ABSTRACT Myeloid cells play a critical role in CNS demyelination and axonal destruction of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The early phase of the disease is characterized by the presence of pathogenic activated macrophages (M1 type), while the recovery phase is associated with alternatively-activated macrophages (M2 type) which release anti-inflammatory cytokines that resolve the pathogenic inflammation. Activated M1 macrophages depend on glycolysis to boost biosynthetic pathways to produce inflammatory mediators. However, anti-inflammatory M2 macrophages rely primarily on mitochondrial respiration. Adenosine monophosphate-activated protein kinase (AMPK) regulates energy metabolism, and thus controls the balance between glycolysis and mitochondrial respiration. We reported previously that AMPKα1 knockout (KO) mice develop severe EAE indicating AMPK activation is protective, yet the molecular mechanism by which AMPK regulates EAE disease progression is not known. AMPKα1-KO macrophages exhibit a hyper- inflammatory phenotype and have a lower rate of metabolism. AMPKα1-KO macrophages also show glycolysis- tricarboxylic acid (TCA) cycle remodeling, which results in an imbalance in the levels of the endogenous metabolites, succinate and itaconate, which regulate pro- and anti-inflammatory macrophage functions, respectively. Their levels are tightly controlled by succinate dehydrogenase (SDH) and immune responsive gene 1 (IRG1), respectively. We hypothesize that the loss of AMPKα1 remodels the glycolytic-TCA pathway causing an imbalance in the levels of succinate and itaconate, which promotes an M1 phenotype over an M2 phenotype. This, in turn, promotes Th17 cells and suppresses T regulatory cells leading to a hyperinflammatory CNS immune response and CNS tissue damage. To test our hypothesis, we have generated monocyte-specific AMPKα1 KO and macrophage-specific, constitutively active AMPKα1T172D transgenic mice. In Aim 1, we will examine how the loss or gain of function of AMPKα1 in macrophages regulates M1 versus M2 macrophage polarization and consequently, Th17 and Tregs differentiation and disease outcomes. Studies under Aim 2 will elucidate the mechanism by which the loss of AMPKα1 reprograms glycolysis-TCA metabolism leading to an imbalance of succinate and itaconate metabolites in macrophages, which in turn, determine the macrophage phenotype. The proposed study is expected to have a positive impact by elucidating the metabolic regulatory mechanism responsible for macrophage plasticity during disease and investigating AMPKα1 as a potential therapeutic target for MS. Our innovative genetic mouse models and precise metabolomics approach will allow us to identify the apparent rewiring of cellular metabolic pathways specific to AMPKα1 in hyperinflammatory cells. Ultimately, this process could be exploited to tailor novel therapeutic strategies to resolve or limit autoimmune inflammation in the CNS.