CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage
CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage
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DOI：
10.1161/strokeaha.120.032749
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发表时间：
2021-05-01
期刊：
影响因子：
8.3
通讯作者：
Zhou, Ming-Sheng
中科院分区：
文献类型：
--
作者：
Liu, Yueyang;Che, Xiaohang;Zhou, Ming-Sheng
背景与目的：钙蛋白酶1（calpain 1）是一种细胞内钙离子调节的半胱氨酸蛋白酶，在脑缺血时可被激活。方法：本研究采用腺相关病毒介导的CAPN 1基因敲低和药物阻断（MDL-28170）技术，在大鼠永久性大脑中动脉闭塞模型和体外缺氧缺糖模型中，研究CAPN 1在自噬-溶酶体通路和神经元损伤中的作用。CAPN 1的遗传和药理学抑制显著减弱了缺血诱导的溶酶体膜透化和随后的体内和体外自噬底物积累。此外，CAPN 1的抑制通过减少自噬调节因子BECN 1（Beclin 1）和ATG（自噬相关基因）5的切割来增加自噬体的形成。重要的是，MDL-28170对缺血性损伤的神经元保护作用可通过与III类-PI 3 K（磷脂酰肌醇3-激酶）抑制剂3-甲基腺嘌呤或溶酶体抑制剂氯喹（氯喹），提示CAPN 1激活介导的自噬流损伤对脑缺血诱导的神经元损伤至关重要。本研究首次证明了缺血诱导的CAPN 1活化损害溶酶体功能并抑制自噬体形成，其有助于底物的积累并加重缺血诱导的神经元细胞损伤。我们的工作突出了CAPN 1在脑缺血介导的自噬-溶酶体途径缺陷和神经元损伤的调节中的重要作用。
Background and Purpose:CAPN1 (calpain1)-an intracellular Ca2+-regulated cysteine protease-can be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined.Methods:In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucose-deprived primary neuron in vitro model.Results:CAPN1 was activated in the cortex of permanent middle cerebral occlusion-operated rats and oxygen-glucose deprivation-exposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage.Conclusions:The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemia-mediated autophagy-lysosomal pathway defects and neuronal damage.