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关键作用。这些细胞发育的缺陷与神经和精神疾病有关。皮质中间神经元的前体通过切向迁移在胚胎皮质中覆盖很大的距离。即使在小鼠的小皮质中，单个中间神经元也要迁移几天才能均匀分散。申请人的工作导致了目前的观点，即趋化因子CXCL 12及其受体CXCR 4和CXCR 7形成了唯一的化学引诱物信号传导模块，其在皮质中的主要迁移路线上引导中间神经元。在该系统中，常规受体CXCR 4通过抑制性G蛋白介导趋化信号。申请人最近的工作表明，CXCR 7作为一种非典型趋化因子受体，可以隔离CXCL 12，以防止CXCR 4在长期切线迁移过程中的过度刺激、下调和脱敏。根据第二种理论，未鉴定的CXCR 7信号，可能是β-Arrestin（β Arr）介导的MAP激酶激活，影响中间神经元迁移。事实上，对于重组表达的七螺旋受体，有充分的证据表明激动剂诱导的C-末端Ser/Thr位点的磷酸化稳定了受体/β Arr相互作用，使受体与G蛋白解偶联，并促进受体内化以及β Arr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中进行研究。该提案解决了受体磷酸化和β Arr如何调节CXCL 12引导的中间神经元迁移。为此，申请人已经产生了独特的基因工程小鼠模型：（1.）编码受体的Cxcr 7等位基因（Cxcr 7STA）的敲入，其中所有潜在的C-末端Ser和Thr磷酸化位点通过Ala置换消除，（2.）一群小鼠以繁殖缺乏一种或两种β Arr同种型的胚胎，以及（3.）携带转基因的Cxcr 7STA和β Arr缺陷小鼠，所述转基因驱动在Cxcl 12启动子控制下的CXCL 12-RFP融合蛋白的表达以使CXCL 12可视化，以及在Cxcr 7启动子控制下的eGFP的表达以使中间神经元可视化。申请人提出使用组织学、神经元培养和成像技术研究来自Cxcr 7 STA/STA和β Arr缺陷胚胎的制备物中的神经元间迁移、受体介导的CXCL 12摄取以及CXCL 12受体的磷酸化、运输和下调。该项目阐明了在相关的体内系统中，磷酸化和β Arr如何调节细胞迁移所需的常规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.