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.

摘要 缝隙连接或“电突触”调节神经元之间的离子流动，因此 对正常的大脑功能是必不可少的。电路活动定义为选择性地放置 特定神经元之间的电性突触，特别是细胞隔间。 尽管已经了解了很多关于直接组装化学物质的机制 在特定神经元之间的突触，对驱动产生 神经元特有的电突触。以其刻板的缝隙连接和 作为基因分析和成像的强大工具，线虫马达电路提供了独特的 研究缝隙连接特异性的机会。Va和Vb运动神经元通过 控制向后驱动的中间神经元(Ava或AVB)的缝隙连接(VA？Ava)或 向前(Vb？AVB)运动。值得注意的是，VA？AVA缝隙连接位于VA轴突上 而VbàAVB缝隙连接位于Vb细胞胞体上。UNC-4转录 因子在VAS中发挥作用以保护VAàAVA电突触；UNC-4突变体采用 VA细胞体上的VA-AVB缝隙连接，因此不能向后移动。因此，北卡罗来纳大学- 4调节一种转录程序，该程序定义了细胞室和神经元- 缝隙连接位置的专一性。我们使用VA特异的RNA-Seq数据来揭示UNC- 4抑制降解cAMP的磷酸二酯酶PDE-1和神经肽的表达 受体，FRPR-17，在对抗cAMP合成的GAO途径中发挥作用。目标1 测试UNC-4抑制特定下游目标以维持cAMP的假设 这反过来又维持了VAàAva的缝隙连接。我们的RNA-Seq数据显示，另一个UNC- 4靶点，非典型运动蛋白VAB-8，在UNC-4突变体VAS中异位表达，其中它 拮抗缝隙连接成分进入VA轴突的正常运输。AIM 2测试 假设VAB-8与微管结合以阻断驱动蛋白的顺行功能 驱动缝隙结运输，从而促进VA？AVB缝隙结的形成 细胞胞体。Aim 3使用单分子成像技术来测试“封锁”模型 VAB-8缺乏ATPase/马达活性，但可与微管结合，从而损害缝隙连接输出 细胞胞体。尽管对培养的哺乳动物细胞的研究涉及cAMP信号转导 以及在缝隙连接组装中的运输，这些通路还没有进行功能测试 在完整神经系统中电突触的神经元特异性定位中的作用。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 控制着人脑中电子突触的形成。