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: 8941488
• 负责人: Dax A Hoffman
• 金额: $ 170.48万
• 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/8941488
• 关键词: Action Potentials; Adult; Affect; Age; Alabama; Alzheimer' s Disease; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Apical; Back; Behavioral; Binding Proteins; Binding Sites; Brain; Brain region; Calcium; Calcium Channel; Calcium Signaling; Calcium Spikes; Cell surface; Cells; Central Nervous System Diseases; Cesium; Cessation of life; Collaborations; Complex; Cysteine; Cysteine-Rich Domain; Data; Dementia; Dendrites; Dendritic Spines; Diagnosis; Dipeptidyl Peptidases; Disulfides; Early Diagnosis; Emotions; Epilepsy; Etiology; Exhibits; Extracellular Domain; Family; Family member; Filopodia; Fragile X Mental Retardation Protein; Fragile X Syndrome; Glutamate Receptor; Goals; Gray unit of radiation dose; Hippocampus (Brain); Human; Individual; Inherited; Integral Membrane Protein; Intellectual functioning disability; Ion Channel; Kinetics; Knowledge; Kv4.2 channel; Mass Spectrum Analysis; Mediating; Memory; Memory Loss; Memory impairment; Messenger RNA; Molecular; Mus; N-terminal; Neuraxis; Neurons; Pathogenesis; Pathway interactions; Patients; Pattern; Physiological; Physiology; Play; Potassium Channel; Prevention; Process; Property; Protein Binding; Proteins; Recovery; Regulation; Research; Role; Senile Plaques; Sodium; Staging; Surface; Synapses; Synaptic plasticity; Synaptosomes; Testing; Time; Transgenic Mice; Translations; Transmembrane Domain; Ubiquitin; Ubiquitination; Universities; Voltage-Gated Potassium Channel; Wild Type Mouse; Work; base; cell type; density; experience; extracellular; hippocampal pyramidal neuron; information processing; long term memory; mRNA Stability; male; multicatalytic endopeptidase complex; network dysfunction; neuron development; neuron loss; neuronal cell body; neuronal excitability; new therapeutic target; novel; overexpression; pre-clinical; protein complex; protein expression; protein folding; research and development; research study; response; signal processing; trafficking; voltage; voltage gated channel

Neuronal excitability in Alzheimer's disease Alzheimers disease (AD), the most common form of dementia, is characterized by progressive neuronal loss, which eventually leads to death. Despite massive efforts over the last few decades, the etiology of AD is not well understood. A major challenge for AD research, and for the development of treatments, is that most AD patients are not diagnosed until neuronal function is irreversibly compromised. Therefore, it is crucial to identify neuronal changes at pre-clinical stages, which could provide a basis for early diagnosis and help to identify novel therapeutic targets. Neuronal hyperexcitability occurs early in the pathogenesis of AD and contributes to network dysfunction in AD patients. Although the Beta-amyloid (Ab) hypothesis suggests AD is caused by extracellular accumulation of insoluble Ab plaques, increasing evidence suggests that synaptic and memory impairments are mediated by soluble Ab. Here, in collaboration with the Roberson lab at the University of Alabama, Ben Throesch, tested the hypothesis that Ab- induced hyperexcitability originates in the dendrites. We found that dendrites, but not somata, of hippocampal neurons were hyperexcitable in mice adult mice overexpressing Aβ. This dendritic hyperexcitability was associated with selective depletion of Kv4.2, a dendritic potassium channel important in regulation of dendritic excitability and synaptic plasticity. In a separate project Eun Young Kim and Jakob Gutzmann investigated synaptic changes in young, 2-month-old transgenic mice that overexpress human amyloid precursor protein (hAPP). At this age, the mice do not exhibit Ab plaque accumulation but show increased soluble Ab levels compared to non-transgenic (NTG) mice. Our findings suggest NMDARs as a possible target for prevention or treatment for memory loss in early stage of AD. We are currently testing the effect of NMDAR subunit antagonists on the progression of AD at both the cellular and behavioral level. The role of DPP6 domain in its localization and function Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K+ channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Lin Lin found that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER. We showed recently (Lin et at., 2013) that DPP6 regulates the formation and stability of dendritic filopodia during early neuronal development, which is independent of Kv4.2. In order to identify additional DPP6 binding proteins, TAP purification approach was employed by Jiahhua Hu to isolate DPP6 protein complex in hippocampal neurons. Mass spectrometry analysis identified known proteins such as Kv4 family members and numerous novel synaptic proteins which Jiahua Hu and Jung Park are currently examining. Dendritic trafficking of voltage-gated calcium channels We are currently investigating the expression and trafficking of the voltage gated calcium channel Cav2.3. Cav2.3 is highly expressed in the dendrites of hippocampal and cortical neurons, where it is capable of generating large calcium spikes in response to both back-propagating action potentials and synaptic activity. Thus, alterations in Cav2.3 mRNA localization and translation could have a dramatic impact on cellular excitability and calcium signaling. Recent evidence suggests that Cav2.3 mRNA can be targeted by the Fragile-X mental retardation protein (FMRP), an mRNA binding protein that regulates translation in dendritic spines. Loss of FMRP results in Fragile X Syndrome, the most common form of inherited intellectual disability in humans. Thus, we are investigating the possibility that FMRP can regulate translation of Cav2.3, and will determine if this regulation may underlie aspects of Fragile X Syndrome. Toward this goal, Ying Liu performed real time PCR on mRNA isolated from the hippocampi or cortex of wild-type and FMRP-KO male mice, and examined the mRNA levels of several dendritic proteins. When comparing hippocampi from FMRP-KO and wild-type mice at 3- or 8-weeks of age, she found no significant difference in the mRNA levels of Cav2.3, Kv4.2, PSD-95, and HCN-1. She will further characterize this regulation by identifying FMRP binding sites on Cav2.3. In conjunction with these experiments, Ivan Trang is determining how FMRP affects Cav2.3 protein expression. From synaptoneurosomes isolated from mouse cortex, Ivan has found that Cav2.3 protein levels are reduced in FMRP-KO mice compared to wild-type mice at 3-weeks of age. In addition, primary neurons cultured from FMRP-KO mice show reduced surface Cav2.3 when compared to levels in wild-type mice. Thus, the loss of FMRP leads to a reduction in both synaptic and surface Cav2.3 protein. To determine how this might affect neuronal physiology, Erin Gray will record Cav2.3-mediated calcium currents as well as basic firing properties from wild-type and FMRP-KO neurons. While FMRP has a clear role in regulating mRNA stability, recent evidence suggests that FMRP may directly regulate the internalization and degradation of voltage gated calcium channels. Little is known about the pathways that underlie Cav2.3 degradation, thus Erin Gray and Joshua Lee have begun experiments aimed at better understanding this process. In heterologous cells overexpressing Cav2.3, Erin and Joshua have shown that Cav2.3 undergoes activity-dependent ubiquitination and degradation by the proteasome. Erin has begun to further investigate a possible role for ubiquitin-mediated alterations in surface levels of Cav2.3, and plans to perform a variety of electrophysiological recordings to determine the physiological consequences of this regulation. Co-regulation of HCN1 and Kv4.2 In CA1 pyramidal neuron dendrites, HCN channels, responsible for Ih, and Kv4.2 channels, responsible for IA, are critically important in signal processing and dendritic integration of synaptic inputs. Both channels show a similar pattern of distribution with an increased density from the soma to the apical dendrite. Using hippocampal primary cultured neurons, Emilie Campanac studied the potential co-regulation of HCN1 and Kv4.2. Results so far indicate reciprocal regulation with overexpression of Kv4.2 being associated with an increase in Ih current density without any change in sodium and calcium current while overexpression of HCN1 leads to an increased in IA current density. Pharmacological blockade of Ih with Cesium (2mM) induced a reduction in both current densities. Our data strongly suggest a homeostatic regulation between IA and Ih currents. We are currently investigating the molecular mechanism underlying this co-regulation.

阿尔茨海默氏病的神经元兴奋性 阿尔茨海默氏病（AD）是痴呆的最常见形式，其特征是进行性神经元丧失，最终导致死亡。 尽管在过去的几十年中竭尽全力，但AD的病因却尚未得到很好的理解。 广告研究和治疗发展的主要挑战是，直到神经元功能不可逆地损害直到神经元功能才被诊断出来。因此，在临床前阶段鉴定神经元变化至关重要，这可以为早期诊断提供基础，并有助于鉴定新的治疗靶标。 神经元过度兴奋性发生在AD发病机理的早期，并导致AD患者的网络功能障碍。 尽管β-淀粉样蛋白（AB）假设表明AD是由不溶性AB斑块的细胞外积累引起的，但越来越多的证据表明突触和记忆障碍是由可溶性AB介导的。 在这里，在与阿拉巴马大学的罗伯森实验室合作的本·托伦施（Ben Throesch）测试了以下假设：诱发的过度兴奋性起源于树突。 我们发现，在过表达Aβ的成年小鼠中，海马神经元的树突（而不是somata）是过度的。这种树突过度兴奋性与Kv4.2的选择性耗竭有关，Kv4.2是一种对树突状兴奋性和突触可塑性调节的重要树突状钾通道。 在另一个项目中，Eun Young Kim和Jakob Gutzmann研究了过表达人类淀粉样蛋白前体蛋白（HAPP）的年轻，2个月大的转基因小鼠的突触变化。 在这个时代，小鼠没有表现出AB斑块的积累，但与非转基因（NTG）小鼠相比，可溶性AB水平升高。 我们的发现表明，NMDAR是预防或治疗AD早期记忆丧失的可能目标。 我们目前正在测试NMDAR亚基拮抗剂对细胞和行为水平上AD进展的影响。 DPP6域在其本地化和功能中的作用 二肽基肽酶样蛋白6（DPP6）是电压门控的K+通道的KV4家族的辅助亚基，已知可增强通道表面表达并有效加速其动力学。 DPP6是单个跨膜蛋白，在结构上以其较大的细胞外结构域而引人注目。 该域中包括一个富含半胱氨酸的基序，其功能未知。 Lin Lin发现，DPP6的这种富含半胱氨酸的结构域是从ER导出并在细胞表面表达的。 以C349/C356和C465/C468形成的二硫键对于增强Kv4.2通道表面表达是必需的，而不是其与KV4.2亚基的相互作用。 DPP6的短细胞内N末端和跨膜结构域与Kv4.2的失活并加速恢复，但是整个细胞外结构域对于增强KV4.2表面表达和稳定是必要的。我们的发现表明，DPP6的富含半胱氨酸的结构域在DPP6的蛋白质折叠中起重要作用，这对于将DPP6/kv4.2复合物从ER中运出所需。 我们最近展示了DPP6在早期神经元发育过程中调节树突状丝霉菌的形成和稳定性，该神经元发展与KV4.2无关。为了鉴定其他DPP6结合蛋白，Jiahhua Hu采用了TAP纯化方法来分离海马神经元中的DPP6蛋白复合物。质谱分析确定了已知的蛋白质，例如KV4家族成员和许多新型突触蛋白，目前正在研究这些蛋白质和Jiahua Hu和Jung Park。 电压门控钙通道的树突状运输 我们目前正在研究电压门控钙通道CAV2.3的表达和运输。 Cav2.3在海马和皮质神经元的树突中高度表达，在该树突中，它能够产生大钙尖峰，以响应后传播的动作电位和突触活性。因此，CAV2.3 mRNA定位和翻译的改变可能会对细胞兴奋性和钙信号传导产生巨大影响。最近的证据表明，CAV2.3 mRNA可以由脆弱的X智力低下蛋白（FMRP）靶向，这是一种调节树突状棘平移的mRNA结合蛋白。 FMRP的丧失导致脆弱的X综合征，X综合征是人类遗传残疾的最常见形式。因此，我们正在研究FMRP可以调节CAV2.3的翻译的可能性，并确定该调节是否可能是脆弱X综合征的方面。 为了实现这一目标，liu对从野生型和FMRP-KO雄性小鼠的海马或皮层分离的mRNA进行了实时PCR，并检查了几种树突状蛋白的mRNA水平。当比较3岁或8周的FMRP-KO和野生型小鼠的海马时，她发现CAV2.3，KV4.2，PSD-95和HCN-1的mRNA水平没有显着差异。她将通过识别CAV2.3上的FMRP结合位点来进一步表征该调节。 与这些实验结合使用，伊万·特兰（Ivan Trang）正在确定FMRP如何影响CAV2.3蛋白表达。从从小鼠皮质中分离出的突触瘤体中，Ivan发现，与3周的野生型小鼠相比，FMRP-KO小鼠的Cav2.3蛋白水平降低。此外，与野生型小鼠的水平相比，从FMRP-KO小鼠培养的原发性神经元显示表面Cav2.3降低。因此，FMRP的丧失导致突触和表面Cav2.3蛋白的降低。为了确定这可能如何影响神经元生理，Erin Gray将记录CAV2.3介导的钙电流以及野生型和FMRP-KO神经元的基本触发特性。 尽管FMRP在调节mRNA稳定性方面具有明显的作用，但最近的证据表明，FMRP可以直接调节电压门控钙通道的内在化和降解。 关于Cav2.3降解的途径知之甚少，因此Erin Gray和Joshua Lee已经开始了旨在更好地理解这一过程的实验。在过表达CAV2.3的异源细胞中，Erin和Joshua表明Cav2.3在蛋白酶体中经历了依赖活性的泛素化和降解。 ERIN已开始进一步研究泛素介导的CAV2.3表面水平改变的可能作用，并计划执行各种电生理记录以确定该调节的生理后果。 HCN1和KV4.2的共同调节 在CA1锥体神经元树突中，负责IH的HCN通道和负责IA的Kv4.2通道在信号处理和突触输入的树突状集成中至关重要。这两个通道均显示出相似的分布模式，密度从躯体到顶端树突的密度增加。使用海马原发性培养神经元，Emilie Campanac研究了HCN1和KV4.2的潜在共同调节。到目前为止，结果表明，kv4.2过表达的相互调控与IH电流密度的增加相关，而钠和钙电流没有任何变化，而HCN1的过表达则导致IA电流密度的增加。用Cesium（2mm）对IH的药理阻滞诱导了两个当前密度的降低。我们的数据强烈表明IA和IH电流之间的稳态调节。 我们目前正在研究该共同调节的分子机制。