Journal: Stroke

Article Title: MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic Flux

doi: 10.1161/STROKEAHA.125.052689

Figure Lengend Snippet: MFN2 (mitofusin-2) knockout or knockdown aggravates cerebral ischemia-reperfusion injury. A , To generate mice with inducible deletion of MFN2 in forebrain neurons, we crossed MFN2 f/f mice with Camk2a-cre mice under the control of the neuron-specific Camk2a promoter, and obtained MFN2 f/f Camk2a-cre (MFN2-cKO) mice and MFN2 f/f mice as controls. Polymerase chain reaction (PCR) analysis on DNA samples was used to confirm the deletion of MFN2 exons in the brains of MFN2-cKO mice. Primer pairs 1 and 2 generated an expected PCR product size of 205 bp represented as wildtype (WT) in MFN2 f/f mice and 574 bp in MFN2-cKO mice, respectively. B , Western blot analysis of MFN2 protein in protein lysates of brain cortex samples obtained from control and MFN2-cKO mice (n=3). β-actin was used as a loading control. C , Male control and MFN2-cKO mice were subjected to 30 minute middle cerebral artery occlusion (n=7). After 24 hour reperfusion, neurological deficits and motor-coordination performance were evaluated using a 48-point deficit score and rotarod tests. Moreover, brain infarcts were stained by 2,3,5-triphenyltetrazolium chloride, and infarct volume was determined. D , Western blot analysis of SY5Y cells transfected with control siRNA (siCTRL) or 3 different siRNAs against MFN2 (siMFN2-1/2/3). GAPDH was used as a loading control. E , Live imaging of apoptotic and necrotic cell death using the RealTime-Glo Annexin-V Apoptosis and Necrosis Assay in SY5Y cells. Cells were transfected with siCTRL or siMFN2-3 and exposed to 24 hours of oxygen-glucose deprivation followed by subsequent reoxygenation. Data are mean±SD values. ns denotes no significant difference between the groups. * P <0.05, ** P <0.01, and *** P <0.001 compared with control. OGD/R indicates oxygen-glucose deprivation/reoxygenation.

Article Snippet: Conditional MFN2 knockout (MFN2 f/f , C57BL/6 background) mice were provided by Cyagen Biosciences (Suzhou, China).

Techniques: Knock-Out, Knockdown, Control, Polymerase Chain Reaction, Generated, Western Blot, Staining, Transfection, Imaging

Journal: Stroke

Article Title: MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic Flux

doi: 10.1161/STROKEAHA.125.052689

Figure Lengend Snippet: MFN2 (mitofusin-2) overexpression attenuates cerebral ischemia/reperfusion injury. A , Neurological deficits and motor-coordination performance of male mice receiving the AAV9-GFP (adeno-associated virus 9 - green fluorescent protein) control or AAV9-MFN2 vector, which were subjected to 30 minute middle cerebral artery occlusion and evaluated using a 48-point deficit score and rotarod tests 24 hours after reperfusion (n=7). Brain sections were stained with 2,3,5-triphenyltetrazolium chloride, and infarct volume was determined (n=7). B , Western blot analysis of MFN2 protein in protein lysates obtained from SY5Y cells transfected with pcDNA3.1 or MFN2 plasmid. After transfection, the cells were exposed to either normoxia (NX) or 24 hour oxygen-glucose deprivation (OGD) followed by 2 hour reoxygenation. GAPDH was used as a loading control. C , Live imaging of apoptotic and necrotic cell death in SY5Y cells using the RealTime-Glo Annexin-V Apoptosis and Necrosis Assay. Cells were transfected with either pcDNA3.1 (control) or MFN2 plasmid, followed by 24 hours of OGD and subsequent reoxygenation. Data are mean±SD values. ns denotes no significant difference between the groups. * P <0.05, ** P <0.01, and *** P <0.001 compared with control. OGD/R indicates oxygen-glucose deprivation/reoxygenation.

Article Snippet: Conditional MFN2 knockout (MFN2 f/f , C57BL/6 background) mice were provided by Cyagen Biosciences (Suzhou, China).

Techniques: Over Expression, Virus, Control, Plasmid Preparation, Staining, Western Blot, Transfection, Imaging

Journal: Stroke

Article Title: MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic Flux

doi: 10.1161/STROKEAHA.125.052689

Figure Lengend Snippet: MFN2 (mitofusin-2) reduces mitochondrial reactive oxygen species (ROS) formation under cerebral ischemia/reperfusion conditions. A , In mice exposed to 30 minute middle cerebral artery occlusion (MCAO), brain tissue samples were harvested from the ischemic cortex, which were digested by trypsin and stained with dichlorodihydrofluorescein diacetate (DCFH-DA). Deacetylated DCFH in cells is oxidized by ROS to fluorescent dichlorofluorescein (DCF), which was measured by a microplate reader. B and C , ROS levels in the previously ischemic cortex of male control and MFN2 f/f Camk2a-cre (MFN2-cKO) mice (in B ; n=3) and AAV9-GFP (adeno-associated virus 9 - green fluorescent protein) control and AAV9-MFN2 mice (in C ; n=3) 24 hours after MCAO. D and E , Mitochondrial oxidative stress assessed using the mitochondria-selective ROS detection agent MitoSOX, a green fluorescent indicator. SY5Y cells were transfected with either control siRNA (siCTRL) or MFN2 siRNA (siMFN2; in D ) or transfected with either control plasmid pcDNA3.1 or MFN2 plasmid (in E ). Cells were exposed to 24 hour oxygen-glucose deprivation (OGD) followed by 2 hour reoxygenation. Scale bar=25 µm. Data are mean±SD values. * P <0.05, *** P <0.001, and **** P <0.0001 compared with control. OGD/R indicates oxygen-glucose deprivation/reoxygenation.

Article Snippet: Conditional MFN2 knockout (MFN2 f/f , C57BL/6 background) mice were provided by Cyagen Biosciences (Suzhou, China).

Techniques: Staining, Control, Virus, Transfection, Plasmid Preparation

Journal: Stroke

Article Title: MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic Flux

doi: 10.1161/STROKEAHA.125.052689

Figure Lengend Snippet: MFN2 (mitofusin-2) is an important modulator of autophagy. A , Transcriptomic analysis was performed in the ischemic cortex of male MFN2 f/f control and MFN2 f/f Camk2a-cre (MFN2-cKO) mice, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (significance level 0.05; n=3). B , Representative transmission electron microscopy images of autophagosomes in the cytoplasm of neurons in the cerebral cortex of ischemic male MFN2 f/f control and MFN2-cKO mice (n=3). Scale bar=20 µm (upper photographs in B ) or 10 µm (lower photographs in B ). C , Western blot analysis of MFN2, BAG6 (BCL2-associated athanogene 6), LC3 (microtubule-associated protein light chain 3), and SQSTM1 / P62 (sequestosome 1) proteins in protein lysates obtained from the cerebral cortex of male control and MFN2-cKO mice, which were exposed to middle cerebral artery occlusion followed by 24 hour reperfusion (n=3). GAPDH was used as a loading control. D , Western blot analysis of MFN2, P62, and LC3 proteins in protein lysates from SY5Y cells, which were transfected with either control siRNA (siCTRL) or MFN2 siRNA (siMFN2) and then exposed to normoxia (NX) or 24 hour oxygen-glucose deprivation (OGD) followed by 2 hour reoxygenation. GAPDH was used as a loading control. E , Autophagy flux analysis in SY5Y cells co-transfected with either LC3-GA120 or LC3-wildtype plasmid and siRNA (siCTRL or siMFN2) or co-transfected with either LC3-GA120 or LC3-wildtype plasmid and pcDNA3.1 or MFN2 plasmid (MFN2). Cells were exposed to NX, 24 h OGD, or 24 hour OGD followed by 2 hour reoxygenation. F , Western blot analysis of DRP1 (dynamin-related protein 1) and p-DRP1 (Ser616) from brain lysates (upper panel) as described in ( C ), with GAPDH used as the loading control. The lower panel shows Western blot analysis of MFN2, DRP1, and p-DRP1 (Ser616) in SY5Y cells treated as described in ( D ), with β-actin used as a loading control. Data are mean±SD values. ns denotes no significant difference between the groups. * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001 compared with control. ECM indicates extracellular matrix; OGD/R, oxygen-glucose deprivation/reoxygenation and TNF, tumor necrosis factor.

Article Snippet: Conditional MFN2 knockout (MFN2 f/f , C57BL/6 background) mice were provided by Cyagen Biosciences (Suzhou, China).

Techniques: Control, Transmission Assay, Electron Microscopy, Western Blot, Transfection, Plasmid Preparation

Journal: Stroke

Article Title: MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic Flux

doi: 10.1161/STROKEAHA.125.052689

Figure Lengend Snippet: MFN2 (mitofusin-2) interacts with BAG6 (BCL2-associated athanogene6) in a TM1 (transmembrane domain 1) domain-dependent way. A , Protein-protein interaction network of autophagy-related proteins in the result of MFN2’s mass spectrometry. B , Immunoprecipitation analysis of MFN2–BAG6 interactions. Human embryonic kidney 293T (HEK293T) cells were transfected with HA-MFN2 (hemagglutinin tagged-MFN2) along with control or DYKDDDDK tagged-BAG6 (FLAG-BAG6), and DYKDDDDK tag (FLAG) pulldown was performed with anti-FLAG beads. Samples were analyzed by immunoblotting analysis. C , Immunofluorescence analysis of the interaction between HA-MFN2 and FLAG-BAG6 in HEK293T cells transfected with HA-MFN2 and FLAG-BAG6. Twenty-four hours after transfection, immunofluorescence analysis was performed. Scale bar=20 µm. D , A series of truncated MFN2 variants was generated based on existing domain information and transfected into HEK293T cells. E , Immunoprecipitation analysis revealed that the TM1 domain of MFN2 is critical for MFN2’s interaction with BAG6.

Article Snippet: Conditional MFN2 knockout (MFN2 f/f , C57BL/6 background) mice were provided by Cyagen Biosciences (Suzhou, China).

Techniques: Mass Spectrometry, Immunoprecipitation, Transfection, Control, Western Blot, Immunofluorescence, Generated

Journal: Stroke

Article Title: MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic Flux

doi: 10.1161/STROKEAHA.125.052689

Figure Lengend Snippet: BAG6 (BCL2-associated athanogene 6 ) overexpression attenuates cerebral ischemia/reperfusion (I/R) injury and potentiates cytoprotective effects of MFN2 (mitofusin-2) post-I/R. A , Neurological deficits and motor-coordination performance of male mice receiving the adeno-associated virus 9-control (AAV9-CON) or adeno-associated virus 9-BAG6 (AAV9-BAG6) vector, which were subjected to 30 minute middle cerebral artery occlusion (MCAO) and evaluated using a 48-point deficit score and rotarod tests 24 hours after reperfusion (n=8). Brain sections collected from mice were stained with 2,3,5-triphenyltetrazolium chloride (TTC), and infarct volume was determined (n=8). Furthermore, reactive oxygen species (ROS) levels in the ischemic cortex of AAV9-CON and AAV9-BAG6 mice were assessed by dichlorodihydrofluorescein diacetate (DCFH-DA) staining 24 hours after MCAO (n=3–4). B , Live imaging of apoptotic and necrotic cell death using the RealTime-Glo Annexin-V Apoptosis and Necrosis Assay in SY5Y cells. Cells were transfected with either pcDNA3.1 (control) or FLAG-BAG6 plasmid, followed by 24 hour oxygen-glucose deprivation (OGD) and subsequent reoxygenation. C , The upper lane is a Western blot analysis of BAG6 overexpression in SY5Y cells treated as described in ( B ) after oxygen-glucose deprivation/reoxygenation (OGD/R; 30 minutes), in which β-actin was used as a loading control. And the lower lane is autophagy flux analysis in SY5Y cells co-transfected with either LC3-GA120 or LC3-wildtype (LC3-WT) plasmid and pcDNA3.1 (control) or FLAG-BAG6 plasmid (BAG6). Cells were exposed to 24 hours of OGD followed by 2 hour reoxygenation. D , SY5Y cells were transfected with either pcDNA3.1 (control) or FLAG-BAG6 plasmid (BAG6), followed by 24 hour OGD and subsequent 2 hour reoxygenation. Mitochondrial membrane potential assessed by tetramethylrhodamine methyl ester intensity (TMRM; red). Cellular ROS levels were assessed using the fluorescent probe CellROX Green, a nonfluorescent dye that becomes fluorescent upon oxidation, which was subsequently quantified. Representative images are shown, and samples were analyzed by ImageJ software. Scale bar=100 µm. E , Male mice receiving the AAV9-MFN2 (control) or AAV9-MFN2+AAV9-BAG6 were subjected to 30 minute MCAO (n=6). After 24 hour reperfusion, neurological deficits and motor-coordination performance were evaluated using a 48-point deficit score and rotarod tests. Brain infarcts were stained by TTC, and infarct volume was determined (n=6). ROS levels in the ischemic cortex assessed by DCFH-DA staining 24 hours after MCAO (n=3). Data are mean±SD values. ns denotes no significant difference between the groups. * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001 compared with control.

Article Snippet: Conditional MFN2 knockout (MFN2 f/f , C57BL/6 background) mice were provided by Cyagen Biosciences (Suzhou, China).

Techniques: Over Expression, Virus, Control, Plasmid Preparation, Staining, Imaging, Transfection, Western Blot, Membrane, Software

Journal: Stroke

Article Title: MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic Flux

doi: 10.1161/STROKEAHA.125.052689

Figure Lengend Snippet: BAG6 (BCL2-associated athanogene 6) functions as an autophagic suppressor, exhibits antioxidant effects, and compensates for MFN2 (mitofusin-2)–mediated anti-apoptotic protection in its absence. A , Live imaging of apoptotic and necrotic cell death using the RealTime-Glo Annexin-V Apoptosis and Necrosis Assay in SY5Y cells. Cells were transfected with either control siRNA (siCTRL) or BAG6 siRNA (siBAG6) and exposed to 24 hours of oxygen-glucose deprivation (OGD) followed by subsequent reoxygenation. B , The upper lane is Western blot analysis in SY5Y cells confirming BAG6 knockdown in normoxia (NX) and OGD followed by 2 hours of reoxygenation (oxygen-glucose deprivation/reoxygenation [OGD/R] 2 hour). And the lower lane is autophagy flux analysis in SY5Y cells co-transfected with either LC3-GA120 or LC3-WT plasmid and either control siRNA (siCTRL) or BAG siRNA (siBAG6). Cells were exposed to 24 hour OGD followed by 2 hour reoxygenation (OGD/R 2h). C , SY5Y cells were treated as described in ( A ). Mitochondrial membrane potential was assessed by tetramethylrhodamine methyl ester intensity (TMRM; red) 2 hours after reoxygenation. Cellular reactive oxygen species (ROS) levels were assessed using the fluorescent dye CellROX Green 2 hours after reoxygenation. Representative images are shown, and samples were analyzed by ImageJ software. Scale bar=100 µm. D , Live imaging of apoptotic and necrotic cell death using the RealTime-Glo Annexin-V Apoptosis and Necrosis Assay in SY5Y cells transfected with a combination of control siRNA with pcDNA3.1 (CTRL), MFN2 siRNA with pcDNA3.1 (KD MFN2), and MFN2 siRNA with FLAG-BAG6 plasmid (KD MFN2+BAG6). Cells were exposed to 24 hour OGD followed by subsequent reoxygenation. E , The left is Western blot analysis of MFN2 and BAG6 proteins from total lysates in SY5Y treated as in ( D ), 2 h after reoxygenation, in which β-actin was used as a loading control. The right is autophagy flux analysis in SY5Y cells co-transfected with either LC3-GA120 or LC3-WT plasmid and the combination of control siRNA with pcDNA3.1 (CTRL), MFN2 siRNA with pcDNA3.1 (KD MFN2), and MFN2 siRNA with FLAG-BAG6 plasmid (KD MFN2+BAG6). Cells were exposed to 24 hour OGD followed by 2 hour reoxygenation (OGD/R 2 h). Data are mean±SD values. ns denotes no significant difference between the groups. * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001 compared with control.

Article Snippet: Conditional MFN2 knockout (MFN2 f/f , C57BL/6 background) mice were provided by Cyagen Biosciences (Suzhou, China).

Techniques: Imaging, Transfection, Control, Western Blot, Knockdown, Plasmid Preparation, Membrane, Software