PMC

Diagram of the protective mechanism of viniferin. Viniferin counteracts mutant Htt-induced depletion of SIRT3 and facilitates its deacetylase activity. SIRT3 deacetylates its substrates Mn-SOD and then enhances its antioxidant activity. SIRT3 also deacetylates LKB1, resulting in activation of AMPK, and activated AMPK promotes mitochondrial biogenesis and homeostasis of energy metabolism, thereby protecting cells from mutant Htt-mediated mitochondrial dysfunction and toxicity. P indicates phosphorylation.

Viniferin decreases mutant Htt toxicity in HD cell models. A, quantification of cell toxicity in N2a cells expressing mutant Htt with or without viniferin (V). #, p < 0.05 versus N63-16Q group, *, p < 0.05 versus vehicle (DMSO) group. B, caspase 3/7 activity assay. #, p < 0.05 versus SThdh^Q7/Q7 (Q7)cells, *, p < 0.05 versus SThdh^Q111/Q111 (Q111) cells by standard Student's t tests. RLU, relative luminescence units. C, representative images of primary cortical neurons transfected with wild-type Htt (N63-16Q (16Q)) or mutant Htt (N63-148Q (148Q)). D, quantification of cell death by chromatin condensation as a marker of dead cells. #, p < 0.05 versus 16Q transfected neurons; *, p < 0.05 versus DMSO-treated mutant Htt (148Q) transfected neurons by standard Student's t tests.

SIRT3 mediates the protective effects of viniferin on mutant Htt. A, STHdh^Q111/Q111 cells were treated with SIRT3 siRNA (10 nm) or scrambled control RNA for 48 h and then collected and analyzed by Western blotting. Note that both the 37-kDa and the 28-kDa bands are decreased by SIRT3 siRNA treatment. B, STHdh^Q111/Q111 cells were pretreated with SIRT3 siRNA or scrambled control RNA for 24 h and then exposed to different treatments, as indicated, for another 24 h; caspase 3/7 activity was measured in a toxicity assay. *, p < 0.05 when compared with the values of DMSO-treated cells in scrambled RNA-pretreated group; **, p < 0.05 when compared with the values of viniferin-treated cells in scrambled RNA-pretreated group by standard Student's t tests. RLU, relative luminescence units.

Viniferin increases AMPK activation, and SIRT3 is required for viniferin-mediated AMPK activation. A, Western blotting of STHdh^Q111/Q111 cells treated with viniferin (V) or vehicle DMSO. The upper panel shows representative blots, and the bottom panel shows quantification of densitometry of Western blots. STHdh^Q111/Q111 cells were collected at 30 min after the treatment, and soluble cell extracts were analyzed by Western blotting with phosphorylated AMPK (p-AMPK) antibody or total AMPK antibody (t-AMPK). β-Actin (Actin) was used as a loading control. *, p < 0.05 versus vehicle-treated group by standard Student's t tests. B, STHdh^Q111/Q111 cells were treated with SIRT3 siRNA (10 nm) for 24 h and then with viniferin at the indicated concentrations or with DMSO for 30 min. Levels of phosphorylated AMPK and total AMPK antibody were determined by Western blot analysis. *, p < 0.05 versus vehicle-treated group by standard Student's t tests.

Viniferin reduces ROS levels in cells expressing mutant Htt (STHdh^Q111/Q111). A, representative CM-H₂DCFDA-loaded cell images in indicated cells. Note the increased fluorescence in SThdh^Q111/Q111 cells, and note that viniferin decreased the fluorescence in these cells, indicating decreased ROS levels by viniferin treatment. B, histograms show that the peak of excitation of dye CM-H₂DCFDA shifted from the left in SThdh^Q7/Q7 (Q7) cells (blue curve) to the right in STHdh^Q111/Q111 (Q111) cells (red curve), indicating the increase of ROS levels in SThdh^Q111/Q111 cells. DMSO (black curve) did not affect the curve shift in SThdh^Q111/Q111 cells, and viniferin treatment (green line) shifted the peak partially to the left, indicating reduced ROS levels in viniferin-treated SThdh^Q111/Q111 cells when compared with that in DMSO-treated SThdh^Q111/Q111 cells. C, quantification of average ROS levels from cells in the indicated groups. #, p < 0.01 versus SThdh^Q7/Q7 cells; *, p < 0.05 versus SThdh^Q111/Q111 or DMSO-treated SThdh^Q111/Q111 cells by standard Student's t tests. MFI, mean fluorescence intensity.

Viniferin decreases mutant Htt-induced hyperacetylation of LKB1, and this action is SIRT3-dependent. A, cells expressing mutant Htt exhibit increased levels of acetylated LKB1. The left panel shows representative blots, and the right panel shows quantification of blots, n = 3. *, p < 0.05 versus SThdh^Q7/Q7 (Q7) cells. Q11, STHdh^Q111/Q111 cells. IP, immunoprecipitation; IB, Western blot. B, viniferin decreases levels of acetylated LKB1 in STHdh^Q111/Q111 cells. The left panel shows the representative bots, and the right panel shows quantification of acetylated LKB1 levels. *, p < 0.05 versus DMSO-treated STHdh^Q111/Q111 cells. C, SIRT3 is required for effects of viniferin on LKB1 deacetylation. Cells were treated with SIRT3 siRNA (10 nm) or scrambled RNA for 24 h before treatment with viniferin at the indicated concentrations or DMSO as vehicle for 2 h, and acetylated LKB1 levels were determined by immunoprecipitation with LKB1 antibody and then blotting with anti-lysine (AcK) antibody. The left panel shows the representative blots, and the right panel shows the quantification of blots. n = 3, mean ± S.D. *, p < 0.05 when compared with the DMSO-treated culture in scrambled RNA-pretreated group by standard Student's t tests.

Viniferin prevents mitochondrial membrane potential loss and promotes mitochondrial biogenesis in STHdh^Q111/Q111 cells. A and B, histograms show that the peak of excitation of TMRE fluorescent dye shifted from the right in SThdh^Q7/Q7 (Q7) cells (blue curve) to the left in STHdh^Q111/Q111 (Q111) cells (red curve), DMSO (black curve) did not affect the peak of TMRE in STHdh^Q111/Q111, and viniferin treatment (green curve) shifted the peak to right, indicating increased fluorescent intensity and mitochondrial membrane potential. C, quantification of average fluorescence intensity by flow cytometry. #, p < 0.01 versus SThdh^Q7/Q7 cells; *, p < 0.05 versus DMSO-treated STHdh^Q111/Q111 cells by standard Student's t tests. MMP, mitochondrial membrane potential; MFI, mean fluorescence intensity. D, ratio of NAD⁺/NADH in the indicated groups. E, the ratio of mitochondrial RNA 12 S rRNA to 18 S rRNA. rRNA levels were measured by quantitative RT-PCR. F, the ratio of mitochondrial gene Cox-2 to cyclophilin A DNA copy numbers was measured by quantitative PCR. #, p < 0.01 versus SThdh^Q7/Q7 cells; *, p < 0.05 versus DMSO-treated STHdh^Q111/Q111 cells by standard Student's t tests. G, viniferin attenuated PGC-1α depletion by mutant Htt. PGC-1α mRNA levels were detected by Q-RT-PCR. #, p < 0.01 versus SThdh^Q7/Q7 cells; *, p < 0.05 versus DMSO-treated STHdh^Q111/Q111 cells by standard Student's t tests.

Viniferin increases levels of SIRT3 protein and its deacetylase activity. A and B, striatal cells expressing mutant Htt (STHdh^Q111/Q111 (Q111)) exhibited lower SIRT3 protein levels than cells expressing normal Htt (STHdh^Q7/Q7 (Q7)) at 24 h following serum withdrawal. Panel A shows representative blots, and panel B shows quantification of densitometry of the 28-kDa band from three independent Western blots. #, p < 0.05 versus values in STHdh^Q7/Q7 cells by standard Student's t tests. W/O, untreated control. C and D, levels of SIRT3 protein (28 kDa) and mitochondrial membrane potential (MMP) in SThdh^Q7/Q7 or SThdh^Q111/Q111 cells following serum withdrawal at the indicated time points. Mean ± S.D., n = 3. *, p < 0.05 versus corresponding STHdh^Q7/Q7 cells by standard Student's t tests. MFI, mean fluorescence intensity. E and F, effects of viniferin and resveratrol on mutant Htt-induced decrease of SIRT3 protein levels in SThdh^Q111/Q111 cells. Cells were treated with viniferin (1 μm) or resveratrol (10 μm) for the indicated times. SIRT3 protein levels were determined by Western blotting. *, p < 0.05 versus SThdh^Q7/Q7 cells, #, p < 0.05 versus DMSO-treated SThdh^Q111/Q111 cells by Standard Student's t tests. G, cells were treated with viniferin for 4 h. Cell extracts were immunoprecipitated (IP) with anti-Mn-SOD antibody, and the immunoprecipitation product was blotted with acetyl-lysine antibody to detect the levels of acetylated Mn-SOD. Note that there is an interaction between Mn-SOD and SIRT3, and viniferin treatment decreased acetylated Mn-SOD levels. The left panel shows representative blots, and the right panel shows quantification of densitometry from three independent Western blots (IB). *, p < 0.05 versus values in vehicle-treated cells by standard Student's t tests.