Fig. 4
From: Targeting SARM1 as a novel neuroprotective therapy in neurotropic viral infections
Neurotropic virus infections induce mitochondrial localization of SARM1 and mitochondrial damage. (A) Immunofluorescence of cells labeled with SARM1 (green) and TOM20 (red) antibodies. Neuro-2a cells were mock-infected or infected with JEV, HSV-1 or RABV (MOI = 1) and fixed at 36 h post-infection. As a positive control, cells were stimulated with FCCP (50 µM) for 45 min, and fixed. Nucleic acids were stained with DAPI (blue). White boxes indicate zoomed areas. Colocalization quantifications were done using the Plot Profile plugin in ImageJ. (B) Immunoblot analysis of SARM1 in the cytosol or mitochondrial fraction of cortical neurons. The cortical neurons were mock-, or JEV-, or HSV-1- or RABV-infected for 36 h. Cox IV and GAPDH were used as loading controls. (C) Neuro-2a cells were mock, or infected with JEV (MOI = 1) for 36 h and either treated with vehicle or 10µM SIC4. FCCP was used as the positive control. Cells were stained with TMRM and MitoTracker Green and analyzed using CytoFLEX S. Flow cytometry plots are representative of three independent experiments. (D) Bar chart is mean ± SEM (n = 3) showing percentage of cells relative to JEV infection vehicle treatment that are MitoTracker Green positive and TMRM negative. (E) The concentration of Neuro-2a cytosolic Ca2+ was determined by measuring fluo-8 fluorescence using flow cytometry (loaded with 1 mg/ml fluo-8-AM for 30 min) during JEV infection for 36 h either treated with vehicle or 10 µM SIC4. FCCP as the Ca2+ mobilization positive control. (F) Relative quantification of the mean fluorescence intensity of fluo-8 for the experiments in panel E. (G) Electron microscope images of cortical nerves from 8-week-old WT and Sarm1KO mice showing representative axonal mitochondria. Morphological changes in the mitochondria associated with JEV infection. TEM images were acquired at 5 dpi from mock- and JEV infected WT and Sarm1KO mice. Data are representative of 5–6 fields for each group. (H) Quantification of axons displaying abnormal mitochondria at 5 dpi from mock- and JEV-infected WT and Sarm1KO mice. (I) Quantification of cristae density of mitochondria in axons. **P < 0.01, ***P < 0.001, ns, no significant difference