Whole-neuron structural input mapping of SRGAP2 regulation of synaptic development

SRGAP2 突触发育调节的全神经元结构输入图谱

• 批准号: 9328662
• 负责人: Daniel Maxim Iascone
• 金额: $ 4.4万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9328662
• 关键词: Adolescent; Adult; Affect; Binding; Brain; Cell division; Cells; Chemosensitization; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Computer software; Confocal Microscopy; Dendrites; Dendritic Spines; Development; Down-Regulation; Electroporation; Equilibrium; Evolution; Excitatory Synapse; Exhibits; Filler; Gene Duplication; Genes; Genome; Human; Human Characteristics; Imagery; Impairment; Individual; Inhibitory Synapse; Institutes; Label; Link; Mammals; Maps; Mediating; Methods; Morphologic artifacts; Morphology; Mus; Neurons; Optics; Plasmids; Play; Population; Positioning Attribute; Proteins; Radiation; Regulation; Resolution; Role; SRGAP2 gene; Scaffolding Protein; Science; Signal Transduction; Software Engineering; Somatosensory Cortex; Synapses; System; Techniques; Testing; Transgenic Mice; Transposase; Trees; Vertebral column; Work; cell type; cognitive function; cortex mapping; density; duplicate genes; experimental study; genetic approach; genome editing; gephyrin; hippocampal pyramidal neuron; in utero; in vivo; insight; neocortical; neuronal cell body; nonhuman primate; organizational structure; overexpression; paralogous gene; postsynaptic; protein aggregate; reconstruction; repaired; software development

Project Summary During development, tightly regulated mechanisms establish the proper balance between excitatory and inhibitory synaptic inputs made onto each neuronal cell type. However, the mechanisms coordinating the development of these two types of synapses are still poorly understood. We recently discovered that SRGAP2 is a postsynaptic protein playing key roles in vivo in promoting the rate of excitatory and inhibitory synaptic maturation and limiting the density of both types of synapses made onto pyramidal neurons in the developing cortex. Additionally, we and others discovered that SRGAP2 has undergone several partial gene duplications specifically in the human lineage. Only one of these gene duplications, called SRGAP2C (the ancestral copy of the human gene was renamed SRGAP2A) has been fixed in the human population and is expressed in the developing human brain. We discovered that SRGAP2C binds to and inhibits the functions of SRGAP2A during synaptic development. When human-specific SRGAP2C is expressed in mouse cortical pyramidal neurons in vivo, it induces significant delay (neoteny) of synaptic maturation and significant increase in both excitatory and inhibitory synaptic density. However, at this point one of the most significant limitations in studying the roles of SRGAP2A or SRGAP2C in synaptic development in vivo is that it is extremely challenging to obtain quantitative information on excitatory and inhibitory synapses across the entire dendritic arbor of well-defined groups of pyramidal neurons. The objective of this application is to create a platform for creating whole-neuron synaptic input maps and to use this platform to investigate how SRGAP2A and SRGAP2C influence synaptic development throughout the dendritic arbor. This application's central hypothesis is that by regulating the scale and timing of synaptic development, SRGAP2A and SRGAP2C control the spatial organization of excitatory and inhibitory synapses across dendritic domains. Aim 1 will enable labeling of inhibitory synapses throughout whole pyramidal neurons using in vivo genome editing with CRISPR-Cas9. I will use in utero electroporation to insert a FLAG tag at the 5’ end of Gephyrin, which encodes an inhibitory synaptic scaffolding protein. In Aim 2, I will use neuron reconstruction software developed in collaboration with the Peng lab to create whole-cell synaptic input maps of cortical pyramidal neurons from transgenic mice expressing SRGAP2C or lacking SRGAP2A. These maps will provide unprecedented insight into the structured organization of synapses throughout the dendritic tree and allow us to determine how SRGAP2A and SRGAP2C regulate global synaptic development.

项目摘要 在发育过程中，严格调节的机制在兴奋和抑制之间建立适当的平衡 突触输入到每种神经元细胞类型。然而，协调发展的机制 这两种类型的突触仍然知之甚少。我们最近发现SRGAP 2是一种突触后蛋白， 一种在体内促进兴奋性和抑制性突触成熟的速率和限制 这两种类型的突触在发育中的皮层锥体神经元上的密度。 此外，我们和其他人发现SRGAP 2经历了几次部分基因复制， 在人类血统中。这些基因中只有一个被称为SRGAP 2C（人类基因的祖先拷贝）。 基因被重新命名为SRGAP 2A）已经在人群中固定，并在发育中的人类中表达。 个脑袋我们发现SRGAP 2C在突触发育过程中与SRGAP 2A结合并抑制其功能。 当人特异性SRGAP 2C在小鼠皮质锥体神经元中体内表达时，其诱导显著的神经元凋亡。 突触成熟延迟（幼态持续），兴奋性和抑制性突触密度显著增加。 然而，在这一点上，研究SRGAP 2A或SRGAP 2C的作用的最重要的限制之一是， 体内突触发育的一个重要问题是，获得关于兴奋性和突触发育的定量信息是极其具有挑战性的。 抑制性突触跨越整个明确定义的锥体神经元群体的树突状结构。的目标 本申请旨在创建用于创建全神经元突触输入映射的平台，并使用该平台来 研究SRGAP 2A和SRGAP 2C如何影响整个树突状乔木的突触发育。 这项应用的中心假设是，通过调节突触发育的规模和时间， SRGAP 2A和SRGAP 2C控制跨树突状细胞的兴奋性和抑制性突触的空间组织 域.目的1将能够标记抑制性突触在整个锥体神经元使用在体内 使用CRISPR-Cas9进行基因组编辑我将使用子宫内电穿孔在Gephyrin的5'末端插入FLAG标签， 其编码抑制性突触支架蛋白。在目标2中，我将使用神经元重建软件 与Peng实验室合作开发，以创建皮质锥体神经元的全细胞突触输入图 来自表达SRGAP 2C或缺乏SRGAP 2A的转基因小鼠。这些地图将提供前所未有的洞察力 进入整个树突树突触的结构化组织，并允许我们确定SRGAP 2A如何 和SRGAP 2C调节全局突触发育。