Research ArticleDRUG TOXICITY

Inflammation, necrosis, and the kinase RIP3 are key mediators of AAG-dependent alkylation-induced retinal degeneration

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Science Signaling  12 Feb 2019:
Vol. 12, Issue 568, eaau9216
DOI: 10.1126/scisignal.aau9216
  • Fig. 1 Alkylation damage and the BER pathway.

    AP, apurinic/apyrimidinic site; OH, 3′OH terminus; XRCC1, x-ray repair cross-complementing protein 1; LigIII, ligase III; ATP, adenosine triphosphate; AIF, apoptosis-inducing factor.

  • Fig. 2 MMS induces AAG- and sex-dependent PR degeneration, vacuolated RPE cells, and subretinal cell infiltrates as early as 3 days after treatment.

    (A) Representative hematoxylin and eosin (H&E)–stained images of retinas from WT and Aag−/− mice either untreated (CTR) or at 3, 5, and/or 7 days after injection with MMS (MMS D3, D5, and D7; 75 mg/kg). Scale bars, 50 μm (left, 200×) and 20 μm (right, 400×). INL, inner nuclear layer; GCL, ganglion cell layer; white arrowheads, cellular infiltrates; green asterisks, vacuolated and swollen RPE cells. (B) Quantification of rows of PR nuclei in the ONL of WT and Aag−/− mice at 3, 5, and/or 7 days after MMS treatment (75 mg/kg). (C) Number of subretinal cell infiltrates per section in WT and Aag−/− mice at 3 and 5 days after MMS (75 mg/kg). All data are means ± SEM; numbers of animals are indicated atop the bars (♂, males; ♀, females); *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 by two-way analysis of variance (ANOVA) followed by Tukey’s test.

  • Fig. 3 MMS-induced PR cell death is associated with necrotic morphology.

    (A) Electron microscopic photomicrographs of the ONL of WT male and female mice either untreated (CTR) or at day 3 after MMS (MMS D3; 75 mg/kg) injection; A, apoptotic nuclei; N, necroptotic nuclei. Scale bar, 2 μm. (B) Quantification of apoptotic and necrotic PR cell nuclei in the ONL of WT male and female mice at day 3 after MMS. (C) Electron microscopic photomicrographs of IS/OS of WT male mice either untreated or at day 3 after MMS injection. Scale bars, 2 μm (top) and 800 nm (bottom). (D and E) Representative images of in vivo PI staining and quantification of PI-positive PRs in WT male and female mice and Aag−/− male mice either untreated or on day 3 after MMS treatment (75 mg/kg). Scale bar, 50 μm. All data are means ± SEM; numbers of animals are indicated atop the bars; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 by two-way ANOVA followed by Tukey’s test. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 4 MMS induces overexpression of Rip1 and Rip3 in the neuroretina of WT male mice.

    Quantitative real-time PCR analysis for Rip1 and Rip3 expression in the neuroretina of WT male, WT female, and Aag−/− male mice at day 3 after MMS treatment (MMS D3; 75 mg/kg). Data are means ± SEM fold change (Δ) relative to untreated controls (CTR); n = 3 to 4 mice; **P ≤ 0.01 and ***P ≤ 0.001 by unpaired t tests.

  • Fig. 5 MMS induces overproduction of ROS in PRs.

    Representative immunofluorescence staining of 8-oxoG, an oxidative stress marker, in WT and Aag−/− male retina sections either untreated (CTR) or at day 3 after MMS treatment (MMS D3; 75 mg/kg). Scale, bar 50 μm. Data are representative of n = 4 mice. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 6 MMS induces PARP activity in PR cells and the release of HMGB1 from PR nuclei.

    Representative immunofluorescence images of PAR and/or HMGB1 on retinal sections from WT and Aag−/− male mice at day 3 after MMS treatment (MMS D3; 75 mg/kg). Scale bar, 50 μm. Data are representative of n = 3 mice.

  • Fig. 7 MMS induces reactive gliosis and macrophage infiltration into the outer retina.

    (A) Representative whole-mount immunofluorescence for IBA1 (microglial/macrophage marker) on WT and Aag−/− male retinas at day 5 after MMS treatment (MMS D5; 75 mg/kg). OPL, outer plexiform layer. Scale bar, 50 μm. (B and C) Representative immunofluorescence staining for GFAP (activated Müller glial marker; B) and for F4/80 (macrophage marker; C) on retinal sections from WT male and female and Aag−/− male mice at day 3 after MMS treatment (MMS D3; 75 mg/kg). White arrowheads, macrophages. Scale bars, 50 μm. Data are representative of n = 3 mice.

  • Fig. 8 MMS induces overexpression of inflammatory cytokines and chemokines in the neuroretina of WT male mice.

    Quantitative real-time PCR analysis for proinflammatory markers Tnf-α, Mcp-1, and Il1β and anti-inflammatory marker Il10 in the neuroretinas of WT male, WT female, and Aag−/− male mice at day 3 after MMS treatment (MMS D3; 75 mg/kg). Data are means ± SEM fold change (Δ) relative to untreated controls (CTR); n = 3 to 5 mice; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 by unpaired t tests.

  • Fig. 9 RIP3 deficiency protects against MMS-induced RD and subretinal cell infiltration.

    (A) Representative H&E-stained images of retinas from Rip3−/− and WT male and female mice either untreated (CTR) or at 3 and 7 days after MMS treatment (MMS D3 and D7; 75 mg/kg). Scale bar, 20 μm. (B and C) Number of rows of PR nuclei (B) in the ONL and number of subretinal cell infiltrates per section (C) in retinas from mice described in (A). Data are means ± SEM; numbers of animals (n) are indicated below the graphs; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 by two-way ANOVA followed by Tukey’s test.

  • Fig. 10 Deficiency of IL-10 increases AAG-dependent, MMS-induced RD in both male and female mice.

    (A and B) Representative H&E-stained images (A) and number of PR nuclei (B) in retinas from male and female WT, Il10−/−, and Aag−/−/Il10−/− mice either untreated (CTR) or at day 7 after MMS treatment (MMS D7; 75 mg/kg). Scale bars, 50 μm. Data are means ± SEM; numbers of animals (n) are indicated below the graphs; *P ≤ 0.05 and ***P ≤ 0.001 by two-way ANOVA followed by Tukey’s test.

  • Fig. 11 Treatment with Q-VD-Oph, a pan-caspase inhibitor, protects WT female, but not male, mice from alkylation-induced RD.

    (A and B) Representative H&E-stained images (A) and number of rows of PR nuclei (B) in the ONL of retinas from male and female WT mice either untreated (CTR) or at day 7 after MMS (MMS D7; 75 mg/kg) and/or Q-VD-Oph (10 mg/kg) treatment, as indicated. Scale bar, 50 μm. Data are means ± SEM; numbers of animals (n) are indicated below the graphs; ***P ≤ 0.001 by one-way ANOVA followed by Tukey’s test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/568/eaau9216/DC1

    Fig. S1. MMS treatment results in abnormal RPE structure and topography but not RPE cell loss.

    Fig. S2. MMS induces overexpression of RIP3 and activation of MLKL in the retina of WT male mice.

    Fig. S3. MMS treatment induces PARP activation in the PR cells.

    Fig. S4. Rip3−/− mice show reduced necrosis and no overexpression of proinflammatory markers after MMS treatment.

    Fig. S5. Treatment with Q-VD-Oph, a pan-caspase inhibitor, does not rescue Rip3−/− male mice from alkylation-induced RD.

  • This PDF file includes:

    • Fig. S1. MMS treatment results in abnormal RPE structure and topography but not RPE cell loss.
    • Fig. S2. MMS induces overexpression of RIP3 and activation of MLKL in the retina of WT male mice.
    • Fig. S3. MMS treatment induces PARP activation in the PR cells.
    • Fig. S4. Rip3−/− mice show reduced necrosis and no overexpression of proinflammatory markers after MMS treatment.
    • Fig. S5. Treatment with Q-VD-Oph, a pan-caspase inhibitor, does not rescue Rip3−/− male mice from alkylation-induced RD.

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