ORAI1/STIM1 INTERACTION INTERVENES IN STROKE AND IN NEUROPROTECTION INDUCED BY ISCHEMIC PRECONDITIONING THROUGH ER Ca2+ REFILLING

Disturbance of Ca2+ homeostasis in endoplasmic reticulum (ER) causes neuronal cell injury in stroke. On the other hand ischemic preconditioning (IPC), a brief non-lethal ischemic episode affording tolerance to a subsequent ischemic insult, restores ER Ca2+ homeostasis. Under physiological conditions, ER content is continuously refilled by the interaction between the ER-located Ca2+ sensor stromal interacting molecule 1 named STIM1 and the plasma membrane channel ORAI1, both underlying the store-operated calcium entry (SOCE) mechanism. However, the role played by ORAI1 and STIM1 in stroke and in IPC-induced neuroprotection during stroke remains unknown. Therefore, we explored whether ORAI1 and STIM1 might be involved in stroke pathogenesis and in IPC-induced neuroprotection. To this aim primary cortical neurons were subjected to OGD+reoxygenation (Rx) to reproduce in vitro brain ischemia. Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion (tMCAO). Expression of ORAI1 and STIM1 transcripts and proteins and immunosignals were detected by qRT-PCR, Western blot and immunocytochemistry, respectively. SOCE and Ca2+ release activated Ca2+ (CRAC) currents (ICRAC) were measured by Fura-2AM videoimaging and patch-clamp electrophysiology in whole cell configuration, respectively. The results of the present study showed that STIM1 and ORAI1 protein expression and immunosignals decreased in the ipsilesional temporoparietal cortex of rats subjected to tMCAO followed by reperfusion. Analogously, in primary hypoxic cortical neurons there was a reduction of STIM1 and ORAI1 transcripts and proteins accompanied by a decrease in SOCE and ICRAC. By contrast, IPC induced SOCE and ICRAC upregulation, preventing STIM1 and ORAI1 downregulation induced by OGD+Rx. Interestingly, the silencing of STIM1 or ORAI1 prevented IPC-induced tolerance and caused ER-stress as measured by GRP78 and caspase-3 upregulation. Collectively ORAI1 and STIM1 which participate to SOCE take part to stroke pathophysiology and play an important role in the neuroprotection induced by IPC.