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: 10266491
• 负责人: Dax A Hoffman
• 金额: $ 198.1万
• 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/10266491
• 关键词: Affect; Age; Alzheimer' s Disease; Alzheimer’s disease biomarker; Amyloid; Anti-Inflammatory Agents; Area; Astrocytes; Axon; Behavior; Behavioral; Binding; Binding Sites; Brain; Brain region; C-terminal; CRISPR/Cas technology; Calcium; Cells; Central Nervous System Diseases; Characteristics; Chromogranin A; Circadian Dysregulation; Cognitive; Complex; Data; Dendrites; Detection; Development; Disease; Dissociation; Down-Regulation; EF Hand Motifs; Electron Microscopy; Electrophysiology (science); Emotions; Environment; Epilepsy; Exhibits; Filopodia; Fluorescence Resonance Energy Transfer; Fragile X Syndrome; Glutamate Receptor; Hippocampus (Brain); Hour; Human; Huntington Disease; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Impairment; Individual; Injections; Ion Channel; Isomerase; Kainic Acid; Knock-in Mouse; Knockout Mice; Knowledge; Kv4 channel; Lead; Learning; Light; MAP Kinase Gene; Mediating; Memory; Microglia; Microtubules; Molecular; Mus; Mutation; Nerve Degeneration; Neuraxis; Neurites; Neurodevelopmental Disorder; Neurons; Pathology; Pathway interactions; Peripheral; Pharmacology; Phenotype; Phosphorylation; Phosphotransferases; Physiology; Play; Potassium Channel; Predisposition; Process; Property; Proteins; Pyramidal Cells; Regulation; Reporting; Research; Reversal Learning; Role; Seizures; Series; Serum; Signal Transduction; Site; Slice; Specificity; Structural defect; Structure; Surface; Swelling; Symptoms; Synapses; Synaptic plasticity; Synaptophysin; System; Techniques; Testing; Therapeutic Intervention; Time; Vertebral column; Weight; Whole-Cell Recordings; aged; autism spectrum disorder; base; brain size; cell type; cognitive enhancement; common symptom; cytokine; diagnostic biomarker; doctoral student; experience; experimental study; flexibility; hippocampal pyramidal neuron; hyperphosphorylated tau; improved; information processing; insight; long term memory; mathematical model; morris water maze; neuroinflammation; neuron loss; neuronal excitability; novel; novel therapeutic intervention; p38 Mitogen Activated Protein Kinase; patch clamp; presynaptic; protein degradation; response; targeted treatment; tau-1; trafficking; voltage; voltage gated channel

Isomerase regulation of potassium channel trafficking and function. The transient voltage-gated K+ current (IA) mediated by Kv4.2 in CA1 hippocampal pyramidal neurons regulates dendritic excitability, synaptic plasticity, and learning. Weve recently identified a novel molecular cascade initiated by the activation of p38 kinase and subsequent isomerization of a C-terminal motif (T607) in Kv4.2 that triggers dissociation from its auxiliary subunit DPP6, a reduction IA and increase of neuronal excitability. The phosphorylation of Kv4.2 T607 site is induced by novel environment exposure or seizure and is mediated by P38 MAPK but not ERK MAPK. To investigate the consequences of this cascade on behavior and neuronal physiology, we used Crispr-Cas9 techniques to generate a knockin mouse in which the isomerase binding site is specifically abolished (Kv4.2TA). The mice are viable and appear normal although activity-dependent dissociation of the Kv4.2-DPP6 complex is impaired. Dr. Cole Malloy used patch clamp electrophysiology in pyramidal cells of hippocampal slices from Kv4.2TA and WT mice to decipher the role of p38-Pin1-mediated regulation of Kv4.2 on neuronal excitability. He found that Kv4.2TA cells displayed a reduction in AP firing relative to WT in response to somatic current injections. This reduced excitability is traced to increased Kv4.2-mediated current in Kv4.2TA cells in outside-out somatic patches. Pharmacological block of both p38 kinase and Pin1 in WT recapitulated the impact of the mutation on neuronal firing properties and IA, confirming the specificity of this cascade underlying these effects. To detect how these alterations in neuronal physiology may manifest in behavioral changes, Dr. Jiahua Hu performed a battery of tests probing seizure susceptibility and learning and memory capability. In response to IP kainic acid injection, Kv4.2TA mice exhibited reduced seizure intensity over an hour-long period relative to WT mice. The reduced seizure intensity, too, could be recapitulated in WT with pharmacological block of p38 kinase. Therefore, we have identified a novel signaling cascade that can be a target for therapeutic intervention to mitigate seizure intensity in epilepsy by reducing Kv4.2 downregulation. Furthermore, Kv4.2TA mice exhibit normal initial learning and memory in the Morris Water Maze however they exhibited better 'reversal' learning in Morris Water Maze than did WT mice. In the operant reversal lever press, the Kv4.2TA mice displayed improved reversal learning. The 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 to a changing environment and is impaired in various neurodevelopmental disorders such as the autism spectrum disorder. In light of the findings that Kv4.2TA mice exhibit enhanced cognitive flexibility, ongoing experiments have utilized whole-cell recordings from pyramidal neurons in hippocampal slice to investigate potential differences in synaptic properties between WT and Kv4.2TA mice. Collectively, these experiments will reveal the cellular mechanisms underlying the reversal learning phenotype in Kv4.2TA mice and will provide further insight into mechanisms impacting cognitive flexibility. Ca2+ regulation of potassium channel function. Dr. Jonathan Murphy found that Ca2+ entry mediated by the voltage-gated Ca2+ channel subunit Cav2.3 regulates Kv4.2 function both in a heterologous expression system and endogenously in CA1 pyramidal neurons through Ca2+ binding auxiliary subunits known as K+ channel interacting proteins (KChIPs). KChIPs are calcium-sensing molecules containing four EF-hands which are dysregulated in a number of diseases and disorders including epilepsy, Huntingtons disease, and Alzheimers disease. He characterized a KChIP-independent interaction between Cav2.3 and Kv4.2 using immunofluorescence colocalization, coimmunoprecipitation, electron microscopy, FRAP, and FRET. We found that Ca2+-entry via Cav2.3 increases Kv4.2-mediated whole-cell current due in part to an increase in Kv4.2 surface expression. In hippocampal neurons, pharmacological block of Cav2.3 reduced whole-cell IA 33%. We also found an 20% reduction in whole-cell IA in Cav2.3 knockout mouse neurons with a loss of the characteristic dendritic IA gradient. Furthermore, the Cav2.3-Kv4.2 complex was found to regulate the size of synaptic currents and spine Ca2+ transients. These results reveal an intermolecular Cav2.3-Kv4.2 complex impacting synaptic integration in CA1 hippocampal neurons. KChIP protein, but not mRNA expression, has been shown to be reduced in Kv4.2 knock-out mouse brains, suggesting increased KChIP protein degradation in the absence of Kv4.2. We hypothesized that KChIP protein degradation is dependent on binding to Kv4.2 and that there is increased KChIP protein degradation in the absence of Kv4.2. We aimed to elucidate the undetermined molecular mechanism of KChIP protein degradation and its effect on Kv4.2 protein levels and function. Joe Krzeski has identified the pathway through which KChIP is degraded and a novel function for KChIP regulation of Kv4.2 in HEK293 cells. A mechanistic understanding of KChIP protein degradation is important as it may lead to new therapeutic strategies to treat diseases in which KChIPs are dysregulated. DPP6 plays a role in Brain Development, Function and Behavior We have previously shown that the Kv4 auxiliary subunit DPP6 has a novel function in regulating dendritic filopodia formation and stability, affecting synaptic development and function (Lin et al. 2013). In 2018, we have reported that DPP6-KO mice are impaired in hippocampus-dependent learning and memory, with smaller brain size and weight (Lin et al. 2018). Recently, using immunofluorescence and electron microscopy, in a project lead by Dr. Lin Lin, we have discovered a novel structure in hippocampal area CA1 that was significantly more prevalent in DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appeared as clusters of large puncta that colocalized NeuN, synaptophysin, and chromogranin A. Electron microscopy revealed that these structures are abnormal, enlarged presynaptic swellings filled with mainly fibrous material with occasional peripheral, presynaptic active zones forming synapses. We found diagnostic biomarkers of Alzheimers disease present in abnormal levels in DPP6-KO mice including accumulation of amyloid and APP in the hippocampal CA1 area and a significant increase in expression of hyper-phosphorylated tau. The amyloid and phosphorylated tau pathologies were associated with neuroinflammation characterized by activation of microglia and astrocytes. Multiplex cytokine array detection with WT and DPP6-KO mouse blood serum showed that levels of proinflammatory or anti-inflammatory cytokines increased in aged DPP6-KO mice. We also found that activated astrocytes and microglia were significantly increased in DPP6-KO brain sections. We show that DPP6-KO mice display circadian dysfunction, a common symptom of Alzheimer disease. Together these results indicate that DPP6-KO mice show symptoms of enhanced neurodegeneration reminiscent of Alzheimers disease associated with a novel structure resulting from synapse loss and neuronal death. 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.

钾通道运输和功能的异构酶调节。 CA1 海马锥体神经元中 Kv4.2 介导的瞬态电压门控 K+ 电流 (IA) 调节树突兴奋性、突触可塑性和学习。 我们最近发现了一种新的分子级联反应，该级联反应是由 p38 激酶的激活和随后 Kv4.2 中 C 端基序 (T607) 的异构化引发的，从而触发其辅助亚基 DPP6 的解离，从而减少 IA 并增加神经元兴奋性。 Kv4.2 T607 位点的磷酸化是由新的环境暴露或癫痫发作诱导的，并且由 P38 MAPK 而不是 ERK MAPK 介导。为了研究这种级联对行为和神经元生理学的影响，我们使用 Crispr-Cas9 技术生成了异构酶结合位点被特异性消除的敲入小鼠 (Kv4.2TA)。 尽管 Kv4.2-DPP6 复合物的活性依赖性解离受损，但小鼠仍能存活并表现正常。 Cole Malloy 博士在 Kv4.2TA 和 WT 小鼠海马切片的锥体细胞中使用膜片钳电生理学来破译 p38-Pin1 介导的 Kv4.2 调节对神经元兴奋性的作用。他发现 Kv4.2TA 细胞响应体细胞电流注射而表现出相对于 WT 的 AP 放电减少。这种兴奋性的降低可归因于外部体细胞斑块中 Kv4.2TA 细胞中 Kv4.2 介导的电流增加。 WT 中 p38 激酶和 Pin1 的药理学阻断概括了突变对神经元放电特性和 IA 的影响，证实了这些效应背后的级联的特异性。 为了检测神经元生理学的这些变化如何在行为变化中体现出来，胡家华博士进行了一系列测试，探讨癫痫易感性以及学习和记忆能力。与 WT 小鼠相比，腹腔注射红藻氨酸后，Kv4.2TA 小鼠在一个小时内表现出癫痫发作强度降低。 WT 中通过药物阻断 p38 激酶也可以重现癫痫发作强度的降低。因此，我们发现了一种新的信号级联反应，可以作为治疗干预的目标，通过减少 Kv4.2 下调来减轻癫痫发作强度。 此外，Kv4.2TA 小鼠在莫里斯水迷宫中表现出正常的初始学习和记忆，但它们在莫里斯水迷宫中表现出比 WT 小鼠更好的“逆转”学习。在操作性反转杠杆按压中，Kv4.2TA 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的行为的能力，并且在各种神经发育障碍（例如自闭症谱系障碍）中受到损害。 鉴于 Kv4.2TA 小鼠表现出增强的认知灵活性，正在进行的实验利用海马切片中锥体神经元的全细胞记录来研究 WT 和 Kv4.2TA 小鼠之间突触特性的潜在差异。 总的来说，这些实验将揭示 Kv4.2TA 小鼠逆转学习表型的细胞机制，并将进一步深入了解影响认知灵活性的机制。 Ca2+ 调节钾通道功能。 Jonathan Murphy 博士发现，电压门控 Ca2+ 通道亚基 Cav2.3 介导的 Ca2+ 进入可通过称为 K+ 通道相互作用蛋白 (KChIP) 的 Ca2+ 结合辅助亚基，在异源表达系统和 CA1 锥体神经元中内源性调节 Kv4.2 功能。 KChIP 是含有四个 EF 手的钙感应分子，这些分子在癫痫、亨廷顿病和阿尔茨海默病等多种疾病和病症中失调。他利用免疫荧光共定位、免疫共沉淀、电子显微镜、FRAP 和 FRET 表征了 Cav2.3 和 Kv4.2 之间不依赖于 KChIP 的相互作用。我们发现，通过 Cav2.3 进入 Ca2+ 会增加 Kv4.2 介导的全细胞电流，部分原因是 Kv4.2 表面表达的增加。在海马神经元中，Cav2.3 的药理学阻断使全细胞 IA 降低 33%。我们还发现 Cav2.3 敲除小鼠神经元的全细胞 IA 减少了 20%，并且特征性树突 IA 梯度丧失。此外，发现 Cav2.3-Kv4.2 复合物可以调节突触电流和脊柱 Ca2+ 瞬变的大小。这些结果揭示了分子间 Cav2.3-Kv4.2 复合物影响 CA1 海马神经元的突触整合。 Kv4.2 敲除小鼠大脑中 KChIP 蛋白（而非 mRNA 表达）减少，表明在 Kv4.2 缺失的情况下 KChIP 蛋白降解增加。我们假设 KChIP 蛋白降解依赖于与 Kv4.2 的结合，并且在缺少 Kv4.2 的情况下 KChIP 蛋白降解会增加。我们的目的是阐明 KChIP 蛋白降解的未确定分子机制及其对 Kv4.2 蛋白水平和功能的影响。 Joe Krzeski 确定了 KChIP 降解的途径以及 KChIP 在 HEK293 细胞中调节 Kv4.2 的新功能。对 KChIP 蛋白降解的机制理解非常重要，因为它可能会带来新的治疗策略来治疗 KChIP 失调的疾病。 DPP6 在大脑发育、功能和行为中发挥作用 我们之前已经证明，Kv4 辅助亚基 DPP6 在调节树突丝状伪足的形成和稳定性、影响突触发育和功能方面具有新功能（Lin et al. 2013）。 2018年，我们报道DPP6-KO小鼠的海马依赖性学习和记忆能力受损，大脑尺寸和重量更小（Lin et al. 2018）。 最近，在Lin Lin博士领导的一个项目中，我们利用免疫荧光和电子显微镜发现了海马CA1区的一种新结构，与同龄的WT小鼠相比，该结构在DPP6-KO小鼠中显着更常见，并且这些结构在DPP6-KO小鼠的发育早期就观察到了。这些新颖的结构表现为大点簇，与 NeuN、突触素和嗜铬粒蛋白 A 共存。电子显微镜显示这些结构是异常的、扩大的突触前肿胀，主要充满纤维材料，偶尔有外周、突触前活性区形成突触。我们发现 DPP6-KO 小鼠中阿尔茨海默病的诊断生物标志物水平异常，包括海马 CA1 区淀粉样蛋白和 APP 的积累以及过度磷酸化 tau 蛋白表达的显着增加。 淀粉样蛋白和磷酸化 tau 蛋白病理与以小胶质细胞和星形胶质细胞激活为特征的神经炎症相关。使用 WT 和 DPP6-KO 小鼠血清进行的多重细胞因子阵列检测表明，老年 DPP6-KO 小鼠中促炎或抗炎细胞因子的水平增加。我们还发现 DPP6-KO 脑切片中活化的星形胶质细胞和小胶质细胞显着增加。 我们发现 DPP6-KO 小鼠表现出昼夜节律功能障碍，这是阿尔茨海默病的常见症状。 这些结果共同表明，DPP6-KO 小鼠表现出神经退行性增强的症状，让人想起与突触丢失和神经元死亡导致的新结构相关的阿尔茨海默病。 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。