Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT‐3 regulates mitochondrial function and morphology via K711 acetylation of DRP1. A) Co‐immunoprecipitation assay showing the interaction between DRP1 and SIRT3 in HeLa cells. B) Bimolecular fluorescence complementation (BiFC) assay confirmed the interaction between DRP1 and SIRT3 in HeLa cells. Fusion constructs of DRP1‐HA and SIRT3‐FLAG were co‐expressed, and green fluorescence indicates protein interaction. Scale bar: 20 µm. C) The schematic diagram of the FLIM – FRET experiment principle for CFP – DRP1 and YFP – SIRT3. Representative images showing the lifetime distribution. D)Time‐correlated single‐photon counting‐fluorescence lifetime imaging microscopy (TCSPC FLIM) with Förster resonance energy transfer (FRET) further confirmed the interaction between DRP1 and SIRT3. Comparison of the decay data and fitting curves of cells expressing CFP alone and those co‐expressing CFP and EYFP confirmed FRET, indicating the interaction between DRP1 and SIRT3. Scale bar: 0.03 mm. E) Co‐immunoprecipitation experiments using shSIRT3 or SIRT3‐overexpressing cell lines co‐transfected with DRP1‐Flag revealed the regulatory role of SIRT3 in DRP1 acetylation. F–H) Western blotting analysis showing the impact of SIRT3 overexpression or knockdown on the acetylation levels of DRP1 at K711 in HeLa cells under the condition of H₂O₂‐induced oxidative stress. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. I) After being co‐transfected with the SIRT3 and K711Q plasmids, the CCK‐8 assay was conducted to show the protective effects of SIRT3 against H₂O₂‐induced cell death in HeLa cells. Data are represented as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. J–M) After being co‐transfected with the SIRT3 and K711Q plasmids and subsequently treated with H₂O₂, Western blotting analysis was performed to detect the expression levels of cleaved caspase‐3, Bax, and Bcl‐2 in HeLa cells, which confirmed the protective effects of SIRT3 against H₂O₂‐induced apoptosis in HeLa cells. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. N,O) Immunofluorescence staining with Tom20 antibody revealed the impact of SIRT3 on mitochondrial morphology in HeLa cells. Quantification of mitochondrial AR and FF is shown. Scale bar: 10 µm. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. Statistical analysis results are presented in Table (Supporting Information).

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Co-Immunoprecipitation Assay, Bimolecular Fluorescence Complementation Assay, Construct, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer, Comparison, Expressing, Immunoprecipitation, Transfection, Western Blot, Over Expression, Knockdown, CCK-8 Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT3 protects against MPP + ‐induced apoptosis by regulating DRP1 K711 acetylation. A–D) Western blotting analysis of the effects of SIRT3 overexpression on acetylated lysine, DRP1 K711 , and SIRT3 levels in SH‐SY5Y cells after MPP + treatment. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus indicated group; ns , not significant. E–G) Mitochondrial morphology in SH‐SY5Y cells after MPP + treatment was assessed via MitoTracker staining. Quantification of mitochondrial AR and FF is shown. Scale bars: 10 µm; 5 µm (zoomed). Data are represented as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 versus indicated group; ns , not significant. H,I) Mitochondrial membrane potential (ΔΨm) in SH‐SY5Y cells after MPP+ treatment evaluated via TMRE staining. Scale bar: 10 µm. Data are represented as the mean ± SD (n = 3). **P < 0.01 and ***P < 0.001 versus indicated group. J,K) ROS generation in MPP+ treated SH‐SY5Y cells assessed via CellROX Deep Red staining. Scale bar: 80 µm. Data are represented as the mean ± SD (n = 3). ***P < 0.001 and ****P < 0.0001 versus indicated group. L) TUNEL analysis of MPP+ treated SH‐SY5Y cell apoptosis. Scale bar: 160 µm. M) Quantification of TUNEL‐positive cells. Data are represented as the mean ± SD (n = 3). ***P < 0.001 versus indicated group. Statistical analysis results are presented in Table (Supporting Information).

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Western Blot, Over Expression, Staining, Membrane, TUNEL Assay

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT3 agonist HKL protects dopaminergic neurons and alleviates motor dysfunction in the MPTP‐induced PD mouse model by modulating DRP1 acetylation. A) Experimental timeline for the administration of HKL and MPTP to establish a PD mouse model and subsequent behavioral testing. B–F) Western blotting analysis of DRP1 K711 , DRP1, TH, and SIRT3 levels in SNc brain tissue samples of MPTP‐induced PD model mice treated with the SIRT3 agonist, HKL. Data are represented as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, and ****P < 0.0001 versus indicated group. G–I) Immunofluorescence staining and analysis of TH and SIRT3 expression levels in the SNc region of HKL‐treated MPTP‐induced C57BL/6J PD model mice. Data are represented as the mean ± SD (n = 3). ****P < 0.0001 versus indicated group. J) Rotarod test analysis of motor function in MPTP‐induced PD mice treated with HKL. Data are represented as the mean ± SD (n = 8). *P < 0.05 and ***P < 0.001 versus indicated group. K) Test diagram from the Catwalk system. The system shows green paw prints and records parameters after recognition. L–N) Catwalk gait analysis of cadence, duration, and average speed in HKL‐treated MPTP‐induced PD mice. Data are represented as the mean ± SD (n = 8). *P < 0.05 and **P < 0.01 versus indicated group.

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT3 knockout exacerbates dopaminergic neuron loss and motor deficits in a PD mouse model. A) Generation of TH‐specific SIRT3 ‐knocked‐out mice via stereotaxic injection of pAAV‐TH‐Cre‐WPRE‐hGHpA virus into the SNc of SIRT3 flox/flox mice. MPTP was administered to establish a PD mouse model, which was subjected to behavioral testing. B–D) Western blotting analysis of DRP1 and total acetylation levels in the SNc brain tissues of MPTP‐induced SIRT3 ‐knocked‐out PD mice. Data are represented as the mean ± SD (n = 6). **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus indicated group. E–G) Western blotting analysis of SIRT3 and DRP1 K711 levels in the SNc brain tissues of MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 6). ***P < 0.001 and ****P < 0.0001 versus indicated group. H,I) Immunofluorescence analysis of TH and SIRT3 expression levels in the SNc region of MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus indicated group. J) Rotarod test analysis of motor function in MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 5). *P < 0.05, **P < 0.01, and ****P < 0.0001 versus indicated group. K) Test diagram from the Catwalk system. The system shows green paw prints and records parameters after recognition. L–N) Catwalk gait analysis of cadence, duration, and average speed in MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 5). *P < 0.05, ***P < 0.001, and ****P < 0.0001 versus indicated group. Statistical analysis results are presented in Table (Supporting Information).

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Knock-Out, Injection, Virus, Western Blot, Immunofluorescence, Expressing

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: Schematic illustration of SIRT3‐mediated DRP1 acetylation is a cause of mitochondrial dysfunction and subsequent damage to dopaminergic neurons in Parkinson's disease.

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: