Research ArticleSYSTEMS PHARMACOLOGY

Synergistically acting agonists and antagonists of G protein–coupled receptors prevent photoreceptor cell degeneration

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Science Signaling  26 Jul 2016:
Vol. 9, Issue 438, pp. ra74
DOI: 10.1126/scisignal.aag0245
  • Fig. 1

    Disruption of the OPL and damage of photoreceptor synaptic terminals in bright light–exposed retinas of Abca4−/−Rdh8−/− mice. (A) Schematic of the experimental protocol. Four- to 6-week-old male and female pigmented Abca4−/−Rdh8−/− mice or BALB/c mice were exposed to bright light at 10,000 lux for 30 min, followed by the procedures as indicated. IHC, immunohistochemistry; OCT, optical coherence tomography; SLO, scanning laser ophthalmoscopy; ERG, electroretinogram; TPM, two-photon microscopy; RNA-seq, RNA sequencing. (B) After 4′,6-diamidino-2-phenylindole (DAPI) staining, retinal cryosections from Abca4−/−Rdh8−/− mice were observed under a fluorescence microscope. Retinas were collected from Abca4−/−Rdh8−/− mice at the time points indicated including 3 hours, 1 day, and 7 days after light exposure. Red arrows mark areas showing the thinning of the OPL. (C) Toluidine blue–stained retinal sections from Abca4−/−Rdh8−/− mice unexposed to bright light or at 3 hours after light exposure. White lines outline the OPL. (D) Electron microscopy of retinal sections from Abca4−/−Rdh8−/− mice unexposed to bright light or at 3 hours after light exposure. M, mitochondrion; R, synaptic ribbon in photoreceptor synaptic terminal. Red arrows, cytoplasmic vacuolation.

  • Fig. 2

    Impaired photoreceptor synaptic terminals, horizontal cell morphology, bipolar cell dendrites, and retinal gliosis in bright lightexposed Abca4−/−Rdh8−/− mice. Retinal cryosections were obtained from Abca4−/−Rdh8−/− mice either unexposed to bright light or after exposure to light at the indicated days. (A to D) Abundance of synaptophysin, calbindin D, PKCα (protein kinase Cα), and GFAP (glial fibrillary acidic protein) (all of which are green) was then revealed by immunohistochemistry and observed by fluorescence microscopy. DAPI counterstaining (blue) was performed to visualize the retinal structure in (B) and (D). IPL, inner plexiform layer; GC, ganglion cell; NFL, nerve fiber layer. White arrows in (A) and (B) indicate representative areas with diminished synaptophysin or calbindin D. Asterisks in (B) identify areas where calbindin D immunoreactivity was barely detected.

  • Fig. 3

    Retina protection conferred by monotherapy. (A) Retinal expression of PNA (green) along with DAPI counterstaining (blue) was examined in cryosections collected from light-exposed Abca4−/−Rdh8−/− mice pretreated with DMSO, BRM, or MTP. (B) Autofluorescent spots in the retinas of Abca4−/−Rdh8−/− mice imaged after exposure to bright light. Abca4−/−Rdh8−/− mice were pretreated as indicated and exposed to bright light, and then SLO was performed to visualize autofluorescence in retinas of Abca4−/−Rdh8−/− mice. (C to E) Total RNA from the indicated experimental groups (n = 3 per group) was isolated from Abca4−/−Rdh8−/− mice 1 day after light exposure, along with mice unexposed to bright light. Total RNA was then subjected to RNA-seq analysis. (C) Three-dimensional (3D) PCAs of all expressed transcripts are shown for the control mice unexposed to bright light (C), light-exposed, DMSO-pretreated mice (L), and mice pretreated with either BRM (B), MTP (M), DOX (D), or TAM (T). Small shapes represent individual samples, and large shapes are the centroid representations for each group. (D) Pearson’s correlation plots of differentially expressed transcripts from retinas of mice unexposed to bright light and the indicated pretreatment groups. Scale bar indicates correlation coefficient with identity orange (correlation coefficient, 1). (E) Gene expression clustering of the differentially expressed transcripts is shown for mice with the indicated pretreatments along with clustering from mice unexposed to light and DMSO-pretreated mice exposed to light. Data are shown for the three independent samples in each treatment. Scale bar represents the Z score indicating up-regulation (orange) and down-regulation (blue).

  • Fig. 4

    Characterizing the activity of BRM, MTP, DOX, and TAM on dopamine and adrenaline receptors. (A) Schematic representation of the BRET assay used to evaluate actions of drugs on selected GPCRs. Activation of a GPCR by agonist leads to the dissociation of inactive heterotrimeric G proteins into active GTP-bound Gα and Venus-Gβγ subunits. The free Venus-Gβγ then interacts with the Gβγ-effector mimetic masGRK3ct-Nluc to produce the BRET signal. GDP, guanosine diphosphate; GRK, G protein–coupled receptor kinase; Nluc, nanoluciferase. (B) Left: Representative BRET signal traces recorded upon activation of D4R with indicated drugs in cells expressing Gαo and Venus-Gβγ, illustrating the agonism assay. Right: Representative traces of D2R responses to 1 μM dopamine in the absence (control) or presence of indicated drugs, illustrating antagonism assay. Note that, in the antagonism assay shown, the black trace overlaps with the red trace and therefore is not visible. (C) Top: Quantification of agonistic activity of ligands on D2R, D4R, D1R, and ADRA1A. D2R-Go, D4R-Go, D1R-Gs, and ADRA1A-Gq signaling were reconstituted in HEK293T/17 cells by transient transfection separately. Maximum amplitudes induced by dopamine (DA; 100 μM), noradrenaline (NA; 100 μM), BRM (25 μM), MTP (100 μM), DOX (50 μM), and TAM (100 μM) were plotted as bar graphs. Bottom: Quantification of antagonistic activity of ligands. To examine the antagonistic activity, cells were preincubated with each of the ligands, and then 1 μM dopamine for dopamine receptors or 1 μM noradrenaline for the adrenaline receptor was applied. In these experiments, only ligands that did not show any agonistic effect were examined for each receptor–G protein combination. The activity is plotted as percent inhibition. Data are means ± SD. Results are representative of two independent experiments each performed with 12 replicates.

  • Fig. 5

    Combination pretreatments improve retinal morphological protection against bright light exposure in Abca4−/−Rdh8−/− mice. (A) BRM, MTP, or TAM was administered either individually at the indicated subeffective dose (in mg/kg bw) to Abca4−/−Rdh8−/− mice or as a combined pretreatment as indicated, each at its subeffective dose. The mice were then exposed to bright light, and OCT imaging was performed 7 days later. Percentages of mice manifesting complete protection of retinal structures were calculated for each condition. For individual drug treatment, combination treatment with BRM and MTP, and combination treatment with MTP and TAM, n = 12 mice per group. For combination of BRM and TAM, n = 21 per group. For combination of BRM, MTP, and TAM, n = 24 per group. (B) Retinal OCT images of Abca4−/−Rdh8−/− mice either unexposed to bright light or exposed to bright light after pretreatment with DMSO or the indicated drug combinations at the subeffective doses for each. Images were obtained 7 days after light exposure. Asterisk indicates impaired ONL structure. (C) The thickness of the ONL was determined from OCT images at 0.45 mm away from the ONH in the region of the retinas in the direction of the temples from five mice 7 days after light exposure. *P < 0.05, compared to no light; #P < 0.05, compared to DMSO (independent samples t test). (D) SLO was performed to image autofluorescent spots in the retinas of Abca4−/−Rdh8−/− mice unexposed to bright light or exposed to light after the indicated pretreatment. SLO imaging was performed 9 days after light exposure. (E) Gross morphology of retinas was examined after H&E staining of paraffin sections collected from light-exposed Abca4−/−Rdh8−/− mice pretreated as indicated. Retinal tissue was obtained 10 days after light exposure. Asterisk indicates severely diminished ONL. All morphological analyses in (B) to (E) were performed with at least five mice.

  • Fig. 6

    Combination pretreatment preserves OPL morphology and retinal function in light-exposed Abca4−/−Rdh8−/− mice. Seven days after bright light exposure, cryosections were prepared from the eye cups of light-exposed Abca4−/−Rdh8−/− mice pretreated with DMSO or a combination of BRM (0.1 mg/kg bw), MTP (1 mg/kg bw), and TAM (0.05 mg/kg bw) (B + M + T). (A to C) The abundance of synaptophysin, PKCα, and calbindin D (each shown in green) was examined by immunohistochemistry. DAPI counterstaining (blue) was performed in (A) and (B) to visualize the retinal structure. White arrows in (B) and (C) denote regions of diminished staining of PKCα or calbindin D. Asterisk indicates a representative area showing diminished ONL and residual staining for synaptophysin (A) or the absence of calbindin D immunoreactivity (C). (D and E) Scotopic and photopic b-wave amplitudes were analyzed after ERG recordings were performed in Abca4−/−Rdh8−/− mice either unexposed to bright light or exposed to bright light and pretreated with either DMSO, a combination of BRM (0.1 mg/kg bw), MTP (1 mg/kg bw), and TAM (0.05 mg/kg) (B + M + T), or a combination of BRM (0.1 mg/kg bw), MTP (1 mg/kg bw), and DOX (1 mg/kg) (B + M + D). Data are means ± SD from five mice.

  • Fig. 7

    RNA-seq analysis of retinal gene expression in Abca4−/−Rdh8−/− mice pretreated with drug combinations. Total RNA from the indicated experimental groups (n = 3 per group) was isolated from Abca4−/−Rdh8−/− mice 1 day after light exposure, along with mice unexposed to bright light. Total RNA was then subjected to RNA-seq analysis. (A) 3D PCAs of all expressed transcripts are shown for the control mice unexposed to bright light (C), light-exposed, DMSO-pretreated mice (L), and mice pretreated with combinations of either BRM, MTP, and TAM (B + M + T) or BRM, MTP, and DOX (B + M + D). (B) Pearson’s correlation plots of the differentially expressed transcripts from retinas of mice unexposed to bright light or subjected to combination therapy before light exposure. Scale bar represents correlation coefficient. (C) Gene expression clustering of differentially expressed transcripts is shown for mice with the indicated combined pretreatments along with clustering from mice unexposed to light and DMSO-pretreated mice exposed to light. Scale bar represents the Z score indicating up-regulation (orange) and down-regulation (blue).

  • Fig. 8

    Pharmacological treatments protect against bright lightinduced alterations in the retinal transcriptome and retinal degeneration. Bright light exposure causes retinal degeneration that is associated with perturbation of retinal transcriptome homeostasis manifested as dysregulation of multiple gene sets, including but not limited to down-regulation of the phototransduction pathway and up-regulation of the apoptosis pathway, p53 signaling, cytokine-cytokine receptor interactions, and chemokine signaling. Pretreatment with individual drugs that act as antagonists at Gs-coupled GPCRs, agonists at Gi-coupled GPCRs, or antagonists at Gq-coupled GPCRs results in retinal protection. We found that, when each drug is delivered at lower subeffective doses, combinations of these drugs acting synergistically upon different GPCRs protected retinas from bright light–induced degeneration. The retinal protection by different treatment modalities, namely, monotherapy at high doses or combined treatments consisting of subeffective doses of drugs, not only prevents light-induced changes in common gene sets but also affects gene sets specific to each treatment. Combined treatment results in improved preservation of the retinal transcriptome compared to that conferred by monotherapy and may also offer the benefit of reduced side effects because of lower doses required for effectiveness.

  • Table 1 Expression of GPCR genes encoding potential therapeutic targets for retinal degeneration in the Abca4−/−Rdh8−/− mouse model of bright light–induced retinopathy.

    FPKM, normalized fragments per kilobase of exon per million mapped reads.

    Gene*Mouse retina (FPKM)Human retina (FPKM)ReceptorG protein signalingAgonistAntagonist
    Drd4241.78139.49Dopamine receptor D4Gi+
    Gabbr140.2435.38γ-Aminobutyric acid B receptor 1Gi+
    Drd223.126.33Dopamine receptor D2Gi+
    Adora118.2613.55Adenosine A1 receptorGi+
    Crhr16.4912.76Corticotropin-releasing hormone receptor 1Gs+
    S1pr111.2111.78Sphingosine-1-phosphate receptor 1Gi+
    Drd1a9.498.45Dopamine receptor D1aGs+
    Smo6.355.91SmoothenedGi+
    Ednrb1.945.77Endothelin receptor type BGq+
    Grm13.524.97Metabotropic glutamate receptor 1Gq+
    Adrb120.183.84β1-Adrenergic receptorGs+
    Adora2b2.533.83Adenosine A2B receptorGs and Gq+
    Hrh319.123.75Histamine receptor H3Gi+
    Grm44.933.1Metabotropic glutamate receptor 4Gi+
    Grm86.432.04Metabotropic glutamate receptor 8Gi+
    Grm73.951.16Metabotropic glutamate receptor 7Gi+
    Adra1a0.450.20α1A-Adrenergic receptorGq
    Adra1b1.601.12α1B-Adrenergic receptorGq
    Adra1d1.721.08α1D-Adrenergic receptorGq
    Adrb21.030.98β2-Adrenergic receptorGs+
    Tacr12.060.95Tachykinin receptor 1Gq+
    Ptger114.880.94Prostaglandin E receptor 1Gq+
    S1pr21.130.63Sphingosine-1-phosphate receptor 2Gq, Gs, and Gi

    *Genes in bold, italicized font encode receptors targeted by the drugs displaying efficacy in this study.

    †Data from previous study (23).

    • Table 2 Summary of GPCR-modulating compounds that conferred retinal morphological and functional protection in bright light–exposed Abca4−/−Rdh8−/− mice.

      Morphological protection was assessed as morphological preservation of the retina by OCT imaging 7 days after exposure to bright light. Complete protection represented the retinas with intact ONL morphology similar to those of mice unexposed to bright light, with ONL thicknesses of ≥50 μm at 0.45 mm away from the optic nerve head (ONH) in the temporal retina. Functional protection was assessed by ERG analyses of retinal function in Abca4−/−Rdh8−/− mice 10 days after bright light exposure. Protection data were collected from at least five mice from each experimental group unless otherwise specifically indicated. ERG data are means ± SD, and statistical analyses were performed with either Student’s t test or analysis of variance (ANOVA). P < 0.05 was considered statistically significant.

      AgentMajor action(s)G protein category
      SCH 23390 hydrochlorideDopamine receptor D1 and D5 antagonistGs
      Rotigotine hydrochlorideDopamine receptor D2 and D3 agonistGi
      2-Bromo-α-ergocryptine methanesulfonate saltDopamine receptor D2 and D3 agonistGi
      Sumanirole maleateDopamine receptor D2 agonistGi
      B-HT 920Dopamine receptor D2, α2-adrenergic receptor agonistGi
      Ro 10-5824 dihydrochlorideDopamine receptor D4 agonistGi
      YM 202074mGluR1 receptor antagonistGq
      Cinnabarinic acidmGluR4 receptor agonistGi
      Doxazosin*Adrenergic receptor α1 antagonistGq
      Tamsulosin*Adrenergic receptor α1 antagonistGq
      MTP tartrateAdrenergic receptor β1 antagonistGs
      ICI 118,551 hydrochlorideAdrenergic receptor β1 antagonistGs
      GS 6201Adenosine A2B receptor antagonistGs
      PSB 1115Adenosine A2B receptor antagonistGs
      SC 19220EP1 prostanoid receptor antagonistGq
      CP 154526Corticotropin-releasing factor 1 receptor antagonistGs
      L-733060Tachykinin NK1 receptor antagonistGq
      CP 96345Tachykinin NK1 receptor antagonistGq

      *Compounds identified from previous study (23).

      • Table 3 Retinal protection conferred by individual and combination therapies targeting GPCRs.

        Either BRM, MTP, or TAM was administered individually at a subeffective dose to Abca4−/−Rdh8−/− mice or in a combined pretreatment consisting of two or three of these compounds, each at its subeffective dose. Doses for either single or combined pretreatments were 0.1, 1, and 0.05 mg/kg bw for BRM, MTP, and TAM, respectively. Protection was assessed as morphological preservation of the retina by OCT imaging 7 days after exposure to bright light. Complete protection represented the retinas with intact ONL morphology similar to those of mice unexposed to bright light, with ONL thicknesses of ≥50 μm at 0.45 mm away from the ONH in the temporal retina. No protection represented retinas exhibiting morphology similar to that of light-exposed and vehicle-treated mice, with ONL thickness of ≤20 μm at 0.45 mm away from the ONH in the temporal retina. Partial protection defined retinas manifesting a reduction in the thickness of the ONL between 20 and 50 μm measured 0.45 mm away from the ONH in the temporal retina. This was followed by exposure to bright light and OCT imaging performed 7 days later. Percentages of mice manifesting no protection of retinal structures were calculated for each condition.

        DrugPercent of mice with complete protectionPercent of mice with no protection
        BRM16.766.7
        MTP2541.7
        TAM16.758.3
        DOX0100
        BRM + MTP33.325
        BRM + TAM42.8642.86
        MTP + TAM41.716.7
        BRM + MTP + TAM87.54.17
        BRM + DOX5016.7
        MTP + DOX33.341.7
        BRM + MTP + DOX805
      • Table 4 Effect of single and combination therapies on gene sets associated with retinal degeneration pathways.

        GSEA identified pathways associated with retinal degeneration in retinas from light-exposed mice. NES, normalized enrichment score.

        PathwaysExperiment 1:
        Change in
        light-exposed
        retinas NES (P)*
        BRM NES
        (P)
        MTP NES
        (P)
        TAM NES
        (P)
        DOX NES
        (P)
        Experiment 2:
        Change in
        light-exposed
        retinas NES (P)*
        BRM +
        MTP +
        TAM
        NES (P)
        BRM +
        MTP + DOX
        NES (P)
        APOPTOSIS1.83 (0)1.75
        (0.0018)
        1.61
        (0.0055)
        1.60
        (0.011)
        1.75
        (0.0018)
        1.88 (0)1.81 (0)1.89 (0)
        P53 SIGNALING2.29 (0)2.06 (0)2.04 (0)1.98 (0)2.18 (0)1.71 (0.0039)1.98 (0)1.69 (0.0064)
        CYTOKINE-CYTOKINE
        RECEPTOR
        INTERACTIONS
        2.50 (0)2.31 (0)2.30 (0)2.29 (0)2.42 (0)2.18 (0)2.13 (0)2.04 (0)
        CHEMOKINE
        SIGNALING
        1.62 (0.0042)1.67 (0.0018)1.44 (0.02)1.39 (0.0199)1.41 (0.018)1.71 (0)1.57 (0.0102)1.67 (0.00144)
        TOLL-LIKE RECEPTOR
        SIGNALING
        2.14 (0)2.20 (0)2.05 (0)1.83 (0)1.99 (0)2.10 (0)2.10 (0)2.21 (0)

        *Comparison of the transcriptomes from vehicle-treated and light-exposed retinas against those from the mice unexposed to bright light.

        †Comparison of the transcriptomes from vehicle-treated and light-exposed retinas against those from the mice treated by indicated drug(s).

        • Table 5 Effect of single and combination therapies on gene sets in pathways down-regulated in retinas from DMSO-treated, light-exposed mice.

          Pathways were identified by GSEA. Only those pathways down-regulated in at least 50% of treatments are included.

          PathwaysExperiment 1:
          Change in
          light-exposed
          retinas NES (P)*
          BRM NES
          (P)
          MTP NES
          (P)
          TAM NES
          (P)
          DOX NES
          (P)
          Experiment 2: Change
          in
          light-exposed
          retinas NES (P)*
          BRM +
          MTP + TAM
          NES (P)
          BRM +
          MTP + DOX
          NES (P)
          PHOTOTRANSDUCTION−2.71 (0)−2.42 (0)−2.74 (0)−2.79 (0)−2.74 (0)−2.70 (0)−2.78 (0)−2.31 (0)
          OXIDATIVE
          PHOSPHORYLATION
          −1.78 (0)−1.52
          (0.010)
          −1.40
          (0.019)
          −1.63
          (0.0022)
          −1.33
          (0.0362)
          −1.38
          (0.0229)

          *Comparison of the transcriptomes from vehicle-treated and light-exposed retinas against those from the mice unexposed to bright light.

          †Comparison of the transcriptomes from vehicle-treated and light-exposed retinas against those from the mice treated by indicated drug(s).

          • Table 6 Genes of core enrichment in the apoptosis pathway of different experimental groups.

            Both of the DMSO vs. no light conditions produced an increase in expression of all of the listed genes in the first condition compared to the second. All other conditions produced a decrease in expression of all of the listed genes. In particular, the “DMSO vs. no light” columns represented controls for each set of experiments and DMSO induced an increase in the expression of the listed genes compared with the no light control.

            DMSO vs.
            no light
            BRM vs.
            DMSO
            MTP vs.
            DMSO
            TAM vs.
            DMSO
            DOX vs.
            DMSO
            DMSO vs.
            no light*
            BRM + MTP +
            TAM vs. DMSO
            BRM + MTP +
            DOX vs. DMSO
            FasMyd88FasBidFasMyd88Myd88Tnfrsf1a
            Ripk1Ripk1Myd88Il1r1Myd88Tnfrsf1aFasMyd88
            Myd88Il1r1BidTnfrsf1aIl1r1Il1r1Tnfrsf1aRipk1
            Il1r1Tnfrsf1aRipk1Casp6Ripk1Ripk1Ripk1Fas
            Tnfrsf1aBidIl1r1TraddTrp53Irak1Il1r1Il1r1
            BidTrp53Trp53Myd88Tnfrsf1aRelaNtrk1Prkx
            Trp53PrkxTnfrsf1aTrp53TraddTrp53Trp53Rela
            Capn2FasPrkxAkt2Tnfrsf10bBidBidMap3k14
            PrkxTraddCapn2FasIrak1Akt2RelaTrp53
            Apaf1Casp3Irak2Ripk1BidPrkxMap3k14Ntrk1
            TraddApaf1Casp6Il1rapChukFasTraf2Bid
            Casp6Irak1NfkbiaCasp3Casp3Map3k14Akt2Akt2
            Ntrk1Birc2TraddRelaCasp6Ntrk1BaxIrak1
            Tnfrsf10bIkbkbIl1rapNtrk1Il1rapTraddNfkbiaIkbkb
            Il1rapNfkbiaRelaIrak2Apaf1Apaf1PrkxBax
            Prkar2bNtrk1Irak1Nfkb1Capn2NfkbiaBcl2l1Apaf1
            Irak1Il1rapCasp3NfkbiaPrkar2bTnfrsf10bTnfrsf10bCasp3
            RelaCapn2Akt2Capn2PrkxCapn2Irak1Nfkbia
            Casp3RelaNtrk1Prkar2bNfkbiaAkt1BadTraf2
            NfkbiaPik3cbIrak2BaxAkt1Bcl2l1
            ChukPik3cbTraf2TraddAifm1
            Ppp3caIl1rapIrak2Pik3r3
            PrkacbCapn1
            Capn2

            *The controls are from two separate experiments (single and combined treatment) that were performed. Each experiment has its own control and light-exposed mice. The differences represent the variation of light damage from one experiment to the other.

            • Table 7 Genes of core enrichment in the phototransduction pathway of different experimental groups.

              Both of the DMSO vs. no light conditions produced an increase in expression of all of the listed genes in the first condition compared to the second. All other conditions produced a decrease in expression of all of the listed genes. In particular, the “DMSO vs. no light” columns represented controls for each set of experiments and DMSO induced an increase in the expression of the listed genes compared with the no light control.

              DMSO vs.
              no light
              BRM vs.
              DMSO
              MTP vs.
              DMSO
              TAM vs.
              DMSO
              DOX vs.
              DMSO
              DMSO vs.
              no light
              BRM + MTP +
              TAM vs. DMSO
              BRM + MTP +
              DOX vs. DMSO
              Grk1Grk1Gucy2eGuca1bGucy2eGucy2eGuca1bGuca1b
              Gucy2eCnga1Grk1Pde6gGrk1Grk1Gucy2eGrk1
              Cnga1Rgs9Guca1bGucy2eCnga1Guca1bRcvrnRgs9
              Guca1bPde6aCnga1Guca1aRgs9Guca1aGrk1Rcvrn
              Pde6aSlc24a1Pde6aGrk1Gngt1Rgs9RhoPde6a
              Rgs9Gngt1Rgs9RcvrnPde6aPde6aGnat1Slc24a1
              Gngt1Guca1bRcvrnGngt1Slc24a1Slc24a1Guca1aGnat1
              Guca1aRcvrnGuca1aPde6bPde6bRcvrnRgs9Guca1a
              RcvrnPde6bSlc24a1Pde6aGuca1bRhoPde6gRho
              Slc24a1Gnb1Pde6bRgs9Guca1aGnat1Slc24a1Cnga1
              Pde6bGucy2fGngt1Cnga1Gucy2fGucy2fPde6aPde6b
              RhoGnat1Gnat1RhoGnb1Cnga1Gngt1Gucy2f
              Pde6gGuca1aGnat2Gnat1RcvrnPde6gCnga1Pde6g
              Gnat1Gnat2RhoSlc24a1Pde6gGnat2Pde6bGnat2
              Gucy2fRhoPde6gGnb1Gnat1Pde6bGucy2fGngt1
              Gnb1Pde6gGucy2fGnat2RhoGngt1Gnat2Gnb1
              Gnat2Gnb1Gucy2fGnat2Gnb1Gnb1
            • Table 8 Effect of different therapies on signaling pathways associated with the altered retinal transcriptome in response to light-induced retinal degeneration.

              The transcriptome-associated pathways in light-exposed Abca4−/−Rdh8−/− mice were compared to those in Abca4−/−Rdh8−/− mice that had not been exposed to bright light. NOM P, nominal P value.

              TreatmentGene set nameNESNOM P
              BRMP53 SIGNALING PATHWAY1.750.00277
              PHOTOTRANSDUCTION−2.740
              MTPP53 SIGNALING PATHWAY1.500.01504
              PHOTOTRANSDUCTION−2.320
              TAMP53 SIGNALING PATHWAY1.510.01966
              PHOTOTRANSDUCTION−1.940.00166
              DOXAPOPTOSIS1.570.01075
              P53 SIGNALING PATHWAY1.950
              PHOTOTRANSDUCTION−2.490
              BRM, MTP, and DOXAPOPTOSIS1.400.04657
              PHOTOTRANSDUCTION−1.830.00301

            Supplementary Materials

            • www.sciencesignaling.org/cgi/content/full/9/438/ra74/DC1

              Fig. S1. Abca4−/−Rdh8−/− mice exposed to bright light exhibit pyknosis of photoreceptor cells, diminished synaptophysin in the OPL, and altered horizontal cell morphology.

              Fig. S2. Pharmacological pretreatments targeting different GPCRs preserve retinal structure in bright light–exposed Abca4−/−Rdh8−/− mice.

              Fig. S3. Variable preservation of retinal morphology by pharmacological pretreatments targeting different GPCRs in bright light–exposed Abca4−/−Rdh8−/− mice.

              Fig. S4. Pharmacological pretreatment affecting various GPCRs preserves retinal function in bright light–exposed Abca4−/−Rdh8−/− mice.

              Fig. S5. BRM or MTP pretreatment protects retinas of BALB/c mice from bright light–induced degeneration.

              Fig. S6. Transcriptome analysis of the retina.

              Fig. S7. Dose-dependent protection of retinal morphology in Abca4−/−Rdh8−/− mice by BRM, MTP, and TAM pretreatment.

              Fig. S8. Combined pretreatments improve retinal morphological protection against bright light–exposed Abca4−/−Rdh8−/− mice.

              Fig. S9. Combined treatments with subeffective doses of individual drugs exhibit improved retinal morphological protection in bright light–exposed Abca4−/−Rdh8−/− mice.

              Fig. S10. Combined pretreatments protect retinas of BALB/c mice from bright light–induced photoreceptor degeneration.

              Table S1. Retinal gene sets significantly altered in response to bright light exposure in Abca4−/−Rdh8−/− mice.

              Table S2. Retinal gene sets beneficially regulated by BRM pretreatment.

              Table S3. Retinal gene sets beneficially affected by MTP pretreatment.

              Table S4. Retinal gene sets beneficially affected by TAM pretreatment.

              Table S5. Retinal gene sets beneficially affected by DOX pretreatment.

              Table S6. Significantly altered retinal gene sets as a result of bright light exposure in Abca4−/−Rdh8−/− mice.

              Table S7. Retinal gene sets beneficially affected as a result of combined pretreatment with BRM, MTP, and TAM.

              Table S8. Retinal gene sets beneficially affected as a result of combined pretreatment with BRM, MTP, and DOX.

            • Supplementary Materials for:

              Synergistically acting agonists and antagonists of G protein–coupled receptors prevent photoreceptor cell degeneration

              Yu Chen,* Grazyna Palczewska, Ikuo Masuho, Songqi Gao, Hui Jin, Zhiqian Dong, Linn Gieser, Matthew J. Brooks, Philip D. Kiser, Timothy S. Kern, Kirill A. Martemyanov, Anand Swaroop, Krzysztof Palczewski*

              *Corresponding author. Email: chenyu6639{at}hotmail.com (Y.C.); kxp65{at}case.edu (K.P.)

              This PDF file includes:

              • Fig. S1. Abca4−/−Rdh8−/− mice exposed to bright light exhibit pyknosis of photoreceptor cells, diminished synaptophysin in the OPL, and altered horizontal cell morphology.
              • Fig. S2. Pharmacological pretreatments targeting different GPCRs preserve retinal structure in bright light–exposed Abca4−/−Rdh8−/− mice.
              • Fig. S3. Variable preservation of retinal morphology by pharmacological pretreatments targeting different GPCRs in bright light–exposed Abca4−/−Rdh8−/− mice.
              • Fig. S4. Pharmacological pretreatment affecting various GPCRs preserves retinal function in bright light–exposed Abca4−/−Rdh8−/− mice.
              • Fig. S5. BRM or MTP pretreatment protects retinas of BALB/c mice from bright light–induced degeneration.
              • Fig. S6. Transcriptome analysis of the retina.
              • Fig. S7. Dose-dependent protection of retinal morphology in Abca4−/−Rdh8−/− mice by BRM, MTP, and TAM pretreatment.
              • Fig. S8. Combined pretreatments improve retinal morphological protection against bright light–exposed Abca4−/−Rdh8−/− mice.
              • Fig. S9. Combined treatments with subeffective doses of individual drugs exhibit improved retinal morphological protection in bright light–exposed Abca4−/−Rdh8−/− mice.
              • Fig. S10. Combined pretreatments protect retinas of BALB/c mice from bright light–induced photoreceptor degeneration.
              • Table S1. Retinal gene sets significantly altered in response to bright light exposure in Abca4−/−Rdh8−/− mice.
              • Table S2. Retinal gene sets beneficially regulated by BRM pretreatment.
              • Table S3. Retinal gene sets beneficially affected by MTP pretreatment.
              • Table S4. Retinal gene sets beneficially affected by TAM pretreatment.
              • Table S5. Retinal gene sets beneficially affected by DOX pretreatment.
              • Table S6. Significantly altered retinal gene sets as a result of bright light exposure in Abca4−/−Rdh8−/− mice.
              • Table S7. Retinal gene sets beneficially affected as a result of combined pretreatment with BRM, MTP, and TAM.
              • Table S8. Retinal gene sets beneficially affected as a result of combined pretreatment with BRM, MTP, and DOX.

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              Citation: Y. Chen, G. Palczewska, I. Masuho, S. Gao, H. Jin, Z. Dong, L. Gieser, M. J. Brooks, P. D. Kiser, T. S. Kern, K. A. Martemyanov, A. Swaroop, K. Palczewski, Synergistically acting agonists and antagonists of G protein–coupled receptors prevent photoreceptor cell degeneration. Sci. Signal. 9, ra74 (2016).

              © 2016 American Association for the Advancement of Science

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