Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance

Abstract

Background and purpose: Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain.

Methods: We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions.

Results: Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1, accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro.

Conclusions: Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders.

Keywords: brain; glucose; metabolomics; neuroprotection; resveratrol; sirtuin 1; stroke.

Fig 1. Generation of inducible, neuron-specific Sirt1 knockout mice (Sirt1^neu−/−)

(A–C) Confocal images of immunostaining from tissue sections of non-induced Sirt1^neu−/−. (A) Hipp – hippocampus; Ctx – cortex; scale bar = 400 µm. (B) Str – striatum, scale bar = 70 µm. (C) Ctx – cortex, scale bar = 20 µm. (D) Western blot for Sirt1 reveals efficient deletion upon tamoxifen treatment in major and stroke-affected brain areas but not heart. Notice the smaller size band of the mutant Sirt1 protein (Δex4 – Sirt1 mutant) in Sirt1^neu−/−; Hipp – hippocampus. Tx – tamoxifen.

Fig 2. Neuronal Sirt1 is required for RPC-induced ischemic tolerance

(A) Representative TTC-stained brain sections following tMCAo (left). Right – infarct quantified as a percentage of the total ipsilateral hemisphere (Control-Veh [n=8], Control-RPC [n=6], Sirt1^neu−/−-Veh [n=9], Sirt1^neu−/−-RPC [n=5],* = p<0.05, ns = not significant, two-way ANOVA – Bonferroni post-test). (B) Neurological scoring, where a lower value indicates better function (* = p<0.05, ns = not significant, two-way ANOVA – Bonferroni post-test). (C) Cerebral blood flow measured by laser doppler flowmetry (ns = not significant, two-way ANOVA – Bonferroni post-test). BL – baseline; ISC – ischemia; REP – reperfusion; Tx – tamoxifen.

Fig 3. Altered metabolic pathways in Sirt1^neu−/− mouse brain

(A) Biochemical analysis of non-targeted, primary metabolomics from control and Sirt1^neu−/−. Metabolites are linked based structural similarity, where color represents fold change from control and shape denotes chemical class. (B–D) mRNA levels from control and Sirt1^neu−/− hippocampi shown as fold change of control (* = p<0.05, ** = p<0.01, t-test, n=5–9). (B) Glucose transporters. (C) Glycolysis and branch point enzymes. (D) Transcription factors known to regulate glycolysis. Tx – tamoxifen.

Fig 4. Glycolytic ATP production is impaired in Sirt1^neu−/− mouse brain

Example field population spikes (fPS) stimulated maximally from control and Sirt1^neu−/− acute hippocampal slices (B) Top – examples traces of the anoxic depolarization (AD) event under different experimental conditions (IAA = iodoacetate, glycolysis inhibitor). Bottom – quantification of latency to AD, used as a readout of glycolytic function when glucose is present under anoxia (*** = p<0.001, two-way ANOVA, Bonferroni post-test, n=3–4; n/m = not measured). Tx – tamoxifen.

Fig 5. RPC increases glycolytic rate in a Sirt1-dependent manner in neurons

Measurement of glycolytic rate (ECAR – extracellular acidification rate) by Seahorse XFp in primary neuronal-enriched cultures. Energetic stress cocktail: mitochondrial uncoupling – FCCP (2 µM); Complex V inhibition – oligomycin (1 µM); arrow indicates application. (A) Naïve cells treated with RPC, Veh, Sirt1-specific inhibitor EX-527 or a combination. Measurements were taken in normal glucose (20 mmol/l) (* = p<0.05, ** = p<0.01, *** = p<0.001, ns = not significant, one-way ANOVA, Bonferroni post-test, n=5). (D) RPC treatment of naïve cells exposed to 25% of normal glucose (5 mmol/l, *** = p<0.001, ns = not significant, t-test, n=4).