Potassium Channels and Dendritic Function in Hippocampal Pyramidal Neurons

海马锥体神经元的钾通道和树突功能

基本信息

批准号：
10007496
负责人：
Dax A Hoffman
金额：
$ 178.03万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10007496
关键词：
Action Potentials Acute Address Affect Affinity Chromatography Alzheimer&apos s Disease Animals Apical Axon Behavior Behavioral Binding Binding Proteins Binding Sites Biochemical Biological Assay Brain Brain region Bungarotoxins CRISPR/Cas technology Calcium Calcium Channel Cells Central Nervous System Diseases Cerebellum Closure by clamp Co-Immunoprecipitations Cognitive Complex Cues Data Dendrites Dependence Development Dipeptidyl Peptidases Disease Down-Regulation Electrophysiology (science)Emotions Environment Epilepsy Exhibits Family member Filopodia Fragile X Syndrome Frequencies Glutamate Receptor Hippocampus (Brain)Human Image Impairment In Vitro Individual Investigation Ion Channel Isomerase Knock-in Knock-in Mouse Knockout Mice Knowledge Kv4 channel Learning Light Location Long-Term Potentiation Magnetic Resonance Imaging Mass Spectrum Analysis Mediating Memory Memory impairment Microcephaly Microtubules Modeling Molecular Molecular Conformation Mus Nerve Degeneration Neuraxis Neurites Neurodevelopmental Disorder Neurons Pathology Peptides Performance Pharmacology Phosphorylation Phosphotransferases Physiological Play Postdoctoral Fellow Potassium Potassium Channel Process Property Protein Family Proteins Proteomics Regulation Reporting Research Reversal Learning Role Scanning Scientist Seizures Series Site Slice Source Speed Structure Surface Synapses Synaptic plasticity Techniques Time Weight Work autism spectrum disorder base brain size cell type conditioned fear density doctoral student experience experimental study extracellular flexibility hippocampal pyramidal neuron improved in vivo information processing insight long term memory mathematical model morris water maze neuropsychiatry new therapeutic target novel object recognition patch clamp protein complex synaptic function trafficking two-photon unpublished works voltage voltage gated channel

项目摘要

Isomerase regulation of potassium channel trafficking and function. Kv4.2 channels are key determinants of dendritic excitability and integration, spike timing-dependent plasticity and long-term potentiation. Downregulation of Kv4.2 channel expression occurs following hippocampal seizures and in epilepsy suggesting A-type currents as targets for novel therapeutics. To identify Kv4.2 binding proteins, staff scientist Jiahua Hu employed a tandem affinity purification approach (TAP)to isolate the Kv4.2 protein complex from hippocampal neurons. Mass-spectrometry analysis identified known proteins such as KChIP family members and DPP6/10. The TAPMS assay also identified an isomerase as a binding partner of Kv4.2. The binding was confirmed by brain co-immunoprecipitation, co-expression in HEK293T cells, and peptide pull down in vitro. The isomerase binds to a specific Kv4.2 site, and the association is regulated by neuronal activity and seizure. To determine if and how the isomerase regulates the trafficking of Kv4.2, postbac Travis Tabor generated bungarotoxin binding site-tagged Kv4.2 at the second extracellular loop for visualizing Kv4.2 in live neurons. The bungarotoxin binding site-tagged Kv4.2 showed similar channel properties as WT Kv4.2 in biochemical and electrophysiological assays. The isomerizing activity may also regulate Kv4.2 binding to its auxiliary subunits. These data suggested that the isomerase plays a role in regulating Kv4.2 function. To further study the physiological function of isomerase and Kv4.2 channel, we generated a knockin (KI) mouse in which the isomerase binding site is specifically abolished using Crispr-Cas9 techniques. These mice are viable and appear normal. They showed normal initial learning and memory in Morris Water Maze. However, these Kv4.2 KI mice showed better reversal learning in Morris Water Maze than WT. In the operant reversal lever press, the KI mice displayed improved reversal learning. These data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility. Cognitive flexibility is the ability to appropriately adjust ones behavior according to a changing environment. Cognitive flexibility is impaired in various neurodevelopmental disorders such as autism spectrum disorder (ASD). In light of these findings, postdoc Cole Malloy investigated how isomerization of Kv4.2 impacts neuronal function using whole-cell patch clamp electrophysiology in acute hippocampal slices. He utilized current-clamp recordings to detect alterations in action potential firing properties in the knock-in mice. Pharmacological manipulation of isomerase and kinase activity addressed the dependence of phosphorylation and conformation change induced by the isomerase to gain further insights into the molecular cascade impacting Kv4.2 function. Furthermore, given the behavioral results showing altered cognitive flexibility, experiments investigating synaptic function and plasticity in the KI mice are underway. Ca2+ regulation of potassium channel function. In addition to pore forming Kv4 subunits, native hippocampal A-type currents require non-conducting modulatory auxiliary subunits known as K-channel interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). Both KChIPs and DPLPs work in concert to enhance Kv4 function. Interestingly, in recent unpublished work we have identified a mechanism by which Kv4.2 current density is regulated by Ca2+ via R-type voltage gated Ca2+ channels (Cav2.3). Ca2+ regulation of Kv4.2 channels occurs despite an apparent lack of the structural determinants of the canonical Ca+-activated K+ channels. Proteomic and subcellular localization studies suggest, that Cav2.3-containing voltage gated calcium channels could be a potential calcium source for a modulatory effect on Kv4.2-mediated A-type K currents (IA) in CA1 hippocampal neurons. Postdoc Jakob Gutzmann established that apical dendrites from CA1 pyramidal neurons in Cav2.3 KO animals show a severe reduction in the typical somato-dendritic gradient of Kv4.2 current density, and used 2-photon calcium-imaging to investigate the functional consequence to this lack of dendritic potassium-current in the Cav2.3 KO animals. Further investigation revealed that individual action potentials showed profound changes in waveform. DPP6 plays a role in Brain Development, Function and Behavior DPP6 is well known as an auxiliary subunit of Kv4.2 which has been associated with numerous developmental and intellectual disorders and neuropsychiatric pathologies, especially ASD. We have reported previously, a novel role for DPP6 in regulating dendritic filopodia formation and stability, affecting synaptic development and function. This year we found DPP6 knockout mice are impaired in learning and memory. Results from the Morris water maze, T-maze, Objects spatial location, Novel Object Recognition and Cued and fear conditioning tasks showed that DPP6-KO mice exhibit slower learning and reduced memory performance. We continued to study DPP6-KO mice in behavioral tasks, and found that DPP6-KO mice are impaired in hippocampus dependent learning and memory and have lower brain weight (Lin et al 2018). To determine which regions effected the smaller brain size, we performed in vivo MRI to scan the live mouse brain, the results showed live DPP6-KO mice display significantly decreased volume specifically in the hippocampus and Cerebellum. Our findings indicate DPP6-loss drives microcephaly and learning and memory impairment in DPP6-KO mice, hallmarks of Alzheimers Disease. We continue to investigate DPP6 in neurodegeneration. Kv4.2 trafficking MD/PhD student Adriano Bellotti has discovered quantitative and qualitative differences in microtubule-based transport of Kv4.2 in axons versus dendrites. He characterized these differences by recording time series of over 500 neurites, and has validated an unexpected result using mathematical models of cargo transport. He developed a deterministic model that corroborates differences in cargo frequency and a stochastic model that validates differences in puncta speed, superdiffusivity, and frequency in axons vs dendrites.

钾通道运输和功能的异构酶调节。 Kv4.2 通道是树突兴奋性和整合、尖峰时间依赖性可塑性和长期增强的关键决定因素。海马癫痫发作后和癫痫中 Kv4.2 通道表达下调，表明 A 型电流可作为新疗法的靶点。为了鉴定 Kv4.2 结合蛋白，研究员胡家华采用串联亲和纯化方法 (TAP) 从海马神经元中分离出 Kv4.2 蛋白复合物。质谱分析鉴定了已知的蛋白质，例如 KChIP 家族成员和 DPP6/10。 TAPMS 测定还鉴定出一种异构酶作为 Kv4.2 的结合伴侣。通过脑免疫共沉淀、HEK293T 细胞共表达和体外肽下拉证实了这种结合。异构酶与特定的 Kv4.2 位点结合，并且这种关联受到神经元活动和癫痫发作的调节。为了确定异构酶是否以及如何调节 Kv4.2 的运输，postbac Travis Tabor 在第二个细胞外环处生成了金环蛇毒素结合位点标记的 Kv4.2，以便在活神经元中可视化 Kv4.2。在生化和电生理学测定中，金环蛇毒素结合位点标记的 Kv4.2 显示出与 WT Kv4.2 相似的通道特性。异构化活性还可能调节 Kv4.2 与其辅助亚基的结合。这些数据表明异构酶在调节 Kv4.2 功能中发挥作用。为了进一步研究异构酶和 Kv4.2 通道的生理功能，我们构建了一种敲入 (KI) 小鼠，其中异构酶结合位点使用 Crispr-Cas9 技术被特异性消除。这些小鼠能够存活并且看起来正常。他们在莫里斯水迷宫中表现出正常的初始学习和记忆能力。然而，这些 Kv4.2 KI 小鼠在莫里斯水迷宫中表现出比 WT 更好的逆转学习能力。在操作性反转杠杆按压中，KI 小鼠表现出改善的反转学习能力。这些数据有力地支持了 Kv4.2 的活动依赖性调节在认知灵活性中发挥重要作用的观点。认知灵活性是指根据不断变化的环境适当调整自己行为的能力。各种神经发育障碍（例如自闭症谱系障碍（ASD））的认知灵活性都会受到损害。根据这些发现，博士后 Cole Malloy 使用全细胞膜片钳电生理学在急性海马切片中研究了 Kv4.2 异构化如何影响神经元功能。他利用电流钳记录来检测敲入小鼠动作电位放电特性的变化。异构酶和激酶活性的药理学操作解决了异构酶诱导的磷酸化和构象变化的依赖性，以进一步了解影响 Kv4.2 功能的分子级联。此外，鉴于行为结果显示认知灵活性发生改变，研究 KI 小鼠突触功能和可塑性的实验正在进行中。 Ca2+ 调节钾通道功能。除了孔形成 Kv4 亚基之外，天然海马 A 型电流还需要称为 K 通道相互作用蛋白 (KChIP) 和二肽基肽酶样蛋白 (DPLP) 的非传导调节辅助亚基。 KChIP 和 DPLP 协同工作以增强 Kv4 功能。有趣的是，在最近未发表的工作中，我们发现了一种机制，通过该机制，Kv4.2 电流密度由 Ca2+ 通过 R 型电压门控 Ca2+ 通道 (Cav2.3) 调节。尽管明显缺乏典型 Ca+ 激活的 K+ 通道的结构决定因素，但 Kv4.2 通道的 Ca2+ 调节仍然发生。蛋白质组学和亚细胞定位研究表明，含有 Cav2.3 的电压门控钙通道可能是对 CA1 海马神经元中 Kv4.2 介导的 A 型 K 电流 (IA) 产生调节作用的潜在钙源。博士后 Jakob Gutzmann 证实 Cav2.3 KO 动物 CA1 锥体神经元的顶端树突显示出 Kv4.2 电流密度的典型体细胞树突梯度严重降低，并使用 2 光子钙成像来研究其功能后果Cav2.3 KO 动物中缺乏树突状钾电流。进一步的研究表明，个体动作电位的波形发生了深刻的变化。 DPP6 在大脑发育、功能和行为中发挥作用众所周知，DPP6 是 Kv4.2 的辅助亚基，与许多发育和智力障碍以及神经精神病理，尤其是自闭症谱系障碍有关。我们之前报道过，DPP6 在调节树突状丝状伪足形成和稳定性、影响突触发育和功能方面具有新作用。今年我们发现DPP6基因敲除小鼠的学习和记忆能力受损。 Morris 水迷宫、T 迷宫、物体空间位置、新物体识别以及提示和恐惧调节任务的结果表明，DPP6-KO 小鼠表现出学习速度减慢和记忆性能下降。我们继续研究 DPP6-KO 小鼠的行为任务，发现 DPP6-KO 小鼠海马依赖性学习和记忆受损，脑重量较低 (Lin et al 2018)。为了确定哪些区域影响了较小的大脑尺寸，我们对活体小鼠大脑进行了体内 MRI 扫描，结果显示活体 DPP6-KO 小鼠的海马体和小脑体积显着减小。我们的研究结果表明，DPP6 缺失会导致 DPP6-KO 小鼠出现小头畸形以及学习和记忆障碍，这是阿尔茨海默病的标志。我们继续研究 DPP6 在神经退行性疾病中的作用。 Kv4.2 贩运医学博士/博士生 Adriano Bellotti 发现轴突与树突中基于微管的 Kv4.2 运输存在定量和定性差异。他通过记录 500 多个神经突的时间序列来描述这些差异，并使用货物运输的数学模型验证了意想不到的结果。他开发了一个确定性模型来证实货物频率的差异，并开发了一个随机模型来验证轴突与树突中的点速度、超扩散性和频率的差异。