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：
10913896
负责人：
Dax A Hoffman
金额：
$ 203.11万
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/10913896
关键词：
Address Adhesions Affect Affinity Aging Alleles Alzheimer&apos s Disease Alzheimer&apos s disease pathology Angelman Syndrome Anxiety Behavior Binding Binding Proteins Biochemical Biological Assay Biotin Biotinylation Brain Brain region C-terminal Cell Adhesion Molecules Cells Central Nervous System Central Nervous System Diseases Circadian Dysregulation Cognition Cognitive Complex Computer software Data Dementia Dendrites Development Disease Electroencephalography Electrophysiology (science)Emotions Environment Epilepsy Exhibits Follow-Up Studies Fragile X Syndrome Frequencies Genes Glutamate Receptor Hippocampus Home Human Human Chromosomes Impairment Implant In Vitro Incidence Individual Injections Ion Channel Isomerase Isomerism Kainic Acid Knock-in Mouse Knockout Mice Knowledge Kv4.2 channel Lead Learning Locomotion Mass Spectrum Analysis Mediating Membrane Memory Molecular Motor Mus Mutation Nerve Degeneration Nervous System Nesting Behavior Neurodevelopmental Disorder Neurons Operative Surgical Procedures Partner in relationship Pathology Pathway interactions Pattern Peptidylprolyl Isomerase Performance Phenotype Phosphorylation Phosphorylation Site Physiological Play Potassium Channel Process Property Proteins Purkinje Cells Regulation Reporting Research Reversal Learning Role Schizophrenia Seizures Sleep Sleep Disorders Slow-Wave Sleep Surface Synapses Synaptic plasticity Telemetry Testing Therapeutic Time UBE3A gene Ubiquitination age related autism spectrum disorder behavior test cell type cognitive enhancement density design detection platform drug testing experience flexibility hippocampal pyramidal neuron implantation imprint improved in vivo information processing insight interest long term memory loss of function morris water maze mouse model mutant neonatal mice neural circuit neuronal circuitry novel p38 Mitogen Activated Protein Kinase patch clamp prevent protein degradation protein function repetitive behavior response trafficking voltage gated channel

项目摘要

Kv4.2 complex regulation and its role in cognitive flexibility We have recently identified a novel molecular cascade that regulates the potassium channel Kv4.2 association with the auxiliary subunit DPP6 and membrane surface expression neurons. This cascade is initiated by various patterns of activity patterns impinging on the neuron, triggering activation of p38 mitogen-activated protein kinase, which phosphorylates the C-terminal motif T607 in Kv4.2 in an activity-dependent manner. This phosphorylation by p38 initiates subsequent isomerization by a prolyl isomerase, Pin1, that selectively binds to and isomerizes phosphor-Ser/Thr-Pro bonds. Pin1 is a ubiquitous isomerase that has been implicated in a growing number of nervous system pathologies, including Alzheimers disease, where it may protect against age-dependent neurodegeneration. To address the role of the p38-Pin1-Kv4.2 in neuronal and neural circuit function, we developed a mutant knock-in mouse model with a Thr607 to Ala substitution at the activity-induced p38 phosphorylation site (Kv4.2TA). This mutation significantly reduces p38 phosphorylation and Pin1 isomerization of this motif, and we observed impaired Kv4.2-DPP6 dynamics and loss of activity-induced internalization of Kv4.2 in these mice. Furthermore, we identified a reduction in intrinsic excitability of hippocampal CA1 pyramidal neurons using whole-cell patch clamp recordings in Kv4.2TA mice relative to WT. This reduction in excitability is traced to an increase in the density of Kv4.2-mediated outward K+ current (A-current), supporting biochemical analysis suggesting loss of Kv4.2 internalization in the Kv4.2TA mice (increased surface Kv4.2). The hypoexcitability in individual neurons observed within the hippocampus of Kv4.2TA mice extends to the circuit/network level, as we identified reduced kainic acid-induced seizure intensity and progression in these mice as well. We have found that Kv4.2TA mice exhibit normal initial learning and memory in the Morris Water Maze and Lever Press, two tests of hippocampal-dependent learning and memory. However, they exhibited better 'reversal' learning in both tests than did WT mice. This improvement in reversal learning indicates an enhancement in cognitive flexibility. These data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility- the ability to appropriately adjust ones behavior to a changing environment and is impaired in various neurodevelopmental disorders such as the autism spectrum disorder. Considering the finding that Kv4.2TA mice demonstrate enhanced cognitive flexibility Dr. Cole Malloy is pursuing the mechanisms underlying this phenotype. We are focusing on potential differences in synaptic properties between WT and Kv4.2TA mice. Results to date have revealed a novel meta-plasticity mechanism in a Kv4.2 mouse model that may provide insights into cognitive flexibility and be of interest in therapeutic design in treating neurodevelopmental disorders characterized by impairments in cognitive flexibility. Kv4.2 K+ channels are a Ube3A substrate and contributes to cognition in Angelman syndrome (AS) AS is a severe debilitating neurodevelopmental disorder with an estimated incidence of 1 in 20,000. It is caused by loss of function of imprinted genes on human chromosome 15q1113 or by mutations in the Ube3A gene, which resides in this region. Imprinting of this gene results in the exclusive expression of the maternal allele in hippocampal neurons and cerebellar Purkinje cells. Deficits of Ube3A lead to accumulation of its target proteins and thus dysregulate neuronal function. Using a TAP-MS screen of Kv4.2 interacting proteins that we developed previously, we identified Ube3A as a Kv4.2 binding protein. Follow up studies led by Dr. Hu confirmed the interaction and demonstrated that Kv4.2-Ube3A binding is activity-dependent. We show that Ube3A binds to Kv4.2 at its N-terminus, and ubiquitinates residue K103 using in vitro ubiquitination assay. Ubiquitination of a substrate by Ube3a usually causes the substrate degradation. We, therefore, examined if Kv4.2 K103 ubiquitination affects Kv4.2 protein level. The result showed that mutation of K103 significantly delayed protein loss compared to un-mutated Kv4.2 in response to AMPA treatment in cultured hippocampal neurons, suggesting K103 is required for activity induced Kv4.2 protein loss. In addition, we showed that Ube3A is associated with internalized Kv4.2. To further study the Kv4.2s role in AS, we imported a mouse model of AS where Ube3A is deleted. We find that Kv4.2 protein level and A-type K+ current are significantly elevated in hippocampus of AS mice compared to WT littermates. Seizure or neuronal activity leads to Kv4.2 protein degradation. We examined if Ube3A is required for Kv4.2 protein degradation. We find that seizure-induced Kv4.2 protein loss is abolished in AS, suggesting that seizure-induced Kv4.2 degradation requires Ube3A. Moreover, using patch clamp electrophysiology, we find deficits in mEPSC frequency and spike-timing-dependent LTP in AS mice. To further study the physiological function of Kv4.2 in AS, we generated CRE-dependent conditional Kv4.2 KO mice and crossed with Emx1-CRE mice to obtain conditional Kv4.2 KO mice (Kv4.2cKO). We then mated AS mice with Kv4.2cKO mice for Drs. Malloy and Welch to examine if electrophysiological deficits in AS mice can be rescued. A behavioral test battery for mouse models of Angelman syndrome has been developed to assess phenotypes in the domains of motor performance, repetitive behavior, anxiety and to test drugs and novel Ube3A mutants. We examined the battery in WT littermates, AS mice, Kv4.2cKO mice and AS/Kv4.2cKO DKO mice and found that locomotion and nesting behaviors can be partially rescued in the DKO mice. In learning and memory tests, AS mice showed impairments in initial learning and reversal learning. However, the deficits in AS mice in reversal learning can be rescued by DKO mice. These findings reveal a novel Ube3A downstream pathway regulating plasticity and cognitive behaviors, and provide potential targets for the treatment of AS. DPP6 impacts brain development, function, and Alzheimers disease/dementia In 2022, we reported that DPP6-KO mice show enhanced neurodegeneration associated with AD pathology. We also found that aging DPP6-KO mice display circadian dysfunction by home-cage tasks. To further study if DPP6-KO mice have sleep disorders related to AD/dementia, we used an in vivo detection system by surgical implantation of HD-XO2 implantable telemetry, and recorded EEG/EMG/ activity from aging DPP6-KO mice brains. Electrophysiological data were collected for 5 days. We used software to analyze the sleep/wake time and perform power spectral analysis. From preliminary data, we found that 12-month-old DPP6-KO mice show less total sleep time, less slow-wave sleep duration, and more wake duration compared to WT. To continue our examination of DPP6 function and its novel roles in preventing neurodegeneration diseases like AD/dementia, we are working on another in vivo assay of proximity-dependent biotin identification by ICV injection in neonatal mice with AAV-DPP6-BioID, to identify other protein that can form dynamic DPP6-binding complexes, including those involved in transient interactions during cell trafficking as well as components of synaptic adhesion. Biotinylated proteins are isolated by affinity capture and identified by mass spectrometry. We have found some interesting binding partner candidates for further confirmation and functional study. These include, for example, the cell adhesion proteins that function in synapse maturation and are involved in autism spectrum disorders, schizophrenia, and neurodegeneration diseases such as AD.

Kv4.2复合体调节及其在认知灵活性中的作用我们最近发现了一种新的分子级联，可以调节钾通道 Kv4.2 与辅助亚基 DPP6 和膜表面表达神经元的关联。该级联是由冲击神经元的各种活动模式启动的，触发 p38 丝裂原激活蛋白激酶的激活，该激酶以活性依赖性方式磷酸化 Kv4.2 中的 C 末端基序 T607。 p38 的这种磷酸化启动随后由脯氨酰异构酶 Pin1 进行的异构化，该酶选择性地结合并异构化磷酸-Ser/Thr-Pro 键。 Pin1 是一种普遍存在的异构酶，与越来越多的神经系统病理有关，包括阿尔茨海默病，它可以预防年龄依赖性神经变性。为了解决 p38-Pin1-Kv4.2 在神经元和神经回路功能中的作用，我们开发了一种突变型敲入小鼠模型，在活性诱导的 p38 磷酸化位点 (Kv4.2TA) 处将 Thr607 替换为 Ala。这种突变显着降低了该基序的 p38 磷酸化和 Pin1 异构化，并且我们在这些小鼠中观察到 Kv4.2-DPP6 动力学受损和活性诱导的 Kv4.2 内化丧失。此外，我们利用 Kv4.2TA 小鼠的全细胞膜片钳记录，相对于 WT 小鼠，发现海马 CA1 锥体神经元的内在兴奋性降低。这种兴奋性的降低可追溯到 Kv4.2 介导的外向 K+ 电流（A 电流）密度的增加，支持生化分析表明 Kv4.2TA 小鼠中 Kv4.2 内化的丧失（表面 Kv4.2 增加）。 Kv4.2TA 小鼠海马内观察到的单个神经元的低兴奋性延伸到了电路/网络水平，因为我们发现这些小鼠中红藻氨酸诱导的癫痫发作强度和进展也有所减少。我们发现 Kv4.2TA 小鼠在莫里斯水迷宫和杠杆压力机（海马依赖性学习和记忆的两项测试）中表现出正常的初始学习和记忆。然而，它们在两项测试中都表现出了比 WT 小鼠更好的“逆转”学习能力。逆向学习的这种改进表明认知灵活性的增强。这些数据有力地支持了这样的观点，即 Kv4.2 的活动依赖性调节在认知灵活性（即根据不断变化的环境适当调整行为的能力）中发挥着重要作用，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。考虑到 Kv4.2TA 小鼠表现出增强的认知灵活性这一发现，Cole Malloy 博士正在研究这种表型背后的机制。我们关注 WT 和 Kv4.2TA 小鼠之间突触特性的潜在差异。迄今为止的结果揭示了 Kv4.2 小鼠模型中的一种新的元可塑性机制，该机制可能提供对认知灵活性的见解，并对治疗以认知灵活性受损为特征的神经发育障碍的治疗设计感兴趣。 Kv4.2 K+ 通道是 Ube3A 底物，有助于天使综合征 (AS) 的认知 AS 是一种严重的神经发育障碍，发病率估计为两万分之一。它是由人类染色体 15q1113 上的印记基因功能丧失或位于该区域的 Ube3A 基因突变引起的。该基因的印记导致母体等位基因在海马神经元和小脑浦肯野细胞中独家表达。 Ube3A 的缺陷会导致其靶蛋白的积累，从而导致神经元功能失调。使用我们之前开发的 Kv4.2 相互作用蛋白的 TAP-MS 筛选，我们将 Ube3A 鉴定为 Kv4.2 结合蛋白。胡博士领导的后续研究证实了这种相互作用，并证明 Kv4.2-Ube3A 结合具有活性依赖性。我们发现 Ube3A 在其 N 末端结合 Kv4.2，并使用体外泛素化测定泛素化残基 K103。 Ube3a 对底物的泛素化通常会导致底物降解。因此，我们检查了 Kv4.2 K103 泛素化是否影响 Kv4.2 蛋白水平。结果表明，在培养的海马神经元中，与未突变的 Kv4.2 相比，K103 突变显着延迟了 AMPA 处理后的蛋白质损失，表明 K103 是活性诱导的 Kv4.2 蛋白质损失所必需的。此外，我们还发现 Ube3A 与内化 Kv4.2 相关。为了进一步研究 Kv4.2s 在 AS 中的作用，我们导入了 Ube3A 被删除的 AS 小鼠模型。我们发现与 WT 同窝小鼠相比，AS 小鼠海马中的 Kv4.2 蛋白水平和 A 型 K+ 电流显着升高。癫痫发作或神经元活动会导致 Kv4.2 蛋白降解。我们检查了 Kv4.2 蛋白降解是否需要 Ube3A。我们发现癫痫引起的 Kv4.2 蛋白丢失在 AS 中被消除，这表明癫痫引起的 Kv4.2 降解需要 Ube3A。此外，利用膜片钳电生理学，我们发现 AS 小鼠的 mEPSC 频率和尖峰时间依赖性 LTP 存在缺陷。为了进一步研究Kv4.2在AS中的生理功能，我们生成了CRE依赖性条件Kv4.2 KO小鼠，并与Emx1-CRE小鼠杂交获得条件Kv4.2 KO小鼠（Kv4.2cKO）。然后我们将 AS 小鼠与 Kv4.2cKO 小鼠交配以供博士使用。马洛伊和韦尔奇研究是否可以挽救 AS 小鼠的电生理缺陷。已开发出用于 Angelman 综合征小鼠模型的行为测试组，用于评估运动表现、重复行为、焦虑等领域的表型，并测试药物和新型 Ube3A 突变体。我们检查了 WT 同窝小鼠、AS 小鼠、Kv4.2cKO 小鼠和 AS/Kv4.2cKO DKO 小鼠的电池，发现 DKO 小鼠的运动和筑巢行为可以得到部分挽救。在学习和记忆测试中，AS小鼠表现出初始学习和逆转学习的障碍。然而，AS 小鼠的逆转学习缺陷可以通过 DKO 小鼠来弥补。这些发现揭示了一条新的 Ube3A 下游通路调节可塑性和认知行为，并为 AS 的治疗提供了潜在的靶点。 DPP6 影响大脑发育、功能和阿尔茨海默病/痴呆 2022 年，我们报道 DPP6-KO 小鼠表现出与 AD 病理相关的神经变性增强。我们还发现，衰老的 DPP6-KO 小鼠因居家笼养任务而表现出昼夜节律功能障碍。为了进一步研究 DPP6-KO 小鼠是否患有与 AD/痴呆相关的睡眠障碍，我们通过手术植入 HD-XO2 植入式遥测技术使用体内检测系统，并记录衰老 DPP6-KO 小鼠大脑的 EEG/EMG/ 活动。收集5天的电生理数据。我们使用软件来分析睡眠/唤醒时间并执行功率谱分析。从初步数据来看，我们发现与 WT 相比，12 个月大的 DPP6-KO 小鼠的总睡眠时间较短，慢波睡眠持续时间较短，觉醒持续时间较长。为了继续研究 DPP6 的功能及其在预防 AD/痴呆等神经退行性疾病中的新作用，我们正在开展另一种体内试验，通过 ICV 注射 AAV-DPP6-BioID 的新生小鼠进行邻近依赖性生物素识别，以鉴定其他可以形成动态 DPP6 结合复合物的蛋白质，包括那些参与细胞运输过程中短暂相互作用的蛋白质以及突触粘附的成分。通过亲和捕获分离生物素化蛋白质并通过质谱法鉴定。我们发现了一些有趣的结合伙伴候选者，用于进一步确认和功能研究。例如，这些蛋白包括在突触成熟中起作用并参与自闭症谱系障碍、精神分裂症和 AD 等神经退行性疾病的细胞粘附蛋白。

项目成果

期刊论文数量（24）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Effects of genetic deletion of the Kv4.2 voltage-gated potassium channel on murine anxiety-, fear- and stress-related behaviors.

DOI：
10.1186/2045-5380-2-5
发表时间：
2012-03-02
期刊：
Biology of mood & anxiety disorders
影响因子：
0
作者：
Kiselycznyk C;Hoffman DA;Holmes A
通讯作者：
Holmes A

Idiopathic Autism: Cellular and Molecular Phenotypes in Pluripotent Stem Cell-Derived Neurons.

DOI：
10.1007/s12035-016-9961-8
发表时间：
2017-08
期刊：
MOLECULAR NEUROBIOLOGY
影响因子：
5.1
作者：
Liu, Xiaozhuo;Campanac, Emilie;Cheung, Hoi-Hung;Ziats, Mark N.;Canterel-Thouennon, Lucile;Raygada, Margarita;Baxendale, Vanessa;Pang, Alan Lap-Yin;Yang, Lu;Swedo, Susan;Thurm, Audrey;Lee, Tin-Lap;Fung, Kwok-Pui;Chan, Wai-Yee;Hoffman, Dax A.;Rennert, Owen M.
通讯作者：
Rennert, Owen M.

Protein kinase a mediates activity-dependent Kv4.2 channel trafficking.

DOI：
10.1523/jneurosci.1951-08.2008
发表时间：
2008-07-23
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
0
作者：
Hammond RS;Lin L;Sidorov MS;Wikenheiser AM;Hoffman DA
通讯作者：
Hoffman DA

KCNJ5 mutations in the National Institutes of Health cohort of patients with primary hyperaldosteronism: an infrequent genetic cause of Conn's syndrome.

美国国立卫生研究院原发性醛固酮增多症患者队列中的 KCNJ5 突变：康恩综合征的罕见遗传原因。

DOI：
10.1530/erc-12-0022
发表时间：
2012-06
期刊：
Endocrine-related cancer
影响因子：
3.9
作者：
Xekouki P;Hatch MM;Lin L;Rodrigo de A;Azevedo M;de la Luz Sierra M;Levy I;Saloustros E;Moraitis A;Horvath A;Kebebew E;Hoffman DA;Stratakis CA
通讯作者：
Stratakis CA

Editorial.

社论。