Molecular mechanisms for neuron-specific assembly of electrical synapses

电突触神经元特异性组装的分子机制

• 批准号: 10390339
• 负责人: DAVID M MILLER
• 金额: $ 37.25万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10390339
• 关键词: ATP phosphohydrolase; Address; Adenylate Cyclase; Adopted; Adoption; Animal Model; Axon; Binding; Biological Assay; Brain; Caenorhabditis elegans; Cells; Chemical Synapse; Chemicals; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Electrical Synapse; Exhibits; Functional disorder; G-Protein-Coupled Receptors; Gap Junctions; Genes; Genetic; Genetic Transcription; Human; Image; Imaging Techniques; Impairment; Interneurons; Ions; Kinesin; Locomotion; Mammalian Cell; Mediating; Methods; Microtubules; Modeling; Molecular; Motor; Motor Activity; Motor Neurons; Movement; Nematoda; Nervous system structure; Neurons; Neuropeptide Receptor; Pathway interactions; Positioning Attribute; Process; Proteins; Role; Signal Transduction; Specific qualifier value; Specificity; Spinal Cord; Spinal cord injury; Synapses; Testing; Transcript; Vesicle; Work; anterograde transport; biochemical model; experimental study; genetic analysis; imaging modality; in vitro Assay; in vivo; live cell imaging; member; mutant; neuronal cell body; novel; optogenetics; phosphoric diester hydrolase; preservation; prevent; programs; single molecule; therapy development; tool; trafficking; transcription factor; transcriptome sequencing

SUMMARY Gap junctions or “electrical synapses” mediate the flow of ions between neurons and are thus essential to normal brain function. Circuit activity is defined by the selective placement of electrical synapses between specific neurons and in particular cellular compartments. Although much has been learned about the mechanisms that direct assembly of chemical synapses between specific neurons, little is known of the pathways that drive the creation of neuron-specific electrical synapses. With its stereotypical placement of gap junctions and powerful tools for genetic analysis and imaging, the C. elegans motor circuit offers a unique opportunity to investigate gap junction specificity. VA and VB motor neurons are connected via gap junctions to command interneurons (AVA or AVB) that drive backward (VAàAVA) or forward (VBàAVB) locomotion. Notably, VAàAVA gap junctions are placed on the VA axon whereas VBàAVB gap junctions are positioned on VB cell soma. The UNC-4 transcription factor functions in VAs to preserve VAàAVA electrical synapses; unc-4 mutants adopt VAàAVB gap junctions on VA cell bodies and are thus unable to move backward. Thus, UNC- 4 regulates a transcriptional program that defines both the cellular compartment and neuron- specificity of gap junction placement. We used VA-specific RNA-Seq data to reveal that UNC- 4 blocks expression of a phosphodiesterase, PDE-1, that degrades cAMP, and a neuropeptide receptor, FRPR-17, that functions in a GaO pathway that antagonizes cAMP synthesis. Aim 1 tests the hypothesis that UNC-4 represses specific downstream targets to maintain cAMP which in turn sustains VAàAVA gap junctions. Our RNA-Seq data revealed that another UNC- 4 target, the atypical kinesin VAB-8, is ectopically expressed in unc-4 mutant VAs where it antagonizes normal trafficking of gap junction components into the VA axon. Aim 2 tests the hypothesis that VAB-8 binds to microtubules to block the anterograde function of kinesins that drive gap junction transport, thus, facilitating the formation of VAàAVB gap junctions on VA cell soma. Aim 3 uses single molecule imaging techniques to test a “blockade” model in which VAB-8 lacks ATPase/motor activity but binds to microtubules to impair gap junction export from the cell soma. Although studies in cultured mammalian cells have implicated cAMP signaling and trafficking in gap junction assembly, these pathways have not been tested for functional roles in neuron-specific placement of electrical synapses in an intact nervous system. Thus, our work with a model organism could provide important clues to fundamental processes governing the formation electrical synapses in the human brain.

概括 间隙连接或“电气突触”介导神经元之间的离子流动，因此 正常的大脑功能必不可少的。电路活动由选择性放置定义 特定神经元和特别的细胞室之间的电突触。 尽管已经了解了直接组装化学物质的机制 特定神经元之间的突触，对推动创造的途径知之甚少 神经特异性电突触。其刻板印象的间隙连接处和 遗传分析和成像的强大工具，秀丽隐杆线索电路提供了独特的 调查间隙连接特异性的机会。 VA和VB运动神经元通过 向后行驶（Vaàava）或 向前（vbàavb）运动。值得注意的是，Vaàava间隙连接位于VA轴突上 而VBàAvb间隙连接位置位于VB细胞体内。 UNC-4转录 VAS中的因子功能以保留Vaàava电气突触； UNC-4突变体采用 Vaàavb缝隙连接在VA细胞主体上，因此无法向后移动。那，unc- 4调节转录程序，该程序既定义细胞室和神经元 - 间隙连接位置的特异性。我们使用VA特异性RNA-Seq数据来揭示unc- 4阻断磷酸二酯酶PDE-1的表达，降解营地和神经肽的表达 受体，FRPR-17，在拮抗CAMP合成的GAO途径中起作用。目标1 检验了UNC-4反映特定的下游目标以维护cAMP的假设 这反过来维持了vaàava间隙连接。我们的RNA-seq数据表明，另一个UNC- 4靶标，非典型的驱动蛋白VAB-8，在unc-4突变体VAS中表达 拮抗间隙连接组件正常运输到VA轴突中。 AIM 2测试 VAB-8与微管结合以阻止驱动素的顺行函数的假设 因此，驱动间隙连接运输，支持VA上的Vaàavb间隙连接的形成 细胞体。 AIM 3使用单分子成像技术来测试“封锁”模型 VAB-8缺乏ATPase/运动活动，但与微管结合以损害从 细胞体。尽管对培养的哺乳动物细胞的研究已经实施了cAMP信号传导 以及在间隙连接组件中进行的贩运，这些途径尚未测试功能 在完整的神经系统中电气突触的神经特异性放置中的作用。那， 我们与模型生物体的工作可以为基本过程提供重要的线索 管理人脑中的地层电气突触。