Stroke is the third largest cause of death in the United States and a leading cause of long-term disability. As such, an understanding of the molecular mechanisms underlying the injury process may provide novel avenues for acute intervention. One potential approach for acute intervention is to alter brain energy balance to create a favorable cellular environment to limit cellular injury. In this regard, a dysregulation of cellular energy balance has not only been implicated in neurodegeneration following ischemic stroke, but also in classical degenerative diseases (Alzheimer's and Parkinson's). Given the importance of the central nervous system in sensing, integrating, and coordinating the response to metabolic signals, an understanding of central metabolic sensors and regulators is critical. Our previous work has shown that 5'-adenosine monophosphate-activated kinase (AMPK) is one sensor that is involved in the brain response to stroke. We have found that pharmacological inhibition of AMPK significantly limits stroke injury in vivo. The overall goal of this proposal is to understand the molecular mechanisms by which AMPK is activated in stroke and how it mediates its detrimental effects. Based on previous studies, it was found that genetic deletion of the a2 catalytic subunit of AMPK provides significant protection in stroke models. Our overall hypothesis is that over-activation of AMPK in the presence of oxidative and metabolic stress as occurs in stroke, elicits """"""""metabolic failure"""""""" which selectively exacerbates neuronal cell death. This is due to inherent differences in the response to metabolic stress under ischemic conditions between neurons and astrocytes. To test this hypothesis, both in vitro (oxygen-glucose deprivation; OGD) and in vivo stroke models (middle cerebral artery occlusion; MCAO) will be utilized. To further clarify the signaling mechanisms by which AMPK activation contributes to cell death in the brain, two specific aims are proposed. The experiments in Aim 1 will utilize astrocytes and neuron cultures from AMPK a2 null mice. Using the OGD model, cell death and metabolic pathways mediated by AMPK will be studies. A variety of techniques including, Western blotting, immunocytochemistry and assays for metabolites will be utilized. Pharmacological inhibitors of proteins in the cell stress response pathway will be utilized to gain insight into signaling mechanisms.
Aim 2 will utilize transgenic mice with a targeted deletion of either the astrocyte or neuron AMPK a2 isoform. These mice will be subjected to MCAO and reperfusion. Stroke injury and outcome are assessed by histological and behavioral tests. Western blotting analysis will be performed to analyze the biochemical mediators of ischemic cell injury. Collectively, these experiments utilizing complementary in vitro and in vivo techniques and will provide insight into the molecular mechanisms and consequences of AMPK activation in stroke. ? ? ?
