Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects
- Neurobiology, Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, United States
- National Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, United States
- Electron Microscopy Laboratory, National Cancer Institute, Center for Cancer Research, Leidos Biomedical Research, Frederick National Laboratory, United States
- Department of Ophthalmology, Scheie Eye Institute, Perelman School of Medicine, University of Pennsylvania, United States
Figures
Figure 1 with 2 supplements
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 …
Figure 1—figure supplement 1
Karyotypes of induced pluripotent stem cell lines.
(A) Mouse. (B) Human. Cells were prepared for G-band stain. Fifteen unique metaphases were analyzed.
Figure 1—figure supplement 2
Phenotypes of retinal organoids differentiated from induced pluripotent stem cells of Nrl-GFP rd16 mice.
(A) Deletion of myosin tail homology domain of the CEP290 protein in rd16 mice compared to the wild-type (WT). (B) Morphology of induced pluripotent stem cells (iPSC) colonies, (C) proliferation …
Figure 2
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 …
Figure 3 with 4 supplements
Effect of reserpine (RSP) on LCA10 patient retinal organoids.
(A) Timeline for RSP treatments and harvest of patient organoids. (B) Immunostaining of rhodopsin (RHO, green) and ARL13B (red). Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). …
Figure 3—figure supplement 1
Genotype and phenotype of the CEP290-LCA family in this study.
(A) Genotypes of the subjects including a heterozygous but phenotypically normal control and two CEP290-LCA patients. (B) Location of the stop codons caused by point mutations in CEP290-LCA …
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Figure 3—figure supplement 1—source data 1
Overlay of the bright field and chemiluminescence images indicating the signal of CEP290.
The size of the protein ladders, CEP290, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 1—source data 2
Overlay of the bright field and chemiluminescence images indicating the signal of GAPDH.
The size of the protein ladders, GAPGH, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig3-figsupp1-data2-v2.zip
Figure 3—figure supplement 2
Effect of reserpine at various conditions in CEP290-LCA retinal organoids.
Rod cell marker rhodopsin (RHO, green) and ciliary marker ARL13B (red) were shown in (A) LCA1 organoids treated with 30 μM reserpine (RSP) at differentiation day (D) 125 (upper) and with 10 μM RSP …
Figure 3—figure supplement 3
Transmission electron microscopy analyses of control, and untreated and treated CEP290-LCA retinal organoids.
(A) Morphology of photoreceptor primary cilium. (B) Stacks of disc-like structures observed in treated patient organoids. Arrowheads indicate relevant structures.
Figure 3—figure supplement 4
Various cell types in reserpine (RSP)-treated CEP290-LCA retinal organoids.
Immunostaining of structural or cell type-specific markers including those for L/M cones (OPN1LMW, green), S-cones (OPN1SW, red), ribbon synapses (BASSOON, red), presynaptic vesicles (SYNAPTOPHYSIN, …
Figure 4 with 1 supplement
Transcriptomic upregulation of proteasomal components induced by reserpine in patient retinal organoids.
(A) Principal component analysis (PCA) diagram of patient and control organoids shows altered retinal transcriptomes after reserpine treatment. (B) Drug-induced genes in patient organoids displayed …
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Figure 4—source data 1
Differentially expressed genes in reserpine-treated retinal organoids.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Reserpine treatment alters expression of retinal pathway genes.
(A) Timeline of treatment and sample harvest. (B) Different retinal cell types appeared to respond uniquely to reserpine. (C) Phototransduction genes were globally downregulated except for specific …
Figure 5 with 3 supplements
Proteostasis Network in patient organoid cultures in response to reserpine treatment.
(A) Schematic diagram of autophagy.(B) Immunoblot analyses and quantification of autophagy cargo adaptor p62 and autophagosome marker LC-II in control and patient organoids treated with reserpine …
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Figure 5—source data 1
Overlay of the bright field and chemiluminescence images indicating the signal of p62.
RSP stands for reserpine. The size of the protein ladders, p62, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data1-v2.zip
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Figure 5—source data 2
Overlay of the bright field and chemiluminescence images indicating the signal of LC3.
RSP stands for reserpine. The size of the protein ladders, LC3, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data2-v2.zip
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Figure 5—source data 3
Overlay of the bright field and chemiluminescence images indicating the signal of β-Actin.
RSP stands for reserpine. The size of the protein ladders, β-Actin, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data3-v2.zip
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Figure 5—source data 4
Overlay of the bright field and chemiluminescence images indicating the signal of 20S proteosome and β-Actin.
RSP stands for reserpine. The size of the protein ladders, β-Actin, 20S proteosome, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data4-v2.zip
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Figure 5—source data 5
Overlay of the bright field and chemiluminescence images indicating the signal of IFT88.
RSP stands for reserpine. The size of the protein ladders, IFT88, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data5-v2.zip
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Figure 5—source data 6
Overlay of the bright field and chemiluminescence images indicating the signal of BBS6.
RSP stands for reserpine. The size of the protein ladders, BBS6, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data6-v2.zip
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Figure 5—source data 7
Overlay of the bright field and chemiluminescence images indicating the signal of CEP164.
RSP stands for reserpine. The size of the protein ladders, CEP164, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data7-v2.zip
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Figure 5—source data 8
Overlay of the bright field and chemiluminescence images indicating the signal of OFD1.
RSP stands for reserpine. The size of the protein ladders, OFD1, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data8-v2.zip
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Figure 5—source data 9
Overlay of the bright field and chemiluminescence images indicating the signal of HDAC6.
RSP stands for reserpine. The size of the protein ladders, HDAC6, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data9-v2.zip
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Figure 5—source data 10
Overlay of the bright field and chemiluminescence images indicating the signal of β-Actin.
RSP stands for reserpine. The size of the protein ladders, β-Actin, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-data10-v2.zip
Figure 5—figure supplement 1
Effect of autophagy inhibitors on patient organoids.
(A) Schematic diagram of autophagy with annotation of the drug effect of autophagy inhibitors. (B) Timeline of treatment and sample harvest. (C) Immunostaining of rhodopsin (green), S-opsin (red), …
Figure 5—figure supplement 2
Bafilomycin A1 (BafA1) and Tubastatin A treatment on patient organoids.
(A) Timeline of BafA1 treatment. (B) Western blot and quantification of p62, LC3-II, and LC3-II/LC3-I ratio upon bafilomycin A1 (BafA1) treatment. The drug vehicle dimethylsulfoxide (DMSO) was added …
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Figure 5—figure supplement 2—source data 1
Overlay of the bright field and chemiluminescence images indicating the signal of LC3.
BafA1 stands for Bafilomycin A1. The size of the protein ladders, LC3, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-figsupp2-data1-v2.zip
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Figure 5—figure supplement 2—source data 2
Overlay of the bright field and chemiluminescence images indicating the signal of p62.
BafA1 stands for Bafilomycin A1. The size of the protein ladders, p62, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-figsupp2-data2-v2.zip
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Figure 5—figure supplement 2—source data 3
Overlay of the bright field and chemiluminescence images indicating the signal of β-Actin.
BafA1 stands for Bafilomycin A1. The size of the protein ladders, b-Actin, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-figsupp2-data3-v2.zip
Figure 5—figure supplement 3
LCA2-derived retinal organoids in response to reserpine (RSP) treatment.
(A) Western blot analyses of p62 and LC3 in dimethylsulfoxide (DMSO)- and RSP-treated LCA2 organoids. β-Actin was used as the loading control. (B) Quantification of immunoblots of p62 and LC3. The …
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Figure 5—figure supplement 3—source data 1
Overlay of the bright field and chemiluminescence images indicating the signal of p62.
The size of the protein ladders, p62, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-figsupp3-data1-v2.zip
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Figure 5—figure supplement 3—source data 2
The size of the protein ladders, p62, and relevant sample identity are labeled.
The size of the protein ladders, b-Actin, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-figsupp3-data2-v2.zip
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Figure 5—figure supplement 3—source data 3
Overlay of the bright field and chemiluminescence images indicating the signal of OFD1.
The size of the protein ladders, OFD1, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-figsupp3-data3-v2.zip
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Figure 5—figure supplement 3—source data 4
Overlay of the bright field and chemiluminescence images indicating the signal of HDAC6.
The size of the protein ladders, HDAC6, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig5-figsupp3-data4-v2.zip
Figure 6 with 3 supplements
Neuroprotective effect of reserpine (RSP) in rd16 mouse retina.
(A) Timeline of in vivo intravitreal injection for quantification of outer nuclear layer (ONL), morphology, and immunohistochemistry studies. The eyes used for subsequent analyses were highlighted …
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Figure 6—source data 1
Individual values of a- and b-wave amplitudes (µV) of scotopic and photopic electroretinogram responses in DMSO- and RSP-treated rd16 mice.
RSP stands for reserpine.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig6-data1-v2.zip
Figure 6—figure supplement 1
Evaluation of reserpine (RSP) injection on photoreceptor layer in rd16 mouse retina.
Evaluation of the impact of reserpine on outer nuclear layer (ONL) thickness of the central and peripheral retina. Each dot in the bee swamp plot indicates the ONL thickness of one retina from one …
Figure 6—figure supplement 2
Evaluation of reserpine (RSP) injection on photoreceptor structure and function in the wild-type (WT) mouse retina.
(A) Immunostaining of rod phototransduction protein rhodopsin (Rho, green), Pde6β (red), rod cell marker Reep6 (green), and cone opsin Opn1sw (red). Nuclei were stained by 4',6-diamidino-2- …
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Figure 6—figure supplement 2—source data 1
Individual values of a- and b-wave amplitudes (µV) of scotopic and photopic electroretinogram responses in DMSO- and RSP-treated WT mice.
RSP stands for reserpine.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig6-figsupp2-data1-v2.zip
Figure 6—figure supplement 3
Effect of reserpine (RSP)-injection on distinct cell types in rd16 mouse retina.
Retinal structures and cell types are shown by (A) rod phototransduction protein Pde6β (red) and rhodopsin (Rho, magenta), (B) PKCα (red) for bipolar cells (red) and Calretinin (magenta) for …
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Figure 6—figure supplement 3—source data 1
Overlay of the bright field and chemiluminescence images indicating the signal of Gfap.
The size of the protein ladders, Gfap, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig6-figsupp3-data1-v2.zip
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Figure 6—figure supplement 3—source data 2
Overlay of the bright field and chemiluminescence images indicating the signal of g-Tubulin.
The size of the protein ladders, g-Tubulin, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig6-figsupp3-data2-v2.zip
Figure 7
Rd16 mouse retina in response to reserpine (RSP) treatment.
(A) p62 level in dimethylsulfoxide (DMSO)- and RSP-treated photoreceptors shown by immunostaining. (B) Quantification of p62 puncta. At least three non-overlap regions were quantified in at least …
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Figure 7—source data 1
Overlay of the bright field and chemiluminescence images indicating the signal of 20S proteosome.
WT and RSP stand for wild-type and reserpine respectively. The size of the protein ladders, 20 S proteosome, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig7-data1-v2.zip
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Figure 7—source data 2
Overlay of the bright field and chemiluminescence images indicating the signal of γ-Tubulin.
WT and RSP stand for wild-type and reserpine respectively. The size of the protein ladders, γ-Tubulin, and relevant sample identity are labeled.
- https://cdn.elifesciences.org/articles/83205/elife-83205-fig7-data2-v2.zip
Figure 8
Action mechanisms of reserpine in photoreceptor survival.
CEP290 mutations lead to defects in the outer segment biogenesis and consequently, ciliary vesicles carrying building blocks of the primary cilium and ciliary proteins are accumulated in patient …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (M. musculus) | Nrl-GFP WT | PMID: 27667917 | Reprogrammed from mouse embryonic fibroblasts by lentivirus carrying Yamanaka factors | |
| Cell line (M. musculus) | Nrl-GFP rd16 line 1 | PMID: 30332642 | Reprogrammed from mouse embryonic fibroblasts by lentivirus carrying Yamanaka factors | |
| Cell line (M. musculus) | Nrl-GFP rd16 line 2 | PMID: 30332642 | Reprogrammed from mouse embryonic fibroblasts by lentivirus carrying Yamanaka factors | |
| Cell line (Homo-sapiens) | Control | PMID: 28700940 | Reprogrammed from control fibroblasts by Sendai virus carrying Yamanaka factors | |
| Cell line (Homo-sapiens) | LCA-1 | PMID: 28700940 | Reprogrammed from control fibroblasts by Sendai virus carrying Yamanaka factors | |
| Cell line (Homo-sapiens) | LCA-2 | In this paper | Reprogrammed from control fibroblasts by Sendai virus carrying Yamanaka factors and maintained in Swaroop lab in National Eye Institute, National Institutes of Health, United States | |
| Cell line (Homo-sapiens) | LCA-1-clone C | In this paper | Reprogrammed from control fibroblasts by Sendai virus carrying Yamanaka factors and maintained in Swaroop lab in National Eye Institute, National Institutes of Health, United States | |
| Cell line (Homo-sapiens) | LCA-2-clone F | PMID: 28700940 | ||
| Antibody | anti-RHO (mouse monoclonal) | Gift of Dr. Robert Molday, University of British Columbia | IF(1:500) | |
| Antibody | anti-OPN1SW (Goat polyclonal) | Santa Cruz | Cat#: sc-14363 | IF(1:500) |
| Antibody | anti-GT335 (Mouse monoclonal) | AdipoGen | Cat#: AG-20B- 0020 | IF(1:500) |
| Antibody | anti-Rootletin (Chicken polyclonal) | PMID: 12427867 | IF (1:200) | |
| Antibody | anti-ARL13B (Rabbit polyclonal) | Proteintech | Cat#: 17711–1-AP | IF (1:500) |
| Antibody | anti-OPN1LMW (Rabbit polyclonal) | Millipore | Cat#: AB5405 | IF (1:500) |
| Antibody | anti-CEP290 (Rabbit monoclonal) | Bethyl laboratories | Cat#: A301-659A | WB (1:500) |
| Antibody | anti-GAPDH (Mouse monoclonal) | Millipore | Cat#: G8795 | WB (1:1000) |
| Antibody | anti-PDE6b (Rabbit polyclonal) | PMID: 33107904 | IF (1:500) | |
| Antibody | anti-Synaptophysin (Mouse monoclonal) | Abcam | ab8049 | IF (1:200) |
| Antibody | anti-p62 (Rabbit polyclonal) | Abcam | ab109012 | IF (1:200) WB (1:500) |
| Antibody | anti-LC3A/B (D3U4C) (Rabbit monoclonal) | Cell Signaling | 12741 | For human retinal organoids, WB (1:1000) |
| Antibody | anti-LC3B (Rabbit polyclonal) | Abcam | ab51520 | For mouse retina, WB (1:1000) |
| Antibody | anti-b-Actin (Mouse monoclonal) | Millipore | A5316 | WB (1:5000-1:10000) |
| Antibody | anti-20S (Rabbit polyclonal) | Enzo Life Sciences | BML-PW8155-0100 | WB (1:500) |
| Antibody | anti-IFT88 (Rabbit polyclonal) | Proteintech | 13967–1-AP | WB (1:500) |
| Antibody | anti-BBS6 (Rabbit polyclonal) | Michel Leroux | #17 | WB (1:500) |
| Antibody | anti-CEP164 (Rabbit polyclonal) | GeneTex | GTX85298 | WB (1:500) |
| Antibody | anti-OFD1 (Rabbit polyclonal) | Thermo | PA5-115684 | WB (1:500) |
| Antibody | anti-HDAC6 (Rabbit polyclonal) | Abgent | AP1106A | WB (1:1000) |
| Antibody | anti-RP1 (Chicken polyclonal) | PMID: 11773008 | IF (1:2000) | |
| Antibody | anti-Bassoon (Rabbit monoclonal) | Cell Signaling | 6897 S | IF (1:200) |
| Antibody | anti-CHX10 (Sheep polyclonal) | Abcam | ab16141 | IF (1:200) |
| Antibody | anti-PKCa (Rabbit monoclonal) | Thermo Fisher | MA1-157 | IF (1:1000) |
| Antibody | anti-CRALBP (Mouse monoclonal) | Abcam | ab15051 | IF (1:200) |
| Antibody | anti-GFAP (Rabbit polyclonal) | Dako | Z0334 | IF (1:500) WB (1:1000) |
| Antibody | anti-Calretinin (Rabbit monoclonal) | Millipore | MAB1568 | IF (1:200) |
| Commercial kits | Proteasome 20 S Activity Assay Kit | Sigma-Aldrich | MAK172-1KT | |
| Chemical compound, drug | Reserpine | Sigma Aldrich | 06859 | |
| Chemical compound, drug | MRT68921 | Sigma Aldrich | SML1644 | |
| Chemical compound, drug | Lys05 | Sigma Aldrich | SML2097 | |
| Chemical compound, drug | Chloroquine | Sigma Aldrich | C6628 | |
| Chemical compound, drug | Hydroxychloroquine | Sigma Aldrich | H0915 | |
| Chemical compound, drug | ROC-325 | Selleck | S8527 | |
| Chemical compound, drug | Bafilomycin A1 | Sigma Aldrich | 19–148 | |
| Chemical compound, drug | Tubastatin A | Sigma Aldrich | SML0044 | |
| Software, algorithm | Photoshop CC 2019 | Adobe | ||
| Software, algorithm | FIJI | FIJI | ||
| Software, algorithm | R | R-core team | ||
| Software, algorithm | Biowulf Linux cluster | National Institutes of Health | http://biowulf.nih.gov | |
| Other | DAPI stain | Invitrogen | D1306 | (1 µg/mL) |
