Analysis of the mechanism of HIF-1alpha activation in adenosine-mediated protection

Constant oxygen supply is ultimatively required for mammalian brain. In case of reduced oxygen and energy level, sophisticated control mechanisms are initiated that should enable cells to adapt to hypoxia. Amongst them is the production of purine nucleosides e.g. adenosine that accumulates and acts as a powerful endogenous neuroprotectant by increasing cerebral blood flow, decreasing neuronal metabolism and playing important roles as intracellular messenger. These effects are coupled to the activation of specific adenosine receptors, which are highly expressed in the brain. However the detailed signaling mechanism remains ill defined. During my employment in the FWF projects 15823 and 19578 I was able to establish the protective effect of adenosine on viability and neurite outgrowth in hypoxic PC12 cells and in primary cerebellar granule neurons. My intention was then to study the molecular mechanisms of purine-mediated protection. Treating cells with adenosine resulted in an increase of p42/44 MAPK phosphorylation and subsequently in a higher HIF-1alpha protein stability. By using siRNA-mediated knockdown of p42/44 MAPK and HIF-1alpha, the adenosinergic protective effect was completely abolished. New preliminary results showed that (1) Pharmacological inhibition of MAPK by PD098059 or reduction of p42/44 MAPK by siRNA hampered adenosine-mediated HIF-1alpha stability indicating the tight connection of these two signaling molecules and (2) Pharmacological inhibition of A2A adenosine receptor by CSC (caffeine) reduced p42/44 MAPK phosphorylation and also HIF-1alpha stability, supporting the view that adenosine receptors are important mediators for purine-mediated neuroprotection. The overall goal of this project is to focus on the innovative advances in understanding the hypoxia-induced cellular response and will be achieved by addressing following specific aims: Specific aim 1: Study of the molecular signaling mechanism of HIF-1alpha in adenosine receptor-mediated "adenosinergic" neuroprotection pathway. Specific aim 2: Study of post-translational modifications and proteosomal degradation of HIF-1alpha in adenosine-mediated neuroprotection. Specific aim 3: Study of HIF-1alpha target molecules in adenosine-mediated neuroprotection. Specific aim 4: Study of cytoskeletal changes in response to stress induced by hypoxia and adenosine-mediated neuroprotection. Long-term goal: Study of neuroprotective mechanisms of purines in organotypic mouse/rat brain cultures: an alternative ischemia/reperfusion model. These insights could contribute to a better knowledge of the adenosine receptor-mediated "adenosinergic" neuroprotection pathway and should provide a basis for developing therapies for disorders caused by oxygen deprivation.

For proper brain function a permanent oxygen supply is necessary. In the case of reduced oxygen (hypoxia) and energy level, sophisticated control and protection mechanisms are initiated. Those enable cells to adapt to reduced conditions and preserve the organism from damage. Amongst them is the production of purine nucleosides e.g. adenosine that accumulates and acts as a powerful endogenous neuroprotectant by increasing cerebral blood flow, decreasing neuronal metabolism and playing important roles as an intracellular messenger. These effects are coupled to the activation of specific adenosine receptors. The aim of this project T421-B18, was to clarify the hypoxia-induced adenosine-mediated adenosinergic protection pathway by investigation of the molecular mechanisms of hypoxia- inducible factor-1alpha (HIF-1alpha).Experiments were done in neuronal PC12 cells (A2A adenosine receptor positive cells) and an incubator with controlled oxygen supply. Former studies of my working group showed the involvement of two key signaling modules in the adenosinergic pathway: i) the mitogen activated protein kinase p42/44 MAPK (Tomaselli B 2008) and 2) hypoxia-inducible factor1alpha (HIF-1alpha) (zur Nedden S. 2008). By using inhibitors and siRNA-knock down techniques I could show that adenosine is as important for p42/44 MAPK phosphorylation as for stabilization of HIF-1alpha, which again confirmed the tight alliance of the two signaling molecules. Pharmacological inhibition of A2A adenosine receptor reduced p42/44 MAPK phosphorylation and also HIF-1alpha stability, supporting the view that adenosine receptors are important mediators for purine-mediated neuroprotection.These insights could contribute to a better knowledge of the adenosine receptor-mediatedadenosinergic neuroprotection pathway and should provide a basis for developing therapies for disorders caused by oxygen deprivation.