According to the European Cardiovascular Disease Statistics 2012 edition, over a third of deaths from cardiovascular disease in the EU are from coronary heart disease and just over a quarter are from stroke. But there is a lack of effective treatment for most stroke patients. Despite an expanding understanding of the cellular and molecular mechanisms underlying ischemia/reperfusion injury, 1000+ experimental compounds have failed at some stage of development. Thus, innovative therapeutic approaches are needed to develop an effective treatment for stroke. Preconditioning (in which a stimulus below the threshold of damage is applied, leading to tissue resistance to the same, or different stimuli given beyond damage threshold) is an attractive experimental strategy to identify endogenous protective mechanisms that could be therapeutically implemented. The proposed studies will identify a key molecular interaction underlying endogenous protective mechanisms induced by preconditioning and its potential to be exploited therapeutically.The lipid mediator sphingosine 1-phosphate (S1P) plays a role in preconditioning, protecting the heart and kidney against ischemia/reperfusion injury. Sphingosine kinase 2 (SK2) is the major S1P-synthezising enzyme in brain and is up-regulated in neurons in response to ischemia. SK2 is a BH3-only protein, that can interact with the cell death-related protein Bcl-xL, suggesting that this enzyme isoform has S1P-independent actions. Two specific aims will test in vivo and in vitro the hypothesis that various preconditioning stimuli induce tolerance to brain ischemia by a novel common mechanism involving both S1P-mediated autocrine/paracrine mechanisms (HIF1alpha - SK2 - S1P receptors - Akt - nitric oxide synthase) and intracellular protein-protein interactions inducing protective autophagy.This project will identify novel therapeutic targets and will provide a route for translation to clinical research.