SIRT1 deacetylates GAPDH to drive microglial glycolysis and neuroinflammation.

Introduction: Microglial activation drives neuroinflammation through a metabolic switch from oxidative phosphorylation to aerobic glycolysis; however, the molecular mechanisms governing this transition remain poorly defined. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sirtuin 1 (SIRT1), lipopolysaccharide (LPS), and interferon-gamma (IFN-γ) are central to this study; GAPDH plays plays a key regulatory role in this switch, and its activity is modulated by reversible acetylation at lysine 254 (K254). It remains unclear whether sirtuin deacetylases regulate this modification in microglia. Methods: Here, we demonstrate that SIRT1 physically associates with GAPDH in murine microglia and deacetylates K254 under basal conditions. Inflammatory activation using LPS/IFN-γ reduced SIRT1 protein levels and deacetylase activity by approximately 50%, leading to a 2.5-fold increase in K254 acetylation. Pharmacological activation of SIRT1 (SRT1720) reversed this modification and enhanced glycolytic output, mimicking the effects of the deacetylation-mimetic K254R mutant. To isolate the causal role of K254, we replaced endogenous GAPDH with K254R or acetylation-mimetic (K254Q) mutant proteins. Results: K254R microglia exhibited approximately 35% higher GAPDH enzymatic activity, 40% greater glycolytic flux, and 1.6- to 2.2-fold higher secretion of TNF-α, IL-1β, IL-6, and IL-12p70 than K254Q cells. Glycolytic inhibition with 2-deoxyglucose reduced most of the excess cytokines, confirming enhanced flux as the causal factor in K254-driven inflammatory amplification. Discussion: Thus, SIRT1-GAPDH signaling represents a post-translational axis linking sirtuin activity directly to glycolytic enzyme function, distinct from SIRT1's traditional transcriptional roles and serving as a viable molecular checkpoint in microglial immunometabolism.