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: 8736870
• 负责人: Dax A Hoffman
• 金额: $ 148.9万
• 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/8736870
• 关键词: Action Potentials; Adaptor Signaling Protein; Adult; Affect; Alzheimer' s Disease; Amyloid; Animals; Apical; Axon; Back; Binding; Biological Assay; Biophysics; Brain; Brain region; C-terminal; Calcium; Cell Adhesion; Cell Line; Central Nervous System Diseases; Chronic; Clathrin Adaptors; Cocaine; Code; Collaborations; Communication; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Dendrites; Dendritic Spines; Development; Disease; Distal; Dominant-Negative Mutation; Dopamine Receptor; Drug Addiction; Emotions; Epilepsy; Exhibits; Extracellular Domain; Extracellular Matrix; Fibronectins; Filopodia; Fluorescence Recovery After Photobleaching; Frequencies; Gene Targeting; Genetic; Genomics; Glutamate Receptor; Hippocampus (Brain); Image; Impairment; In Situ Hybridization; Information Storage; Injection of therapeutic agent; Interneurons; Investigation; Journals; Knock-out; Knockout Mice; Knowledge; Kv4 channel; Kv4.2 channel; Lead; Learning; Long-Term Potentiation; Measures; Medial; Mediating; Membrane; Memory; Molecular; Movement; Mus; Myosin ATPase; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; N-terminal; Nature; Neuraxis; Neuroglia; Neurons; Neurosciences; Nucleus Accumbens; Paper; Pathway interactions; Pattern; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Play; Population; Postdoctoral Fellow; Potassium Channel; Prefrontal Cortex; Preparation; Probability; Property; Protein Subunits; Proteins; Publishing; Rattus; Receptor Activation; Recording of previous events; Relative (related person); Reporting; Research Personnel; Resistance; Role; Shapes; Signal Transduction; Surface; Synapses; Synaptic plasticity; System; Tail; Techniques; Testing; The Sun; Ubiquitin; Variant; Vertebral column; Virus; Withdrawal; Work; Yeasts; autism spectrum disorder; cell motility; cocaine exposure; density; dopamine transporter; electrical property; experience; follow-up; graduate student; hippocampal pyramidal neuron; in vivo; mutant; neuron development; neuronal cell body; neurophysiology; overexpression; patch clamp; postsynaptic; protein complex; psychostimulant; receptor; receptor expression; research study; response; small hairpin RNA; trafficking; transmission process; ubiquitin ligase; voltage; voltage gated channel

Kv4.2 trafficking in CA1 pyramidal neuron dendrites. We previously reported that neuronal stimulation results in a redistribution of Kv4.2 channels away from dendritic spines to the dendritic shaft. This activity-dependent redistribution of Kv4.2 required activation of NMDA-type glutamate receptors and calcium influx, two requirements shared with synaptic plasticity, which is thought to underlie learning and memory. Given the nonuniform distribution of Kv4.2 channels in CA1 dendrites, Mike Nestor performed experiments to test the hypothesis that Kv4.2 channels are differentially trafficked at different regions along the apical dendrite during basal activity and upon stimulation in CA1 neurons. Proximal (50-150 um from the soma, primary and oblique) and distal (>200 um) apical dendrites were selected. The fluorescence recovery after photobleaching (FRAP) technique was used to measure basal cycling rates of EGFP-tagged Kv4.2 (Kv4.2g). We found that the cycling rate of Kv4.2 channels was one order of magnitude slower at both primary and oblique dendrites between 50-150 um from the soma. Kv4.2 channel cycling increased significantly at 200-250 um from the soma. Expression of a Kv4.2 mutant lacking a phosphorylation site for protein kinase-A (Kv4.2gS552A) abolished this distance-dependent change in channel cycling; demonstrating that phosphorylation by PKA underlies the increased mobility in distal dendrites. These results indicate that distance-dependent Kv4.2 mobility is regulated by activity-dependent phosphorylation of Kv4.2 by PKA. Postbac Josh Lee has followed up this project, aiming to uncover the pathway of internalized Kv4.2 as well as proteins that may be involved in directing its path. To date, he has shown that ubiquitin and ubiquitin ligase Nedd4-2 interact with Kv4.2 when overexpressed in a non-neuronal cell line. In the same cell line, he has shown that ubiquitin facilitates degradation of Kv4.2 compared to the basal rate of degradation. We are simultaneously conducting experiments to visually track the movement of Kv4.2. Colocalizing Kv4.2 with markers of well characterized endosomal compartments will reveal its pathway. In a collaboration with the Juan Bonifacino lab, Postbac Laura Long is examining the interaction of Kv4.2 with the clathrin-associated adaptor protein complex AP-1A. AP-1A is responsible for intracellular trafficking and has recently been implicated in determining receptor polarity in neurons. Molecular approaches have confirmed that Kv4.2 does indeed interact with AP-1A, and coimmunoprecipitation and yeast in situ hybridization assays suggest that this binding occurs on both the N terminal and C terminal tails of Kv4.2. She is currently investigating the functional implications of this interaction through imaging and shRNA knockouts. Functional role of the Kv4.2 auxiliary subunits: DPP6 Studies in heterologous expression systems have shown that Kv4.2 subunits interact with transmembrane DPP6 proteins to regulate channel trafficking and properties. The DPP6 auxiliary subunit protein, which is expressed in CA1 neurons, has recently been identified in large copy-number variants screens from some populations as an Autism Spectrum Disorder and ALS target gene. DPP6 enhances the opening probability of Kv4 channels and increases channel surface expression in heterologous systems. In dendritic recordings from DPP6 knock out mice, graduate student Wei Sun discovered that DPP6 is critical for generating the A-type K+ current gradient observed in CA1 dendrites. The loss this gradient led to hyper-excitable dendrites, with implications for information storage and coding. This year, researcher Lin Lin showed in a Nature Communications paper that knockdown and genetic deletion of DPP6 reveals its importance for the formation and stability of dendritic filopodia during early neuronal development. Additionally, hippocampal neurons lacking DPP6 showed a sparser dendritic branching pattern along with fewer spines throughout development and into adulthood. In electrophysiological and imaging experiments we showed that these deficits lead to fewer functional synapses and occur independently of the potassium channel subunit Kv4.2. We report that the extracellular domain of DPP6 interacts with a filopodia-associated myosin as well as with fibronectin in the extracellular matrix. DPP6 therefore plays an unexpected but important role in cell-adhesion and motility, impacting hippocampal synaptic development and function. Role of Kv4.2 channels in synaptic plasticity and development We have found that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced A-type K+ current (IA) showed a decrease in relative synaptic NR2B/NR2A subunit composition and do not exhibit a form of synaptic plasticity called long-term potentiation or LTP. Conversely, reducing IA by expression of a Kv4.2 dominant negative or through genomic knockout of Kv4.2 led to an increased fraction of synaptic NR2B/NR2A and enhanced LTP. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDA receptor activation to control synaptic NMDA receptor expression and plasticity. Additional advances from postdoc Eunyoung Kim included an investigation into the role of Kv4.2 in controlling the expression of synaptic NMDA receptors in vivo and during development. Synaptic NR2B fraction is developmentally regulated with implications for synaptic plasticity and learning and memory as well as diseases associated with learning impairments. Dr. Kim published a 2013 Journal of Neuroscience paper showing that in vivo injection of virus to alter Kv4.2 expression levels bidirectionally regulates NR2B subunit expression throughout development. Dendritic intrinsic excitability changes in disease Intrinsic excitability changes have been observed upon drug addiction. Cocaine is an addictive drug with psychostimulant effects that are attributed to inhibition of the dopamine transporter, which increases dopaminergic transmission. Chronic exposure to cocaine leads to neurodaptations in several voltage membrane conductances of neurons localized in the medial prefrontal cortex (mPCF) and nucleus accumbens. Postdoc Emily Campanac published a paper this year showing repeated cocaine exposure increases fast-spiking interneuron excitability in the rat mPFC. After cocaine withdrawal, interneurons showed an increase in action potential firing, increased input resistance, and decreased hyperpolarization-activated current. We also observed a reduction in miniature excitatory postsynaptic currents, whereas miniature inhibitory postsynaptic current activity was unaffected. In animals with cocaine history, dopamine receptor D(2) activation was less effective in increasing interneuron intrinsic excitability. Interestingly, these alterations are only observed 1 wk or more after the last cocaine exposure. This suggests that the dampening of D(2)-receptor-mediated response may be a compensatory mechanism to rein down the excitability of interneurons. Neuronal hyperexcitability is an early feature of Alzheimers disease. The underlying cellular mechanisms are unclear however. Postbac Ben Throesch directly examined dendritic excitability by patch-clamp recordings on dendrites of hippocampal neurons in mice expressing increased levels of amyloid-β. In a paper in preparation, he describes his results showing that neuronal dendrites were hyper-excitable due to a decrease in Kv4.2 expression, while the soma showed normal firing.

Kv4.2 在 CA1 锥体神经元树突中的运输。 我们之前报道过神经元刺激导致 Kv4.2 通道从树突棘重新分布到树突轴。 Kv4.2 的这种活动依赖性重新分布需要 NMDA 型谷氨酸受体的激活和钙流入，这两个要求与突触可塑性相同，而突触可塑性被认为是学习和记忆的基础。 鉴于 CA1 树突中 Kv4.2 通道的分布不均匀，Mike Nestor 进行了实验来检验以下假设：在基础活动期间和 CA1 神经元受到刺激时，Kv4.2 通道在沿顶端树突的不同区域有差异性传输。选择近端（距体体50-150 um，初级和斜向）和远端（> 200 um）顶端树突。光漂白后荧光恢复 (FRAP) 技术用于测量 EGFP 标记的 Kv4.2 (Kv4.2g) 的基础循环率。我们发现，在距体体 50-150 um 之间的初生树突和斜树突处，Kv4.2 通道的循环速率均慢一个数量级。 Kv4.2 通道循环在距离体细胞 200-250 um 处显着增加。 缺乏蛋白激酶 A 磷酸化位点的 Kv4.2 突变体 (Kv4.2gS552A) 的表达消除了通道循环中这种距离依赖性的变化；证明 PKA 磷酸化是远端树突活动性增加的基础。 这些结果表明，距离依赖性 Kv4.2 迁移性是通过 PKA 对 Kv4.2 的活性依赖性磷酸化来调节的。 Postbac Josh Lee 一直在跟进这个项目，旨在揭示内化 Kv4.2 的途径以及可能参与指导其途径的蛋白质。迄今为止，他已经证明，当在非神经元细胞系中过度表达时，泛素和泛素连接酶 Nedd4-2 会与 Kv4.2 相互作用。在同一细胞系中，他证明与基础降解率相比，泛素促进 Kv4.2 的降解。我们同时进行实验以直观地跟踪 Kv4.2 的运动。将 Kv4.2 与充分表征的内体区室标记物共定位将揭示其途径。 Postbac Laura Long 与 Juan Bonifacino 实验室合作，正在研究 Kv4.2 与网格蛋白相关衔接蛋白复合物 AP-1A 的相互作用。 AP-1A 负责细胞内运输，最近被认为参与确定神经元中受体的极性。分子方法已经证实Kv4.2确实与AP-1A相互作用，并且免疫共沉淀和酵母原位杂交分析表明这种结合发生在Kv4.2的N端和C端尾部。她目前正在通过成像和 shRNA 敲除研究这种相互作用的功能影响。 Kv4.2辅助亚基的功能作用：DPP6 异源表达系统的研究表明，Kv4.2 亚基与跨膜 DPP6 蛋白相互作用，调节通道运输和特性。 DPP6 辅助亚基蛋白在 CA1 神经元中表达，最近在一些人群的大拷贝数变异筛选中被鉴定为自闭症谱系障碍和 ALS 靶基因。 DPP6 增强 Kv4 通道的开放概率并增加异源系统中的通道表面表达。 在 DPP6 敲除小鼠的树突记录中，研究生 Wei Sun 发现 DPP6 对于产生在 CA1 树突中观察到的 A 型 K+ 电流梯度至关重要。 该梯度的损失导致树突过度兴奋，这对信息存储和编码产生影响。 今年，研究人员Lin Lin在《自然通讯》的一篇论文中表明，DPP6的敲除和基因删除揭示了它对于早期神经元发育过程中树突状丝状伪足的形成和稳定性的重要性。 此外，缺乏 DPP6 的海马神经元在整个发育和成年期表现出更稀疏的树突分支模式以及更少的棘。 在电生理学和成像实验中，我们表明这些缺陷会导致功能性突触减少，并且独立于钾通道亚基 Kv4.2 发生。 我们报道 DPP6 的细胞外结构域与丝状伪足相关的肌球蛋白以及细胞外基质中的纤连蛋白相互作用。 因此，DPP6 在细胞粘附和运动中发挥着意想不到但重要的作用，影响海马突触的发育和功能。 Kv4.2 通道在突触可塑性和发育中的作用 我们发现改变功能性 Kv4.2 表达水平会导致 CA1 突触的快速、双向重塑。表现出增强的 A 型 K+ 电流 (IA) 的神经元表现出相对突触 NR2B/NR2A 亚基组成的减少，并且不表现出称为长时程增强或 LTP 的突触可塑性形式。相反，通过表达 Kv4.2 显性失活或通过基因组敲除 Kv4.2 来减少 IA 会导致突触 NR2B/NR2A 比例增加和 LTP 增强。 我们的数据表明，A 型 K+ 通道是突触复合体的组成部分，突触复合体通过自发 NMDA 受体激活来调节 Ca2+ 信号传导，从而控制突触 NMDA 受体的表达和可塑性。 博士后 Eunyoung Kim 的其他进展包括对 Kv4.2 在体内和发育过程中控制突触 NMDA 受体表达的作用的研究。 突触 NR2B 部分受到发育调节，对突触可塑性、学习和记忆以及与学习障碍相关的疾病有影响。 Kim 博士在 2013 年《神经科学杂志》上发表了一篇论文，表明体内注射病毒来改变 Kv4.2 表达水平可以在整个发育过程中双向调节 NR2B 亚基表达。 疾病中的树突内在兴奋性变化 药物成瘾时观察到内在兴奋性变化。可卡因是一种具有精神兴奋作用的成瘾药物，其归因于抑制多巴胺转运蛋白，从而增加多巴胺能传递。长期接触可卡因会导致位于内侧前额皮质 (mPCF) 和伏核的神经元的几种电压膜电导发生神经适应。博士后 Emily Campanac 今年发表的一篇论文表明，反复接触可卡因会增加大鼠 mPFC 中快速峰值的中间神经元兴奋性。 可卡因戒断后，中间神经元表现出动作电位放电增加、输入电阻增加和超极化激活电流减少。我们还观察到微型兴奋性突触后电流减少，而微型抑制性突触后电流活动不受影响。在有可卡因历史的动物中，多巴胺受体 D(2) 激活在增加中间神经元内在兴奋性方面效果较差。有趣的是，这些变化仅在最后一次接触可卡因后 1 周或更长时间才能观察到。这表明 D(2) 受体介导的反应的抑制可能是抑制中间神经元兴奋性的一种补偿机制。 神经元过度兴奋是阿尔茨海默病的早期特征。然而，潜在的细胞机制尚不清楚。 Postbac Ben Throesch 通过膜片钳记录表达淀粉样蛋白-β 水平增加的小鼠海马神经元树突，直接检查了树突兴奋性。在一篇正在准备的论文中，他描述了他的结果，表明神经元树突由于 Kv4.2 表达的减少而过度兴奋，而体细胞则显示出正常的放电。