CXCL12-dependent development of neuronal structures under the control of the atypical chemokine receptor CXCR7

gaba能中间神经元在大脑皮层神经元回路的活动调节中起着关键作用。这些细胞发育中的缺陷与神经和精神疾病有关。皮层中间神经元的前体通过切向迁移在胚胎皮层中覆盖较大的距离。即使在小鼠的小皮层中，单个中间神经元也会迁移数天以均匀分散。申请人的工作导致了目前的观点，即趋化因子CXCL12及其受体CXCR4和CXCR7形成了唯一的趋化信号传导模块，指导中间神经元在皮层中的主要迁移路线。在该系统中，常规受体CXCR4通过抑制性G蛋白介导趋化信号。申请人最近的工作表明，CXCR7作为一种非典型趋化因子受体，可隔离CXCL12，以防止CXCR4在长时间切向迁移过程中过度刺激、下调和脱敏。根据第二种理论，一个未知的CXCR7信号，可能是ß-Arrestin （ßArr）介导的MAP激酶激活，影响神经元间迁移。事实上，有充分的证据表明，在重组表达的七螺旋受体中，激动剂诱导的c端丝氨酸/苏氨酸位点磷酸化稳定了受体/ßArr相互作用，使受体与G蛋白分离，促进受体内化以及ßArr依赖性MAP激酶活化。迄今为止，这些过程对神经元迁移的影响尚未在哺乳动物生物体中进行研究。这一建议解决了受体磷酸化和ßArr如何调节cxcl12引导的神经元间迁移。为此，申请人产生了独特的基因工程小鼠模型：(1)。编码受体的Cxcr7等位基因（Cxcr7STA）敲入，其中所有潜在的c端丝氨酸和苏氨酸磷酸化位点被Ala替换消除，(2)一群小鼠繁殖缺乏一个或两个ßArr亚型的胚胎；Cxcr7STA和ß arr缺陷小鼠携带转基因，在Cxcl12启动子控制下驱动Cxcl12 - rfp融合蛋白的表达，以显示Cxcl12，在Cxcr7启动子控制下表达eGFP，以显示中间神经元。申请人拟利用组织学、神经元培养和成像技术研究Cxcr7STA/STA和ß arr缺陷胚胎制剂中神经元间迁移、受体介导的CXCL12摄取，以及CXCL12受体的磷酸化、转运和下调。该项目阐明了在一个相关的体内系统中，磷酸化和ßArr如何调节细胞迁移所需的传统和非典型趋化因子受体的相互作用。

GABAergic interneurons play a critical role in activity modulation of neuronal circuits in the cerebral cortex. Defects in the development of these cells have been linked to neurological and mental disorders. Precursors of cortical interneurons cover large distances in the embryonic cortex by tangential migration. Even in the small cortex of mice, individual interneurons migrate several days to become evenly dispersed. Work of the applicant led to the current notion that the chemokine CXCL12 and its receptors CXCR4 and CXCR7 form the only chemoattractant signaling module guiding interneurons on their main migration routes in the cortex. In this system, the conventional receptor CXCR4 mediates chemotactic signals through inhibitory G proteins. The applicant´s recent work showed that CXCR7 acts as an atypical chemokine receptor that sequesters CXCL12 to prevent excessive stimulation, down-regulation and desensitization of CXCR4 during the long-lasting tangential migration process. According to a second theory, an unidentified CXCR7 signal, probably ß-Arrestin (ßArr)-mediated MAP Kinase activation, influences interneuron migration. In fact, there is ample evidence with recombinant expressed heptahelical receptors that agonist-induced phosphorylation at C-terminal Ser/Thr sites stabilizes receptor/ßArr interactions, uncouples receptors from G proteins and promotes receptor internalization as well as ßArr-dependent MAP Kinase activation. To date, the influence of these processes on neuronal migration has not been investigated in mammalian organisms. This proposal addresses how receptor phosphorylation and ßArr regulate CXCL12-guided interneuron migration. To this end, the applicant has generated unique genetically engineered mouse models: (1.) A knockin of a Cxcr7 allele (Cxcr7STA) encoding for a receptor in which all potential C-terminal Ser and Thr phosphorylation sites are eliminated by Ala replacement, (2.) a colony of mice to breed embryos lacking one or both ßArr isoforms and (3.) Cxcr7STA and ßArr-deficient mice carrying transgenes that drive expression of a CXCL12-RFP fusion protein under control of the Cxcl12 promoter for visualization of CXCL12 and expression of eGFP under the Cxcr7 promoter for visualization of interneurons. The applicant proposes to study interneuron migration, receptor-mediated CXCL12 uptake, as well as phosphorylation, trafficking and down-regulation of CXCL12 receptors in preparations from Cxcr7STA/STA and ßArr-deficient embryos using histological, neuronal culture and imaging techniques. This project elucidates in a relevant in vivo system, how phosphorylation and ßArr regulate the interplay of a conventional and an atypical chemokine receptor necessary for cell migration.