Microglial K+ Channels in Ischemic Stroke

缺血性中风中的小胶质细胞 K 通道

• 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
• 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

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.

缺血性卒中引发以大量小胶质细胞激活为特征的强烈神经炎症反应。 然而，小胶质细胞不仅通过释放促炎细胞因子和活性氧来引起损伤 物种，它们也可以发挥有益的功能。与巨噬细胞类似，小胶质细胞可以呈现典型的 活化的（M1样）或替代地活化的（M2样）表型。虽然M2样小胶质细胞可能是 神经保护性和抗炎性，并已被描述为在啮齿动物模型中相对较早达到峰值， 缺血性中风时，M1极化的小胶质细胞开始出现在梗死区，尤其是边缘区， 并扩大神经元损伤。因此，有效的中风抗炎治疗不应该是一种 但是相反地，通过优先地抑制小胶质细胞， 针对促炎性小胶质细胞我们小组在免疫系统中研究K+通道有很长的历史， 系统和以前开发的电压门控KV1.3和Ca 2+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 小胶质细胞在它们的K+通道表达谱上有显著差异，这里提出测试KV1.3是否 阻断剂可以优先抑制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：在目标1下，我们 将研究K+通道在培养的M1和M2小胶质细胞中的表达谱和功能作用 和巨噬细胞。在Aim-2中，我们将在更“自然的环境”中研究小胶质细胞，并使用器官型切片 暴露于缺氧/无血糖或来自Cx 3cr 1GFP/+小鼠的急性切片， 动脉闭塞（MCAO）以使用全细胞膜片钳测定K+通道表达和功能， 免疫组织化学、qPCR和流式细胞术。作为这些实验的一部分，我们将描述 K+通道和M1和M2标志物表达的过程，并将它们与脑细胞因子谱相关联， 病理平行的免疫组化实验将在中风患者的脑切片上进行 在人类M1和M2标志物的背景下评估K+通道表达。最后，在Aim-3中， 提出测试我们的假设，即选择性靶向M1样小胶质细胞与KV1.3阻滞剂是有益的， 通过评估KV1.3基因敲除和药物阻断对缺血性卒中的影响， 阻断剂PAP-1。这些实验将包括PAP-1给药将与对照组相匹配的研究。 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封锁将 保留有益的小胶质细胞功能，如吞噬碎片和产生神经营养因子， 优先靶向有害的促炎性小胶质细胞功能。这一战略可能非常有益于 缺血性中风，但也可以适用于其他神经炎性脑疾病，其中 神经炎症在病理学上是显著的。

项目成果

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.

• DOI: 10.1080/19336950.2020.1853943
• 发表时间: 2021-12
• 期刊: Channels (Austin, Tex.)
• 作者: Fomina AF;Nguyen HM;Wulff H
• 通讯作者: Wulff H

Immunocytoprotection after reperfusion with Kv1.3 inhibitors has an extended treatment window for ischemic stroke.

• DOI: 10.3389/fphar.2023.1190476
• 发表时间: 2023
• 期刊: Frontiers in pharmacology
• 影响因子: 5.6