Decision letter: Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects
2022-11-09

Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration. Editor's evaluation This work provides an important pipeline for high-throughput screening platform to be used for drug discovery. The current data are complete. Validation of human patients-derived iPSC clones and functional assays in mice further strengthens the current conclusion. https://doi.org/10.7554/eLife.83205.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Leber congenital amaurosis (LCA) is an inherited disease that affects the eyes and causes sight loss in early childhood, which generally gets worse over time. Individuals with this condition have genetic mutations that result in the death of light-sensitive cells, known as photoreceptors, in a region called the retina at the back of the eye. Patients carrying a genetic change in the gene CEP290 account for 20-25% of all LCA. At present, treatment options are only available for a limited number of patients with LCA. One option is to use small molecules as drugs that may target or bypass the faulty processes within the eye to help the photoreceptors survive in many different forms of LCA and other retinal diseases. However, over 90% of new drug candidates fail the first phase of clinical trials for human diseases. This in part due to the candidates having been developed using cell cultures or animal models that do not faithfully reflect how the human body works. Recent advances in cell and developmental biology are now enabling researchers to use stem cells derived from humans to grow retina tissues in a dish in the laboratory. These tissues, known as retinal organoids, behave in a more similar way to retinas in human eyes than those of traditional animal models. However, the methods for making and maintaining human retinal organoids are time-consuming and labor-intensive, which has so far limited their use in the search for new therapies. To address this challenge, Chen et al. developed a large-scale approach to grow retinal organoids from rd16 mutant mice stem cells (which are a good model for LCA caused by mutations to CEP290) and used the photoreceptors from these organoids to screen over 6,000 existing drugs for their ability to promote the survival of photoreceptors. The experiments found that the drug reserpine, which was previously approved to treat high blood pressure, also helped photoreceptors to survive in the diseased organoids. Reserpine also had a similar effect in retinal organoids derived from human patients with LCA and in the rd16 mice themselves. Further experiments suggest that reserpine may help patients with LCA by partially restoring a process by which the body destroys and recycles old and damaged proteins in the cells. The next steps following on from this work will be to perform further tests to demonstrate that this use of reserpine is safe to enter clinical trials as a treatment for LCA and other similar eye diseases. Introduction Once considered vestigial, the primary cilium has emerged as a key microtubule-based organelle that senses the external environment and modulates diverse signaling pathways in multiple tissues. Aberrant cilium biogenesis and/or ciliary transport and functions lead to numerous diseases, collectively termed ciliopathies, which manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies including aberrant kidney morphogenesis, brain malformation, and congenital retinal degeneration (Reiter and Leroux, 2017). The mammalian retina is an extension of the central nervous system that is specialized for vision (London et al., 2013). The visual information is captured by rod and cone photoreceptors, integrated and processed by interneurons, and transmitted to the cortex through the retinal ganglion cells. Inability of the retinal photoreceptors to detect and/or transmit light-triggered signals is a major cause of vision impairment in retinal and macular degenerative diseases (Veleri et al., 2015; Wright et al., 2010; Verbakel et al., 2018). Mutations in over 200 genes can lead to inherited retinal diseases (IRDs) (RetNet, https://sph.uth.edu/retnet/), with a combined prevalence of 1/3–4000 individuals (Verbakel et al., 2018; Hanany et al., 2020). Among them, up to 20% of IRD-causing genes are involved in primary cilium biogenesis or functions (Zelinger and Swaroop, 2018). Due to extensive clinical and genetic heterogeneity, high inter- and intra-family variability, and incomplete penetrance (Farrar et al., 2017), treatment options for IRD are limited (Garafalo et al., 2020). Only one gene therapy drug is currently approved by FDA for Leber congenital amaurosis (LCA, MIM204000) caused by RPE65 mutations (Pierce and Bennett, 2015). While the initial clinical outcomes were promising, the long-term data of this gene therapy drug are less encouraging (Gardiner et al., 2020). Furthermore, the development of individualized gene therapy protocols for divergent mutations in a multitude of genes for rare IRDs would be time-consuming, expensive, and labor-intensive (Tambuyzer et al., 2020). Thus, gene-agnostic paradigms are being developed for retinal and macular diseases using model organisms and/or stem cell-based approaches (Scholl et al., 2016). Small-molecule or antibody drugs represent a relatively affordable and scalable option (Tambuyzer et al., 2020). However, over 90% of drug candidates fail in Phase I clinical trials due to the lack of effective model systems which faithfully recapitulate the pathophysiology of human diseases (Horvath et al., 2016). The limited number of cells in the retina and challenges in the maintenance of primary retinal cultures have also hindered the progress of therapeutic development. LCA is a clinically severe and genetically heterogeneous group of IRDs, leads to vision loss in early childhood (Cremers et al., 2018). LCA10 caused by mutations in the cilia-centrosomal gene CEP290 (also called NPHP6) is one of the most common types, accounting for over 20% of patients (den Hollander et al., 2008). Mutations in CEP290 exhibit pleiotropy with phenotypes ranging from LCA (affecting vision and other sensory systems) to nephronophthisis, and Joubert and Meckel syndromes involving multiple organ systems (Sayer et al., 2006; Valente et al., 2006; McEwen et al., 2007; Coppieters et al., 2010; Drivas et al., 2015). The large 290 kDa centrosome-cilia protein CEP290 is ubiquitously expressed and localized to the Y-links of the transition zone of primary cilia, where it acts as a hub for connecting major protein complexes and likely performs a gating function (Sayer et al., 2006; Craige et al., 2010; Rachel et al., 2012; Drivas et al., 2013). The rd16 mouse carries an in-frame deletion in the myosin tail of CEP290, which causes a malformed connecting cilium (equivalent to the transition zone) and rudimentary outer segment (the primary cilium of photoreceptor) structure, leading to rapid degeneration of photoreceptors (Chang et al., 2006; Rachel et al., 2015). Additional studies have also demonstrated the critical role of CEP290 in cilia biogenesis (Drivas et al., 2013; Tsang et al., 2008; Barbelanne et al., 2015; Wu et al., 2020; Prosser et al., 2022). Thus, LCA10 is considered a retinal ciliopathy caused by hypomorphic mutations (den Hollander et al., 2006; Cideciyan et al., 2007) that eliminate some functions of CEP290 (Shimada et al., 2017). Vision impairment in early childhood of LCA10 patients imposes a considerable burden on families and society (Leroy et al., 2021). Notably, these patients demonstrate sparing of the foveal cones even at late stages of life, providing an attractive target for therapy (Cideciyan et al., 2011). Gene replacement using an AAV vector is difficult because of the large coding region of CEP290. Thus, other strategies including antisense oligonucleotides are currently under investigation (Collin et al., 2012; Gerard et al., 2012; Burnight et al., 2014; Dooley et al., 2018; Dulla et al., 2018; Kim et al., 2018). However, no approved treatment is currently available for alleviating vision loss due to CEP290 defects. Generation of three-dimensional tissue organoids from induced pluripotent stem cells (iPSCs) (Nakano et al., 2012; Sasai, 2013) has revolutionized biological and disease-modeling investigations and created opportunities for high-throughput screening (HTS) to design novel treatment paradigms (Welsbie et al., 2017). Further refinements of human retinal organoid culture protocols have permitted high efficiency and yield, developmental staging, and higher reproducibility (Zhong et al., 2014; Capowski et al., 2019; Kaya et al., 2019; Regent et al., 2022). Biogenesis of diverse cell types in retinal organoid cultures largely recapitulates structural and temporal development of in vivo human retina and can display intrinsic light responses mimicking those of primate fovea (Saha et al., 2022; Cowan et al., 2020; Sridhar et al., 2020). Patient iPSC-derived human retinal organoids demonstrate disease-associated phenotypes and are being used to evaluate various therapeutic approaches (Bell et al., 2020; Kruczek and Swaroop, 2020). However, long and tedious generation protocols spanning 150+ days, inherent variability, and lack of compatible HTS assays still pose challenges to the application of human retinal organoids for developing therapies (Struzyna and Watt, 2021). In this study, we aimed to establish a reliable drug discovery pipeline using an organoid-based HTS platform with the goal to identify drug candidates for maintaining photoreceptor survival in retinopathies, focusing initially on LCA10. For primary HTS of over 6000 bioactive small molecules, we designed survival assays using rd16 mouse iPSC-derived retinal organoids, which showed compromised photoreceptor development and viability. An FDA-approved small molecule drug reserpine was identified as an efficacious lead compound, which also enhanced photoreceptor development/survival in LCA10 patient iPSC-derived retinal organoids as well as in the rd16 retina in vivo. Transcriptomic analyses of drug-treated patient organoids indicated modulation of cell survival pathways including p53 and proteostasis by reserpine. Further examination validated the mis-regulation of autophagy in patient organoids and rd16 retina and demonstrated partial restoration of proteostasis and improved ciliogenesis after reserpine treatment. Our study thus establishes a cross-species drug discovery pipeline using organoid-based HTS and identifies a repurposing drug candidate for maintaining photoreceptor survival in LCA10 patients. As the action mechanisms of reserpine are partially through the modulation of proteostasis, which could be a common pathological impact of ciliopathies, reserpine, and its derivatives could potentially serve as an effective treatment for patients with other retinal ciliopathies. Results Establishment of an organoid-based HTS platform We designed an unbiased HTS assay to identify drug candidates that might improve photoreceptor development and/or survival. For a primary screen, we chose mouse retinal organoids because of a much shorter photoreceptor differentiation time and efficient generation of rudimentary outer segments using our modified HIPRO protocol (Chen et al., 2016a). We decided to use a karyotypically normal iPSC line derived from the Nrl-GFP containing rd16 mice (Figure 1—figure supplement 1A), which expresses green fluorescent protein (GFP) in all rods and carries an in-frame deletion in the myosin tail homology domain of CEP290 (Figure 1—figure supplement 2A). The rd16 mice are a model of photoreceptor degeneration observed in LCA10 patients (Chang et al., 2006). The iPSC lines derived from rd16 mice displayed similar morphology, proliferation rate, and stem cell properties as those derived from the wild-type (WT) (Figure 1—figure supplement 2B and C). WT and rd16 retinal organoids also showed comparable morphology at the early stages of differentiation (Figure 1A). However, at later stages, rod photoreceptors in rd16 organoids displayed aberrant morphology, with malformed or missing ciliary axoneme, connecting cilium and ciliary rootlets (Figure 1—figure supplement 2D). We then performed flow cytometry analyses to evaluate the differentiation of GFP+ rod photoreceptors in the rd16 organoid cultures, with the goal to identify quantifiable phenotypes for HTS. The rd16 organoids demonstrated as much as 50% lower organoid viability and almost 60% fewer GFP+ rod photoreceptors at day (D) 32 (Figure 1B), the end stage of differentiation in mouse retinal organoids. Figure 1 with 2 supplements see all Download asset Open asset Drug discovery pipelines to identify drug candidates. (A) Morphology of Nrl-GFP wild-type (WT) and rd16 retinal organoids differentiated from mouse-induced pluripotent stem cells (iPSC) at various differentiation time points. (B) Flow cytometry analysis of GFP+ rod photoreceptors and viability at different developmental stages. Each data point summarized at least three batches of independent experiments, each of which included at least 10 organoids. One-way ANOVA was performed to compare the %GFP+ cells and viability of organoids from four different cell lines. *p<0.05; **p<0.01. (C) Fluorescent images of dissociated day (D) 28 WT and rd16 cells stained by 4',6-diamidino-2-phenylindole (DAPI) and anti-GFP antibody. (D) Schematic outline of the drug discovery strategy. The phenotypes in GFP+ rd16 rod photoreceptors permitted the development of an HTS screening platform to identify bioactive small-molecule candidates for augmenting rod cell differentiation and/or survival (Figure 1—figure supplement 2E). To avoid the impact of organoid variability on drug effects, we dissociated rd16 organoids into single cells at D25 and performed the treatment using two-dimensional cultures from D26 to D28. These cultures recapitulated the phenotypes detected in non-dissociated organoids, and rd16 cells displayed reduced viability and fewer GFP+ rod cells at D28 compared to the WT (Figure 1C). Our drug discovery pipeline is illustrated in Figure 1D. In the primary screens, rd16 retinal organoid-derived cells were treated with over 6000 small molecules; of these, 114 compounds revealed higher GFP signal intensity, indicating a positive effect on rod photoreceptor survival. We then eliminated the compounds that showed toxicity or autofluorescence even in the absence of GFP expression (false positives). Fourteen small molecule compounds were then selected for further screening to identify a lead compound. Selection of the lead compound We then treated rd16 retinal organoids with the 14 selected small molecules in a secondary assay, which started at D22 and lasted for three days to maximize the drug effect without causing toxicity (Figure 2A). WT, untreated, and treated rd16 organoids were harvested at D29 to allow a sufficient period for the restoration of photoreceptors. The lead compound was selected based on the efficacy of the small molecule drugs to improve photoreceptor development and/or survival. Immunostaining of rhodopsin and S-opsin, markers of rod and cone photoreceptors respectively, was performed to quantify the drug effect (Figure 2B). Rhodopsin is highly expressed and polarized to the apical side of rod photoreceptors in the neural retina of WT organoids. In contrast, rd16 photoreceptors exhibited lower expression of rhodopsin, and the polarity was considerably diminished. Two of the compounds, B05 and to some extent B03, were able to enhance rhodopsin expression, with B05 improving the polarity of expression in the neural retina as well. We note that S-cone photoreceptors are difficult to be maintained in mouse organoid culture (DiStefano et al., 2018); nonetheless, compounds B01 and B05 were able to augment the expression and polarity of S-opsin. Three of the 5 compounds (B01, B03, B05) that showed no toxicity revealed a dramatic increase in rod and/or cone photoreceptors in D29 rd16 organoids (Figure 2C); of these, B05 was the most effective molecule and chosen as the lead compound. Interestingly, the other four molecules (B01-B04) were found to be derivatives of B05, further validating our results. B05 was identified as reserpine, a small-molecule drug that has been approved by the FDA for the treatment of hypertension and schizophrenia (Armitage et al., 1956; Kalliomäki and Kasanen, 1969), thereby holding a potential for drug repurposing (Figure 2D). Figure 2 Download asset Open asset Identification of reserpine as the lead compound. (A) Timeline for hit validation in rd16 retinal organoids. (B) Immunostaining of rod cell marker rhodopsin (RHO, green) and cone cell marker S-opsin (OPN1SW, red) in wild-type (WT) and rd16 organoids treated with non-toxic positive hits (B01–B05). Drug vehicle dimethylsulfoxide (DMSO) was used as a control. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). Arrowheads indicate relevant staining. Images were representative of at least three independent experiments, each of which had at least three organoids. (C) Bee swarm plots show the quantification of fluorescence intensity of rhodopsin (upper) and S-opsin (lower) staining in the validation. The shape of the plot indicates the distribution of data points, which are shown by colorful circles in the center. The black diamond indicates the mean, and the error bar reveals the standard error of the mean. The pink dash line shows the mean fluorescence intensity of DMSO-treated organoids. The plot summarizes at least three independent experiments with at least three organoids in each batch. One-way ANOVA followed by the Bonferroni test was performed. *p<0.05; **p<0.01. (D) Chemical structure depiction of the selected lead compound reserpine. Validation of reserpine on LCA10 patient organoids To further examine the efficacy of the lead compound reserpine, we utilized iPSC-derived retinal organoids from a previously-reported LCA10 family (Shimada et al., 2017), which comprised of a heterozygous unaffected mother (referred to as control henceforth) and two affected compound heterozygous children (LCA-1 and LCA-2; Figure 3—figure supplement 1A and B). Immunoblot analysis revealed a profound reduction of full-length CEP290 in patient organoids compared to controls (Figure 3—figure supplement 1C). Defects in rod development and outer segment biogenesis were evident in patient organoids from D120 as revealed by the loss of connecting cilium marker and absence or of protein rhodopsin (Figure 3—figure supplement In with the of central cones in LCA10 cone photoreceptors were only compromised even at a late stage of differentiation in patient organoids. that outer segment biogenesis D120 in organoids et al., and aberrant phenotypes in patient organoids were at this stage (Figure 3—figure supplement reserpine was to patient organoids at at a of 10 or based on the effective in the primary (Figure We could detect improved connecting cilium and rod development as early as with the of reserpine (Figure 3—figure supplement 2A). using the we observed more polarized rhodopsin and connecting cilium at the apical side of photoreceptors in treated patient organoids, a effect of the drug (Figure 3—figure supplement 2A). in drug on organoids derived from the two patients could be reserpine treatment the fluorescence intensity of rhodopsin in retinal organoids derived from two patients (Figure an of rod photoreceptor development. To avoid we also reserpine on of each these retinal organoids showed an positive of rhodopsin staining reserpine treatment (Figure 3—figure supplement C). could be to variability in response to and/or treatment of retinal organoids derived from various iPSC lines. We then performed to structural of the primary cilium in photoreceptors of patient organoids after treatment. Reserpine the of mother ciliary in patient organoids (Figure and Figure 3—figure supplement which were missing in a of LCA photoreceptors (Shimada et al., 2017). organoids demonstrated almost 50% more ciliary after reserpine treatment ciliary were of the of the ciliary indicated a increase in patient organoids after treatment (Figure Notably, could be in treated (Figure 3—figure supplement a positive impact of reserpine treatment on photoreceptor primary cilium development. Figure with supplements see all Download asset Open asset of reserpine on LCA10 patient retinal organoids. (A) Timeline for treatments and of patient organoids. (B) Immunostaining of rhodopsin (RHO, green) and Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). Arrowheads indicate relevant staining. Images were representative of at least three independent experiments, each of which had at least three organoids. (C) Bee swarm plots show the quantification of fluorescence intensity of rhodopsin staining. The shape of the plot indicates the distribution of data points, which are shown by colorful circles in the center. The black diamond indicates the mean, and the error bar reveals the standard error of the mean. The plot summarizes at least three independent experiments with at least one in each batch. was performed to compare and treated (D) analysis of untreated, and treated patient organoids. Arrowheads indicate relevant staining. of the number of the ciliary ciliary axoneme, and aberrant cilia as well as the of the primary cilia in and patient organoids. Aberrant cilia were as mother without ciliary or ciliary The data summarized at least four batches of independent experiments, each of which has at least two organoids and mother The shape of the plot indicates the distribution of data points, which are shown by colorful circles in the center. The black diamond indicates the mean, and the error bar reveals the standard error of the mean. was performed to compare and treated We note that reserpine with the nervous system by the transport of into et al., even at no effect was detected on the development of cone photoreceptors, cells, and in treated patient organoids (Figure 3—figure supplement of cell survival and proteostasis pathways in organoids To the of action of reserpine and to insights into photoreceptor cell death in we performed analyses of control and patient retinal organoids. The organoids were harvested after the treatment with reserpine at (Figure supplement 1A). with the response of patient organoid to reserpine (Figure 3—figure supplement we not identify sufficient expressed genes between and treated of for analyses not and thereby only on analysis revealed in patient organoid compared to control and after reserpine treatment (Figure The for up to of the and is likely due to the drug treatment. of the and to be by the disease Notably, the group was to the control compared to the group in of genes were expressed between and treated organoids at of and change (Figure data with the analysis indicated that genes in treated patient organoids showed a from the group the control (Figure However, compared to the most of the genes those involved in in organoids had an even higher expression drug treatment. the expression of genes in was even more than the controls by reserpine treatment. treatment of organoids with 10 or reserpine not sufficient genes for further analyses not Figure with 1 supplement see all Download asset Open asset Transcriptomic of induced by reserpine in patient retinal organoids. (A) analysis of patient and control organoids shows retinal after reserpine treatment. (B) genes in patient organoids displayed compared to control organoids. (C) plot summarizes gene expression in organoids. (D) analysis of showed genes to protein metabolism, and signaling The highlights the plot and of of the change of genes reserpine treatment. to reserpine treatment. and two key of protein showed expression after reserpine treatment. Figure data 1 expressed genes in retinal organoids. Download We first the expression of genes to different retinal cell types and those involved in and photoreceptor outer segment Reserpine treatment the expression of marker genes for retina cell types, including (Figure supplement rod and cone photoreceptor genes and showed lower expression (Figure Figure supplement Figure data with cilia genes et al., in expression (Figure supplement As reserpine not to the expression of key involved in disease we the genes to pathways and created an a of three genes with and the