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，何哈胡（Jiahua Hu）采用串联亲和力纯化方法（TAP）将KV4.2蛋白复合物与海马神经元分离。质谱分析确定了已知的蛋白质，例如Kchip家族成员和DPP6/10。 TAPMS分析还确定了异构酶是KV4.2的绑定伙伴。结合通过脑共免疫沉淀，HEK293T细胞中的共免疫沉淀和肽降低体外。异构酶与特定的Kv4.2位点结合，并且该关联受神经元活性和癫痫发作的调节。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上生成了Bungarotoxin结合位点标记的KV4.2，以可视化活神经元中的Kv4.2。 Bungarotoxin结合位点标记的KV4.2在生化和电生理测定中表现出与WT KV4.2相似的通道特性。异构活性还可以调节Kv4.2与其辅助亚基结合。这些数据表明，异构体ePlays在调节KV4.2功能中起作用。为了进一步研究异构酶和KV4.2通道的生理功能，我们产生了一种敲击小鼠蛋白，使用CRISPR-CAS9技术特别废除了同酶结合位点。这些小鼠是可行的，看起来正常。我们现在正在研究学习和与记忆有关的行为，包括新颖的客观识别，莫里斯水迷宫和恐惧调节。 DPP6删除导致小鼠记忆力障碍 DPP6作为KV4的辅助亚基起重要作用，而DPP6基因与人类的神经发育障碍相关。 我们发现DPP6缺失会导致成年小鼠的身体和脑体重较小的识别，空间学习和记忆的行为障碍。 此外，我们发现海马CA1区域神经元突触中的突触结构缺陷。 DPP6诱导突触形成，并通过神经元中的实时图像和共培养测定法调节稳定。 在脆弱X综合征的小鼠模型中的调节CAV2.3表达的丧失 脆弱的X综合征（FXS）是人类中智障的一种严重形式，它是由于脆弱X智力低下蛋白（FMRP）的丧失而产生的，这是一种mRNA结合蛋白，可调节I组替代性谷氨酸受体下游的翻译（GPI MGLURS）。 FMRP的丧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的表达的可能性。在我们的最初研究中，我们表明小鼠中FMRP的损失改变了Cav2.3皮层和海马中的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FMRP免疫沉淀从脑组织中也取下了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。 FMRP依赖性的CAV2.3 mRNA似乎会影响CAV2.3通道的表达，因为我们先前的数据表明，FMRP KO小鼠在皮质区域和海马中的Cav2.3表达增强。 Cav2.3表达的这种增加会影响神经元生理。与野生型动物相比，在从FMRP KO小鼠中分离的培养的海马神经元中，CAV2.3的电流增强。因此，似乎FMRP结合通常在基础条件下抑制CAV2.3翻译和FMRP的丢失会导致膜中Cav2.3蛋白的增加。 我们还研究了FMRP对CAV2.3表达抑制的可能性，可以通过GPI mGlurs的上游活性来调节。为了支持这一想法，我们以前的数据表明，刺激GPI mGlurs会增加WT神经元中Cav2.3的局部突触翻译和表达，而不是缺乏FMRP的神经元中。为了确定GPI MGLUR激活下游的CAV2.3是否在突触可塑性中起作用，我们通过刺激Wildtype和Cav2.3 KO小鼠的海马切片中的GPI MGLURS（MGLUR-LTD）诱导长期突触抑制。令人惊讶的是，我们发现来自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在基础条件下和以下活动中充当Cav2.3表达的关键转化调节剂，这对于MGLUR依赖性的可塑性形式可能至关重要。 FMRP KO小鼠中神经元多动症和异常钙尖峰的丧失可能是FMRP小鼠的神经元多动症和异常的钙峰值的基础，并有助于FXS，这可能是未来治疗策略的新目标。 FMRP与CAV2.3 mRNA相互作用 liu表明，FMRP KO神经元中Cav2.3 mRNA水平发生了变化，而Cav2.3蛋白水平在FMRP KO中显着增强。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。我们的数据显示，在转染的HEK293细胞以及小鼠皮层和海马中，FMRP与CAV2.3 mRNA相互作用。我们的结果表明，FMRP直接或间接地与CAV2.3 mRNA结合以抑制CAV2.3翻译并调节神经元兴奋性。 在相关工作中，乔恩·墨菲（Jon Murphy）正在研究使用FMRP KO小鼠在海马神经元的树突中调节Cav2.3和Kv4.2的树突状FMRP和蛋白质表达。最近的进展主要集中在WT和FMRP敲除小鼠神经元神经元树突中的mRNA定位和调节总蛋白翻译的调节。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的蛋白质在FMRP KO小鼠的整个树突状arbor中具有增加的丰度。已知FMRP通过CAMKII mRNA的直接结合来抑制CAMKII翻译，这表明CAMKII mRNA在FMRP敲除小鼠中更高度转录，它不再在抑制原位杂交的mRNA蛋白质复合物中被隔离。相反，在WT和FMRP KO神经元中，CAV2.3 mRNA信号在整个树突中均低。 KV4.2在WT和FMRP敲除神经元中的MRNA定位正在进行中。 Cav2.3 Kv4.2的钙调节来源吗？ 蛋白质组学和亚细胞定位研究表明，含Cav2.3的含Cav2.3的钙通道可能是对CA1海马神经元中KV4.2介导的A型A型K电流（IA）的调节作用的潜在钙来源。 Jakob Gutzmann将野生型与Cav2.3敲除神经元进行了比较，并显着降低了体细胞。现在正在分析从体细胞和树突附着的斑块记录中分析IA，以研究cav2.3损失对IA沿着野生型Ca1锥体神经元的顶端树突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的相互作用，从而塑造CA1锥体细胞电气行为。