Journal: Advanced Science

Article Title: Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas‐Sting Axis in Spinal Cord Injury

doi: 10.1002/advs.202305442

Figure Lengend Snippet: Mitochondria swelling and expression of mitofusin decreased after SCI. a) Representative ultrastructure of microglia in the perilesional tissues at 7 d after SCI (Scale bar: 1 µm (a1,a2) and 0.5 µm (a3,a4)). b) Western blot analysis of the Drp1, Mfn1, and Mfn2 levels in microglia of Control and LPS. c) Western blot analysis of cGas, Sting, Drp1, and Mfn2 levels in microglia of Control and LPS, LPS+MASM7, and LPS+Mdivi‐1. d) Representative of immunofluorescence staining of M1 state (iNOS) in microglia (Scale bar: 20 µm). e) Levels of pro‐inflammatory cytokines, including IL‐1β, IFN‐β, and TNF‐α in the medium of microglia. f) Flow cytometric analysis on the expression levels of M1 ratio (F4/80/ CD86+). g) The neuron death rate measured by Annexin V and PI staining. n = 6 for each group. h) Mfn2 /Iba1 double immunostaining in the perilesional tissues 7 d after SCI (Scale bar: 50 µm). Error bars denote mean ± SEM, ns, no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: [ ] To activate Mfn2, microglia were treated with 1 μ m MASM7 (MedChemExpress, USA).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining, Double Immunostaining

Journal: Advanced Science

Article Title: Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas‐Sting Axis in Spinal Cord Injury

doi: 10.1002/advs.202305442

Figure Lengend Snippet: Mitochondrial fission‐induced mtDNA release mediates activation of Sting signaling in LPS‐induced microglial. a) Representative of immunofluorescence staining of Mfn2 in microglia in different groups (Scale bar = 20 µm). b) Western blot and quantitative analysis of Mfn2, cGas, Sting, Irf3, p‐Irf3, P65 and p‐P65 in microglia of Control, LPS, LPS + vehicle, and LPS + MASM7. c) dsDNA and HSP60 double immunostaining microglia in different groups (Scale bar: 20 µm). d) Representative MitoTracker fluorescence images illustrating mitochondrial morphology in microglia (Scale bar: 20 µm). e) Representative fluorescence staining of JC‐1 aggregates (red)/JC‐1 monomers (green) illustrating the MMP (Scale bar: 20 µm). f) Representative MitoSOX fluorescence images of mitochondria‐derived ROS (Scale bar: 20 µm). Error bars denote mean ± SEM, ns, no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: [ ] To activate Mfn2, microglia were treated with 1 μ m MASM7 (MedChemExpress, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Control, Double Immunostaining, Fluorescence, Derivative Assay

Journal: Advanced Science

Article Title: Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas‐Sting Axis in Spinal Cord Injury

doi: 10.1002/advs.202305442

Figure Lengend Snippet: Schematic process of the activation of cGas‐Sting signaling mediated by Mfn2‐dependent release of mtDNA in microglia in SCI.

Article Snippet: [ ] To activate Mfn2, microglia were treated with 1 μ m MASM7 (MedChemExpress, USA).

Techniques: Activation Assay

Journal: Nature communications

Article Title: G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion.

doi: 10.1038/ncomms1099

Figure Lengend Snippet: Figure 4 | G2 requires Mfn1 for its effect on mitochondrial dynamics. (a) Representative images of wild type or Mfn1 − / − and/or Mfn2 − / − MEF cells transfected with Flag-tagged Gβ2. Mitochondria in cells were stained with MitoTracker Red and Flag- Gβ2 were immunostained with a Flag-specific antibody. Scale bar, 10 µm. (b) Mitochondria in Mfn2 − / − cells, and Mfn1 − / − cells are shown in the representative images of Mfn1 − / − or Mfn2 − / − MEFs transfected with Gβ2 shRNA (red). The box area A (tranfected cell) and B (untransfectd cell) in the left panel show the different mitochondrial size at higher magnifications. Scale bar, 10 µm. (c) The quantification of mitochondrial size scored by Image J (Wayne Rasband, NIH). Filled box represents mitochondrial size in Mfn2 − / − MEFs and open box represents in Mfn2 − / − MEFs transfected with Gβ2 shRNA. The data represent the mean s.d., n = 10–15. Differences in the mean mitochondrial average area between mfn2 − / − MEFs and mfn2 − / − MEFs with Gβ2 shRNA were statistically significant at *P < 0.05. (d) Mfn1-YFP was co-transfected into HeLa cells with either Flag- Gβ2 (•, black) or Gβ2 shRNA (, red) and the mitochondrial Mfn1 mobility was measured by FRAP. Fis1 (, green) was used as a control of fast membrane mobility as previously reported28 and Mfn1 alone (, blue) was used as baseline control. Each line represents the mean of ≥20 measurements. (e) The fluorescence recovery of Mfn1-YFP in HeLa cells and the cells co-transfected with either Flag-Gβ2 or Gβ2 shRNA was measured by FRAP. Differences in the mean fluorescence recovery between Mfn1-YFP in HeLa cells and Mfn1-YFP in HeLa cells co-transfected with Flag-Gβ2, or with Gβ2 shRNA were statistically significant *P < 0.05 and **P < 0.005; Error bars represent the s.e.m., n = 21. (f) Mobility of Mfn1-GFP, or Mfn1-GFP (∆241–350), Mfn1-GFP (∆1–387) in HeLa cells and the cells co-transfected with DsRed-Gβ2 shRNA was measured by FRAP. The fluorescence recovery of Mfn1-GFP, Mfn1-GFP (∆241–350) and Mfn1-GFP (∆1–387) in HeLa cells and the cells co-transfected with Gβ2 shRNA was measured by FRAP. *P < 0.05 and NS means nonsignificant. Data represent the mean s.e.m., n = 20.

Article Snippet: Antibodies to Mfn1 and Mfn2 monoclonal were from Novus, the concentrations of both are 500 μg ml − 1.

Techniques: Transfection, Staining, shRNA, Control, Membrane, Fluorescence

Journal: Behavioural brain research

Article Title: Mitophagy activation by rapamycin enhances mitochondrial function and cognition in 5×FAD mice.

doi: 10.1016/j.bbr.2024.114889

Figure Lengend Snippet: Fig. 3. RAPA alleviates mitochondrial morphology and functional impairments in 5 ×FAD mice. (A) Representative images of mitochondria morphology in the hippocampal CA1 region under TEM. The red arrowheads indicate mitochondria. Scale bar = 500 nm; (B-D) Quantification of specific surface area (B), numeric density (C), and volume densities of mitochondria (D). n = 3 per group. (E) Relative expressions of proteins related to mitochondrial dynamics determined by western blotting in the cortices homogenate. (F)-(H) Relative quantification analysis of p-Drp1(ser637) (F), MFN2 (G), and OPA1 (H). Oxidative stress injury was evaluated based on the MDA level (I) and SOD activity (J) in the cortex. Energy metabolism was determined based on the ATP level (K) and respiratory chain complex I activity (L) in the cortex. (M) The mitochondrial membrane in the cortex. n = 3 per group.

Article Snippet: The membranes were blocked with QuickblockTM Western blocking buffer (cat. P0252, Beyotime Biotechnology) for 30 min, then subjected to an overnight incubation at 4 ◦C with various primary antibodies at a 1:1000 dilution: β-actin (cat. A2103, Abcam), anti-synapsin (cat. 2312 S, CST) and PSD95 (cat. GB11277, Servicebio), OPA1 (cat. 80741 S, CST) and MFN2 (cat. 11925 S, CST) and DRP1 (cat. 8570 S, CST), Phospho-DRP1(ser637) (cat. 4867 S, CST) and PINK1 (cat. 23274–1-AP, Proteintech) and Parkin (cat. ab77924, Abcam), LC3 (cat. SAB5701328, Sigma), Beclin-1 (cat. 207612, Abcam) and mTOR (cat. 2972 S, CST), and SQSTM1/p62 (cat. ab109012, Abcam).

Techniques: Functional Assay, Western Blot, Quantitative Proteomics, Activity Assay, Membrane

Journal: Behavioural brain research

Article Title: Mitophagy activation by rapamycin enhances mitochondrial function and cognition in 5×FAD mice.

doi: 10.1016/j.bbr.2024.114889

Figure Lengend Snippet: Fig. 7. 3-MA counteracts the effect of rapamycin in improving mitochondrial structure and function in 5 ×FAD mice. (A) Representative TEM images of the mitochondrial morphology in the CA1 region. Red arrowheads indicate mitochondria. Scale bar = 500 nm. (B-D) Quantification of the specific surface area (B), numeric density (C), and volume density of mitochondria (D). n = 3 per group. (E) Relative expression of proteins related to mitochondrial dynamics in the cortical homogenate as determined by western blotting. (F)-(H) Relative quantification of p-Drp1(ser637) (F), MFN2 (G), and OPA1 (H). Oxidative stress injury was determined based on MDA level (I) and SOD activity (J), and energy metabolism was determined based on ATP level (K), respiratory chain complex I activity (L), and mitochondrial membrane (M) in the cortices.

Article Snippet: The membranes were blocked with QuickblockTM Western blocking buffer (cat. P0252, Beyotime Biotechnology) for 30 min, then subjected to an overnight incubation at 4 ◦C with various primary antibodies at a 1:1000 dilution: β-actin (cat. A2103, Abcam), anti-synapsin (cat. 2312 S, CST) and PSD95 (cat. GB11277, Servicebio), OPA1 (cat. 80741 S, CST) and MFN2 (cat. 11925 S, CST) and DRP1 (cat. 8570 S, CST), Phospho-DRP1(ser637) (cat. 4867 S, CST) and PINK1 (cat. 23274–1-AP, Proteintech) and Parkin (cat. ab77924, Abcam), LC3 (cat. SAB5701328, Sigma), Beclin-1 (cat. 207612, Abcam) and mTOR (cat. 2972 S, CST), and SQSTM1/p62 (cat. ab109012, Abcam).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Activity Assay, Membrane

Journal: Frontiers in Microbiology

Article Title: Zika Virus-Induced Neuronal Apoptosis via Increased Mitochondrial Fragmentation

doi: 10.3389/fmicb.2020.598203

Figure Lengend Snippet: MFN2 protein level was reduced in ZIKV-infected cells. (A) Immunofluorescent images of ZIKV-infected NSCs and SNB-19 cells (multiplicity of infection, MOI = 2) stained for ZIKV MR766 ENV protein (red), PRVABC59 ENV protein (green), and nuclei (blue). Scale bar, 50 μm. (B) Western blot for MFN2, MFN1, OPA1, FIS1, DNM1L, TOMM20, and ENV from NSC and SNB-19 cell lysates 24 h after infection with Mock, MR766, or PRVABC59 (MOI = 2) Beta-actin (ACTB) is the loading control. (C) Quantification of MFN2 protein in the western blot in (B) (upper panel). Real-time PCR for MFN2 mRNA expression after 24 h of infection (lower panel). All values represent the mean ± the standard deviation (SD) ( n ≥ 3 replicates). (D) Fluorescence images of SNB-19 infected with either ZIKV MR766 or PRVABC59 and stained for MFN2 (green), ZIKV NS1 (white), nuclei (blue), and mitochondria (red). Scale bar, 10 μm. (E) Average immunostaining intensity of MFN2 in (D) . (F) The violin plot shows the mean branch length in (D) ( n = 12). (G) The histogram shows the percentage of the mitochondrial network in (D) ( n = 12). (H) Pearson correlation analysis shows the relationship between the percentage of mitochondrial individual and average MFN2 intensity in (D) . (I) Graphical illustration of ZIKV-induced mitochondrial fragmentation by reduced MFN2 protein resulting in mitochondrial fusion deficient. P -values were calculated by two-way ANOVA with Tukey’s multiple-comparison (C) , one-way ANOVA with Tukey’s multiple-comparison (E–G) . ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: An improved variant of GFP, mGFP, was used tagged to the open reading frame of MFN1 (RC207184L4V, Origene), MFN2 (RC202218L4V, Origene) or control (PS100071V, Origene) in a lentiviral particle plasmid.

Techniques: Infection, Staining, Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Fluorescence, Immunostaining, Comparison

Journal: Frontiers in Microbiology

Article Title: Zika Virus-Induced Neuronal Apoptosis via Increased Mitochondrial Fragmentation

doi: 10.3389/fmicb.2020.598203

Figure Lengend Snippet: ZIKV-induced mitochondrial fragmentation proceeded apoptosis. (A,B) DePsipher staining images of NSCs (A) and SNB-19 cells (B) 24 h after infection with ZIKV MR766 or PRVABC59 at various MOIs (0.5, 1, and 2). Green puncta indicate the DePsipher monomers in the mitochondria. Red puncta indicate the DePsipher aggregates in the mitochondria and normal membrane potential. Scale bar, 10 μm. (C,D) Quantification of red puncta signal intensity in (A) and (B) , respectively. (E) Cell viability, as measured by intracellular ATP assay, of MR766 infected NSCs (left) and SNB-19 (right) at various MOIs (0–5). (F) Cell viability, as measured by intracellular ATP assay, of PRVABC59 infected NSCs (left) and SNB-19 (right) at various MOIs (0–5). (G) Western blot of ZIKV ENV, CASP3, cleaved CASP3, CASP9, cleaved CASP9, CASP7, cleaved CASP7, PARP, cleaved PARP, CASP8, and cleaved CASP8 of NSCs (left) or SNB-19 cells (right) at 24- or 48-h post-infection with ZIKV MR766 or PRVABC59 (MOI = 1). (H) Fluorescence images of SNB-19 treated with 30 μM emricasan and infected with ZIKV MR766 or PRVABC59 for 24 h. Cells were then stained for MFN2 (green), ZIKV NS1 (white), nuclei (blue) and mitochondria (red). Scale bar, 10 μm. (I) Violin plot of the mean branch length in (H) ( n = 12). (J) Quantification of immunostaining intensity of MFN2 in (H) ( n = 12). (K) Pearson correlation analysis shows the relationship between the percentage of mitochondrial individual and average MFN2 intensity in (H) . (L) A time-course schematic diagram of cells after ZIKV infection. P -values were calculated by two- tailed Student’s t -test (C,D) or one-way ANOVA with Tukey’s multiple-comparison test (I,J) . ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: An improved variant of GFP, mGFP, was used tagged to the open reading frame of MFN1 (RC207184L4V, Origene), MFN2 (RC202218L4V, Origene) or control (PS100071V, Origene) in a lentiviral particle plasmid.

Techniques: Staining, Infection, Membrane, ATP Assay, Western Blot, Fluorescence, Immunostaining, Two Tailed Test, Comparison