From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Italy (A.S., T.P., V.T., F.B., A.V., R.C., A.P., P.M., G.P.).
IRCCS SDN, Naples, Italy (L.A.).
Stroke. 2019 May;50(5):1240-1249. doi: 10.1161/STROKEAHA.118.024115.
Background and Purpose- Disturbance of endoplasmic reticulum (ER) Ca homeostasis causes neuronal cell injury in stroke. By contrast, ischemic preconditioning (IPC)-a brief sublethal ischemic episode affording tolerance to a subsequent ischemic insult-restores ER Ca homeostasis. Under physiological conditions, ER calcium content is continuously refilled by the interaction between the ER-located Ca sensor STIM (stromal interacting molecule) 1 and the plasma membrane channel ORAI1 (a structural component of the CRAC calcium channel)-2 key mediators of 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. Methods- Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia and IPC were induced in rats by transient middle cerebral artery occlusion. Expression of ORAI1 and STIM1 transcripts and proteins and their immunosignals were detected by qRT-PCR, Western blot, and immunocytochemistry, respectively. SOCE and Ca release-activated Ca currents (I) were measured by Fura-2 AM video imaging and patch-clamp electrophysiology in whole-cell configuration, respectively. Results- STIM1 and ORAI1 protein expression and immunosignals decreased in the ipsilesional temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion followed by reperfusion. Analogously, in primary hypoxic cortical neurons, STIM1 and ORAI1 transcript and protein levels decreased concurrently with SOCE and Ca release-activated Cacurrents. By contrast, IPC induced SOCE and Ca release-activated Cacurrent upregulation, thereby preventing STIM1 and ORAI1 downregulation induced by oxygen and glucose deprivation+reoxygenation. Silencing of STIM1 or ORAI1 prevented IPC-induced tolerance and caused ER stress, as measured by GRP78 (78-kDa glucose regulated protein) and caspase-3 upregulation. Conclusions- ORAI1 and STIM1, which participate in SOCE, take part in stroke pathophysiology and play an important role in IPC-induced neuroprotection.
背景与目的-内质网（ER）钙稳态紊乱会导致中风时神经元细胞损伤。相比之下，缺血预处理（IPC）-即短暂的亚致死性缺血发作，使机体对随后的缺血性损伤产生耐受-恢复 ER 钙稳态。在生理条件下，ER 钙含量通过 ER 定位钙传感器 STIM（基质相互作用分子）1 与质膜通道 ORAI1（CRAC 钙通道的结构成分）-2 的相互作用得以持续补充，这是储存操作钙内流（SOCE）机制的关键介质。然而，ORAI1 和 STIM1 在中风和中风期间 IPC 诱导的神经保护中的作用尚不清楚。因此，我们探讨了 ORAI1 和 STIM1 是否可能参与中风发病机制和 IPC 诱导的神经保护。方法- 原代皮质神经元氧和葡萄糖剥夺+复氧以复制体外脑缺血。通过短暂性大脑中动脉闭塞诱导大鼠局灶性脑缺血和 IPC。通过 qRT-PCR、Western blot 和免疫细胞化学分别检测 ORAI1 和 STIM1 转录物和蛋白质及其免疫信号。通过 Fura-2 AM 视频成像和全细胞膜片钳电生理学分别测量 SOCE 和 Ca 释放激活的 Ca 电流（I）。结果- 短暂性大脑中动脉闭塞再灌注后继发性大脑中动脉同侧颞顶皮质中 STIM1 和 ORAI1 蛋白表达和免疫信号降低。类似地，在原代缺氧皮质神经元中，STIM1 和 ORAI1 转录物和蛋白水平的降低与 SOCE 和 Ca 释放激活的 Ca 电流同时发生。相比之下，IPC 诱导 SOCE 和 Ca 释放激活的 Ca 电流上调，从而防止由氧和葡萄糖剥夺+复氧诱导的 STIM1 和 ORAI1 下调。沉默 STIM1 或 ORAI1 可阻止 IPC 诱导的耐受，并导致 GRP78（78kDa 葡萄糖调节蛋白）和 caspase-3 上调的 ER 应激。结论- 参与 SOCE 的 ORAI1 和 STIM1 参与中风病理生理学，并在 IPC 诱导的神经保护中发挥重要作用。
