Blockade of HCN2 Channels Provides Neuroprotection Against Ischemic Injury via Accelerating Autophagic Degradation in Hippocampal Neurons
Blockade of HCN2 Channels Provides Neuroprotection Against Ischemic Injury via Accelerating Autophagic Degradation in Hippocampal Neurons
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DOI：
10.1007/s12264-020-00513-7
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发表时间：
2020-06-10
影响因子：
5.6
通讯作者：
Li, Chang-Jun
中科院分区：
文献类型：
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作者：
Chen, Cheng;Liu, Li;Li, Chang-Jun
在中枢神经系统中，超极化激活的环核苷酸门控（HCN）通道对于维持正常神经元功能至关重要。近年来的研究表明，HCN通道可能参与了缺血性脑损伤的病理过程，但具体机制尚不清楚。自噬在脑缺血中被激活，但其在细胞死亡/存活中的作用仍然存在争议。本研究结果表明，HCN通道阻断剂ZD 7288可显著降低缺氧缺糖再灌注（OGD/R）诱导的海马HT 22神经元凋亡率，纠正OGD/R诱导的过度自噬。此外，在OGD/R组中，p-mTOR、p-ULK 1（Ser（757））和p62显著降低，而p-ULK 1（Ser（317））、atg 5和beclin 1显著升高。ZD 7288未改变参与调节自噬体形成的p-ULK 1（Ser（757））、ULK 1（Ser（317））、p62、Beclin 1和atg 5的表达。此外，我们发现OGD/R诱导组织蛋白酶D表达显著增加，但不诱导LAMP-1表达。在OGD/R组中，10 μ mol/L ZD 7288处理未改变组织蛋白酶D和LAMP-1的表达。然而，降低自噬体-溶酶体融合的氯喹（CQ）消除了ZD 7288对过度自噬和神经保护的纠正。此外，HCN 2通道的shRNA敲低可显著减少OGD/R和短暂性全脑缺血（TGCI）模型中LC 3-II的积累，增加神经元存活，CQ也可消除HCN 2-shRNA的作用。此外，我们发现，在暴露于OGD/R或CQ的Con-shRNA转染的HT 22神经元中，与LAMP-1阳性溶酶体共定位的LC 3阳性斑点的百分比降低。在HCN 2-shRNA转染的HT 22神经元中，与LAMP-1阳性溶酶体共定位的LC 3阳性斑点的百分比在OGD/R下增加;然而，将CQ添加到HCN 2-shRNA转染的HT 22神经元中，该百分比显著降低。目前的结果表明，HCN 2通道的阻断通过加速可归因于促进自噬体和溶酶体融合的自噬降解来提供针对OGD/R和TGCI的神经保护。
In the central nervous system, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential to maintain normal neuronal function. Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury, but the mechanisms remain unclear. Autophagy is activated in cerebral ischemia, but its role in cell death/survival remains controversial. In this study, our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) in hippocampal HT22 neurons. Furthermore, in the OGD/R group, p-mTOR, p-ULK1 (Ser(757)), and p62 were significantly decreased, while p-ULK1 (Ser(317)), atg5, and beclin1 were remarkably increased. ZD7288 did not change the expression of p-ULK1 (Ser(757)), ULK1 (Ser(317)), p62, Beclin1, and atg5, which are involved in regulating autophagosome formation. Besides, we found that OGD/R induced a significant increase in Cathepsin D expression, but not LAMP-1. Treatment with ZD7288 at 10 mu mol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1. However, chloroquine (CQ), which decreases autophagosome-lysosome fusion, eliminated the correction of excessive autophagy and neuroprotection by ZD7288. Besides, shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-II and increased neuron survival in the OGD/R and transient global cerebral ischemia (TGCI) models, and CQ also eliminated the effects of HCN2-shRNA. Furthermore, we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNA-transfected HT22 neurons exposed to OGD/R or CQ. In HCN2-shRNA-transfected HT22 neurons, the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R; however, the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons. The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.