Uncovering the molecular mechanisms of ischemic tolerance---the role of ATP in depolarization-induced neuroprotection

Cortical spreading depression (CSD) is a slowly propagating wave of neuronal depolarization which causes alterations in ion homeostasis, blood flow, and energy metabolism without causing irreversible damage to brain tissue. One interesting effect of CSD is that it can provide protection against a subsequent ischemic attack. However, the molecular mechanisms of this neuroprotection are still unclear. The present study was undertaken to further elucidate the molecular mechanisms of the induction of ischemic tolerance by CSD. Using a combination of molecular techniques we have investigated the role of adenosine triphosphate (ATP) in the induction of ischemic tolerance and have identified receptors, signaling pathways, channels and a transcription factor involved in the onset of protection following CSD. One of the genes known to be upregulated following CSD as well as other preconditioning stimuli is early growth response factor-1 (Egr-1). Egr-1 is a transcription factor that may control the overall genomic response to CSD. Therefore, we characterized Egr-1 expression in rat primary cortical neurons in response to conditions thought to occur during CSD. It was found that Egr-1 was upregulated following exposure to depolarization, glutamate, and, to the greatest extent, ATP. Through reverse transcription polymerase chain reaction (RT-PCR) the P2Y purinergic receptors and the protein kinase A (PKA), protein kinase C (PKC) and phospholipase C (PLC) signal transduction pathways were shown to be involved in the upregulation of Egr-1 by extracellular ATP. Rat primary cortical cultures were then exposed to extracellular ATP as a preconditioning stimulus. These cultures developed tolerance to subsequent oxygen glucose deprivation (OGD) as well as a chemical form of ischemia potassium cyanide (KCN). We also found that the P2Y purinergic receptors as well as multiple signal transduction pathways mediate the tolerance induced by ATP with the PKA and PLC pathways contributing the most. Finally, we determined that exposing cultures to potassium chloride (KCl) also elicits protection against OGD and KCN. This protection requires the release of ATP through connexin channels, specifically connexin 36 hemichannels. Through the course of this study we have found that CSD releases ATP into the extracellular space through connexin hemichannels. ATP activates P2Y purinergic receptors that induce signal transduction pathways and lead to the upregulation of transcription factors such as Egr-1, which may control the overall genomic response to preconditioning.