Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects
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eLife assessment
This work provides an important pipeline for high-throughput screening platform to be used for drug discovery. The current data are incomplete. Further validation of human patients-derived iPSC clones and functional assays in mice will strengthen the conclusion.
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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.
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Author Response
Reviewer #1 (Public Review):
The current study proposed a drug discovery pipeline to accelerate the process of identifying drug candidates for LCA10 patients using cells from mouse retinal organoid for initial screening, human patient iPSC-derived retinal organoid for further testing, and then mouse mutants for in vivo validation. Reserpine was identified as the top candidate, possibly through modulating proteostasis and autophagy to promote cilium assembly. The study was with high translational value. However, the rationale using dissociated cells from mouse retinal organoid for initial drug screening needs to be justified. In addition, the consistency of phenotypic characteristics in human patient iPSC-derived retinal organoid needs to be reported. It was unclear if the rescued phenotypic changes were from the drug …
Author Response
Reviewer #1 (Public Review):
The current study proposed a drug discovery pipeline to accelerate the process of identifying drug candidates for LCA10 patients using cells from mouse retinal organoid for initial screening, human patient iPSC-derived retinal organoid for further testing, and then mouse mutants for in vivo validation. Reserpine was identified as the top candidate, possibly through modulating proteostasis and autophagy to promote cilium assembly. The study was with high translational value. However, the rationale using dissociated cells from mouse retinal organoid for initial drug screening needs to be justified. In addition, the consistency of phenotypic characteristics in human patient iPSC-derived retinal organoid needs to be reported. It was unclear if the rescued phenotypic changes were from the drug effects or a result of phenotypic variations in organoids.
We thank the reviewer for the comments and suggestions. Please see the response provided in the “Essential Revisions” earlier. Briefly, the use of single-cell cultures for screening is to compensate for the variations of the Nrl-GFP signal in rd16 organoids so that each compound was present to homogenous cells. In addition, we performed a large-scale screening with 11 concentrations and 2 duplicates of over 6000 compounds. It was thus not feasible to manually perform the screening. We used a semi-automatic electronic dispenser to set up the screens in 1536-well plates and a liquid handling system to add the compounds. Intact mouse retinal organoids are too big to be dispensed and would be damaged during the process. They are also too big to fit into one well of a 1536-well plate or even in a 384-well plate. Therefore, single-cell cultures outweigh intact organoids in this application. We understand the potential pitfalls and thus the positive hits were verified in intact organoids in the secondary assays.
We have now tested reserpine on retinal organoids derived from 2 clones of each (a total of 4) of LCA1 and LCA2 patients. As suggested by the reviewers, we quantified the fluorescence intensity of rod marker rhodopsin staining in multiple sections of at least two batches of differentiation (Figure 3C and Figure 3—figure supplement 2). Although showing variability as predicted, reserpine treatment significantly increased the fluorescence intensity of rhodopsin in retinal organoids differentiated from multiple lines (Figure 3C), further validating the rescue effect of reserpine.
Reviewer #2 (Public Review):
In this manuscript, a drug discovery pipeline was developed using a human iPSC derived organoid-based high-throughput screening platform to be used to identify drug candidates for maintaining photoreceptor survival in LCA10 retinopathies. Reserpine proved effective in patient organoids and in mutant mouse retina in vivo to improve photoreceptor survival and outer segment structure. Protein homeostasis was restored after reserpine treatment by increasing p62 levels, decreasing the 20S proteasome, and increasing proteasome activity. The manuscript is clearly written, contains a large amount of valuable and high-quality data and demonstrates that rebalancing proteostasis can stabilize photoreceptor overall homeostasis in the presence of a mutation that causes retinal degeneration.
The manuscript may lack functional in vivo data on the treatment by reserpine in RD16 mice such as ERG measurements or other functional tests (the authors also refer to it as future direction). Nevertheless, in my view, the study provides a solid and convincing set of data and substantially advances our understanding on the neuroprotective effects of reserpine beyond the scope of the retina and therefore can be expected to have widespread influence on a readership interested in the principles of neuroprotection rebalancing proteostasis.
We sincerely thank the reviewer for the positive comments and suggestions. This study has taken many years to materialize. We agree and have now performed full-field electroretinogram (ERG) of untreated and reserpine-treated rd16 retina (as stated in response to an earlier comment). Scotopic a-wave was only marginally increased, yet scotopic b-wave displayed a significant higher amplitude, suggesting improved rod photoreceptor function (Figure 6D).
Reviewer #3 (Public Review):
Chen et al. perform an innovative screen using retinal organoids derived from rd16 mice to identify small molecules to treat CEP290 hypomorphic mutations linked to ciliopathies such as LCA. The authors identify reserpine which promotes photoreceptor development and viability in retinal organoids derived from LCA patient iPSCs and rd16 mouse retinas. The authors finally propose a mechanistic model where reserpine restores proteostasis thereby improving ciliogenesis.
The authors present a highly effective drug screen that utilizes the benefits of retinal organoids while also accounting for the inherent variability of retinal organoids by performing a screen on 2D cultures derived from the organoids. This is an innovated approach to using retinal organoids in drug screens and is of interest to the greater community. The success of the screen is reflected in the effectiveness of reserpine in the in vivo rd16 mouse retinal model where it promotes photoreceptor survival. However there are multiple issues with the LCA patient organoid screen that must be resolved.
We are grateful to the reviewer for generous comments. We have incorporated the suggestions and performed additional work to resolve the issues, as mentioned earlier in this response as well as below.
The patient derived iPSC lines are not controlled sufficiently enough to make conclusions stated in the manuscript. The authors rely on single iPSC clones from disease patients to perform experiments, and it is not clear whether karyotyping to validate normal chromosomal integrity was performed. In the case of the RNAseq experiment one patient clone does not show any differences calling into question the findings from the other clone. Patient derived iPSC studies would benefit from the use of multiple independently derived iPSC clones per patient, or rescuing the LCA10 mutation using CRISPR editing to validate the correlation of the mutation with the differences observed.
This study could be strengthened by parallel RNAseq studies is the rd16 mouse retina and patient iPSC retinal organoids.
Thanks for the suggestions. As mentioned earlier in “Essential Revisions” and response to other reviewers, we have performed additional experiments using multiple iPSC clones and from three patients (2 each from LCA1 and LCA2). These iPSC lines have been characterized previously (Shimada et al. 2017). We have now provided more details on iPSC derivation, iPSC maintenance, and differentiation. Karyotypes of all human and mouse iPSC lines were provided in Figure 1—figure supplement 1. Retinal organoids were generated using iPSC lines within 10 passages of test cells.
The purpose of the RNA-seq data is to provide primers on the signaling pathways modulated by reserpine treatment. The rescue effect of reserpine suggests that these pathways might be implicated in disease pathogenesis. Based on our RNA-seq data, we have validated the dysregulation of proteostasis pathway in patient-derived retinal organoids and in vivo rd16 retina. Further investigations are needed to validate other pathways but are beyond the scope of this manuscript. Although RNA-seq studies have advantages, more detailed molecular and functional assays are needed to validate the findings of RNA-seq studies and therefore we argue that performing additional RNA-seq on different clones or cell lines or mouse retina would provide more solid information.
According to our quantification of rhodopsin staining intensity (Figure 3C and Figure 3—figure supplement 2), LCA1 organoids are more responsive to reserpine compared to LCA2, which is not surprising based on the variations of patient responsiveness to drug treatments in previous clinical studies. We note that reserpine is not a transcription factor, thus the differentially expressed genes in reserpine treatments are secondary effects and the change of gene profiles upon reserpine treatment could vary in time and intensity, which could explain the few differentially expressed genes observed in LCA-2. Nevertheless, the action mechanisms of reserpine we found based on LCA1 could be validated on LCA2 (Figure 5—figure supplement 3), further strengthening our findings.
The reason why we performed RNA-seq on treated organoids but not treated mice was to identify the signaling pathways modulated by reserpine in a well-controlled manner in order to catch the small changes. Compared to reserpine treatment on organoid cultures, in which the organoids have stable and constant contact with reserpine, intravitreal injection of reserpine into P7 mice is technically challenging and leads to substantial variations. In this case, some small changes might be missed and masked by the variations.
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eLife assessment
This work provides an important pipeline for high-throughput screening platform to be used for drug discovery. The current data are incomplete. Further validation of human patients-derived iPSC clones and functional assays in mice will strengthen the conclusion.
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Reviewer #1 (Public Review):
The current study proposed a drug discovery pipeline to accelerate the process of identifying drug candidates for LCA10 patients using cells from mouse retinal organoid for initial screening, human patient iPSC-derived retinal organoid for further testing, and then mouse mutants for in vivo validation. Reserpine was identified as the top candidate, possibly through modulating proteostasis and autophagy to promote cilium assembly. The study was with high translational value. However, the rationale using dissociated cells from mouse retinal organoid for initial drug screening needs to be justified. In addition, the consistency of phenotypic characteristics in human patient iPSC-derived retinal organoid needs to be reported. It was unclear if the rescued phenotypic changes were from the drug effects or a result …
Reviewer #1 (Public Review):
The current study proposed a drug discovery pipeline to accelerate the process of identifying drug candidates for LCA10 patients using cells from mouse retinal organoid for initial screening, human patient iPSC-derived retinal organoid for further testing, and then mouse mutants for in vivo validation. Reserpine was identified as the top candidate, possibly through modulating proteostasis and autophagy to promote cilium assembly. The study was with high translational value. However, the rationale using dissociated cells from mouse retinal organoid for initial drug screening needs to be justified. In addition, the consistency of phenotypic characteristics in human patient iPSC-derived retinal organoid needs to be reported. It was unclear if the rescued phenotypic changes were from the drug effects or a result of phenotypic variations in organoids.
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Reviewer #2 (Public Review):
In this manuscript, a drug discovery pipeline was developed using a human iPSC derived organoid-based high-throughput screening platform to be used to identify drug candidates for maintaining photoreceptor survival in LCA10 retinopathies. Reserpine proved effective in patient organoids and in mutant mouse retina in vivo to improve photoreceptor survival and outer segment structure. Protein homeostasis was restored after reserpine treatment by increasing p62 levels, decreasing the 20S proteasome, and increasing proteasome activity. The manuscript is clearly written, contains a large amount of valuable and high-quality data and demonstrates that rebalancing proteostasis can stabilize photoreceptor overall homeostasis in the presence of a mutation that causes retinal degeneration.
The manuscript may lack …
Reviewer #2 (Public Review):
In this manuscript, a drug discovery pipeline was developed using a human iPSC derived organoid-based high-throughput screening platform to be used to identify drug candidates for maintaining photoreceptor survival in LCA10 retinopathies. Reserpine proved effective in patient organoids and in mutant mouse retina in vivo to improve photoreceptor survival and outer segment structure. Protein homeostasis was restored after reserpine treatment by increasing p62 levels, decreasing the 20S proteasome, and increasing proteasome activity. The manuscript is clearly written, contains a large amount of valuable and high-quality data and demonstrates that rebalancing proteostasis can stabilize photoreceptor overall homeostasis in the presence of a mutation that causes retinal degeneration.
The manuscript may lack functional in vivo data on the treatment by reserpine in RD16 mice such as ERG measurements or other functional tests (the authors also refer to it as future direction). Nevertheless, in my view, the study provides a solid and convincing set of data and substantially advances our understanding on the neuroprotective effects of reserpine beyond the scope of the retina and therefore can be expected to have widespread influence on a readership interested in the principles of neuroprotection rebalancing proteostasis.
-
Reviewer #3 (Public Review):
Chen et al. perform an innovative screen using retinal organoids derived from rd16 mice to identify small molecules to treat CEP290 hypomorphic mutations linked to ciliopathies such as LCA. The authors identify reserpine which promotes photoreceptor development and viability in retinal organoids derived from LCA patient iPSCs and rd16 mouse retinas. The authors finally propose a mechanistic model where reserpine restores proteostasis thereby improving ciliogenesis.
The authors present a highly effective drug screen that utilizes the benefits of retinal organoids while also accounting for the inherent variability of retinal organoids by performing a screen on 2D cultures derived from the organoids. This is an innovated approach to using retinal organoids in drug screens and is of interest to the greater …
Reviewer #3 (Public Review):
Chen et al. perform an innovative screen using retinal organoids derived from rd16 mice to identify small molecules to treat CEP290 hypomorphic mutations linked to ciliopathies such as LCA. The authors identify reserpine which promotes photoreceptor development and viability in retinal organoids derived from LCA patient iPSCs and rd16 mouse retinas. The authors finally propose a mechanistic model where reserpine restores proteostasis thereby improving ciliogenesis.
The authors present a highly effective drug screen that utilizes the benefits of retinal organoids while also accounting for the inherent variability of retinal organoids by performing a screen on 2D cultures derived from the organoids. This is an innovated approach to using retinal organoids in drug screens and is of interest to the greater community. The success of the screen is reflected in the effectiveness of reserpine in the in vivo rd16 mouse retinal model where it promotes photoreceptor survival. However there are multiple issues with the LCA patient organoid screen that must be resolved.
The patient derived iPSC lines are not controlled sufficiently enough to make conclusions stated in the manuscript. The authors rely on single iPSC clones from disease patients to perform experiments, and it is not clear whether karyotyping to validate normal chromosomal integrity was performed. In the case of the RNAseq experiment one patient clone does not show any differences calling into question the findings from the other clone. Patient derived iPSC studies would benefit from the use of multiple independently derived iPSC clones per patient, or rescuing the LCA10 mutation using CRISPR editing to validate the correlation of the mutation with the differences observed.
This study could be strengthened by parallel RNAseq studies is the rd16 mouse retina and patient iPSC retinal organoids.
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