PROJECT SUMMARY This is our revised competitive renewal application (R01 NS048216-16 A1, 02-04-2005 to 01-28-2021), “Na/H exchanger in microglial metabolism and function”. In this renewal proposal, we will study Na+/H+ exchanger isoform 1 (NHE1) in regulating microglial immunometabolism and cellular function in brain tissue repair. Microglial phagocytosis is important for debris clearance, remyelination, and synapse remodeling for tissue repair in neurodegenerative diseases [stroke, vascular dementia, Alzheimer’s Disease (AD), ADRD, etc.]. Therefore, modulating microglial phagocytosis functions presents a novel therapeutic strategy for neurodegenerative diseases. However, to date, the mechanisms that govern coordinated regulation of microglial energy metabolism and phagocytosis are not completely understood, filling this knowledge gap is the central scientific premise for our study. We reported previously that NHE1 protein-mediated H+ efflux (in exchange of Na+ influx) alkalinizes intracellular pH (pHi) in microglia, which promotes sustained NADPH oxidase (NOX2) activation because the latter is inhibited by low pHi. We obtained the following new findings: 1). selective deletion of microglial Nhe1 in the Cx3cr1-CreER+/- ;Nhe1f/f (cKO) mice reduced pro-inflammatory microglial responses but increased their restorative transformation after ischemic stroke; 2). Nhe1 cKO microglia exhibited increased oxidative phosphorylation (OXPHOS), post- stroke phagocytosis and spine stripping activities; 3). bulk RNAseq transcriptome analysis showed that the cKO microglia displayed significantly elevated genes encoding key rate-limiting enzymes for OXPHOS, the hallmark LXR/RXR-APOE-TREM2 pathway genes for phagocytosis and cholesterol efflux, and genes for phagolysosomal function; 4). the cKO stroke mice concurrently exhibited enhanced oligodendrogenesis and remyelination with improved cognitive functions. These findings strongly suggest that microglial NHE1 activity is involved in regulating microglial immunometabolism and functions in post-stroke brains and emerges as a novel therapeutic target. We will test our hypothesis by investigating: 1) the impact of selective deletion of microglial Nhe1 in cKO mice on phagocytosis transcriptomic and functional changes in post-stroke brains; 2) how Nhe1 cKO microglia enhance phagocytosis and cholesterol efflux for remyelination in post-stroke brains; 3) how Nhe1 cKO microglia boost mitochondrial biogenesis and OXPHOS to support cellular functions in post-stroke brains. Impact, completion of this study will improve our understanding on coordinated regulation of immunometabolism and cellular functions in microglia; and will shed light on NHE1 protein as a therapeutic target for promoting restorative microglial metabolism and functions for brain tissue repair in neurodegenerative diseases.