Department of Physiology, Shenyang Medical College, China (Y.L., Y. Yao, Y. Yang, R.C., M.-S.Z.).
Department of Pharmacology, Shenyang Pharmaceutical University, China (Y.L., X.C., H.Z., X.F., X.Y., J.Y.).
Stroke. 2021 May;52(5):1809-1821. doi: 10.1161/STROKEAHA.120.032749. Epub 2021 Apr 20.
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.
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.
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.
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.
CAPN1（钙蛋白酶 1）是一种细胞内 Ca2+ 调节的半胱氨酸蛋白酶，可在脑缺血时被激活。然而，CAPN1 激活促进脑缺血损伤的机制尚不清楚。
在本研究中，我们使用腺相关病毒介导的 CAPN1 基因敲低和药理学阻断（MDL-28170）来研究 CAPN1 在调节自噬-溶酶体途径和神经元损伤中的作用，该作用在大鼠永久性大脑中动脉闭塞体内模型和氧葡萄糖剥夺原代神经元体外模型中得到了验证。
CAPN1 在永久性大脑中动脉闭塞手术大鼠的皮质和氧葡萄糖剥夺暴露的神经元中被激活。CAPN1 的基因和药理学抑制显著减弱了体内和体外缺血诱导的溶酶体膜通透性增加以及随后的自噬底物积累。此外，抑制 CAPN1 通过减少自噬调节因子 BECN1（Beclin1）和 ATG（自噬相关基因）5 的裂解，增加自噬体的形成。重要的是，用 3-甲基腺嘌呤（class III-PI3K 抑制剂）或氯喹（溶酶体抑制剂）进行联合处理，逆转了 MDL-28170 对缺血性损伤的神经元保护作用，这表明 CAPN1 激活介导的自噬流损伤对于缺血性诱导的神经元损伤至关重要。
本研究首次证明，缺血诱导的 CAPN1 激活会损害溶酶体功能并抑制自噬体的形成，这会导致底物的积累并加重缺血诱导的神经元细胞损伤。我们的工作强调了 CAPN1 在调节脑缺血介导的自噬-溶酶体途径缺陷和神经元损伤中的重要作用。
