SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

Abstract

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.

Figure 1

Upregulation of SIRT1 in mouse models displaying progressive and severe neurodegeneration. (A) Upregulation of SIRT1 in p25 transgenic mice during progressive neurodegeneration (after 2–12 weeks of induction). (B) Quantifications of levels of SIRT1 in p25 transgenic mice. ^**P(T⩽t) two tails: 0.007. (C) Acetylation levels of PCG-1alpha is decreased in forebrains of p25 transgenic mice (10–12 weeks of induction). Densitometry-based analyses of acetylated PCG-1alpha levels is shown in the lower panel. (D) Progressive increase of SIRT1 in mutant SOD1G37R (line 29) peaking at stage of massive neurodegeneration (10–12 months). (E) Quantifications of levels of SIRT1 in SOD1G37R mice. ^***P(T⩽t) two tails: 0.0004. (F) Treatment of primary cortical neurons with low concentrations of ionomycin (1 μM) and H₂O₂ (25 μM) induces rapid upregulation of SIRT1 associated with generation of p25. Time expressed in minutes. Top panels show densitometry-based analyses of SIRT1 levels. (G) Treatment of primary cortical neurons with increasing doses of ionomycin or H₂O₂ for 20 min demonstrates a dose-dependent induction of SIRT1 by these neurotoxic stimuli. Top panels show densitometry-based analyses of SIRT1 levels.

Figure 2

Resveratrol protects against p25 and mutant SOD1 toxicity. (A) No toxicity observed in GFP-transfected neurons treated with resveratrol for 48 h (50–500 nM). For all experiments (A–E), primary rat neurons were transfected at DIV 5–7 with plasmids, and resveratrol was added to the medium at 3 h after transfection. Characterization of neuronal integrity was performed 24–48 h after transfection. Scale bar, 40 μm. (B) Representative confocal images of dying and healthy neurons transfected with p25-GFP, and untreated or treated with resveratrol (250 nM) for 24 h, respectively. Inset in DAPI-only panel is a magnification of the nucleus of the transfected neuron, as indicated by arrow. Scale bar, 20 μm. (C) Quantifications of p25-GFP-induced cell death in neurons untreated or treated with 250 nM resveratrol for 24 h expressed in percent (%) (54±12.2 versus 27±8%; ^**P(T⩽t) two tails: 0.003). (D) Representative confocal images of neurons transfected with SOD1G93A-FLAG, and untreated or treated with 250 nM resveratrol for 48 h. Right panels are magnifications of the boxed area shown in the left panel. Arrowheads indicate regions of SOD1 aggregation. Scale bar, 50 μm. (E) Quantifications of SOD1G93A-FLAG-induced cell death in neurons untreated or treated with 250 nM resveratrol for 48 h expressed in percent (%) (52±3 versus 28±3%; ^***P(T⩽t) two tails: <0.001).

Figure 3

Overexpression of SIRT1 protects against p25 and mutant SOD1 toxicity. (A) Effects of overexpression of SIRT1 or SIRT1 lacking catalytic deacetylase activity (H363Y) on p25 GFP toxicity. Arrows indicate neurons transfected with p25-GFP with or without SIRT1 (red). Inset in DAPI-only panel is a magnification of the nucleus of the transfected neuron, as indicated by arrow. Scale bar, 20 μm. (B) Quantifications of cell death in p25-GFP expressing neurons with or without ectopic expression of SIRT1 or H363Y. a, control versus p25-GFP, P<0.001; b, control versus p25-GFP+SIRT1, P<0.05; c, control versus p25-GFP+H363Y, P<0.001; d, p25-GFP versus p25-GFP+SIRT1, P<0.01; e, p25-GFP+SIRT1 versus p25-GFP+H363Y, P<0.01. p25-GFP versus P25GFP+H363Y is non-significant (P>0.05). One-way ANOVA with Neuman–Keuls Multiple Comparison Test. (C) Unchanged levels of p25-GFP following expression of SIRT1 or H363Y. h, human; m, mouse. Comparison of HEK and CAD cells for SIRT1 expression. (D) Effects of overexpression of SIRT1 or H363Y on SOD1G93A toxicity. Arrows indicate to SOD1 aggregates as detected with FLAG Ab. WT SOD1 is not toxic. Scale bar, 25 μm. (E) Quantifications of cell death in SOD193A and WT SOD1-expressing neurons with or without ectopic expression of SIRT1 or H363Y. a, control versus G93A, P<0.001; b, control versus G93A+SIRT1, P<0.001; c, control versus G93A+H363Y, P<0.001; d, G93A versus G93A+SIRT1, P<0.001; e, G93A+SIRT1 versus G93A+H363Y, P<0.001. G93A versus G93A+H363Y is non-significant (P>0.05). One-way ANOVA with Neuman–Keuls Multiple Comparison Test.

Figure 4

Resveratrol prevents neurodegeneration in p25 transgenic mice. (A) Experimental design for ICV injection of resveratrol (Resv) or vehicle (Veh) in p25 transgenic mice (n=5 for vehicle (Veh); n=9 for resveratrol (Resv)). (B) Acetylation levels of PGC-1alpha is decreased in p25 animals treated with resveratrol, compared to p25 animals injected with vehicle. Shown also is densitometry-based analysis of acetylated PCG-1alpha levels. (C) Downregulation of activated caspase 3 and GFAP, markers of cell death and astrogliosis, in the hippocampus of p25 transgenic mice treated with resveratrol (n=3), compared to p25 animals injected with vehicle (n=2), as revealed by Western blots. Uninjected age-matched WT mice (n=2) were also compared as a control. SIRT1 levels are increased in p25 transgenic mice compared to WT mice, but are similar between resveratrol and vehicle-treated mice. Densitometry values for activated caspase 3, GFAP and SIRT1 are also shown. Actin and FAK are used for loading controls. (D) Reduction of GFAP-expressing cells in CA1 of p25 transgenic mice treated with resveratrol (n=3), compared to p25 animals injected with vehicle (n=2) and uninjected WT controls (n=2) as revealed by immunofluorescence staining. Scale bar, 15 μm. (E) Immunofluorescence staining revealed reduced caspase 3 activation and higher number of p25-GFP expressing cells in CA1 of p25 transgenic mice (n=2) injected with resveratrol versus vehicle (n=2). Scale bar, 50 μm. (F) Two weeks induced p25 transgenic mice were injected ICV with either resveratrol (n=9) or vehicle (n=5) 2–3 × /week for 3 weeks. An additional control group of p25 transgenic mice was not injected with vehicle or resveratrol (n=8). Subsequently all groups and WT mice (n=20) were subjected to contextual fear conditioning. Left: resveratrol had no effect on the total activity and escape response to the electric foot shock during the training procedure. ES, electric foot shock. Right: vehicle treated and non-treated p25 transgenic mice displayed reduced freezing behavior during the memory test when compared to WT littermates (P=0.0032, t_(1,23)= 3.295; P<0.0001, t_(1,26)= 5.048). However, when compared to the vehicle group (P= 0.0109, t_(1,12)=3.009) or non-treated p25 transgenic mice (P= 0.0005 t_(1,15)=4.407) resveratrol-treated p25 transgenic mice showed significantly improved freezing behavior during the memory test. ^* Denotes significant difference; ES, electric foot shock.

Figure 5

Acetylation of p53, a SIRT1 substrate, in p25 transgenic mice reversed by resveratrol. (A) Upregulation of p53 in p25 transgenic mice (n=4) detected by immunoprecipitation followed by Western blot. Densitometry analyses of p53 levels are shown on right. (B) Acetylation of p53 at lysine 382 in p25 transgenic mice (n=3) detected by immunoprecipitation, followed by Western blot. ^* Indicates nonspecific band. (C) P53 knockdown in p25-expressing primary hippocampal neurons rescues p25 neurotoxicity by 25%. ^**P(T⩽t) two tails: 0.001. (D) Efficient knockdown of p53 by RNAi in cell line transfected with p53. (E) Reduced acetylation of p53 at lysine 382 and downregulation of p53 in p25 transgenic mice (n=3) treated with resveratrol. Densitometry analyses of acetylated p53 levels is shown in the bottom panel.

Figure 6

SIRT1 expression prevents neurodegeneration in p25 transgenic mice. (A) Less p25-GFP-positive neurons are present in the CA1 of control virus-injected hemispheres compared to the CA1 of the SIRT1 lentivirus-injected hemispheres. For each animal, the ratio number of neurons in control side: SIRT1 side was calculated, where the SIRT1 side equals 1. Count data are shown in Supplementary Table 1. (B, C) Representative confocal images of CA1 hippocampal GFP-positive neurons in control (left) and SIRT1-injected (right) hemispheres of p25 mouse 806, showing decreased number of GFP-positive neurons in the control hemisphere. Scale bar, 100 μm. (D, E) High-magnification confocal images of CA1 hippocampal GFP-positive neurons in control (left) and SIRT1-injected (right) p25 mouse 807. Neuronal integrity in SIRT1-injected p25 transgenic mice is better preserved when compared to the contralateral control-injected side. Scale bar, 15 μm. (F–H) GFP-positive neurons express SIRT1, as revealed by co-staining with HA antibody. Scale bar, 15 μm.