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.

神经回路在发育过程中积极重组，因为突触在某些情况下被拆除。 在其他地方组装。尽管突触重塑对回路功能的重要性， 基本机制在很大程度上是未知的。为了研究这个问题，我们正在利用DD GABA能 运动神经元C.在幼虫发育过程中经历突触重塑的秀丽线虫。在新孵化的 幼虫，DD突触前终扣最初位于腹侧肌肉上，但随后在约5小时内重新定位 周期到背侧DD神经突的输入背肌。DD重塑是转录调控的， 易洛魁族同源结构域蛋白IRX-1。IRX-1通过上调突触前体细胞凋亡相关蛋白α-SMA-8， 钠上皮通道（ENaC），其触发突触前解体的Ca 2+依赖性机制。 然而，可能需要额外的下游效应物，因为α-8抑制了 突触前成分（RAB 3、v-SNARE、liprin-β、内啡肽），而IRX-1也在平行通路中起作用 去除额外的突触前蛋白（UNC 13、ELKS、Clarinet）。 为了鉴定额外的IRX-1靶点，我们使用单细胞RNA测序（scRNA-Seq）来检测IRX-1靶点。 在重塑DD神经元中上调的转录物。该数据集的RNAi筛选检测到 神经细胞粘附蛋白NCAM-1在DD突触重塑中的必要作用。一种基因突变体， NCAM-1损害腹侧突触的去除以及它们在背侧DD神经突中的重新组装，因此 证实NCAM-1通常促进重塑。考虑到NCAM在以下方面的既定作用： 脊椎动物的神经发育，我对C.线虫NCAM同源物可以揭示保守的 突触可塑性的机制，也在大脑中运作。 我假设NCAM-1介导突触前解体，重塑GABA能神经元， 平行于08。在目标1中，我将使用（A）来测试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调节，（B）ncam-1中GFP标记的单簧管的活细胞成像 突变体，以确定NCAM-1是否与β 2 -8平行发挥功能，和（C）GFP标记的NCAM-1，以确定 其在重塑DD神经元中的位置。目的2：利用基因组工程技术， 突触前拆卸所需的结构域。此外，我将使用细胞特异性RNAi（csRNAi）， 确定DD神经元中是否需要ncam-1功能来进行突触拆卸。最后，在目标3中，我将 确定NCAM-1是否需要光转换的（红色）dendra 2：：RAB-3从腹侧到背侧的再循环 突触重塑DD神经元。因为NCAM-1的脊椎动物同源物介导NCAM-1的去除， GABA能的输入在发育中的皮层，我们认为，潜在的机制可能是保守的， 因此可以通过C.优雅的