Journal: Neural Regeneration Research

Article Title: Microglial CARD19 ameliorates post-stroke neuroinflammation by stabilizing mitochondrial cristae

doi: 10.4103/NRR.NRR-D-24-00923

Figure Lengend Snippet: Microglial CARD19 deficiency accelerates ultrastructural and functional damage to mitochondria in ischemic stroke. (A) IP-MS analysis of CARD19-interacting proteins. (B) Representative immunofluorescence images of HSP60 (green), CARD19 (red), and DAPI (blue) in primary microglia. Scale bar: 5 μm. (C) Representative transmission electron microscope image of mitochondria in microglia from AAV-control– and AAV-shCARD19–injected mice 3 days after MCAO. (D) Quantification of Class I, Class II, and Class III mitochondria in AAV-control– and AAV-shCARD19–injected mice. n = 3/group. (E) Quantification of cristae junctions/mitochondrial cristae in AAV-control– and AAV-shCARD19–injected mice. n = 6/group. (F) Relative TMRE fluorescence values in LV-NC– and LV-shCARD19–treated microglia after OGD/R. (G) Representative transmission electron microscope images of mitochondria in LV-NC– and LV-shCARD19–treated microglia after OGD/R. (H) Representative immunofluorescence images of MitoSOX in LV-NC– and LV-shCARD19–treated microglia after OGD/R. Scale bar: 100 μm. (I) Representative immunofluorescence images of mitotracker (green), dsDNA (red), and DAPI (blue) in LV-NC– and LV-shCARD19–treated microglia after OGD/R. Scale bar: 10 μm. (J, K) qPCR analysis of D-Loop/Tert (J) and ND1/Tert (K) in LV-NC– and LV-shCARD19–treated microglia after OGD/R. n = 3/group. (L, M) qPCR analysis of IFN-α (L) and IFN- β (M) in LV-NC– and LV-shCARD19–treated microglia after OGD/R. n = 3/group. (N, O) qPCR analysis of IFN-α (N) and IFN- β (O) in Vector and Flag-CARD19/group microglia after OGD/R. n = 3/group. (P, Q) Representative images of TTC staining and quantitative analysis of AAV-control– and AAV-shCARD19–injected mice 3 days after MCAO injected with PBS or DNase I. Normal tissues stained red, and infracted tissues remained white. n = 4/group. All experiments were repeated at least three times, with each using a separate brain or cell sample. Data are presented as mean ± SD. In E, one-way ANOVA followed by Bonferroni post hoc test was used. In F, J–O, Q, two-way ANOVA followed by Tukey’s post hoc test was used. * P < 0.05, *** P < 0.001. AAV: Adeno-associated virus; ANOVA: analysis of variance; CARD19: caspase activation and recruitment domain 19; DAPI: 4′,6-diamidino-2-phenylindole; D-loop: displacement loop; HSP60: heat shock protein 60; IFN-α: interferon-α; IFN-β: interferon-β; LV: lentivirus; MCAO: middle cerebral artery occlusion; MitoSOX: mitochondrial superoxide indicator; ND1: NADH dehydrogenase 1; ns: not significant; OGD/R: oxygen–glucose deprivation/reoxygenation; qPCR: quantitative polymerase chain reaction; SD: standard deviation; Tert: telomerase reverse transcriptase; TMRE: tetramethylrhodamine, ethyl ester; TTC: 2,3,5-triphenyltetrazolium chloride.

Article Snippet: The membranes were blocked with 5% nonfat milk for 1.5 hours at room temperature, then incubated with primary antibodies against CARD19 (rabbit, 1:1000, Abclonal, Wuhan, China, Cat# A13196), GAPDH (rabbit, 1:2000, Bioworld Technology, St. Louis Park, MN, USA, Cat# MB66349), β-actin (rabbit, 1:5000, Bioworld Technology, Cat# AP0060), mitochondrial inner membrane protein 19 (MIC19) (rabbit, dilution: 1:1000, Abclonal, Cat# A8584), mitochondrial inner membrane protein 60 (MIC60) (rabbit, 1:1000, Abclonal, Cat# A2751), sorting and assembly machinery component 50 (SAMM50) (rabbit, 1:1000, Abclonal, Cat# A3401), and Flag (rabbit, dilution: 1:1000, Abclonal, Cat# AE063) overnight at 4°C.

Techniques: Functional Assay, Protein-Protein interactions, Immunofluorescence, Transmission Assay, Microscopy, Control, Injection, Fluorescence, Plasmid Preparation, Staining, Virus, Activation Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Reverse Transcription

Journal: Neural Regeneration Research

Article Title: Microglial CARD19 ameliorates post-stroke neuroinflammation by stabilizing mitochondrial cristae

doi: 10.4103/NRR.NRR-D-24-00923

Figure Lengend Snippet: CARD19 interacts with MIB components and stabilizes mitochondrial cristae. (A) IP-MS results showing that CARD19 interacts with Myo18a, MIC60, SAMM50, and MIC19. (B) Representative immunoblot images showing that CARD19 interacts with MIC60, SAMM50, and MIC19 in microglia. (C) Representative immunoblot images showing that CARD19 overexpression partially rescued the binding between MIC19 and SAMM50 after OGD/R in microglia. (D) Representative immunoblot images showing that CARD19 knockdown of exacerbated the loss of binding between MIC19 and SAMM50 after OGD/R in microglia. All experiments were repeated at least three times, with each using a separate cell sample. CARD19: Caspase activation and recruitment domain 19; IP-MS: immunoprecipitation-mass spectrometry; MIB: mitochondrial intermembrane bridge; MIC19: mitochondrial inner membrane protein 19; MIC60: mitochondrial inner membrane protein 60; OGD/R: oxygen–glucose deprivation/reoxygenation; SAMM50: sorting and assembly machinery component 50.

Article Snippet: The membranes were blocked with 5% nonfat milk for 1.5 hours at room temperature, then incubated with primary antibodies against CARD19 (rabbit, 1:1000, Abclonal, Wuhan, China, Cat# A13196), GAPDH (rabbit, 1:2000, Bioworld Technology, St. Louis Park, MN, USA, Cat# MB66349), β-actin (rabbit, 1:5000, Bioworld Technology, Cat# AP0060), mitochondrial inner membrane protein 19 (MIC19) (rabbit, dilution: 1:1000, Abclonal, Cat# A8584), mitochondrial inner membrane protein 60 (MIC60) (rabbit, 1:1000, Abclonal, Cat# A2751), sorting and assembly machinery component 50 (SAMM50) (rabbit, 1:1000, Abclonal, Cat# A3401), and Flag (rabbit, dilution: 1:1000, Abclonal, Cat# AE063) overnight at 4°C.

Techniques: Protein-Protein interactions, Western Blot, Over Expression, Binding Assay, Knockdown, Activation Assay, Immunoprecipitation, Mass Spectrometry, Membrane