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复杂的调节及其在认知灵活性中的作用我们最近确定了一种新型的分子级联反应，该级联对钾通道KV4.2与辅助亚基DPP6和膜表面表达神经元的关联。该级联反应是由撞击神经元上的各种活性模式引发的，从而触发了p38有丝分裂原激活的蛋白激酶的激活，该蛋白激酶在KV4.2中以活性依赖性的方式磷酸化了C末端基序T607。通过p38的这种磷酸化启动了通过PROLYL异构酶PIN1的随后异构化，该异构酶有选择地结合并异构化磷光 - ser/thr-Pro键。 PIN1是一种无处不在的异构酶，与越来越多的神经系统病理相关，包括阿尔茨海默氏病，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函数中的作用，我们在活动诱导的p38磷酸化位点（KV4.2TA）开发了一种用THR607替换为ALA取代的突变型敲入小鼠模型。该突变显着降低了该基序的p38磷酸化和PIN1异构化，我们观察到Kv4.2-DPP6动力学受损，以及在这些小鼠中kv4.2活性诱导的内在化的丧失。此外，我们使用KV4.2TA小鼠中的全细胞贴片夹记录相对于WT，我们确定了海马CA1锥体神经元的内在兴奋性的降低。这种兴奋性的降低可追溯到Kv4.2介导的向外K+电流（A-电流）的密度增加，支持生化分析表明KV4.2 KV4.2 KV4.2TA小鼠的内在化损失（表面KV4.2增加）。在KV4.2TA小鼠的海马中观察到的单个神经元的低脱位性扩展到电路/网络水平，因为我们确定了这些小鼠的海藻酸诱导的癫痫发作强度和进展。我们发现KV4.2TA小鼠在Morris Water Maze和Lever Press中表现出正常的初始学习和记忆，这是海马依赖性学习和记忆的两个测试。但是，他们在两种测试中表现出比WT小鼠更好的“逆转”学习。逆转学习的这种改善表明认知灵活性有所提高。这些数据强烈支持这样的观念：KV4.2的活动依赖性调节在认知灵活性中起着重要作用 - 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。考虑到KV4.2TA小鼠表现出增强的认知灵活性Cole Malloy博士正在追求这种表型的基础机制。我们专注于WT和KV4.2TA小鼠之间突触性质的潜在差异。迄今为止，结果揭示了在KV4.2小鼠模型中的一种新型元塑性机制，该机制可能会提供对认知灵活性的见解，并且在治疗以认知灵活性障碍为特征的神经发育障碍方面对治疗设计具有感兴趣。 Kv4.2 K+频道是UBE3A底物，有助于Angelman综合征的认知（AS）正如一种严重的使人衰弱的神经发育障碍，估计发病率为20,000。它是由于印迹基因在人类15q1113上的功能的丧失或位于该区域的UBE3A基因中的突变引起的。该基因的印记导致海马神经元和小脑Purkinje细胞中母体等位基因的独家表达。 UBE3A的缺陷导致其靶蛋白的积累，从而失调神经元功能。使用以前开发的KV4.2相互作用蛋白的TAP-MS屏幕，我们将UBE3A确定为Kv4.2结合蛋白。 HU博士领导的后续研究证实了这种相互作用，并证明KV4.2-UBE3A结合是活性依赖性的。我们表明，UBE3A在其N末端与Kv4.2结合，并使用体外泛素化测定法泛素将残基K103结合。 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，与未突变的Kv4.2相比，K103的突变显着延迟了蛋白质的损失，以响应培养的海马神经元的AMPA处理，这表明活性引起的KV4.2蛋白质丧失需要K103。此外，我们表明UBE3A与内部化的Kv4.2相关。为了进一步研究AS中的Kv4.2s角色，我们导入了删除ube3a的小鼠模型。我们发现，与WT同窝同els相比，AS小鼠海马的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性导致KV4.2蛋白质降解。我们检查了kv4.2蛋白质降解是否需要UBE3A。我们发现，癫痫发作诱导的KV4.2蛋白损失被取消了AS，这表明癫痫发作诱导的Kv4.2降解需要UBE3A。此外，使用斑块夹电生理学，我们发现MEPSC频率的缺陷和AS小鼠中峰值依赖性LTP。为了进一步研究AS中Kv4.2的生理功能，我们生成了CRE依赖性的条件KV4.2 KO小鼠，并与EMX1-CRE小鼠交叉以获得有条件的KV4.2 KO小鼠（KV4.2CKO）。然后，我们将小鼠与Kv4.2CKO小鼠交配。 Malloy和Welch检查是否可以挽救AS小鼠的电生理缺陷。已经开发了针对Angelman综合征小鼠模型的行为测试电池，以评估运动性能，重复行为，焦虑和测试药物和新型UBE3A突变体领域的表型。我们检查了wt同窝仔的电池，作为小鼠，KV4.2CKO小鼠和AS/KV4.2CKO DKO小鼠，发现运动和筑巢行为可以在DKO小鼠中部分营救。在学习和记忆测试中，由于小鼠在最初的学习和逆转学习中表现出障碍。但是，DKO小鼠可以挽救逆转学习中小鼠的缺陷。这些发现揭示了一种新型的UBE3A下游途径调节可塑性和认知行为，并为AS治疗提供了潜在的靶标。 DPP6影响大脑发育，功能和阿尔茨海默氏病/痴呆症在2022年，我们报道DPP6-KO小鼠显示与AD病理学相关的神经变性增强。我们还发现，老化的DPP6-KO小鼠通过家庭式任务显示昼夜节律功能障碍。为了进一步研究DPP6-KO小鼠是否患有与AD/痴呆有关的睡眠障碍，我们通过手术植入HD-XO2植入式遥测，使用了体内检测系统，并记录了衰老DPP6-KO小鼠大脑的EEG/EMG/活性。收集电生理数据5天。我们使用软件来分析睡眠/唤醒时间并执行功率谱分析。从初步数据中，我们发现12个月大的DPP6-KO小鼠的总睡眠时间较小，缓慢的睡眠持续时间较少，并且与WT相比，唤醒时间更长。为了继续检查DPP6功能及其在预防AD/痴呆等神经退行性疾病中的新作用，我们正在研究另一种在本体内测定的体内测定，即在新生儿小鼠中通过ICV注入近端依赖性生物素鉴定，与AAV-DPP6生物群中的新生儿小鼠在AAV-DPP6-BIOID中，在包括其他蛋白质相互作用的情况下，在包括动态DPP6结合的情况下进行互动，以识别其他相互作用，这些蛋白质在包括动态DPP6链接中，以构成动态dpp6链接的形式。突触粘附。生物素化蛋白通过亲和捕获分离，并通过质谱鉴定。我们发现了一些有趣的结合伙伴候选者，以进一步确认和功能研究。其中包括例如，在突触成熟中起作用并参与自闭症谱系疾病，精神分裂症和神经变性疾病（例如AD）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）

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.

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

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

Kv4.2 block of long-term potentiation is partially dependent on synaptic NMDA receptor remodeling.