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电流（IA）调节树突状兴奋性，突触可塑性和学习。 我们最近发现了通过p38激酶的激活以及随后在KV4.2中C末端基序（T607）的异构化引发的新型分子级联反应，从而触发了与其辅助亚基DPP6的解离，还原性的降低和神经兴奋性。 Kv4.2 T607位点的磷酸化是由新型环境暴露或癫痫发作诱导的，并由p38 MAPK介导，而不是ERK MAPK。为了研究该级联反应对行为和神经元生理的后果，我们使用CRISPR-CAS9技术来生成一种敲击小鼠，其中异构酶结合位点被明确废除（KV4.2TA）。 小鼠是可行的，看起来正常，尽管Kv4.2-DPP6复合物的活性依赖性解离受到了损害。 Cole Malloy博士在从KV4.2TA和WT小鼠的海马切片的锥体细胞中使用了斑块电生理学，以解读p38-PIN1介导的Kv4.2调节KV4.2对神经元兴奋性的作用。他发现KV4.2TA细胞在响应体细胞注射时显示出AP发射相对于WT的降低。这种降低的兴奋性可追溯到外部体细胞斑块中KV4.2TA细胞中Kv4.2介导的电流增加。 WT中p38激酶和PIN1的药理学阻滞概括了突变对神经元放电特性和IA的影响，证实了这些效应基础的级联的特异性。 为了检测这些神经元生理学的改变如何在行为变化中表现出来，Jiahua Hu博士进行了一系列测试，以探测癫痫发作易感性，学习和记忆能力。为了响应IP Kainic Acient，KV4.2TA小鼠相对于WT小鼠，在一个小时的时间内表现出癫痫发作强度降低。癫痫发作强度的降低也可以在WT中用p38激酶的药理学块概括。因此，我们已经确定了一种新型的信号级联，该级联反应可以通过减少KV4.2下调来减轻癫痫的癫痫发作强度的治疗干预措施。 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，但是与WT小鼠相比，它们在莫里斯水迷宫中表现出更好的“逆转”学习。在操作逆转杠杆出版社中，KV4.2TA小鼠显示了改进的逆转学习。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。认知灵活性是能够适当地将行为适当地调整为不断变化的环境的能力，并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的发现，正在进行的实验利用了来自海马片中金字塔神经元的全细胞记录，以研究WT和KV4.2TA小鼠之间突触的潜在差异。 总的来说，这些实验将揭示KV4.2TA小鼠逆转学习表型的基础的细胞机制，并将进一步了解影响认知灵活性的机制。 Ca2+钾通道功能的调节。 乔纳森·墨菲（Jonathan Murphy）博士发现，由电压门控的Ca2+通道亚基CAV2.3介导的Ca2+进入。3调节KV4.2在异源表达系统中均功能和通过Ca1锥体神经元中的CA2+结合辅助亚基在CA1锥体神经元中的内源性功能，称为K+通道相互作用蛋白（Kchips）。 Kchip是钙感应分子，其中包含四个EF手，这些手机在多种疾病和疾病中失调，包括癫痫，亨廷顿氏病和阿尔茨海默氏病。他使用免疫荧光共定位，共免疫沉淀，电子显微镜，FRAP和FRET表征了CAV2.3和Kv4.2之间非Kchip无关的相互作用。我们发现，通过CAV2.3的Ca2+-entry增加了Kv4.2介导的全细胞电流，部分原因是Kv4.2表面表达增加。在海马神经元中，CAV2.3的药理学块降低了全细胞IA 33％。我们还发现，在CAV2.3敲除小鼠神经元中，全细胞IA降低了20％，并且损失了特征性的树突状IA梯度。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的CAV2.3-KV4.2复合物影响CA1海马神经元中的突触整合。 KCHIP蛋白，但不是mRNA表达，已显示在KV4.2敲除小鼠大脑中降低，这表明在没有KV4.2的情况下，Kchip蛋白降解的增加。我们假设Kchip蛋白降解取决于与Kv4.2的结合，并且在没有KV4.2的情况下，Kchip蛋白降解增加。我们旨在阐明Kchip蛋白降解的不确定的分子机制及其对KV4.2蛋白水平和功能的影响。乔·克尔兹基（Joe Krzeski）已经确定了Kchip降解的途径，并确定了HEK293细胞中KCHIP调控Kchip调控的新功能。对Kchip蛋白降解的机械理解很重要，因为它可能导致新的治疗策略来治疗Kchip失调的疾病。 DPP6在大脑发育，功能和行为中起作用 我们先前已经表明，KV4辅助亚基DPP6在调节树突状丝状形成和稳定性方面具有新的功能，从而影响突触发育和功能（Lin等，2013）。在2018年，我们报道了DPP6-KO小鼠在海马依赖性学习和记忆中受到损害，脑大小和体重较小（Lin等，2018）。 最近，使用免疫荧光和电子显微镜，在Lin Lin博士领导的项目中，我们发现了海马面积CA1中的一种新结构，该结构在DPP6-KO小鼠中与相同年龄的WT小鼠相比，该结构在DPP6-KO小鼠中的普遍性明显更高，并且这些结构在DPP6-KO小鼠中的发育早期观察到。这些新型结构似乎是大点的簇，它们共定位于Neun，突触素和铬素A.电子显微镜表明，这些结构是异常，突触前肿胀的异常，突触前肿胀，主要纤维材料，主要是偶有的外周外周，突触前活性Zones形成的，形成合成的synapses。我们发现，在DPP6-KO小鼠中存在异常水平的阿尔茨海默氏病的诊断生物标志物，包括海马CA1区域中淀粉样蛋白和APP的积累，以及高磷酸化的TAU表达的显着增加。 淀粉样蛋白和磷酸化的TAU病理学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。多路复用细胞因子阵列阵列在WT和DPP6-KO小鼠血清中检测表明，老年DPP6-KO小鼠的促炎或抗炎细胞因子的水平增加。我们还发现，在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。 我们表明DPP6-KO小鼠表现出昼夜节律功能障碍，这是阿尔茨海默氏病的常见症状。 这些结果共同表明，DPP6-KO小鼠表现出增强神经变性的症状，让人联想到与突触丧失和神经元死亡有关的新结构相关的阿尔茨海默氏病。 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。