BACKGROUND AND AIMWe recently identified protein kinase N1 (PKN1) as a master regulator of brain development. However, its function in the adult brain has not been clearly established. In this study, we assessed the cerebral energetic phenotype of wildtype (WT) and global Pkn1 knockout (Pkn1-/-) animals under physiological and pathophysiological conditions.METHODSCerebral energy metabolism was analyzed by 13C6-glucose tracing in vivo and real time seahorse analysis of extracellular acidification rates as well as mitochondrial oxygen consumption rates (OCR) of brain slice punches in vitro. Isolated WT and Pkn1-/- brain mitochondria were tested for differences in OCR with different substrates. Metabolite levels were determined by mass spectrometric analysis in brain slices under control and energetic stress conditions, induced by oxygen-glucose deprivation and reperfusion, an in vitro model of ischemic stroke. Differences in enzyme activities were assessed by enzymatic assays, western blotting and bulk RNA sequencing. A middle cerebral artery occlusion stroke model was used to analyze lesion volumes and functional recovery in WT and Pkn1-/- mice.RESULTSPkn1 deficiency resulted in a remarkable upregulation of cerebral energy metabolism, in vivo and in vitro. This was due to two separate mechanisms involving an enhanced glycolytic flux and higher pyruvate-induced mitochondrial OCR. Mechanistically we show that Pkn1-/- brain tissue exhibits an increased activity of the glycolysis rate-limiting enzyme phosphofructokinase. Additionally, glucose-1,6-bisphosphate levels, a metabolite that increases mitochondrial pyruvate uptake, were elevated upon Pkn1 deficiency. Consequently, Pkn1-/- brain slices had more ATP and a greater accumulation of ATP degradation metabolites during energetic stress. This translated into increased phosphorylation and activity of adenosine monophosphate (AMP)-activated protein kinase (AMPK) during in vitro stroke. Accordingly, Pkn1-/- brain slices showed a post-ischemic transcriptional upregulation of energy metabolism pathways and Pkn1 deficiency was strongly protective in in vitro and in vivo stroke models. While inhibition of mitochondrial pyruvate uptake only moderately affected the protective phenotype, inhibition of AMPK in Pkn1-/- slices increased post-ischemic cell death in vitro.CONCLUSIONThis is the first study to comprehensively demonstrate an essential and unique role of PKN1 in cerebral energy metabolism, regulating glycolysis and mitochondrial pyruvate-induced respiration. We further uncovered a highly protective phenotype of Pkn1 deficiency in both, in vitro and in vivo stroke models, validating inhibition of PKN1 as a promising new therapeutic target for the development of novel stroke therapies.