Article Figures & Data
Figures
- Figure 1.
A regimen of 3 month 30% CR reduces neurodegeneration. A, Schematic of the experimental timeline. Three groups of mice were used: CON,AL, CK-p25,AL, and CK-p25,CR. B, Weight development over the experimental timeline of all three groups of mice used in this study. C, CR-mediated preservation of neuronal integrity in CK-p25,CR animals. Immunohistochemical images depicting the number of neurons as evidenced by NeuN-staining and the number of p25-expressing cells by GFP-staining. Scale bars, 200 μm. D, Quantification of NeuN-positive cells under C. One-way ANOVA (F(2,9) = 10.01, p ≤ 0.01), followed by Tukey's post hoc tests; n = 4 mice each. E, Quantification of GFP-positive cells under C (one-tailed t test, t(4) = 3.56; n = 3 each). F, CR-mediated preservation of overall brain mass. One-way ANOVA F(2,30) = 102.6, p ≤ 0.0001, followed by Tukey's post hoc tests; n = 12 for CON,AL and CK-p25,AL; n = 9 for CK-p25,CR. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for Tukey's post hoc comparisons. All values are mean ± SEM.
- Figure 2.
CR protects structural synaptic and dendritic integrity. A, CR-mediated preservation of presynaptic terminals in CK-p25,CR animals as evidenced by SVP immunohistochemistry in the stratum radiatum of hippocampal area CA1. B, Quantification of A [one-way ANOVA (F(2,8) = 8.99, p ≤ 0.01) followed by Tukey's post hoc tests; n = 3 for CON,AL and CK-p25,CR; n = 5 for CK-p25,AL]. C, CR-mediated preservation of the number of synapses in the hippocampus of CK-p25,CR animals as evidenced by transmission electron microscopy. D, Quantification of C. One-way ANOVA (F(2, 6) = 24.84, p ≤ 0.01) followed by Tukey's post hoc tests; n = 3 mice each. E, CR-mediated preservation of the number of spines per given dendritic length in the hippocampus of CK-p25,CR animals as evidenced by Golgi impregnation. F, Quantification of E [one-way ANOVA (F(2,22) = 23.31, p ≤ 0.01) followed by Tukey's post hoc tests; n = 7–10 dendrites per animal, 3 animals each]. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for Tukey's post hoc comparisons. All values are mean ± SEM. Scale bars: A, 100 μm; C, 0.5 μm; E, 2 μm.
- Figure 3.
CR maintains synaptic plasticity and memory. A, Preservation of long-term potentiation by CR. fEPSPs (expressed in percentage of maximal amplitude) in hippocampal area CA1 of CON,AL, CK-p25,AL and CK-p25,CR animals (n = 3–4 slices from 3–4 mice each) before and after TBS. *p ≤ 0.05 by ANOVA for the effect of treatment. Sample traces above the line chart represent fEPSPs at 1 min before (black) and 1 h after (colored) TBS. B, Input–output relationship of comparable baseline synaptic transmission between CON,AL, CK-p25,AL, and CK-p25,CR animals. C, Cued fear conditioning. Left, Freezing responses before the onset of the tone were comparable between CON,AL, CK-p25,AL and CK-p25,CR animals 24 h after training. Right, Freezing responses after the onset of the tone show comparable memory retention between CON,AL and CK-p25,CR animals, but reduced memory for CK-p25,AL mice. One-way ANOVA (F(2,30) = 6.29, p ≤ 0.01) followed by Tukey's post hoc tests; n = 10–12 mice each. D, Contextual fear conditioning. Freezing responses 24 h after training on a contextual fear conditioning task show comparable memory retention between CON,AL and CK-p25,CR animals, but reduced memory for CK-p25,AL mice [one-way ANOVA (F(2,30) = 7.52, p ≤ 0.01) followed by Tukey's post hoc tests; n = 10–13 mice each]. E, Novel object recognition. Discrimination ratio 24 h after training revealing a significantly enhanced object memory in CK-p25,CR compared to CK-p25,AL animals [one-way ANOVA (F(2,19) = 7.16, p ≤ 0.01) followed by Tukey's post hoc tests; n = 7–8 mice each]. *p ≤ 0.05; **p ≤ 0.01 for Tukey's post hoc comparisons. All values are mean ± SEM.
- Figure 4.
SIRT1 activation by the CR regimen. A, Increased SIRT1 expression following CR in hippocampal area CA1 as evidenced by SIRT1 immunohistochemistry. B, Quantification of A (one-tailed t test, t(4) = 2.68; n = 3 each). C, Decreased AcH3K56 following CR in hippocampal area CA1 as evidenced by AcH3K56 immunohistochemistry. D, Quantification of C (one-tailed t test, t(4) = 3.84; n = 3 each). E, Increased SIRT1 deacetylase activity following CR. SIRT1 deacetylase activity in CK-p25,AL and CK-p25,CR hippocampi. One-tailed t test, t(5) = 2.03; n = 3 for CK-p25,AL; n = 4 for CK-p25,CR. F, Decreased acetylation of p53 following CR. Left, Representative images of Western blot analysis following immunoprecipitation of acetylated (top) and total (bottom) p53 levels in CK-p25,AL and CK-p25,CR hippocampi. Right, Quantification of the ratio of acetylated/total p53 protein levels (one-tailed t test, t(13) = 2.04; n = 6 for CK-p25,AL; n = 9 for CK-p25,CR). *p ≤ 0.05; **p ≤ 0.01 for one-tailed t tests. All values are mean ± SEM. Scale bars: 100 μm.
- Figure 5.
SRT3657 activates SIRT1 in CK-p25 mice. A, Schematic of the experimental timeline. Two groups of mice were used: CK-p25,VEH and CK-p25,SRT3657. B, Increased SIRT1 activity following SRT3657 treatment. SIRT1 deacetylase activity in CK-p25,VEH and CK-p25,SRT3657 hippocampi (one-tailed t test, t(8) = 2.65; n = 4 for CK-p25,VEH; n = 6 for CK-p25,SRT3657). C, Decreased AcH3K56 following SRT3657 treatment in hippocampal area CA1 as evidenced by AcH3K56 immunohistochemistry. Scale bar, 100 μm. D, Quantification of C. One-tailed t test, t(8) = 2.61; n = 4 for CK-p25,VEH; n = 6 for CK-p25,SRT3657. E, Decreased acetylation of p53 following SRT3657 treatment. Top, Representative images of Western blot analysis following immunoprecipitation of acetylated (top) and total (bottom) p53 levels in CK-p25,VEH and CK-p25,SRT3657 hippocampi. Bottom, Quantification of the ratio of acetylated/total p53 protein levels (one-tailed t test, t(8) = 2.61; n = 4 for CK-p25,VEH; n = 6 for CK-p25,SRT3657). *p ≤ 0.05 for one-tailed t tests. All values are mean ± SEM.
- Figure 6.
SRT3657 recapitulates the beneficial effects of CR. A, SRT3657-mediated increase in presynaptic terminals in the stratum radiatum of hippocampal area CA1 in CK-p25,SRT3657 animals compared to CK-p25,VEH mice as evidenced by SVP immunohistochemistry. B, Quantification of A [one-way ANOVA (F(2,11) = 5.77, p ≤ 0.05) followed by Tukey's post hoc tests; n = 4–5 mice each]. C, SRT3657-mediated increase in the number of neurons in hippocampal area CA1 in CK-p25,SRT3657 animals compared to CK-p25,VEH mice as revealed by NeuN-immunohistochemistry. D, Quantification of C [one-way ANOVA (F(2,12) = 29.99, p ≤ 0.0001) followed by Tukey's post hoc tests; n = 3–6 mice each]. E, SRT3657-mediated preservation of overall brain mass. One-way ANOVA (F(2,11) = 19.68, p ≤ 0.001) followed by Tukey's post hoc tests; n = 4–7 mice each. F, Cued fear conditioning. Left, Freezing responses before the onset of the tone were comparable between CK-p25,VEH and CK-p25,SRT3657 animals 24 h after training. Right, Freezing responses after the onset of the tone show improved memory retention in CK-p25,SRT3657 mice [one-way ANOVA (F(2,23) = 7.47, p ≤ 0.01) followed by Tukey's post hoc tests; n = 8–10 mice each]. G, Contextual fear conditioning. Freezing responses 24 h after training on a contextual fear conditioning task show improved memory retention in CK-p25,SRT3657 mice [one-way ANOVA (F(2,22) = 5.28, p ≤ 0.05) followed by Tukey's post hoc tests; n = 8–10 mice each]. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for Tukey's post hoc comparisons. All values are mean ± SEM. Scale bars: A, 50 μm; C, 20 μm.
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