Microglial K+ Channels in Ischemic Stroke

缺血性中风中的小胶质细胞 K 通道

基本信息

批准号：
9886291
负责人：
HEIKE WULFF
金额：
$ 33.44万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9886291
关键词：
Acute Adult Affect Anti-Inflammatory Agents Area B-Lymphocyte Subsets B-Lymphocytes Blood Brain Brain Diseases Brain-Derived Neurotrophic Factor Cells Data Development Electrophysiology (science)Environment Exposure to Expression Profiling Female Flow Cytometry Genetic Grant Head Human Hypoxia Immune system Immunoglobulin D Immunohistochemistry Immunomodulators Immunosuppressive Agents Infarction Inflammatory Interferons Interleukin-1 Interleukin-10 Interleukin-4 Ischemic Stroke Knock-out Lead Mediating Microglia Middle Cerebral Artery Occlusion Minocycline Mus Neonatal Neuronal Injury Neurons Pathologic Pathology Phagocytosis Pharmacology Phenotype Play Potassium Channel Production Reactive Oxygen Species Recording of previous events Recovery Reducing Agents Reperfusion Therapy Reverse Transcriptase Polymerase Chain Reaction Rodent Model Role Sex Differences Signal Transduction Slice T memory cell T-Lymphocyte Testing Therapeutic Intervention Time Voltage-Gated Potassium Channel Work base cytokine design experimental study macrophage male monocyte mouse model neuroinflammation neurotrophic factor novel therapeutics patch clamp preservation response small molecule inhibitor stroke model stroke patient stroke therapy therapeutic evaluation voltage

项目摘要

Ischemic stroke elicits a strong neuroinflammatory response characterized by massive microglia activation. However, microglia do not only cause damage by releasing pro-inflammatory cytokines and reactive oxygen species, they can also exert beneficial functions. Similar to macrophages, microglia can assume a classically activated (M1-like) or alternatively activated (M2-like) phenotype. While M2-like microglia are presumably neuroprotective and anti-inflammatory and have been described to peak relatively early in rodent models of ischemic stroke, M1-polarized microglia begin to appear later in the infarct area, especially in the border zone, and expand neuronal injury. An effective anti-inflammatory treatment for stroke should therefore not be a general immunosuppressant but instead suppress microglia in a subtype specific manner by preferentially targeting pro-inflammatory microglia. Our group has a long history of studying K+ channels in the immune system and previously developed small molecule inhibitors for the voltage-gated KV1.3 and the Ca2+-activated KCa3.1 channel as immunomodulators. We recently obtained exciting new data showing that M1 and M2 microglia significantly differ in their K+ channel expression profiles and here propose to test whether KV1.3 blockers can preferentially inhibit M1-like microglia functions and preserve beneficial M2-like functions. We propose to test this therapeutic hypothesis with three interrelated Specific Aims: Under Aim-1 we will investigate the expression profile and the functional role of K+ channels in cultured M1 and M2 microglia and macrophages. In Aim-2 we will study microglia in a more “natural environment” and use organotypic slices exposed to hypoxia/aglycemia or acute slices from Cx3cr1GFP/+ mice subjected to reversible middle cerebral artery occlusion (MCAO) to determine K+ channel expression and function using whole-cell patch-clamp, immunohistochemistry, qPCR and flow cytometry. As part of these experiments we will characterize the time courses of K+ channel and M1 and M2 marker expression and correlate them with brain cytokine profiles and pathology. Parallel immunohistochemical experiments will be performed on brain sections from stroke patients to evaluate K+ channel expression in the context of M1 and M2 markers in humans. Finally, in Aim-3 we are proposing to test our hypothesis that selective targeting of M1-like microglia with KV1.3 blockers is beneficial in ischemic stroke by evaluating the effect of KV1.3 knockout and pharmacological blockade with our KV1.3 blocker PAP-1 in MCAO. These experiments will include studies where PAP-1 administration will match the time-course of the presence of KV1.3 on microglia in the infarct. Overall, we expect that KV1.3 blockade will spare beneficial microglia functions such as phagocytosis of debris and production of neurotrophic factors and preferentially target detrimental pro-inflammatory microglia functions. This strategy could be very beneficial for ischemic stroke but could also be applied to other neuroinflammatory brain disorders, where neuroinflammation is pathologically significant.

缺血性中风会引发强烈的神经炎症反应，其特征是大量小胶质细胞激活。然而，小胶质细胞不仅通过释放促炎细胞因子和活性氧造成损伤与巨噬细胞类似，小胶质细胞也可以发挥经典的作用。激活（M1 样）或替代激活（M2 样）表型，而 M2 样小胶质细胞可能是。神经保护和抗炎作用，据描述在啮齿类动物模型中相对较早达到峰值缺血性中风，M1极化小胶质细胞较晚开始出现在梗塞区域，特别是在边界区，因此，有效的抗炎治疗不应成为中风的治疗方法。一般免疫抑制剂，而是通过优先以亚型特异性方式抑制小胶质细胞我们的团队在研究免疫中的 K+ 通道方面有着悠久的历史。系统和先前开发的用于电压门控 KV1.3 和 Ca2+ 激活的小分子抑制剂 KCa3.1 通道作为免疫调节剂，我们最近获得了令人兴奋的新数据，表明 M1 和 M2。小胶质细胞的 K+ 通道表达谱存在显着差异，此处建议测试 KV1.3 是否阻滞剂可以优先抑制 M1 样小胶质细胞功能并保留有益的 M2 样功能。我们建议用三个相互关联的具体目标来检验这一治疗假设：在 Aim-1 下，我们将研究培养的 M1 和 M2 小胶质细胞中 K+ 通道的表达谱和功能作用在 Aim-2 中，我们将在更“自然的环境”中研究小胶质细胞并使用器官切片。暴露于缺氧/血糖或来自接受可逆中脑损伤的 Cx3cr1GFP/+ 小鼠的急性切片动脉闭塞 (MCAO)，使用全细胞膜片钳测定 K+ 通道表达和功能，作为这些实验的一部分，我们将表征时间。 K+ 通道以及 M1 和 M2 标记物表达的过程，并将它们与脑细胞因子谱相关联将对中风患者的脑切片进行平行免疫组织化学实验。评估人类 M1 和 M2 标记背景下的 K+ 通道表达。提议检验我们的假设，即用 KV1.3 阻滞剂选择性靶向 M1 样小胶质细胞有益于通过评估 KV1.3 敲除和用我们的 KV1.3 进行药物阻断的效果来治疗缺血性中风这些实验将包括 PAP-1 给药与 MCAO 中的阻断剂 PAP-1 相匹配的研究。梗死区小胶质细胞上 KV1.3 存在的时间过程总体而言，我们预计 KV1.3 阻断将会发生。保留有益的小胶质细胞功能，例如吞噬碎片和产生神经营养因子优先针对疼痛促炎性小胶质细胞功能，这种策略可能非常有益。缺血性中风，但也可应用于其他神经炎症性脑部疾病，其中神经炎症具有病理学意义。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4-mediated calcium entry in microglia.

DOI：
10.1002/glia.23847
发表时间：
2020-11
期刊：
Glia
影响因子：
6.2
作者：
Nguyen HM;di Lucente J;Chen YJ;Cui Y;Ibrahim RH;Pennington MW;Jin LW;Maezawa I;Wulff H
通讯作者：
Wulff H

Kv1.3 inhibition attenuates neuroinflammation through disruption of microglial calcium signaling.