Table_2_TMEM16F Aggravates Neuronal Loss by Mediating Microglial Phagocytosis of Neurons in a Rat Experimental Cerebral Ischemia and Reperfusion Model.DOCX

Cerebral ischemia is a severe, acute condition, normally caused by cerebrovascular disease, and results in high rates of disability, and death. Phagoptosis is a newly recognized form of cell death caused by phagocytosis of viable cells, and has been reported to contribute to neuronal loss in brain tissue after ischemic stroke. Previous data indicated that exposure of phosphatidylserine to viable neurons could induce microglial phagocytosis of such neurons. Phosphatidylserine can be reversibly exposed to viable cells as a result of a calcium-activated phospholipid scramblase named TMEM16F. TMEM16F-mediated phospholipid scrambling on platelet membranes is critical for hemostasis and thrombosis, which plays an important role in Scott syndrome and has been confirmed by much research. However, few studies have investigated the association between TMEM16F and phagocytosis in ischemic stroke. In this study, a middle-cerebral-artery occlusion/reperfusion (MCAO/R) model was used in adult male Sprague-Dawley rats in vivo, and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate cerebral ischemia-reperfusion (I/R) injury in vitro. We found that the protein level of TMEM16F was significantly increased at 12 h after I-R injury both in vivo and in vitro, and reversible phosphatidylserine exposure was confirmed in neurons undergoing I/R injury in vitro. Additionally, we constructed a LV-TMEM16F-RNAi transfection system to suppress the expression of TMEM16F during and after cerebral ischemia. As a result, TMEM16F knockdown alleviated motor function injury and decreased the microglial phagocytosis of viable neurons in the penumbra through inhibiting the “eat-me” signal phosphatidylserine. Our data indicate that reducing neuronal phosphatidylserine-exposure via deficiency of TMEM16F blocks phagocytosis of neurons and rescues stressed-but-still-viable neurons in the penumbra, which may contribute to reducing infarct volume and improving functional recovering.

脑缺血是一种严重且急性的病症，通常由脑血管疾病引起，导致高比例的残疾和死亡。噬亡是一种新近被识别的细胞死亡形式，由活细胞被吞噬引起，据报道，它可能参与缺血性卒中后脑组织中神经元的损失。先前的研究表明，磷脂酰丝氨酸暴露于活神经元可以诱导小胶质细胞对这些神经元的吞噬。磷脂酰丝氨酸可以通过钙激活的磷脂外翻酶TMEM16F可逆地暴露于活细胞。TMEM16F介导的血小板膜上的磷脂外翻对于止血和血栓形成至关重要，这在Scott综合征中起着重要作用，并被大量研究所证实。然而，很少有研究调查TMEM16F与缺血性卒中中吞噬作用之间的关联。在本研究中，我们利用成年雄性Sprague-Dawley大鼠的中脑动脉闭塞/再灌注（MCAO/R）模型，在体内进行培养，将神经元暴露于氧糖剥夺/再氧合（OGD/R）以模拟体外脑缺血/再灌注（I/R）损伤。我们发现，在体内和体外，TMEM16F蛋白水平在I-R损伤后12小时显著增加，并在体外确认了神经元在I/R损伤过程中可逆地暴露磷脂酰丝氨酸。此外，我们构建了一个LV-TMEM16F-RNAi转染系统，以抑制脑缺血期间及之后的TMEM16F表达。结果发现，TMEM16F敲低减轻了运动功能损伤，并减少了半影区中存活神经元的微胶质细胞吞噬，这通过抑制‘吞噬我’信号磷脂酰丝氨酸实现。我们的数据表明，通过TMEM16F缺乏减少神经元磷脂酰丝氨酸暴露，可以阻断神经元的吞噬，并挽救半影区中受压但仍存活的神经元，这可能会减少梗死体积并改善功能恢复。