, ordinary one-way ANOVA with Tukey’s post-hoc test.

Figure 4.. Opsin accumulates in the outer nuclear layer of CEP290 LCA10 retinal organoids and is rescued by eupatilin.

A, Representative images of CEP290 WT (control) and CEP290 LCA10 retinal organoids at D180 treated with eupatilin (20 μM) or vehicle for 30 days (from D150), stained for rhodopsin (magenta) and L/M opsin (green). Photoreceptor nuclei were stained with DAPI (blue). Scale bar: 10 μm. B, Quantification of RHO and L/M opsin (C) immunofluorescence intensity in the photoreceptor outer nuclear layer (ONL). Each data point represents the mean of 3 measurements from one organoid, bar represents mean ± SD. * p<0.05 ^** p<0.01, ^*** P<0.001.

Figure 5.. Transcriptome analysis of eupatilin treated control retinal organoids.

A, The effect of eupatilin on of CRX, ARR3, RCVRN, RHO, OPN1LW-OPN1MW gene expression by qPCR. Day 250 control retinal organoids were treated with vehicle (DMSO) or eupatilin at the indicated concentrations for 14 days and harvested or allowed to recover for 14 days with no treatment (‘pulse’), untreated organoids are shown at D250 for comparison. qPCR analyses. Means ± SD n=3 independent retinal organoids, * p<0.05 ^** p<0.01, one-way ANOVA with Tukey’s post-hoc test. Effect of eupatilin on the transcriptome by. Bulk RNA sequencing was performed on day 240 organoids after 1 month eupatilin treatment. B, PCA analyses showing separation between the eupatilin treated organoids and the vehicle treated organoids by PC2 variance. C, Volcano plot showing the most differentially expressed genes. D, Dotplot showing the top 20 gene ontology (GO) cellular components by over-representation analysis. E, Netplot showing the relationship between the top 10 most enriched gene ontology terms and their genes.