Voltage-dependent potassium channel Kv4.2 promotes neurogenesis and alleviates ischemic stroke impairments

OBJECTIVE Stroke has become the top ten leading cause of death in China. Ischemic stroke accounts for 85% of stroke cases,and insufficiency of cerebral blood supply caused by atherosclerosis is one of the important causes of ischemic stroke.Therefore, it is of positive significance to study the molecular mechanism of stroke injury caused by hypoperfusion in the search for drug targets. Voltage-dependent potassium channels are a family of potassium channels widely expressed in the central nervous system. However, their roles in neurogenesis after stroke insults have not been clearly illustrated. The purpose of this experiment is to explore the expression changes of different subfamilies of voltage-dependent potassium channels after the occurrence of ischemic stroke and their influence on neuroregeneration, to study the molecular mechanism of stroke injury caused by hypoperfusion, and to find potential targets for drug therapy of ischemic stroke. METHODS C57 BL/6 mice aged 7-8 weeks and C17.2 cells were used in vivo and in vitro in the experiment.The mice in the experimental group were suffered from bilateral common carotid artery occlusion（BCCO） for 1 h and reperfusion for 7 d. In the control group, bilateral carotid artery was dissected without occlusion. Behavioral assay of suspension test were performed to assess the motor deficits of the mice. In this assay, the time of the first drop（latency）, the number of drops within one minute（frequency）, and the final scores were recorded as the results of athletic ability. A lower score indicated more severe motor damage of the mice. TTC staining was used to observe the cerebral infarction areas caused by ligation of bilateral common carotid arteries. After seven days, mice were sacrificed and brain tissue protein samples were collected for real-time quantitative PCR（RT-PCR） and Western blotting test to detect the changes of potassium channel subfamily expression levels in different brain regions. Neuronal injuries in all brain regions were detected using Nissl staining methods 7 d following model establishment. To detect the effects and the underlying mechanism of the related potassium channel on neurogenesis, recombinant plasmids of the potassium channels were transfected in cultured C17.2 neural stem cells. Afterwards, oxygen glucose deprivation experiments were performed. RESULTS Behavioral tests showed that BCCO can cause impaired motor performance. TTC staining showed that cerebral infarction existed in the striatum region, and the motor function decline caused by the injury in this region was consistent with the behavioral experiment results which verified the effectiveness of our surgical operation.Nissl staining revealed a large amount of neuronal cell necrosis in the cortex and striatum regions,and dense neuronal cells in the lateral ventricular limbic region, suggesting that neurogenesis may have occurred in this region. The results of realtime quantitative RT-PCR showed that among the detected potassium channels distributed in the measured nervous system, the expression of voltage-dependent potassium channel Kv4.2 decreased significantly in all brain regions after stroke, suggesting that it may be involved in the pathological process of stroke. Immunohistochemical staining showed that there was neurogenesis in the subgranular zone（SGZ） and subventricular zone（SVZ） of the mice, and Kv4.2 expression was significantly changed in the regions, suggesting that it may be involved in the regulation of neuro regeneration after stroke.The transfected Kv4.2 plasmid enhanced the differentiation of the C17.2 neural stem cells to neurons and astrocytes under normoxia and the oxygen-glucose deprivation, suggesting that Kv4.2 may induce the differentiation of neural stem cells after stroke. Kv4.2 could induce the neural stem cells to differentiate into neurons in vitro and in vivo, and Western blotting assay showed that Kv4.2 could up-regulate the expression level of ERK1/2, p-ERK1/2, p-STAT3, NGF, p-TtkA,and BDNF. Moreover, the calcium ions and CAMK Ⅱ was also increased by Kv4.2 in vitro.CONCLUSION BCCO insults can induce the expressions of the potassium channels in the brains, among which the expression of Kv4.2 is down-regulated in the cerebral cortex, hippocampus and striatum. In vitro experiments confirmed that Kv4.2 can induce the differentiation of C17.2 neural stem cells into neurons and astrocytes under the condition of normoxia and oxygenglucose deprivation. We concluded that Kv4.2 possibly promoted neurogenesis through ERK1/2/STAT3, NGF/TrkA, and Ca;/CAMK Ⅱ signal pathways after stroke. Regulating the physiological functions of Kv4.2 channel might contribute to the rehabilitation of neuronal damage after stroke.