Sirtuin modulators control reactive gliosis in an in vitro model of Alzheimer's disease

Abstract

Among neurodegenerative disorders, Alzheimer's disease (AD) represents the most common cause of dementia in the elderly. Several genetic and environmental factors have been identified; however, aging represents the most important risk factor in the development of AD. To date, no effective treatments to prevent or slow this dementia are available. Sirtuins (SIRTs) are a family of NAD(+)-dependent enzymes, implicated in the control of a variety of biological processes that have the potential to modulate neurodegeneration. Here we tested the hypothesis that activation of SIRT1 or inhibition of SIRT2 would prevent reactive gliosis which is considered one of the most important hallmark of AD. Primary rat astrocytes were activated with beta amyloid 1-42 (Aβ 1-42) and treated with resveratrol (RSV) or AGK-2, a SIRT1 activator and a SIRT2-selective inhibitor, respectively. Results showed that both RSV and AGK-2 were able to reduce astrocyte activation as well as the production of pro-inflammatory mediators. These data disclose novel findings about the therapeutic potential of SIRT modulators, and suggest novel strategies for AD treatment.

Keywords: AGK-2; Alzheimer’s disease; astrocyte; beta-amyloid; reactive gliosis; resveratrol; sirtuins.

FIGURE 1

Resveratrol (RSV) and AGK-2 affect astrocyte viability and proliferation induced by Aβ 1-42 challenge. Cells were challenged with 0.23 μM Aβ 1–42 in the presence or absence of one of the following substances: RSV (2 – 10 – 50 μM), a potent SIRT1 activator; AGK-2 (0.35 – 3.5 – 35 μM), a selective SIRT2 inhibitor. 24 h later cell viability was assessed by neutral red uptake assay (A,C). 74 h after treatments cell proliferation was evaluated by trypan blu assay (B,D). Results are expressed as cell viability-fold increase versus unchallenged (open bars) or Aβ-challenged cells (black bars). Results are the mean ± SEM of four experiments in triplicate. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison test. p < 0.01 Aβ-challenged versus unchallenged cells; *p < 0.05; **p < 0.01; ***p < 0.001 for multiple comparison among groups.

FIGURE 2

Effect of RSV and AGK-2 on GFAP and S100B expression. 24 h after treatments, astrocytes were lysated and protein expression was evaluated. Representative Western blots for GFAP and S100B proteins in lysates from astrocytes challenged with Aβ 1-42 (0.23 μM) in the presence of RSV (2 – 10 – 50 μM; A) or AGK-2 (0.35 – 3.5 – 35 μM; C). Densitometric analyzes normalized to β-actin loading controls (B,D for RSV and AGK-2, respectively). Results are the mean ± SEM of four experiments in triplicate. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison test. p < 0.01 Aβ-challenged versus unchallenged cells; *p < 0.05; **p < 0.01; for multiple comparison among groups.

FIGURE 3

Effect of RSV and AGK-2 on iNOS and COX-2 expression. Astrocytes were treated with Aβ 1-42 (0.23 μM) in the presence of RSV (2 – 10 – 50 μM) or AGK-2 (0.35 – 3.5 – 35 μM). Western blot experiments were carried out 24 h after treatments. Representative immunoblots for iNOS and COX-2 proteins (A,C for RSV and AGK-2, respectively). Densitometric analyzes normalized to β-actin loading controls (B,D for RSV and AGK-2, respectively). Results are the mean ± SEM of four experiments in triplicate. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison test. p < 0.001 and p < 0.05 Aβ-challenged versus unchallenged cells; *p < 0.05; **p < 0.01; for multiple comparison among groups.