SIRT1 deacetylates GAPDH to drive microglial glycolysis and neuroinflammation

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.

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.

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.

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.