Inhibiting SirT1 deacetylase increases oxidative stress resistance in neurons. Cortical neurons were cultured in 96-well plates (A–E) or 10 cm dishes (F). (A, B, C, D) 10–14 DIV neurons were treated with nicotinamide (A, B; Nico) or sirtinol (C, D) at indicated concentrations for 48 hrs. Oxidative stress was then induced by 400 μM of hydrogen peroxide (A, C) or 7.5 μM of menadione (B, D), respectively. 24 hrs later cell viability was measured by MTT assay. The ODs are normalized to respective controls (H₂O₂/menadione relative to vehicle-treated control; H₂O₂+Nico/sirtinol relative to Nico/sirtinol alone; menadione+Nico/sirtinol relative to Nico/sirtinol alone). (E) 5 DIV neurons were transfected with control or SirT1 siRNA. 2 days later some neurons were subjected to H₂O₂ or menadione, followed by MTT assay 24 hrs later. (F) 5 DIV neurons were transfected with control or SirT1 siRNA and 2 days later SirT1 deacetylase activity was measured. Data represent mean±SEM.

Inhibition of SirT1 deacetylase decreases Ras/ERK_1/2 activation in cultured neurons and in vivo. (A) Representative blots showing the effect of SirT1 inhibitor sirtinol on Ras activation. 10–14 DIV neurons were treated with vehicle or 60 μM sirtinol for 4 hrs and subjected to Ras activation assay or immunoblotted with anti-Ras. (B, C) Representative blots showing the effect of SirT1 inhibitors nicotinamide (B) and sirtinol (C) on ERK_1/2 signaling. 10–14 DIV neurons were treated with Nico for 48 hrs or sirtinol for 4 hrs. Total cell lysates were collected for SDS-PAGE and blotted with anti-phospho-ERK_1/2 and anti-ERK_1/2, respectively. (D) Quantification of immunoblots showing the effect of sirtinol on ERK_1/2 activation. (E) 7 DIV cortical neurons were transfected with U6Pro-SirT1-siRNA+GFP or dominant-negative GFP-SirT2 (SirT2DN) and 48 hrs later were immunostained with anti-phospho-ERK_1/2. (F) Quantification of immunodensity from 12 transfected and 12 nearby non-transfected cells for SirT1 siRNA and SirT2DN, respectively. (G, H) Representative blots (G) and quantification (H) showing the effect of SirT1 deacetylase on ERK_1/2 signaling in mice hippocampus. (I, J, K, L) Representative blots (I, K) and quantification (J, L) showing the effect of sirtinol on ERK_1/2 signaling in HEK cells. HEK cells were starved for 15 hrs in media containing 0.5% FBS and incubated with 60 μM sirtinol for 4 hrs and then treated with IGF-1 or PMA for 5 min. Cell lysates were subjected to SDS-PAGE and blotted with anti-phospho-ERK_1/2 or anti-ERK_1/2. Quantifications are shown as mean±SEM.

Subcellular localization of SirT1 in the brain. (A, B) Forebrains from postnatal 3 days (P3, A) and adult (B) mice were homogenized and separated into nuclear, cytosol and membrane fractions through sucrose-gradient centrifugation and immunoblotted with SirT1, NeuN, GAPDH, GluR_2/3, respectively. In P3 brain SirT1 is localized in both nucleus and cytosol, while it is found predominantly in cytosol in the adult brain. (C) The specificity of SirT1 immunoblot was confirmed with SirT1−/− mice.

SirT1 regulates Ras/ERK_1/2 signaling via deacetylation of IRS-2. (A) Physical interaction between SirT1 and IRS-2. HEK293 cells were transfected with V5-SirT1 and 48 hrs later cell lysates were immunoprecipitated with V5 antibody or control IgG and probed with anti-IRS1 or anti-IRS2, respectively. (B) SirT1 deacetylates IRS-2 in vitro. HEK cell lysates were precipitated with anti-IRS2, divided to 3 equal parts and incubated with 0, 1, or 2 μg SirT1 deacetylase respectively for 1.5 hr. They were then subjected to SDS-PAGE and blotted with anti-acetylated-lysine (AcK). (C, D) Representative blots (C) and quantification (D) showing that inhibition of SirT1 deacetylase in HEK cells increased acetylation of IRS-2 and decreased phosphorylation of IRS-2 while having no effect on IRS-1. (C) HEK cells were treated as indicated and cell lysates were precipitated with anti-IRS1 or anti-IRS2 and blotted with anti-AcK or anti-phosphotyrosine (PY). Cell lysates were also blotted for IRS1 or IRS2. (D) Quantification from 4–5 blots (mean±SEM). (E) Representative blots showing the effect of SirT1 on IRS-2 deacetylation in neurons. 5 DIV neurons were transfected with either control or SirT1 siRNA. 2 days later cell lysates were immunoprecipitated with anti-IRS2 and blotted for AcK or PY. Cell lysates were also blotted for IRS2, P-ERK_1/2 and ERK_1/2, respectively.

Inhibiting ERK_1/2 protects neurons against oxidative stress. Neurons were cultured in 96-well plates (A, D, E) or glass coverslips in 6-well plates (B). 10–14 DIV neurons were pretreated with ERK_1/2 inhibitors U0126 or SL327 for 4 hr. Oxidative stress was induced by H₂O₂ or menadione. Viability was measured by MTT (A) or live/dead assay (B). (C) % live was calculated from the number of live (green) and dead (red) cells. (D, E) ERK_1/2 inhibition primarily attenuates necrosis. Neurons were transfected with control or SirT1 siRNA, or incubated with SL327 before exposed to 400 μM H₂O₂ or 7.5 μM menadione. 24 hrs later culture media were collected for LDH assay and cells were lysed for apoptosis assay. Data represent mean±SEM.

Reduced oxidative stress in SirT1−/− mice brain and the reduced life span of SirT1−/− mice. (A, B) Reduced oxidative stress in SirT1 knockout mice brain. Protein carbonyl content (A; nmol protein carbonyl per mg protein, mean±SEM) and lipid peroxidation (B, TBARS assay; nmol malondialdehyde [MDA] equivalent per mg protein) were measured in 18 month old SirT1 +/+ and −/− mice brains. (C, D) Calorie restriction does not extend life span of SirT1 −/− mice. (C) Survival curves for SirT1 +/+ (n=16), +/−(n=15), −/− mice (n=14) on ad-lib diet (P<0.05, log-rank test). (D) Survival curves for SirT1 +/+ (n= 13), +/− (n=18), −/− (n=12) mice on 40% reduced calorie diet (P<0.001, log-rank test). Mice were 2–5 months old at the onset of CR.