Precise assembly of retinal circuitry through rejection of inappropriate synaptic partners

通过拒绝不适当的突触伙伴来精确组装视网膜电路

• 批准号: 10320054
• 负责人: Jeremy N Kay
• 金额: $ 44.17万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320054
• 关键词: Adhesives; Axon; Binding; Biochemical; Biological Assay; Cell Surface Proteins; Cell surface; Cells; Cues; Data; Dendrites; Development; Disease; Event; Exclusion; Failure; Family; Goals; Growth; Image; Impairment; Inner Plexiform Layer; Interneurons; Knock-out; Knowledge; Lead; Ligands; Location; Logic; Mediating; Modeling; Molecular; Mus; Mutant Strains Mice; Neuraxis; Neurites; Neurodegenerative Disorders; Neurons; Neuropil; Phenocopy; Physiological; Process; Property; Proteins; Public Health; Retina; Retinal Degeneration; Signal Transduction; Specificity; Synapses; Testing; Therapeutic; Time; Vision; Visual; Visual Perception; Work; base; cell type; ganglion cell; genetic approach; in vivo; insight; loss of function; mutant; neural circuit; novel therapeutics; prevent; rational design; receptor; regenerative therapy; retinal neuron; starburst; starburst amacrine cell; synaptogenesis

During development, retinal neurons make exquisitely precise connections with specific synaptic partners. These synaptic choices impact the computational capacity of retinal circuits, and thereby influence visual per- ception. Cell-surface recognition molecules mediate synaptic choices by encoding two kinds of trans-cellular signals: 1) attractive signals that connect neurons with their circuit partners; 2) repulsive signals that shun non- target cells. Both types of cues are needed for precise retinal wiring, but the molecular mechanisms underlying rejection of inappropriate synaptic partners are unknown. The objective here is to identify recognition mecha- nisms that prevent connections between inappropriate synaptic partners. Our central hypothesis is that FLRT and UNC5 families of cell-surface molecules mediate repulsive receptor-ligand interactions that prevent cross- circuit synapse formation. The rationale for this work is that it will reveal a new class of synaptic choice recog- nition molecules that act through repulsive mechanisms. Understanding how the wrong synapses are avoided is a necessary step towards ultimately deciphering the molecular logic underlying synaptic partner choice. To this end, the following Specific Aims are proposed: 1) Identify ligands that prevent retinal neurons from se- lecting inappropriate synaptic partners. Retinal circuits occupy parallel sublayers within the inner plexiform layer (IPL) neuropil. This arrangement facilitates synapse specificity by bringing together arbors of circuit part- ners in a defined location where they are segregated from non-target cells. In preliminary studies using the mouse direction-selective (DS) circuit as a model, we obtained preliminary evidence that the UNC5C cell sur- face protein is a repulsive ligand that confines DS circuit arbors to their appropriate sublayers. This hypothesis will be tested using Unc5c mutant mice and Unc5c misexpression in vivo. 2) Identity receptor-mediated mo- lecular mechanisms that enforce synaptic specificity. Preliminary studies led us to hypothesize that the cell surface protein FLRT2, which is expressed by DS circuit neurons, serves as an UNC5C receptor that con- fines DS circuit arbors to their appropriate sublayers. This hypothesis will be tested using biochemical and in vivo genetic approaches. 3) Determine cellular mechanisms by which retinal neurons shun inappropriate synaptic partners. During dendrite growth, many exploratory branches are eliminated. Our preliminary data suggest that elimination of mistargeted arbors is impaired in Flrt2 and Unc5c mutants. We therefore hypothe- size that UNC5C-FLRT2 repulsion eliminates errant branches to prevent neurons from accessing inappropriate synaptic partners. This idea will be tested by time-lapse imaging of nascent DS circuit dendrites and synapses in Flrt2 and Unc5c mutants. Completion of these Aims is expected to define cellular and molecular mecha- nisms by which neurons avoid incorrect synaptic choices. This contribution will be significant because, once repulsive mechanisms for synapse specificity are known, it will become possible to comprehend how repulsion and attraction work together to produce the overarching molecular logic of synaptic partner choice.

在发育过程中，视网膜神经元与特定的突触伙伴建立极其精确的连接。 这些突触选择影响视网膜回路的计算能力，从而影响视觉性能。 构思。细胞表面识别分子通过编码两种跨细胞的 信号：1）连接神经元与其电路伙伴的吸引信号; 2）避开非 靶细胞这两种类型的线索都是精确的视网膜布线所必需的，但其背后的分子机制 不适当的突触伴侣的排斥是未知的。我们的目标是识别识别机制- 阻止不合适的突触伴侣之间连接的突触。我们的中心假设是FLRT 和UNC 5家族的细胞表面分子介导排斥性受体-配体相互作用，阻止交叉， 回路突触形成。这项工作的基本原理是，它将揭示一类新的突触选择识别， 通过排斥机制起作用的点火分子。了解如何避免错误的突触 是最终破译突触伴侣选择背后的分子逻辑的必要步骤。到 为此，提出了以下具体目标：1）鉴定阻止视网膜神经元自噬的配体， 选择不合适的突触伴侣视网膜回路在内部网状结构中占据平行的亚层 层（IPL）神经元。这种排列通过将电路部分的主干聚集在一起，促进了突触的特异性。 在一个确定的位置，它们与非靶细胞隔离。在初步研究中， 以小鼠方向选择性（DS）回路为模型，我们获得了初步证据，表明UNC 5C细胞表面 表面蛋白是排斥配体，其将DS电路主干限制在它们的适当亚层。这一假设 将使用Unc 5c突变小鼠和Unc 5c体内错误表达进行测试。2)识别受体介导的 强化突触特异性的神经机制。初步研究使我们假设 细胞表面蛋白FLRT 2，由DS回路神经元表达，作为一种UNC 5C受体， 将DS电路芯轴细化到其适当的子层。这一假设将使用生物化学和 体内遗传学方法。3)确定视网膜神经元避免不适当的细胞机制 突触伴侣在枝晶生长期间，许多探索性分支被消除。我们的初步数据 表明在Flrt 2和Unc 5c突变体中消除错误启动的Arbors受损。因此，我们假设- 确定UNC 5C-FLRT 2排斥作用大小可消除错误分支，以防止神经元进入不适当的通路， 突触伴侣这一想法将通过对新生的DS回路树突和突触进行延时成像来验证 在Flrt 2和Unc 5c突变体中。这些目标的完成有望定义细胞和分子机制， 神经元避免错误突触选择的机制。这一贡献将是巨大的，因为一旦 突触特异性的排斥机制是已知的，它将成为可能理解排斥如何 和吸引力共同作用，产生了突触伴侣选择的总体分子逻辑。