Silent information regulator 1 (SIRT1), an NAD+-dependent deacetylase, is involved in the regulation of gene transcription, energy metabolism, and cellular aging and has become an important therapeutic target across a range of diseases. Recent research has demonstrated that SIRT1 possesses neuroprotective effects; however, it is unknown whether it protects neurons from NMDA-mediated neurotoxicity. In the present study, by activation of SIRT1 using resveratrol (RSV) in cultured cortical neurons or by overexpression of SIRT1 in SH-SY5Y cell, we aimed to evaluate the roles of SIRT1 in NMDA-induced excitotoxicity. Our results showed that RSV or overexpression of SIRT1 elicited inhibitory effects on NMDA-induced excitotoxicity including a decrease in cell viability, an increase in lactate dehydrogenase (LDH) release, and a decrease in the number of living cells as measured by CCK-8 assay, LDH test, and Calcein-AM and PI double staining. RSV or overexpression of SIRT1 significantly improved SIRT1 deacetylase activity in the excitotoxicity model. Further study suggests that overexpression of SIRT1 partly suppressed an NMDA-induced increase in p53 acetylation. These results indicate that SIRT1 activation by either RSV or overexpression of SIRT1 can exert neuroprotective effects partly by inhibiting p53 acetylation in NMDA-induced neurotoxicity.
Silent information regulator 1 (SIRT1), an NAD+-dependent deacetylase, is known to deacetylate histone and nonhistone proteins such as transcription factors. It participates in a variety of physiopathological processes such as health maintenance in development, gametogenesis, homeostasis, longevity, and several neurodegenerative diseases as well as age-related disorders [
Glutamate is a primary excitatory amino acid neurotransmitter and activation of glutamate receptors including NMDA receptor plays crucial roles in the central nervous system. However, overactivation of NMDA receptor may cause intracellular calcium overload, leading to an enzymatic cascade of events resulting ultimately in cell death known as excitotoxicity [
The present study was designed to investigate the neuroprotection of SIRT1 in NMDA-induced excitotoxicity by activation of SIRT1 using resveratrol (RSV) in cultured cortical neurons or by overexpression of SIRT1 in the SH-SY5Y cell line. The neuroprotective role of SIRT1 activity
Neurobasal/B27, DMEM/F-12, and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). Lipofectamine 2000 transfection reagents were obtained from life technologies (St. Louis, MO, USA). Poly-D-lysine (MW 150,000–300,000), trypsin, arabinoside cytosine, Calcein-AM, propidium iodide (PI), RSV, Sirtinol, NMDA, MK-801, and SIRT1 assay kit were all purchased from Sigma-Aldrich (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK-8) was from Dojindo, and the kit of LDH was from Njjcbio. The polyclonal antibody to SIRT1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Two polyclonal antibodies to p53 and Ace-p53 were obtained from Cell Signaling Technology (Beverly, MA, USA).
Primary cortical cells were isolated from 1–3-day-old Wistar rats and were cultured as previously described [
The human neuroblastoma SH-SY5Y cell, obtained from the Chinese Academy of Sciences Institute of Cell Resource Center, Shanghai, China, was maintained under a DMEM/F12 medium with 10% FBS in 5% CO2 incubator. They were washed by PBS buffer before adding 0.25% Trypsin-EDTA, followed by incubation for 5 min at room temperature. Then, the cells were detached, resuspended in medium, counted, and seeded into plates at the density of 1 × 105.
After overnight incubation allowing the cells to reach 80% confluency, cells were treated with NMDA-containing Mg2+-free Locke’s buffer for 2 h. RSV was added to cultures 12 h prior to NMDA induction. Sirtinol was added 2 h before NMDA treatment. MK-801 and NMDA was simultaneously added to Mg2+-free Locke’s buffer in the NMDA + MK-801 group. Control cells were incubated with drug-free Mg2+-free Locke’s buffer and grown at 37°C in an atmosphere containing 5% CO2.
The expression vector expressing human wild-type SIRT1 (WT-SIRT1) and the dominant-negative form of human SIRT1 (DN-SIRT1) was constructed by Genecopoeia. The plasmids were extracted with a Plasmid Midi Kit (Omega, GA, USA). The SH-SY5Y cells were seeded into plates at a density of 1 × 105, and after 24 h, the plasmids were transfected into the cells with a Lipofectamine 2000 Transfection Reagent.
Cells were seeded in 96-well plates, and cell viability was assayed 24 h after NMDA exposure. Administration of 10
LDH is released from cells into a culture medium upon cell lysis. The cells were plated in 24-well plates. At 24 h after NMDA exposure, the supernatant was collected to measure LDH release according to the manufacturer’s instructions.
Calcein-AM solution (20
To measure SIRT1 activity, the protein was extracted from cells. The enzyme activity of SIRT1 was measured using a SIRT1 assay kit (CS1040; Sigma-Aldrich) based on the fleur de Lys-SIRT1 substrate peptide. The fluorescence intensity was measured with a microplate reader (Packard, Meridien, MS), and the excitation wavelength was 365 nm, and the emission wavelength was 460 nm.
Total RNA from SH-SY5Y cell was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instruction. Reverse transcription was performed with High-Capacity cDNA Archive Kit (Applied Biosystem). qRT-PCR primers were synthesized by the software of Primer Premier according to the following sequences:
The SH-SY5Y cells were collected at 24 h after exposure to NMDA. Then, cells were lysed in a lysis buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1% Triton X-100). Cleared cell lysates were obtained after centrifugation at 10000 ×g for 30 min at 4°C. After measurement of protein concentration using a BCA Protein Assay kit, cell lysates (30 ~ 50
The data were expressed as means ± S.E.M. of at least three independent experiments. One-way analysis of variance (ANOVA) with Bonferroni post hoc test was used for statistical comparisons.
Our previous study showed that the optimal excitotoxicity was induced 24 h after NMDA (100
Effects of RSV on NMDA-induced decrease in cell viability in primary neurons. (a) Pretreatment of RSV (10
As shown in Figure
After treatment of NMDA, LDH levels rose by 116.03% compared with those of the control group (
Effects of RSV on NMDA-induced LDH release in primary neurons. RSV (25
Exposure to NMDA resulted in a significant decrease in the cell survival rate estimated by Calcein-AM and PI staining (Figure
Effects of RSV on NMDA-induced decrease in the number of living cells in primary neurons. (a) Representative micrographs showing the suppression of RSV (25
As shown in Figure
Effects of RSV on NMDA-induced decrease of SIRT1 deacetylase activity in primary neurons. RSV (25
To better characterize NMDA-induced neuronal insults of the SH-SY5Y cell line, the administration of NMDA at different concentrations (10
Effects of different concentrations (10–1000
NMDA significantly decreased the level of SIRT1 mRNA (Figure
Overexpression of SIRT1 (WT-SIRT1 or DN-SIRT1) increased the levels of SIRT1 mRNA and protein reduced by NMDA (500
As shown in Figure
Effects of SIRT1 overexpression on the deacetylase activity in NMDA-induced excitotoxicity of SH-SY5Y cell. WT-SIRT1 overexpression significantly reversed the deacetylase activity decreased by NMDA (
Figure
Effects of SIRT1 overexpression on p53 acetylation in NMDA-induced excitotoxicity of SH-SY5Y cell. WT-SIRT1 overexpression partially inhibited NMDA-stimulated p53 acetylation (
Figure
Effects of SIRT1 overexpression on cell viability reduced by NMDA in the SH-SY5Y cell line. WT-SIRT1 overexpression reversed NMDA-induced decrease in cell viability (
As shown in Figure
Effects of SIRT1 overexpression on NMDA-induced the amount of LDH release the in SH-SY5Y cell line. WT-SIRT1 overexpression reduced NMDA-induced LDH release (
Calcein-AM and PI staining results (Figure
Effects of SIRT1 overexpression on the number of living cells reduced by NMDA in the SH-SY5Y cell line. (a) Representative micrographs showing the suppression of WT-SIRT1 overexpression on NMDA-induced decrease of living cells (
The present study provided the following three important findings. First, activation of SIRT1 or overexpression of SIRT1 protected against NMDA-mediated excitotoxicity; second, the neuroprotective effects of SIRT1 on NMDA-induced excitotoxicity were attributed to its deacetylase activity; and third, inhibition of p53 acetylation might be one of the mechanisms underlying SIRT1-mediated neuroprotection.
In this study, we found that either preincubation of cortical neurons with RSV or overexpression of WT-SIRT1 in the SH-SY5Y cell line prevented NMDA-induced excitotoxicity including a decrease in cell viability, an increase in LDH release, and an increase in cell death, suggesting that SIRT1 has neuroprotection in NMDA-induced excitotoxicity. As has been reported, activation of SIRT1 using RSV has protection against disorders of the nervous system, for example, brain ischemia reperfusion injury [
Further observation shows that RSV significantly ameliorated NMDA-reduced SIRT1 deacetylase activity in primary neurons, and this amelioration was prevented when SIRT1 activity was inhibited by Sirtinol. Therefore, it raises the possibility that the deacetylase activity is required for SIRT1’s neuroprotection in the excitotoxicity model. In addition, we observed that overexpression of WT-SIRT1 reversed NMDA-induced reduction of SIRT1 mRNA, SIRT1 protein level, and SIRT1 deacetylase activity and inhibition of NMDA-induced insults of SH-SY5Y cell. However, overexpression of DN-SIRT1 increased the levels of SIRT1 mRNA and protein reduced by NMDA but had no effect on NMDA-induced decrease in the deacetylase activity and also did not inhibit subsequent excitotoxic cell death. These results clearly indicated that SIRT1 deacetylase activity is crucial to the neuroprotective effects of SIRT1 in NMDA-induced insults. A previous work by a number of other laboratories has also established that RSV potentiates SIRT1 activity and provides neuroprotection in recurrent stroke models [
Additionally, we found that overexpression of WT-SIRT1 significantly inhibited NMDA-induced p53 acetylation and subsequent neurotoxicity. However, DN-SIRT1 overexpression has no such effect. The findings suggest that SIRT1 might provide potent neuroprotection against NMDA insult through regulating p53 acetylation. As a deacetylase, SIRT1 is known to deacetylate and modulate the activity of key transcription factors, such as P53, NF-
SIRT1 is an endogenous neuroprotective factor and mediates protection through different pathways. The mechanisms of the neurotoxic effects of NMDA are very complex including calcium overload, oxidative stress, mitochondrial dysfunction, cell necrosis, and apoptosis [
In summary, a growing body of evidence has confirmed the neuroprotective effects of SIRT1. The finding of the present study suggests that SIRT1 might be a therapeutic target for certain neurological diseases related to NMDA-mediated excitotoxicity.
Silent information regulator 1
Resveratrol
Lactate dehydrogenase
Fetal bovine serum
Propidium iodide
Acetylated p53
Alzheimer’s disease
Huntington’s disease.
There are no conflicts of interest to declare.
Xiaorong Yang and Peipei Si have contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (no. 31000481, no. 31171023, and no. 81601167), the Natural Science Foundation of Shanxi Province, China (no. 2011011040-2), the Fund for Shanxi Key Subjects Construction (FSKSC) and the Students’ Innovation and Entrepreneurship Training Program of Shanxi Province (no. 2015141).