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: 10609808
• 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/10609808
• 关键词: ATP phosphohydrolase; Address; Adenylate Cyclase; Adopted; Adoption; Axon; Binding; Biological Assay; Brain; Caenorhabditis elegans; Cells; Chemical Synapse; 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; Homologous Gene; Human; Image; Imaging Techniques; Impairment; Interneurons; Ions; Kinesin; Learning; Locomotion; Mammalian Cell; Mediating; Methods; Microtubules; Modeling; Molecular; Motor; Motor Activity; Motor Neurons; Movement; Nematoda; Nervous System; Neurons; Neuropeptide Receptor; Pathway interactions; Positioning Attribute; Process; Proteins; Repression; 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; model organism; mutant; neuronal cell body; novel; optogenetics; phosphoric diester hydrolase; preservation; prevent; programs; protein purification; 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.

总结 间隙连接或“电突触”介导神经元之间的离子流动，因此是神经元之间的电连接。 对正常的大脑功能至关重要电路活动是由选择性放置 特定神经元之间的电突触，特别是细胞区室。 尽管人们已经对化学物质直接组装的机制有了很多了解， 突触之间的特定神经元，很少有人知道的途径，驱动创建 神经元特异性电突触由于缝隙连接的典型位置， 强大的遗传分析和成像工具，C。elegans电机电路提供了一个独特的 研究缝隙连接特异性的机会。VA和VB运动神经元通过 间隙连接命令中间神经元（AVA或AVB）向后驱动（VAàAVA）或 向前（VBàAVB）运动。值得注意的是，VA间隙连接位于VA轴突上， 而VB索马间隙连接位于VB细胞体上。《四库全书》抄本 因子在VA中的功能是保护VAàAVA电突触; unc-4突变体采用 VA细胞体上的VA <$AVB间隙连接，因此不能向后移动。因此， 4调节一个转录程序，定义细胞区室和神经元， 缝隙连接位置的特异性。我们使用VA特异性RNA-Seq数据来揭示， 4阻断磷酸二酯酶PDE-1（降解cAMP）和神经肽的表达 受体FRPR-17，其在拮抗cAMP合成的GaO途径中起作用。要求1 检验了以下假设：β-4抑制特异性下游靶点以维持cAMP水平 从而维持VAàAVA间隙连接。我们的RNA-Seq数据显示，另一种生物- 非典型驱动蛋白VAB-8在unc-4突变体VA中异位表达， 拮抗缝隙连接组分进入VA轴突的正常运输。目标2测试 假设VAB-8与微管结合以阻断驱动蛋白的顺行功能， 驱动间隙连接运输，从而促进VA上VAàAVB间隙连接的形成 细胞索马。AIM 3使用单分子成像技术来测试一种“阻断”模型， VAB-8缺乏ATP酶/运动活性，但与微管结合以损害间隙连接输出， 细胞索马。尽管在培养的哺乳动物细胞中的研究已经暗示cAMP信号传导 和运输间隙连接组装，这些途径尚未被测试的功能 在完整的神经系统中电突触的神经元特异性放置中的作用。因此，在本发明中， 我们对模式生物的研究可以为基本过程提供重要线索 控制着人类大脑中电突触的形成。