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Small molecule Photoregulin3 prevents retinal degeneration in the RhoP23H mouse model of retinitis pigmentosa

  1. Paul A Nakamura
  2. Andy A Shimchuk
  3. Shibing Tang
  4. Zhizhi Wang
  5. Kole DeGolier
  6. Sheng Ding
  7. Thomas A Reh  Is a corresponding author
  1. University of Washington, School of Medicine, United States
  2. University of California, San Francisco, United States
Short Report
Cite as: eLife 2017;6:e30577 doi: 10.7554/eLife.30577
4 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
PR3 reduces rod gene expression via Nr2e3.

(A) Chemical structure of Photoregulin3 (PR3). (B) Dose-response relationship of PR1 and PR3 on Rhodopsin expression in dissociated retinal cell cultures (n = 3 for each concentration for each compound; value graphed is the mean ±SEM of 3 biological replicates). Example scans of Rhodopsin and ToPro3 staining from retinal cell cultures are shown on the right. (C) Confirmation of PR3’s potency by qPCR from intact retinal explant cultures from P4 mice treated with DMSO or 0.3 μM PR1 or PR3 for 2 days (n = 3–4 for each compound; value graphed is the mean ±SEM of the biological replicates, *p=0.0075 for DMSO vs. PR3, two-tailed t-test assuming equal variance using ΔCT values). (D) Intact retinas from P11 mice were explanted in media containing DMSO or 0.3 μM PR3 for 3 DIV and then stained for S Opsin in a whole-mount preparation. Scale bar represents 50 μm. (E) PR3-treated retinas had more S Opsin+ cells per 100 μm x 100 μm field in the ventral, but not dorsal, retina compared to DMSO-treated retinas (n = 3 biological replicates; value graphed is the mean ±SEM of the biological replicates, *p=0.00063, two-tailed t-test assuming equal variance). (F) Isothermal titration calorimetric study of PR3 binding to Nr2e3. (G) Intact retinas from adult (P23–P35) wild type and Rd7 were explanted in media containing DMSO or 1 μM PR3 for 2 days and Rhodopsin expression was measured by qPCR. PR3 decreased Rhodopsin in wild type retinas but not in Rd7 retinas (n = 4 biological replicates; value graphed is the mean ±SEM of the biological replicates, *p=0.033, two-tailed t-test assuming equal variance for wild type and p=0.13, two-tailed t-test assuming equal variance for Rd7 using ΔCT values).

https://doi.org/10.7554/eLife.30577.003
Figure 1—figure supplement 1
Effects of PR3 on photoreceptor promoter activation and co-activator binding.

(A) HEK 293 cells were transfected with NR2E3, CRX, and NRL and BR-225Luc (firefly luciferase driven by the bovine Rhodopsin promoter) and pRL-CMV (renilla luciferase driven by the CMV promoter; internal transfection control) and then treated with DMSO or 1 μM PR3 for 2 days. PR3 decreased Rhodopsin promoter activity after transfection of NR2E3, CRX, and NRL (n = 4 separate transfections per condition, values graphed are the mean ±SEM of the biological replicates, p<0.001, two-tailed t-test assuming equal variance). (B) HEK 293 cells were transfected with NR2E3 and NRL and then treated with DMSO or 1 μM PR3 for 2 days. Lysates were immunoprecipitated with an anti-Nr2e3 antibody and then analyzed by Western blot analysis. (C) PR3 treatment increased the binding between Nr2e3 and Nrl (n = 3 separate transfections per condition, values graphed are the mean ±SEM of the biological replicates, p=0.013, two-tailed t-test assuming equal variance). (D) HEK 293 cells were transfected with RORb, CRX, and S Opsin Luc (firefly luciferase driven by the S Opsin promoter) and pRL-CMV (renilla luciferase driven by the CMV promoter; internal transfection control) and then treated with DMSO or 1 μM PR3 for 2 days. PR3 decreased S Opsin promoter activity (n = 4 separate transfections per condition, values graphed are the mean ±SEM of the biological replicates, p=0.0014, two-tailed t-test assuming equal variance). (E) Retinas from P0 mice were explanted in media containing DMSO or 0.3 μM PR3 for 2 days. PR3 treatment reduced Rhodopsin expression (n = 4 biological replicates, values graphed are the mean ±SEM of the biological replicates, p=0.00081, two-tailed t-test assuming equal variance), but not Prdm1 expression, a direct target of RORb (n = 4 biological replicates, values graphed are the mean ±SEM of the biological replicates, p=0.41, two-tailed t-test assuming equal variance).

https://doi.org/10.7554/eLife.30577.004
In vivo characterization of PR3 treatment.

(A) RNA sequencing results plotting log2FoldChange against log2Expression (RPKM) of wild type mice treated with DMSO vehicle or 10 mg/kg PR3 shows robust reduction in rod photoreceptor genes. (n = 2 mice per condition; values graphed are the mean of the two biological replicates) (B) Gene ontology analysis (http://geneontology.org/page/go-enrichment-analysis) results for largest changes (top 100) in gene expression assessed by RNA sequencing. (C) Whole mount S Opsin staining of retinas from mice treated with 10 mg/kg PR3 or DMSO vehicle for 3 days. Scale bar represents 25 μm. (D) Retinas from mice treated with PR3 had more S Opsin+ cells per 100 μm x 100 μm field in the ventral, but not dorsal, retina compared to controls (n = 4 biological replicates; value graphed is the mean ±SEM of the biological replicates, *p=0.0086, two-tailed t-test assuming equal variance). (E) Electron microscope micrographs of retinal sections of wild type mice treated with DMSO or 10 mg/kg PR3 (n = 2 mice per condition). Compared to DMSO controls, PR3 retinas have arrested outer segment development (arrows indicate outer segments in DMSO treated mice). Scale bare represents 10 μm.

https://doi.org/10.7554/eLife.30577.007
PR3 slows the progression of photoreceptor degeneration in the RhoP23H mouse model of RP.

(A) Timeline of photoreceptor degeneration in the RhodopsinP23H mouse and experimental design. (B) Immunofluorescence staining for Rhodopsin, S Opsin, Otx2, and Cone Arrestin on retinal sections from RhodopsinP23H mice demonstrate preservation of photoreceptors with PR3 treatment. Scale bar represents 50 μm. (C) Counts for rows of DAPI+ cells in the central and peripheral ONL show greater survival of photoreceptors with PR3 treatment (n = 7 mice for DMSO treatment and 8 mice for PR3; values graphed are the mean ±SEM of the biological replicates, p=0.0014 for center and 0.015 for periphery, two-tailed t-test assuming equal variance). (D) qPCR on whole retinas from RhodopsinP23H mice treated with DMSO or PR3 shows greater expression of photoreceptor genes Recoverin, Rhodopsin, and S Opsin with PR3 treatment (n = 6 mice for DMSO and 7 mice for PR3; values graphed are the mean ±SEM of the biological replicates, p=1.73E-05 for Recoverin, 3.13E-05 for Rhodopsin, and 0.076 for S Opsin, two-tailed t-test assuming equal variance on ΔCT values).

https://doi.org/10.7554/eLife.30577.009
PR3 preserves visual function in the RhodopsinP23H mouse.

(A) Scotopic b-wave amplitudes from P21 RhodopsinP23H mice treated with 10 mg/kg PR3 or DMSO vehicle (n = 4 mice for DMSO treatment and 8 mice for PR3 treatment, values graphed are the mean ±SEM of the biological replicates, p=0.025, 0.012, 0.032, 0.021, and 0.015 for 0, 10, 20, 30, and 50 Decibels (dB) respectively, two-tailed t-test assuming unequal variance). (B) Representative scotopic ERG waveforms from a single DMSO vehicle mouse. (C) Representative scotopic ERG waveforms from a single PR3–treated mouse. (D) Photopic b-wave amplitudes from P21 RhodopsinP23H mice treated with 10 mg/kg PR3 or DMSO vehicle (n = 4 mice for DMSO treatment and 8 mice for PR3 treatment, values graphed are the mean ±SEM of the biological replicates, p=0.18, 0.025, 0.048, and 0.021 for 0, 30, 50, and 100 dB respectively, two-tailed t-test assuming unequal variance). (E) Representative photopic ERG waveforms from a single control mouse. (F) Representative photopic ERG waveforms from a single PR3-treated mouse.

https://doi.org/10.7554/eLife.30577.011

Tables

Key resource table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Transgenic mice (mus
musculus), both sexes
Rhodopsin-Pro23HisThe Jackson LaboratoryJackson Stock No: 017628;
RRID:IMSR_JAX:017628
Transgenic mice (mus
musculus), both sexes
Nr2e3-Rd7The Jackson LaboratoryJackson Stock No: 004643;
RRID:IMSR_JAX:004643
C57Bl6J mice (mus
musculus), both sexes
Wild typeThe Jackson LaboratoryJackson Stock No: 000664;
RRID:IMSR_JAX:000664
Chemical compoundPhotoregulin3 (PR3)ChemDiv, and then
synthesized in Ding Lab
PubChem CID: 2092851
Chemical compoundPhotoregulin1 (PR1)ChemDiv, and then
synthesized in Ding Lab
PubChem CID: 7901316
AntibodyAnti- Rhodopsin (Rho4D2),
mouse monoclonal
Dr. Robert Molday, UBCRRID:AB_23152731:250
AntibodyAnti-S Opsin, goat polyclonalSCBTSc-14363; RRID:AB_21583321:400
AntibodyAnti Cone Arrestin,
rabbit polyclonal
MilliporeAB15282; RRID:AB_11633871:1000
AntibodyAnti-Otx2, goat polyclonalR&D SystemsAF1979; RRID:AB_21571721:200
AntibodyAnti-Nr2e3, mouse monoclonalR&D Sysems:PP-H7223-00; RRID:AB_21554811:10,000
AntibodyAnti-Nrl, rabbit polyclonalChemiconAb56931:10,000
AntibodyAnti-beta actin,
mouse monoclonal
AbcamAb6276; RRID:AB_22232101:20,000
Recombinant DNAHsCD00084154 (Nr2e3)DNASUHsCD00084154
Recombinant DNABR-225LucDr. Shiming Chen, Washington
University; PMID: 15689355
Recombinant DNAS Opsin 600 pGL3Dr. Douglas Forrest, NIH;
PMID: 16574740
Recombinant DNApRL-CMVPromegaE6931
Recombinant DNAhNRL-pCMVSport6Open BiosystemsMHS1010-58005
Recombinant DNAhCRX-pCMVSport6Open BiosystemsMHS1010-73672
Recombinant DNAhNR2E3-pcDNA3.1/HisCDr. Shiming Chen, Washington
University; PMID: 15689355
Recombinant DNAHsCD00329674 (RORB)DNASUHsCD00329674

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