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: 10163931
• 负责人: DAVID M MILLER
• 金额: $ 37.16万
• 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/10163931
• 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“电突触”介导神经元之间的离子流动，因此 对正常的大脑功能至关重要。电路活动是通过选择性放置来定义的 特定神经元和特定细胞区室之间的电突触。 尽管人们对直接化学组装的机制已经了解很多 特定神经元之间的突触，人们对驱动突触产生的途径知之甚少 神经元特异性电突触。凭借其典型的间隙连接位置和 作为遗传分析和成像的强大工具，线虫运动电路提供了独特的 研究间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，VAàAVA 间隙连接位于 VA 轴突上 而 VBàAVB 间隙连接位于 VB 细胞胞体上。 UNC-4 转录 VA 中保护 VAàAVA 电突触的因子功能； unc-4突变体采用 VAàAVB 间隙连接在 VA 细胞体上，因此无法向后移动。因此，UNC- 4 调节定义细胞区室和神经元的转录程序 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，该酶可降解 cAMP 和神经肽 受体 FRPR-17，在 GaO 途径中发挥作用，拮抗 cAMP 合成。目标1 测试 UNC-4 抑制特定下游靶标以维持 cAMP 的假设 反过来又维持 VAàAVA 间隙连接。我们的 RNA-Seq 数据显示，另一个 UNC- 4 靶标，即非典型驱动蛋白 VAB-8，在 unc-4 突变体 VA 中异位表达，其中 拮抗间隙连接成分进入 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 细胞体。 Aim 3 使用单分子成像技术来测试“封锁”模型，其中 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 细胞体。尽管对培养的哺乳动物细胞的研究表明 cAMP 信号传导 和间隙连接组装的运输，这些途径尚未经过功能测试 在完整神经系统中电突触的神经元特异性放置中的作用。因此， 我们对模型生物体的研究可以为基本过程提供重要线索 控制人脑中电突触的形成。