Molecular Mechanisms of Developmental Spine Remodeling

前额叶皮质(PFC)中脊椎和突触重构的机制是 工作记忆、认知灵活性和社交能力还没有完全被理解。脊椎消除功能受损- 大脑发育过程中的TION可能导致自闭症谱系中脊椎和突触密度的增加 精神障碍(ASD)。我们发现了一种选择性脊椎修剪的新机制，在这种机制中，Semaphorin-3受体 由L1黏附分子、神经粘连蛋白和丛状蛋白A组成的复合体促进活动依赖型脊柱 通过Rho家族GTP酶在青少年脑中消除。所有L1-CaM都结合Ankyrin B(AnkB)，这是一种肌动蛋白- 由高置信度ASD基因Ank2编码的血影蛋白接头。AnkB在脊柱调节中的作用尚未 因为Ank2基因胚系缺失的小鼠模型是致命的。我们生成了一个 一种新型的可诱导的Ank2小鼠株系(Nex1Cre-ERT2：Ank2flx：EGFP flx)，其中Ank2可在有丝分裂后缺失. IC锥体神经元(PNS)在出生后发育期间，绕过致命性。AnkB有条件MU- Ant类似于多种人类新生ASD变体，缺少两种主要AnkB亚型(220 kDa和440 kDa)。 初步结果显示AnkB突变体的脊柱密度增加和兴奋性神经传递改变 一等兵。我们将研究AnkB在发育中调节脊柱修剪和突触消除的假说。 在PFC网络中，AnkB的遗传缺陷在出生后敏感的雄鹿中破坏了这些机制。 ES影响皮质兴奋性和与ASD相关的行为。 在目标1中，将分析Nex1Cre-ERT2：ANK2FLOX：EGFP-FLOX小鼠，以描绘AnkB基因在DEC中的剂量 发育和成体PNS改变树突棘的调节以及兴奋性和抑制性合成的水平。 在一等兵阵容中倒下。只删除AnkB-440的新小鼠模型Nex1Cre-ERT2：Ank2外显子37flx：EGFP flx将 以区分特定于异构体的角色。在目标2中，一种新的Ank2缺失的分子救助方法 神经元培养将在Semaphorin-3诱导的脊髓重塑、受体聚集中询问AnkB的功能 和细胞内信号。将对受损的人类新Ank2错义突变进行分析 L1-CAM和β-SPECTIN结合和信号导致脊椎修剪。超分辨率显微镜将是 被用来探索AnkB在组织脊椎中的突触纳域方面的潜在新角色。在AIM 3中 AnkB缺乏增加皮质兴奋性的假说将通过电生理记录得到验证- 取正常小鼠和AnkB基因缺陷小鼠的大脑皮质切片，取第2/3层PNS。兴奋性和抑制性- 将对RY传输进行比较，并通过激光扫描光刺激映射突触输入。行为 对Ank2突变小鼠的测试将解决AnkB在社交、反向学习和工作记忆中的作用。 该项目有望产生重大影响，因为它将阐明新的分子机制。 用于调节大脑皮质投射神经元在前额叶微电路发育过程中的兴奋性连接，以及 AnkB缺乏对与自闭症谱系障碍相关的突触病理的影响。

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.