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

• 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
• 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' 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target; novel; object recognition; patch clamp; protein complex; synaptic function; trafficking; two-photon; unpublished works; voltage; voltage gated channel

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通道是树突状兴奋性和整合，尖峰时序依赖性可塑性和长期增强的关键决定因素。 Kv4.2通道表达的下调发生在海马癫痫发作和癫痫病中，表明A型电流是新型治疗剂的靶标。为了识别KV4.2结合蛋白，工作人员科学家Jiahua Hu采用双重亲和力纯化方法（TAP）来隔离海马神经元的KV4.2蛋白质复合物。质谱分析确定了已知的蛋白质，例如Kchip家族成员和DPP6/10。 TAPMS分析还确定了异构酶是KV4.2的绑定伙伴。结合通过脑共免疫沉淀，HEK293T细胞的共免疫沉淀和肽在体外拉下来证实。异构酶与特定的Kv4.2位点结合，并且该关联受神经元活性和癫痫发作的调节。 为了确定异构酶是否以及如何调节Kv4.2的运输，后BAC Travis Tabor产生的Bungarotoxin结合位点标记的Kv4.2在第二个细胞外环上以可视化活神经元中的KV4.2。 Bungarotoxin结合位点标记的KV4.2在生化和电生理测定中表现出与WT KV4.2相似的通道特性。异构活性还可以调节Kv4.2与其辅助亚基结合。这些数据表明异构酶在调节KV4.2功能中起作用。 为了进一步研究异构酶和KV4.2通道的生理功能，我们生成了一种敲击蛋白（Ki）小鼠，其中使用CRISPR-CAS9技术专门废除了同子酶结合位点。这些小鼠是可行的，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。 但是，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。在操作逆转杠杆出版社中，Ki小鼠表现出改进的逆转学习。这些数据强烈支持以下观点：KV4.2的活动依赖性调节在认知灵活性中起着重要作用。 认知灵活性是根据不断变化的环境适当调整行为的能力。在自闭症谱系障碍（ASD）等各种神经发育障碍中，认知灵活性受到损害。 鉴于这些发现，博士后Cole Malloy研究了KV4.2的异构化如何使用急性海马切片中的全细胞贴片夹电生理学影响神经元功能。 他利用电流钳记录来检测敲入小鼠动作电势射击特性的改变。 异构酶和激酶活性的药理操作解决了异构酶引起的磷酸化和构象变化的依赖性，以进一步了解影响Kv4.2功能的分子级联反应。此外，鉴于行为结果表明认知灵活性改变了，研究了Ki小鼠的突触功能和可塑性的实验正在进行中。 Ca2+钾通道功能的调节。 除了形成KV4亚基的孔外，天然海马A型电流还需要非导导调节辅助亚基，称为K通道相互作用蛋白（KCHIPS）和二肽基肽酶样蛋白（DPLPS）。 Kchips和DPLP共同起作用，以增强KV4功能。有趣的是，在最近未发表的工作中，我们确定了一种机制，该机制通过R型电压门控Ca2+通道（CAV2.3）通过Ca2+调节电流密度。尽管明显缺乏规范CA+活化的K+通道的结构决定因素，但KV4.2通道的Ca2+调节仍会发生。蛋白质组学和亚细胞定位研究表明，含Cav2.3的含Cav2.3的钙通道可能是对CA1海马神经元中KV4.2介导的A型A型K电流（IA）的调节作用的潜在钙来源。 dostdoc jakob gutzmann确定，来自Cav2.3动物中Ca1锥体神经元的顶端树突表现出典型的Kv4.2电流密度的somato树突状梯度的严重下降，并使用了2-光子钙的成像，以调查对这种齿状动物的功能，以调查cav2.3 Ko n the Cav2.3 Ko的功能。进一步的研究表明，单个动作电位显示了波形的深刻变化。 DPP6在大脑发育，功能和行为中起作用 DPP6被称为KV4.2的辅助亚基，该亚基与众多发育和智力障碍以及神经精神病学，尤其是ASD有关。 我们之前已经报道了DPP6在调节树突状丝状形成和稳定性中的新作用，影响着突触发育和功能。今年，我们发现DPP6淘汰小鼠在学习和记忆中受到了损害。莫里斯水迷宫，T迷宫，物体空间位置，新颖的对象识别和提示以及恐惧调节任务的结果表明，DPP6-KO小鼠的学习较慢和记忆力下降。 我们继续研究行为任务中的DPP6-KO小鼠，发现DPP6-KO小鼠在海马依赖的学习和记忆中受到损害，并且脑体重较低（Lin等人，2018年）。 为了确定哪些区域影响了较小的大脑大小，我们在体内MRI进行了扫描活小鼠大脑，结果显示活体DPP6-KO小鼠在海马和小脑中特别降低了体积。 我们的发现表明，DPP6损失驱动了小头畸形，并且在阿尔茨海默氏病的标志DPP6-KO小鼠中学习和记忆障碍。 我们继续研究神经变性中的DPP6。 Kv4.2贩运 MD/PhD学生Adriano Bellotti发现了基于微管的Kv4.2在轴突与树突中基于微管的运输方面存在定量和定性差异。他通过记录500多个神经突的记录时间序列来表征这些差异，并使用货物传输的数学模型验证了意外结果。他开发了一个确定的模型，该模型佐证了货物频率的差异和一个随机模型，该模型验证了轴突与树突的轴突速度，超高率和频率的差异。