Molecular Mechanisms of Developmental Spine Remodeling

前额叶皮层（PFC）中的棘突和突触重塑机制是神经元关键功能的基础。 工作记忆、认知灵活性和社交能力尚未完全理解。脊柱受损消除- 大脑发育过程中的紧张可能有助于自闭症谱系中脊柱和突触密度的增加 自闭症（ASD）。我们发现了一种新的选择性脊髓修剪机制，其中脑信号蛋白-3受体 包括L1粘附分子、神经纤毛蛋白和丛蛋白A的复合物促进活性依赖性棘 通过Rho家族GTP酶在幼年脑中消除。所有L1-CAM都结合锚蛋白B（Ank B），一种肌动蛋白- 由高置信度ASD基因Ank 2编码的血影蛋白衔接子。AnkB在脊柱调节中的作用还没有 因为Ank 2基因种系缺失的小鼠模型是致命的。我们产生了 新的可诱导的Ank 2小鼠系（Nex 1Cre-ERT 2：Ank 2flox：EGFPflox），其中Ank 2可以在线粒体后缺失， 在出生后的发育过程中的锥体神经元（PN），规避致死性。AnkB条件mu- 突变体类似于缺乏两种主要AnkB同种型（220和440 kDa）的多种人类新生ASD变体。 初步结果显示AnkB突变体的棘密度增加和兴奋性神经传递改变 PFC.我们将研究AnkB在发育过程中调节脊柱修剪和突触消除的假设。 在PFC网络中，AnkB的遗传缺陷会在敏感的出生后阶段破坏这些机制， 影响皮质兴奋性和与ASD相关的行为。 在目的1中，将分析Nex 1Cre-ERT 2：Ank 2flox：EGFPflox小鼠以描述AnkB基因剂量如何在脱蛋白中起作用。 成年和成年的每种性别的PN改变树突棘的调节和兴奋性和抑制性突触的水平。 一个新的小鼠模型Nex 1Cre-ERT 2：Ank 2 exon 37 flox：EGFPflox只删除AnkB-440， 以区分异构体特异性作用。目的2：一种新的Ank 2缺陷的分子拯救试验， 神经元培养物将询问AnkB在脑信号蛋白-3诱导的脊柱重塑、受体聚集中的功能 和细胞内信号传导。将分析选定的人从头Ank 2错义突变的受损 L1-CAM和β-血影蛋白结合和信号传导导致脊柱修剪。超分辨率显微镜将是 用于探索AnkB在组织棘中的突触纳米结构域中的潜在新作用。在目标3 AnkB缺陷增加皮层兴奋性的假设将通过电生理记录来检验- 在来自正常和AnkB缺陷的每种性别的小鼠的皮质切片中的第2/3层PN。兴奋和抑制- 将比较Ry传输，并通过激光扫描光刺激映射突触输入。行为 对Ank 2突变小鼠的测试将阐明AnkB在社交性、反向学习和工作记忆中的作用。 该项目预计将产生重大影响，因为它将阐明新的分子机制 用于调节皮层投射神经元在发育中的前额微电路的兴奋性连接，和 AnkB缺陷对自闭症谱系障碍相关突触病理学的影响

Mechanisms of spine and synaptic remodeling in the prefrontal cortex (PFC) underlie critical functions of working memory, cognitive flexibility, and sociability yet are incompletely understood. Impaired spine elimina- tion during brain development likely contributes to increased spine and synapse density in autism spectrum disorders (ASD). We identified a novel mechanism of selective spine pruning in which Semaphorin-3 receptor complexes, comprising L1 adhesion molecules, Neuropilins, and PlexinAs, promote activity-dependent spine elimination in the juvenile brain through Rho family GTPases. All L1-CAMs bind Ankyrin B (AnkB), an actin- spectrin adaptor encoded by the high confidence ASD gene Ank2. A role for AnkB in spine regulation has not been explored, because mouse models with germline deletion of the Ank2 gene are lethal. We generated a novel, inducible Ank2 mouse line (Nex1Cre-ERT2:Ank2flox:EGFPflox) in which Ank2 can be deleted in postmitot- ic pyramidal neurons (PNs) during postnatal development, circumventing lethality. The AnkB conditional mu- tant resembles multiple human de novo ASD variants lacking both principal AnkB isoforms (220 and 440 kDa). Preliminary results reveal elevated spine density and altered excitatory neurotransmission in AnkB mutant PFC. We will investigate the hypothesis that AnkB regulates spine pruning and synapse elimination in develop- ing PFC networks, and that genetic deficiency of AnkB disrupts these mechanisms at sensitive postnatal stag- es, impacting cortical excitability and behaviors relevant to ASD. In Aim 1 Nex1Cre-ERT2:Ank2flox:EGFPflox mice will be analyzed to delineate how AnkB gene dosage in de- veloping and adult PNs of each sex alters dendritic spine regulation and levels of excitatory and inhibitory syn- apses in the PFC. A new mouse model Nex1Cre-ERT2:Ank2 exon37flox:EGFPflox deleting only AnkB-440 will be generated to distinquish isoform-specific roles. In Aim 2 a novel molecular rescue assay in Ank2-deficient neuronal cultures will interrogate AnkB function in Semaphorin-3 induced spine remodeling, receptor clustering and intracellular signaling. Selected human de novo Ank2 missense mutations will be analyzed for impaired L1-CAM and β-spectrin binding and signaling leading to spine pruning. Super-resolution microscopy will be employed to explore a potentially new role for AnkB in organizing synaptic nanodomains in spines. In Aim 3 the hypothesis that AnkB deficiency increases cortical excitability will be tested by electrophysiological record- ing of layer 2/3 PNs in cortical slices from normal and AnkB-deficient mice of each sex. Excitatory and inhibito- ry transmission will be compared, and synaptic inputs mapped by laser scanning photo-stimulation. Behavioral testing of Ank2 mutant mice will address roles for AnkB in sociability, reversal learning, and working memory. This project is expected to have significant impact, because it will illuminate novel molecular mechanisms for regulating excitatory connectivity of cortical projection neurons in developing prefrontal microcircuitry, and consequences of AnkB deficiency on synaptic pathology associated with autism spectrum disorders.