NCAM drives synaptic remodeling in developing GABAergic neurons in C. elegans

NCAM 驱动线虫发育中 GABA 能神经元的突触重塑

• 批准号: 10752458
• 负责人: Casey Gailey
• 金额: $ 3.3万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2026-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752458
• 关键词: Address; Adhesives; Brain; Caenorhabditis elegans; Cell Adhesion Molecules; Cells; Cytoskeleton; Data Set; Development; Distal; Dorsal; ELK1 gene; Excision; Fibronectins; Genes; Genetic; Genetic Transcription; Genome engineering; Homeobox; Homeodomain Proteins; Homologous Gene; Impairment; Larva; Location; Mediating; Molecular; Motor Neurons; Muscle; NCAM1 gene; Nerve; Neural Cell Adhesion Molecules; Neuralized-like Protein; Neurites; Neurons; Pathway interactions; Positioning Attribute; Presynaptic Terminals; Proteins; RNA Interference; RNA interference screen; Recycling; Role; Side; Signal Transduction; Synapses; Synaptic plasticity; Testing; Transcript; UNC13B gene; epithelial Na+ channel; extracellular; genetic analysis; hatching; live cell imaging; mutant; neural circuit; neurodevelopment; presynaptic; programs; single-cell RNA sequencing; synaptic function; transcription factor; vesicle-associated membrane protein; vesicular SNARE proteins

Neural circuits are actively restructured during development as synapses are dismantled in some locations and assembled in others. Despite the importance of synaptic remodeling to circuit function, the underlying mechanisms are largely unknown. To investigate this question, we are exploiting the DD GABAergic motor neurons in C. elegans which undergo synaptic remodeling during larval development. In newly hatched larvae, DD presynaptic boutons are initially positioned on ventral muscles but are then relocated over a ~5 hr period to the dorsal DD neurite for input to dorsal muscles. DD remodeling is transcriptionally regulated by the Iroquois-type homeodomain protein, IRX-1. IRX-1 directs synaptic remodeling by upregulating UNC-8, a sodium epithelial channel (ENaC), which triggers a Ca2+-dependent mechanism of presynaptic disassembly. Additional downstream effectors are likely required, however, because UNC-8 dismantles a subset of presynaptic components (RAB3, v-SNARE, liprin-, endophilin) whereas IRX-1 also acts in a parallel pathway to remove additional presynaptic proteins (UNC13, ELKS, Clarinet). To identify additional IRX-1 targets, we used single-cell RNA-Sequencing (scRNA-Seq) to detect transcripts that are upregulated in remodeling DD neurons. An RNAi screen of this dataset detected a necessary role for the neural cell adhesion protein, NCAM-1, in DD synaptic remodeling. A genetic mutant of NCAM-1 impairs both the removal of ventral synapses as well as their reassembly in dorsal DD neurites, thus confirming that NCAM-1 normally promotes remodeling. Considering the established roles of NCAM in vertebrate neural development, my studies of the C. elegans NCAM homolog could reveal conserved mechanisms for synaptic plasticity that also operate in the brain. I hypothesize that NCAM-1 mediates presynaptic disassembly in remodeling GABAergic neurons in parallel to UNC-8. In Aim 1, I will test the role of NCAM-1 as a regulator of presynaptic remodeling using (A) smFISH to determine if ncam-1 is regulated by IRX-1, (B) live-cell imaging of GFP tagged Clarinet in ncam-1 mutants to determine if NCAM-1 functions in a parallel to UNC-8, and (C) GFP-tagged NCAM-1 to determine its location in remodeling DD neurons. In Aim 2, I will use genome-engineering to identify NCAM-1 structural domains that are required for presynaptic disassembly. Additionally, I will use cell-specific RNAi (csRNAi) to determine if ncam-1 function is required in DD neurons for synaptic disassembly. Finally, in Aim 3, I will determine if NCAM-1 is required for recycling of photoconverted (red) dendra2::RAB-3 from ventral to dorsal synapses in remodeling DD neurons. Because the vertebrate homolog of NCAM-1 mediates the removal of GABAergic inputs in the developing cortex, we suggest that the underlying mechanism may be conserved and thus can be molecularly defined by studies in C. elegans.

在开发过程中，神经回路会积极恢复，因为某些突触被拆除 位置并组装在其他地方。尽管突触重塑对电路功能的重要性，但 潜在的机制在很大程度上是未知的。为了调查这个问题，我们正在利用DD Gabaergic 秀丽隐杆线虫中的运动神经元在幼虫发育过程中进行突触重塑。在新孵化 幼虫，dd前胸子最初位于腹部肌肉上，然后在约5小时内重新定位 循环到背DD神经蛋白，以输入背部肌肉。 DD重塑受转录调节 易洛魁型型同源域蛋白，IRX-1。 IRX-1通过上调UNC-8，A指导突触重塑 钠上皮通道（ENAC），它触发了突触前拆卸的Ca2+依赖性机制。 但是，可能需要其他下游效果，因为UNC-8拆除了一部分 突触前成分（RAB3，V-SNARE，LIPIN-，内菲林），而IRX-1也起作用 去除其他突触前蛋白（UNC13，麋鹿，单簧管）。 为了识别其他IRX-1目标，我们使用了单细胞RNA-sequest（SCRNA-SEQ）检测 重塑DD神经元中上调的转录本。该数据集的RNAi屏幕检测到 DD突触重塑中神经细胞粘附蛋白NCAM-1的必要作用。一个遗传突变体 NCAM-1均会损害腹侧突触的去除及其在背dD神经上的重新组装，从而损害 确认NCAM-1通常会促进重塑。考虑到NCAM在 脊椎动物神经元的发育，我对秀丽隐杆线虫NCAM同源物的研究可以揭示保守的 突触可塑性的机制也在大脑中起作用。 我假设NCAM-1在重塑GABA能神经元中介导了突触前拆解 平行于UNC-8。在AIM 1中，我将使用（a）测试NCAM-1作为突触前重塑的调节剂的作用 Smfish以确定NCAM-1是否受IRX-1的调节，（b）NCAM-1中GFP标记的单簧管的活细胞成像 突变体确定NCAM-1是否在平行于UNC-8的平行和（C）GFP标记的NCAM-1中起作用以确定 它的位置在重塑DD神经元中。在AIM 2中，我将使用基因组工程来识别NCAM-1结构 突触前拆卸所需的域。另外，我将使用细胞特异性RNAi（CSRNAI） 确定DD神经元中是否需要进行NCAM-1功能进行突触拆卸。最后，在AIM 3中，我会 确定是否需要NCAM-1回收光电转换（红色）Dendra2 :: Rab-3从腹侧到背侧 重塑DD神经元中的突触。因为NCAM-1的脊椎动物同源物介导了去除 发育中的皮质中的GABA能输入，我们建议潜在机制可以保存，并且 因此，可以通过秀丽隐杆线虫的研究来分子定义。