Article Figures & Data
Figures
- Figure 1.
SIRT1 prolongs the life span of homozygous A53T mouse model—mice expressing α-synuclein gene with A53T mutation. A, Survival analysis of A53T and A53T mice overexpressing SIRT1 (A53T-Tg). n = 32 for each genotype. B, Survival analysis of A53T and A53T mice lacking SIRT1 in brain (A53T-BSKO). n = 24 for each genotype. Graphs show percentage of survival scored monthly. C, A53T α-synuclein RNA levels quantified from whole brains of A53T and A53T-Tg or A53T-F/F and A53T-BSKO mice by qPCR. n = 4 for each indicated genotype. D, Western blotting of SIRT1 in whole-brain extracts from A53T mice and wt littermates. n = 6 for each genotype. Quantification is shown on the right. A representative blot is shown. The statistical analysis in C and D was performed using Student's t test. Error bars in figures represent SEM.
- Figure 2.
SIRT1 decreases α-synuclein aggregates in A53T mouse brain. A, Immunostaining of α-synuclein aggregates in cortical sections of 3.5-month-old A53T and A53T-Tg mice (top panel) and A53T-F/F and A53T-BSKO mice (middle panel). Quantification is shown on the right. n = 6 for each genotype. There were no α-synuclein aggregates observed in wt mice or proteinase K-treated cortical sections of A53T mice (bottom panel, left and middle, respectively). Higher magnification picture of α-synuclein aggregates is also demonstrated (bottom panel, right). The statistical analysis was performed using Student's t test, and significant differences are demonstrated by a single asterisk (*) indicating p < 0.01. Error bars in figures represent SEM. B, Immunostaining of gliosis in cortical sections of A53T, A53T-Tg, and wt mice (top and middle panels) and A53T-F/F and A53T-BSKO mice (bottom panel) is performed by using GFAP antibody. Quantification is shown on the right. n = 6 for each genotype. The statistical analysis was performed by two-way ANOVA in the top panel, and significant differences are demonstrated by single asterisk (*) or pound sign (#) indicating p < 0.01. In the bottom panel, Student's t test was performed for statistical analysis; significant differences are demonstrated by single asterisk (*) indicating p < 0.01. Error bars in figures represent SEM.
- Figure 3.
SIRT1 decreases α-synuclein aggregates in A53T mouse brain A, Immunostaining of α-synuclein aggregates in the brainstem sections of A53T and A53T-Tg mice (top panel) and A53T-F/F and A53T-BSKO mice (bottom panel). Quantification is shown on the right. n = 6 for each genotype. The statistical analysis was performed using Student's t test, and significant differences are demonstrated by single asterisk (*) indicating p < 0.01. Error bars in figures represent SEM. B, Western blotting of detergent-soluble (top panel) and detergent-insoluble (bottom panel) α-synuclein fractions extracted from whole brain of indicated mice (1 mouse per lane). n = 6 for each genotype. Quantification is shown on the right. Representative blots are shown. C, Western blotting of detergent-soluble (top panel) and detergent-insoluble (bottom panel) α-synuclein fractions extracted from whole brain of indicated mice (1 mouse per lane). n = 6 for each genotype. Representative blots are shown. Quantification is shown on the right. The statistical analysis in B and C was performed using Student's t test, and significant differences are demonstrated by a single asterisk (*) indicating p < 0.01. Error bars in figures represent SEM.
- Figure 4.
SIRT1 deacetylates HSF1 and increases Hsp70 levels in brains of A53T mice. A, Left, Whole-brain extracts from A53T mice: A53T-wt, A53T-Tg, A53T-F/F, and A53T-BSKO immunoprecipitated with NRS or anti-HSF1 antibody and blotted with anti-HSF1 or anti-acetyl lysine (H-AcK) antibodies. Right, Whole-brain extracts from mice without A53T α-synuclein gene: wt, Tg, F/F, and BSKO immunoprecipitated with NRS or anti-HSF1 antibody and blotted with anti-HSF1 and anti-acetyl lysine (H-AcK) antibodies. n = 4 for each genotype. Representative immunoblots are shown. Bottom panel, Whole-brain extracts from A53T mice and wt littermates were immunoprecipitated with NRS or anti-HSF1 antibody and blotted with anti-HSF1 and anti-acetyl lysine (H-AcK) antibodies. n = 6 for each genotype. Representative immunoblots are shown. Acetylated bands are quantified by using ImageJ program. B, Hsp70 RNA levels quantified from whole brains of mice by qPCR. n = 6 for each indicated genotype. The statistical analysis is performed by using two-way ANOVA; significant differences are demonstrated by a single asterisk (*) indicating p < 0.01. Error bars in figures represent SEM. C, Western blotting of Hsp70 protein extracted from whole brains of wt, Tg, F/F, and BSKO mice without (top panel) or with the A53T α-synuclein gene (bottom panel). n = 4 for each genotype. Representative immunoblots are shown. Quantification is shown on the right. For the top panel, statistical analysis was performed using Student's t test, and significant differences are demonstrated by a single asterisk (*) indicating p < 0.01. For the bottom panel, statistical analysis was performed by using two-way ANOVA, and significant differences are demonstrated by single asterisk (*) and pound sign (#) indicating p < 0.01. Error bars in figures represent SEM.
- Figure 5.
SIRT1 deacetylates HSF1 and activates Hsp70 in MEFs and protects against α-synuclein toxicity in cells. A, Cell lysates from wt and SIRT1−/− MEFs were immunoprecipitated with NRS or anti-SIRT1 antibody after being subjected to heat shock (HS) and then blotted with anti-SIRT1 and anti-HSF1 antibodies (left panel). The control cells without heat shock (no HS) are shown on the right panel. The two proteins are shown to interact at endogenous levels only under heat shock conditions. B, SIRT1 deacetylates HSF1 only under heat shock conditions. Cell lysates from wt, SIRT1-overxpressing MEFs (mSIRT1), SIRT1−/− MEFs were immunoprecipitated with anti-HSF1 antibody or NRS after being subjected to heat shock (HS) and analyzed by Western blotting with anti-HSF1 or anti-pan acetylated lysine (Ac-K) antibodies (left panel). The control cells without heat shock (no HS) are shown on the right panel. Acetylated bands are quantified by using ImageJ. C, Western blotting of Hsp70 protein from the lysates of wt, SIRT1-overexpressing (mSIRT1), and SIRT1−/− MEFs with (HS) or without being subjected to heat shock. Actin serves as a loading control. The experiment was performed four times. Representative blots are shown. Quantification is shown below. Statistical analysis was performed using two-way ANOVA, and significant differences are demonstrated by a single asterisk (*) indicating p < 0.01. Error bars in figures represent SEM. D, Toxicity levels of H4 cells that express A53T α-synuclein by the inducible tet-off system (1 μg/ml doxycycline). The toxicity levels were increased by removing doxycycline (samples 4–12). Overexpression of SIRT1 (2) or Hsp70 (3) had no effect on control cells (compare 1 with 2 or 3). However, overexpression of SIRT1 in A53T α-synuclein-expressing cells (compare 4, 5) or overexpression of Hsp70 (compare 4, 7) decreased toxicity. SIRT1 shRNA (6) or Hsp70 shRNA (8) increased toxicity (compare 4, 6; or 4, 8). When cells were transfected with Hsp70-shRNA during SIRT1 overexpression, the toxicity level was reduced but not to the level without the Hsp70 knockdown (compare 5, 9; not significant). When the cells were transfected with both SIRT1 shRNA and Hsp70 shRNAs (10), the toxicity level was increased (compare 4, 10) but was not higher than SIRT1 shRNA alone (6) or Hsp70 shRNA alone (8). The analysis was performed using two-way ANOVA. €p < 0.01 indicates control (1) versus vector (4). *p < 0.01 indicates vector (4) versus SIRT1 overexpression (5) or SIRT1 shRNA (6) or Hsp70 overexpression (7) or Hsp70 shRNA (8), or SIRT1 shRNA+Hsp70 shRNA (10). ¥p < 0.01 indicates vector (4) versus Hsp70 shRNA (8) or SIRT1 overexpression plus Hsp70 shRNA (9) or SIRT1 shRNA plus Hsp70 shRNA (10). #p < 0.01 indicates +SIRT1 (5) versus +SIRT1 plus Hsp70 shRNA (9). E, Western blotting of SIRT1 levels from the lysates of A53T-overexpressing H4 cells that are transfected by SIRT1 (left) or SIRT1-shRNA (right). Actin serves as a loading control. F, Western blotting of Hsp70 levels from the lysates of A53T-overexpressing H4 cells that are transfected by Hsp70 (left) or Hsp70-shRNA (right). Actin serves as a loading control.
- Figure 6.
SIRT1 deacetylates HSF1 and activates Hsp70 in A53T α-synuclein-expressing H4 cells. Left, Lysates from A53T-overexpressing H4 cells transfected by SIRT1 (mSIRT1), SIRT1-shRNA, or vector immunoprecipitated with anti-HSF1 antibody or NRS and blotted with anti-HSF1 or anti-pan-acetylated lysine (Ac-K) antibodies. Right, Hsp70 RNA levels quantified from cell lysates of A53T-overexpressing H4 cells transfected by SIRT1 (mSIRT1), SIRT1-shRNA, or vector quantified by qPCR. The analysis was performed using two-way ANOVA, and significant differences are demonstrated by single asterisk (*) or pound sign (#) indicating p < 0.01. Error bars in figures represent SEM.
