Figure 2.
SARM1 mediates RGC and oligodendrocyte death in a neuroinflammatory model of glaucoma.(A) Representative Iba1 immunostaining of WT and SARM1 KO retinas at 3 or 7 d after TNF-α or PBS intravitreal injection. Scale bar, 50 µm. (B) Quantification of microglial soma size at various times after intravitreal injection of PBS or TNF-α of WT or SARM1 KO animals (No inj.: intact eye without injection). Data represent the mean ± SEM; n = 4–6 for each condition; one-way ANOVA with post hoc Tukey test, F(11,56) = 44.8, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (C) Representative CC1 and DAPI immunostaining of WT and SARM1 KO optic nerves (cross-section) 7 d after TNF-α or PBS injection. Scale bar, 50 µm. (D) Quantification of mature oligodendrocytes in WT and SARM1 KO optic nerves at 7 d after PBS or TNF-α treatment. Data are displayed as a percentage of oligodendrocytes present in WT eyes injected with PBS. Data are represented as mean ± SEM; n = 4; One-way ANOVA with post hoc Tukey test, F(3,12) = 41.78, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (E) Quantification of G-ratio. The EM images used in Fig. 1, C and D, were analyzed. Data represent the mean ± SEM; n = 4–5 for each condition; one-way ANOVA with post hoc Tukey test, F(7,30) = 0.7174, P = 0.6580; NS, not significant; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (F) Top: Schematic diagram of AAV vector expressing human SARM1.DN (hSARM1.DN) under control of neuron-specific hSyn. Bottom: Representative retinal cross-section. SARM1.DN protein (green) is present in the RGC layer (triangle) and RGC axons that colabel with TUJ1 (red). Inner nuclear layer (circle), outer nuclear layer (square), cell bodies (DAPI, blue). Scale bar, 0.2 mm. (G) Top: Virus expressing WT SARM1 (AAV.SARM1.WT) or AAV.SARM1.DN is delivered via intravitreal injection into WT mice. 4 wk later, TNF-α is injected intravitreally, and oligodendrocytes are quantified 7 d later. Bottom: Representative CC1 and DAPI immunostaining of SARM1.WT or SARM1.DN-expressing optic nerve 7 d after TNF-α injection. Scale bar, 50 µm. (H) Quantification from G of the number of mature oligodendrocytes following TNF-α injection (displayed as a percentage relative to the number observed in WT nerve following PBS intravitreal injection). Data are represented as mean ± SEM, n = 4; two-tailed unpaired t test, SARM1.WT versus SARM1.DN, P = 0.0007; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (I) Immunoblot for pMLKL from optic nerve extracts derived from WT or SARM1 KO animals 3 d after intravitreal injection of TNF-α or PBS or uninjected control (No_inj). TUJ1 was used as a loading control. (J) Quantification of pMLKL expression from immunoblots. TUJ1 signal was used for normalization. Data represent the mean ± SEM, n = 4; two-tailed unpaired t test, (WT) PBS_3d versus TNF_3d, P = 0.03; (SARM1KO) PBS_3d versus TNF_3d, P = 0.02; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (K) Immunoblot for MLKL, RIPK3, and phosphorylated RIPK3 (pRIPK3) from optic nerve extracts derived from WT or SARM1 KO animals 3 d after intravitreal injection of TNF-α or PBS. TUJ1 was used as a loading control. (L) Quantification of the intensity of MLKL (n = 4), RIPK3 (n = 3), and pRIP3 (n = 4) from immunoblots. TUJ1 signal was used for normalization. Data represent the mean ± SEM; two-tailed unpaired t test; (MLKL, WT) PBS_3d versus TNF_3d, P = 0.014; (MLKL, SARM1 KO) PBS_3d versus TNF_3d, P = 0.005; (RIPK3, WT) PBS_3d versus TNF_3d, P = 0.028; (RIPK3, SARM1 KO) PBS_3d versus TNF_3d, P = 0.018; (pRIPK3, WT) PBS_3d versus TNF_3d, P = 1.9E-06; (pRIPK3, SARM1 KO) PBS_3d versus TNF_3d, P = 0.03; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

SARM1 mediates RGC and oligodendrocyte death in a neuroinflammatory model of glaucoma. (A) Representative Iba1 immunostaining of WT and SARM1 KO retinas at 3 or 7 d after TNF-α or PBS intravitreal injection. Scale bar, 50 µm. (B) Quantification of microglial soma size at various times after intravitreal injection of PBS or TNF-α of WT or SARM1 KO animals (No inj.: intact eye without injection). Data represent the mean ± SEM; n = 4–6 for each condition; one-way ANOVA with post hoc Tukey test, F(11,56) = 44.8, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (C) Representative CC1 and DAPI immunostaining of WT and SARM1 KO optic nerves (cross-section) 7 d after TNF-α or PBS injection. Scale bar, 50 µm. (D) Quantification of mature oligodendrocytes in WT and SARM1 KO optic nerves at 7 d after PBS or TNF-α treatment. Data are displayed as a percentage of oligodendrocytes present in WT eyes injected with PBS. Data are represented as mean ± SEM; n = 4; One-way ANOVA with post hoc Tukey test, F(3,12) = 41.78, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (E) Quantification of G-ratio. The EM images used in Fig. 1, C and D, were analyzed. Data represent the mean ± SEM; n = 4–5 for each condition; one-way ANOVA with post hoc Tukey test, F(7,30) = 0.7174, P = 0.6580; NS, not significant; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (F) Top: Schematic diagram of AAV vector expressing human SARM1.DN (hSARM1.DN) under control of neuron-specific hSyn. Bottom: Representative retinal cross-section. SARM1.DN protein (green) is present in the RGC layer (triangle) and RGC axons that colabel with TUJ1 (red). Inner nuclear layer (circle), outer nuclear layer (square), cell bodies (DAPI, blue). Scale bar, 0.2 mm. (G) Top: Virus expressing WT SARM1 (AAV.SARM1.WT) or AAV.SARM1.DN is delivered via intravitreal injection into WT mice. 4 wk later, TNF-α is injected intravitreally, and oligodendrocytes are quantified 7 d later. Bottom: Representative CC1 and DAPI immunostaining of SARM1.WT or SARM1.DN-expressing optic nerve 7 d after TNF-α injection. Scale bar, 50 µm. (H) Quantification from G of the number of mature oligodendrocytes following TNF-α injection (displayed as a percentage relative to the number observed in WT nerve following PBS intravitreal injection). Data are represented as mean ± SEM, n = 4; two-tailed unpaired t test, SARM1.WT versus SARM1.DN, P = 0.0007; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (I) Immunoblot for pMLKL from optic nerve extracts derived from WT or SARM1 KO animals 3 d after intravitreal injection of TNF-α or PBS or uninjected control (No_inj). TUJ1 was used as a loading control. (J) Quantification of pMLKL expression from immunoblots. TUJ1 signal was used for normalization. Data represent the mean ± SEM, n = 4; two-tailed unpaired t test, (WT) PBS_3d versus TNF_3d, P = 0.03; (SARM1KO) PBS_3d versus TNF_3d, P = 0.02; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (K) Immunoblot for MLKL, RIPK3, and phosphorylated RIPK3 (pRIPK3) from optic nerve extracts derived from WT or SARM1 KO animals 3 d after intravitreal injection of TNF-α or PBS. TUJ1 was used as a loading control. (L) Quantification of the intensity of MLKL (n = 4), RIPK3 (n = 3), and pRIP3 (n = 4) from immunoblots. TUJ1 signal was used for normalization. Data represent the mean ± SEM; two-tailed unpaired t test; (MLKL, WT) PBS_3d versus TNF_3d, P = 0.014; (MLKL, SARM1 KO) PBS_3d versus TNF_3d, P = 0.005; (RIPK3, WT) PBS_3d versus TNF_3d, P = 0.028; (RIPK3, SARM1 KO) PBS_3d versus TNF_3d, P = 0.018; (pRIPK3, WT) PBS_3d versus TNF_3d, P = 1.9E-06; (pRIPK3, SARM1 KO) PBS_3d versus TNF_3d, P = 0.03; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

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