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：
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
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/8941488
关键词：
Action Potentials Adult Affect Age Alabama Alzheimer&apos 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

项目摘要

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.

阿尔茨海默病的神经元兴奋性阿尔茨海默病（AD）是最常见的痴呆症，其特征是进行性神经元丧失，最终导致死亡。尽管过去几十年来付出了巨大的努力，但 AD 的病因仍不清楚。 AD 研究和治疗开发的一个主要挑战是，大多数 AD 患者直到神经元功能受到不可逆转的损害才被诊断出来。因此，在临床前阶段识别神经元的变化至关重要，这可以为早期诊断提供基础，并有助于确定新的治疗靶点。神经元过度兴奋发生在 AD 发病机制的早期，并导致 AD 患者的网络功能障碍。尽管β-淀粉样蛋白 (Ab) 假说表明 AD 是由不溶性抗体斑块在细胞外积聚引起的，但越来越多的证据表明突触和记忆障碍是由可溶性抗体介导的。在这里，Ben Throesch 与阿拉巴马大学 Roberson 实验室合作，测试了 Ab 诱导的过度兴奋起源于树突的假设。我们发现，在过度表达 Aβ 的成年小鼠中，海马神经元的树突而非体细胞过度兴奋。这种树突过度兴奋性与 Kv4.2 的选择性耗尽有关，Kv4.2 是一种树突钾通道，对树突兴奋性和突触可塑性的调节很重要。在另一个项目中，Eun Young Kim 和 Jakob Gutzmann 研究了过度表达人类淀粉样前体蛋白 (hAPP) 的 2 个月大的年轻转基因小鼠的突触变化。在这个年龄，与非转基因 (NTG) 小鼠相比，小鼠没有表现出抗体斑块积累，但表现出可溶性抗体水平增加。我们的研究结果表明 NMDAR 可以作为预防或治疗 AD 早期记忆丧失的可能靶点。我们目前正在细胞和行为水平上测试 NMDAR 亚基拮抗剂对 AD 进展的影响。 DPP6结构域在其定位和功能中的作用二肽基肽酶样蛋白 6 (DPP6) 是电压门控 K+ 通道 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 是一种单跨膜蛋白，其结构因其大的胞外结构域而引人注目。该结构域包含富含半胱氨酸的基序，其功能尚不清楚。 Lin Lin发现DPP6的这个富含半胱氨酸的结构域是其从内质网输出并在细胞表面表达所必需的。在富含半胱氨酸的结构域的 C349/C356 和 C465/C468 处形成的二硫桥对于增强 Kv4.2 通道表面表达是必需的，但不是其与 Kv4.2 亚基的相互作用。 DPP6 的短胞内 N 端和跨膜结构域与 Kv4.2 失活相关并加速其恢复，但整个胞外结构域对于增强 Kv4.2 表面表达和稳定性是必需的。我们的研究结果表明，DPP6 富含半胱氨酸的结构域在 DPP6 的蛋白质折叠中发挥着重要作用，这是将 DPP6/Kv4.2 复合物转运出 ER 所需的。我们最近表明（Lin 等人，2013）DPP6 在早期神经元发育过程中调节树突状丝状伪足的形成和稳定性，这与 Kv4.2 无关。为了鉴定其他 DPP6 结合蛋白，Jiahhua Hu 采用 TAP 纯化方法分离海马神经元中的 DPP6 蛋白复合物。质谱分析鉴定了已知的蛋白质，例如 Kv4 家族成员以及 Jiahua Hu 和 Jung Park 目前正在研究的许多新型突触蛋白质。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，它能够响应反向传播动作电位和突触活动而产生大的钙尖峰。因此，Cav2.3 mRNA 定位和翻译的改变可能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 可以被脆性 X 智力迟钝蛋白 (FMRP) 靶向，FMRP 是一种调节树突棘翻译的 mRNA 结合蛋白。 FMRP 缺失会导致脆性 X 综合征，这是人类遗传性智力障碍最常见的形式。因此，我们正在研究 FMRP 调节 Cav2.3 翻译的可能性，并将确定这种调节是否可能是脆性 X 综合征的某些方面的基础。为了实现这一目标，Ying Liu 对从野生型和 FMRP-KO 雄性小鼠的海马或皮质中分离的 mRNA 进行了实时 PCR，并检查了几种树突蛋白的 mRNA 水平。当比较 3 周或 8 周龄 FMRP-KO 小鼠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。结合这些实验，Ivan Trang 正在确定 FMRP 如何影响 Cav2.3 蛋白表达。 Ivan 从小鼠皮层分离的突触神经体中发现，与 3 周龄的野生型小鼠相比，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 小鼠培养的原代神经元显示出表面 Cav2.3 降低。因此，FMRP 的缺失会导致突触和表面 Cav2.3 蛋白的减少。为了确定这可能如何影响神经元生理学，Erin Gray 将记录 Cav2.3 介导的钙电流以及野生型和 FMRP-KO 神经元的基本放电特性。虽然 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 可能直接调节电压门控钙通道的内化和降解。人们对 Cav2.3 降解的途径知之甚少，因此 Erin Gray 和 Joshua Lee 开始了旨在更好地了解这一过程的实验。 Erin 和 Joshua 证明，在过表达 Cav2.3 的异源细胞中，Cav2.3 会经历活性依赖性泛素化和蛋白酶体降解。 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，并计划进行各种电生理记录以确定这种调节的生理后果。 HCN1 和 Kv4.2 的共同调控在 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整合中至关重要。两个通道显示出相似的分布模式，从胞体到顶端树突的密度增加。 Emilie Campanac 使用海马原代培养神经元研究了 HCN1 和 Kv4.2 的潜在共同调节作用。迄今为止的结果表明，Kv4.2 的过度表达与 Ih 电流密度的增加相关，而钠电流和钙电流没有任何变化，而 HCN1 的过度表达导致 IA 电流密度的增加。用铯 (2mM) 药理学阻断 Ih 会导致两种电流密度降低。我们的数据强烈表明 IA 和 Ih 电流之间存在稳态调节。我们目前正在研究这种共同调节背后的分子机制。