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: 9550351
• 负责人: Dax A Hoffman
• 金额: $ 211.07万
• 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/9550351
• 关键词: Adult; Affect; Affinity Chromatography; Alzheimer' s Disease; Animals; Apical; Behavior; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Brain; Brain region; Bungarotoxins; CRISPR/Cas technology; Calcium; Calcium Channel; Calcium Spikes; Cells; Central Nervous System Diseases; Co-Immunoprecipitations; Coculture Techniques; Complex; Data; Dendrites; Electrophysiology (science); Emotions; Epilepsy; FMR1; Family member; Fluorescent in Situ Hybridization; Fragile X Syndrome; Future; Genes; Glutamate Receptor; Gray unit of radiation dose; Hippocampus (Brain); Human; Hyperactive behavior; Image; Immunoprecipitation; In Situ Hybridization; Individual; Intellectual functioning disability; Ion Channel; Isomerase; Knock-in; Knock-out; Knockout Mice; Knowledge; Kv4 channel; Learning; Long-Term Depression; Mass Spectrum Analysis; Mediating; Membrane; Memory; Memory impairment; Messenger RNA; Metabotropic Glutamate Receptors; Mus; Neuraxis; Neurodevelopmental Disorder; Neurons; Peptides; Physiological; Physiology; Play; Potassium; Potassium Channel; Process; Property; Proteins; Proteomics; Pyramidal Cells; RNA; Receptor Activation; Regulation; Repression; Research; Role; Seizures; Shapes; Signal Transduction; Site; Slice; Source; Structure; Surface; Synapses; Synaptic plasticity; Techniques; Therapeutic; Time; Translations; Weight; Work; behavioral impairment; brain tissue; calmodulin-dependent protein kinase II; cell type; conditioned fear; experience; extracellular; hippocampal pyramidal neuron; information processing; knockout animal; long term memory; morris water maze; mouse model; neuronal excitability; novel; protein complex; protein expression; synaptogenesis; trafficking; voltage; voltage gated channel

Isomerase regulation of potassium channel trafficking and function. To identify Kv4.2 binding proteins, Jiahua Hu employed a tandem affinity purification approach (TAP)to isolate the Kv4.2 protein complex from hippocampal neurons. Mass-spectrometry analysis identified known proteins such as KChIP family members and DPP6/10. The TAPMS assay also identified an isomerase as a binding partner of Kv4.2. The binding was confirmed by brain co-immunoprecipitation, co-expression in HEK293T cells, and peptide pull downin vitro. The isomerase binds to a specific Kv4.2 site, and the association is regulated by neuronal activity and seizure. To determine if and how the isomerase regulates the trafficking of Kv4.2, we generated bungarotoxin binding site-tagged Kv4.2 at the second extracellular loop for visualizing Kv4.2 in live neurons. The bungarotoxin binding site-tagged Kv4.2 showed similar channel properties as WT Kv4.2 in biochemical and electrophysiological assays. The isomerizing activity may also regulate Kv4.2 binding to its auxiliary subunits. These data suggested that the isomeraseplays a role in regulating Kv4.2 function. To further study the physiological function of isomerase and Kv4.2 channel, we generated a knockin mousein whichthe isomerase binding site is specifically abolished using Crispr-Cas9 techniques. These mice are viable and appear normal. We are now working on learning and memory related behaviors including novel objective recognition, Morris Water Maze and fear conditioning. DPP6 deletion leads to memory impairments in mice DPP6 plays an important role as an auxiliary subunit of Kv4, and the DPP6 gene has been associatedwith neurodevelopmental disorders in human. We found that DPP6 deletion leads to behavioral impairments in recognition, spatial learning and memory with less body and brain weights in adult mice. In addition, we found synaptic structure deficits in neuronal synapses in the hippocampal CA1 region. DPP6 induces synapse formation and regulates stabilization by live image and co-culture assay in neurons. Loss of regulated Cav2.3 expression in a mouse model of Fragile X Syndrome Fragile X syndrome (FXS) is a severe form of intellectual disability in humans that arises from the loss of the fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates translation downstream of group I metabotropic glutamate receptors (GpI mGluRs). Loss of FMRP leads to enhanced calcium spiking and neuronal excitability, thus Erin Gray sought to explore the possibility that FMRP regulates expression of the dendritic voltage gated calcium channel Cav2.3. In our initial studies, we showed that loss of FMRP in mice alters Cav2.3 mRNA levels in both the cortex and hippocampus. Considering that FMRP is an mRNA binding protein, we performed an RNA immunoprecipitation (RIP) assay and found that immunoprecipitation of FMRP from brain tissue also pulls down Cav2.3 mRNA. These results suggest that FMRP directly binds Cav2.3 mRNA to regulate its abundance in neurons. FMRP-dependent regulation of Cav2.3 mRNA appears to impact Cav2.3 channel expression as our previous data showed that FMRP KO mice have enhanced expression of Cav2.3 in cortical regions and in the hippocampus. This increase in Cav2.3 expression impacts neuronal physiology; Cav2.3 currents are enhanced in cultured hippocampal neurons isolated from FMRP KO mice compared to wild-type animals. Thus, it appears that FMRP binding normally represses Cav2.3 translation under basal conditions and loss of FMRP leads to an increase of Cav2.3 protein in the membrane. We are also investigating the possibility that repression of Cav2.3 expression by FMRP can be regulated by upstream activity of GpI mGluRs. In support of this idea, our previous data showed that stimulation of GpI mGluRs increases local synaptic translation and expression of Cav2.3 in WT neurons but not in neurons lacking FMRP. To determine if Cav2.3 expression downstream of GpI mGluR activation has a role in synaptic plasticity, we induced long term synaptic depression by stimulating GpI mGluRs (mGluR-LTD) in hippocampal slices from wildtype and Cav2.3 KO mice. Strikingly, we found that hippocampal slices from Cav2.3 KO mice lacked mGluR-LTD, demonstrating the importance of Cav2.3 in mGluR-dependent synaptic plasticity. Thus, FMRP serves as a key translational regulator of Cav2.3 expression under basal conditions and following activity and this may be critical for mGluR-dependent forms of plasticity. Loss of regulated Cav2.3 expression could underlie the neuronal hyperactivity and aberrant calcium spiking in FMRP KO mice and contribute to FXS, potentially serving as a novel target for future therapeutic strategies. FMRP interacts with Cav2.3 mRNA Ying Liu has shown that Cav2.3 mRNA levels were altered in FMRP KO neurons and Cav2.3 protein levels were significantly enhanced in the FMRP KO. To study if Cav2.3 mRNA is one of the targets of FMRP, we performed RNA immunoprecipitation. Our data showed that FMRP interacts with Cav2.3 mRNA in transfected HEK293 cells and in mouse cortex and hippocampus. Our results suggest that FMRP binds to Cav2.3 mRNA directly or indirectly to repress Cav2.3 translation and regulates neuronal excitability. In related work, Jon Murphy is examining whether the dendritic FMRP regulates mRNA trafficking and protein expression of CaV2.3 and KV4.2 in the dendrites of hippocampal neurons using the FMRP KO mouse. Recent progress has centered primarily on analysis of mRNA localization and regulation of total protein translation in neuronal dendrites of WT and FMRP knockout mouse neurons. Using fluorescence in situ hybridization to detect mRNAs for CaV2.3 and CaMKII in neurons, we have found that CaMKII mRNA, a known dendritically synthesized protein has increased abundance throughout the dendritic arbor of FMRP KO mice. FMRP is known to inhibit CaMKII translation through direct binding of CaMKII mRNA suggesting either CaMKII mRNA is more highly transcribed in FMRP knockout mice, it is no longer sequestered in mRNA-protein complexes where in situ hybridization is inhibited, or both. Conversely, CaV2.3 mRNA signals are low throughout dendrites in both WT and FMRP KO neurons. Studies of Kv4.2 mRNA localization in WT and FMRP knockout neurons are ongoing. Is Cav2.3 the source of calcium regulation of Kv4.2? Proteomic and subcellular localization studies suggest, that Cav2.3-containing voltage gated calcium channels could be a potential calcium source for a modulatory effect on Kv4.2-mediated A-type K currents (IA) in CA1 hippocampal neurons. Jakob Gutzmann compared wild type with Cav2.3 knock-out neurons, and saw a significant reduction in somatic . Were now analyzing IA from somatic and dendritic attached patch recordings, to investigate the potential influence of a loss of Cav2.3 on the distinct functional gradient that IA shows along the apical dendrites of wild type CA1 pyramidal neurons. We continue to characterize Cav2.3 knockout animals, to better understand the interplay between calcium and potassium that shape CA1 pyramidal cell electrical behavior.

钾通道运输和功能的异构酶调节。 为了鉴定 Kv4.2 结合蛋白，Jiahua Hu 采用串联亲和纯化方法 (TAP) 从海马神经元中分离 Kv4.2 蛋白复合物。质谱分析鉴定了已知的蛋白质，例如 KChIP 家族成员和 DPP6/10。 TAPMS 测定还鉴定出一种异构酶作为 Kv4.2 的结合伴侣。通过脑免疫共沉淀、HEK293T 细胞共表达和体外肽下拉证实了这种结合。异构酶与特定的 Kv4.2 位点结合，并且这种关联受到神经元活动和癫痫发作的调节。为了确定异构酶是否以及如何调节 Kv4.2 的运输，我们在第二个细胞外环处生成了金环蛇毒素结合位点标记的 Kv4.2，以便在活神经元中可视化 Kv4.2。在生化和电生理学测定中，金环蛇毒素结合位点标记的 Kv4.2 显示出与 WT Kv4.2 相似的通道特性。异构化活性还可能调节 Kv4.2 与其辅助亚基的结合。这些数据表明异构酶在调节Kv4.2功能中发挥作用。为了进一步研究异构酶和Kv4.2通道的生理功能，我们利用Crispr-Cas9技术构建了异构酶结合位点被特异性消除的敲入小鼠。这些小鼠能够存活并且看起来正常。我们现在正在研究学习和记忆相关的行为，包括新颖的目标识别、莫里斯水迷宫和恐惧条件反射。 DPP6缺失导致小鼠记忆障碍 DPP6作为Kv4的辅助亚基发挥着重要作用，DPP6基因与人类神经发育障碍有关。 我们发现 DPP6 缺失会导致成年小鼠的识别、空间学习和记忆方面的行为障碍，并导致其身体和大脑重量减轻。 此外，我们发现海马 CA1 区神经元突触存在突触结构缺陷。 DPP6 通过实时图像和神经元共培养测定诱导突触形成并调节稳定性。 脆性 X 综合征小鼠模型中受调节的 Cav2.3 表达缺失 脆性 X 综合征 (FXS) 是一种严重的人类智力障碍，起因于脆性 X 智力迟钝蛋白 (FMRP) 的缺失，FMRP 是一种调节 I 类代谢型谷氨酸受体 (GpI mGluR) 下游翻译的 mRNA 结合蛋白。 FMRP 的缺失会导致钙尖峰和神经元兴奋性增强，因此 Erin Gray 试图探索 FMRP 调节树突电压门控钙通道 Cav2.3 表达的可能性。在我们的初步研究中，我们发现小鼠 FMRP 的缺失会改变皮质和海马中的 Cav2.3 mRNA 水平。考虑到 FMRP 是一种 mRNA 结合蛋白，我们进行了 RNA 免疫沉淀 (RIP) 测定，发现脑组织中的 FMRP 免疫沉淀也能降低 Cav2.3 mRNA。这些结果表明 FMRP 直接结合 Cav2.3 mRNA 以调节其在神经元中的丰度。 Cav2.3 mRNA 的 FMRP 依赖性调节似乎会影响 Cav2.3 通道的表达，因为我们之前的数据表明 FMRP KO 小鼠皮质区和海马中 Cav2.3 的表达增强。 Cav2.3 表达的增加会影响神经元生理学；与野生型动物相比，FMRP KO 小鼠培养的海马神经元中的 Cav2.3 电流增强。因此，FMRP 结合通常会在基础条件下抑制 Cav2.3 翻译，而 FMRP 的丧失会导致膜中 Cav2.3 蛋白的增加。 我们还在研究 FMRP 对 Cav2.3 表达的抑制是否可以通过 GpI mGluRs 的上游活性来调节。为了支持这一观点，我们之前的数据表明，刺激 GpI mGluRs 会增加 WT 神经元中的局部突触翻译和 Cav2.3 的表达，但在缺乏 FMRP 的神经元中则不会。为了确定 GpI mGluR 激活下游的 Cav2.3 表达是否在突触可塑性中发挥作用，我们通过刺激野生型和 Cav2.3 KO 小鼠海马切片中的 GpI mGluR (mGluR-LTD) 诱导长期突触抑制。引人注目的是，我们发现 Cav2.3 KO 小鼠的海马切片缺乏 mGluR-LTD，这证明了 Cav2.3 在 mGluR 依赖性突触可塑性中的重要性。因此，FMRP 在基础条件下和活性后充当 Cav2.3 表达的关键翻译调节因子，这对于 mGluR 依赖性形式的可塑性可能至关重要。受调节的 Cav2.3 表达的丧失可能是 FMRP KO 小鼠神经元过度活跃和异常钙峰值的基础，并有助于 FXS，可能成为未来治疗策略的新靶点。 FMRP 与 Cav2.3 mRNA 相互作用 Ying Liu 发现 FMRP KO 神经元中 Cav2.3 mRNA 水平发生改变，FMRP KO 神经元中 Cav2.3 蛋白水平显着增强。为了研究 Cav2.3 mRNA 是否是 FMRP 的靶标之一，我们进行了 RNA 免疫沉淀。我们的数据表明，FMRP 与转染的 HEK293 细胞以及小鼠皮质和海马中的 Cav2.3 mRNA 相互作用。我们的结果表明，FMRP 直接或间接与 Cav2.3 mRNA 结合，抑制 Cav2.3 翻译并调节神经元兴奋性。 在相关工作中，Jon Murphy 正在使用 FMRP KO 小鼠检查树突 FMRP 是否调节海马神经元树突中 CaV2.3 和 KV4.2 的 mRNA 运输和蛋白表达。最近的进展主要集中在 WT 和 FMRP 敲除小鼠神经元的神经元树突中 mRNA 定位和总蛋白翻译调节的分析。使用荧光原位杂交检测神经元中 CaV2.3 和 CaMKII 的 mRNA，我们发现 CaMKII mRNA（一种已知的树突合成蛋白）在 FMRP KO 小鼠的整个树突乔木中丰度增加。已知 FMRP 通过直接结合 CaMKII mRNA 来抑制 CaMKII 翻译，这表明 CaMKII mRNA 在 FMRP 敲除小鼠中转录程度更高，或者它不再隔离在原位杂交受到抑制的 mRNA-蛋白质复合物中，或者两者兼而有之。相反，WT 和 FMRP KO 神经元中整个树突的 CaV2.3 mRNA 信号都很低。 Kv4.2 mRNA 在 WT 和 FMRP 敲除神经元中定位的研究正在进行中。 Cav2.3是Kv4.2钙调节的来源吗？ 蛋白质组学和亚细胞定位研究表明，含有 Cav2.3 的电压门控钙通道可能是对 CA1 海马神经元中 Kv4.2 介导的 A 型 K 电流 (IA) 产生调节作用的潜在钙源。 Jakob Gutzmann 将野生型神经元与 Cav2.3 敲除神经元进行比较，发现体细胞显着减少。现在我们从体细胞和树突附着斑块记录中分析 IA，以研究 Cav2.3 缺失对 IA 在野生型 CA1 锥体神经元顶端树突上显示的不同功能梯度的潜在影响。我们继续表征 Cav2.3 敲除动物，以更好地了解钙和钾之间影响 CA1 锥体细胞电行为的相互作用。