Signaling receptor turnover by endocytic pathways in the patterning of the vertebrate nervous system

脊椎动物神经系统模式中内吞途径的信号受体周转

• 批准号: RGPIN-2015-04475
• 负责人: Iulianella, Angelo
• 金额: $ 2.19万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=654349
• 关键词: Signaling; receptor; turnover; endocytic; pathways

The patterning of the vertebrate nervous system is a key event in the generation of cell diversity and the wiring of the brain. It is thought that Sonic Hedgehog (Shh) signaling acts as a "morphogen" gradient specifying distinct cell types based on the concentration threshold of Shh signaling along the dorso-ventral axis. The default model states that Shh is a signaling molecule expressed in a gradient of high to low in the ventral nervous system. It binds to a receptor called Patched1 (Ptch1) at the cell surface in initiation an intracellular series of events that ultimately leads to transcriptional activation and cell fate acquisition. Normally, in the absence of ligand, Ptch1 prevents the activation of a co-receptor called Smoothened, which in turn is required for the activation of Shh pathway through the Gli transcription factors. Ptch1 therefore acts as a negative regulator for Shh pathway activation and the binding of Shh to Ptch1 relieves this inhibition. However, it is unclear how a graded response to Shh signaling is achieved in growing tissues such as the developing neural tube. What remains to be determined is how Ptch1 helps create a graded response to the Shh morphogen field in the developing vertebrate nervous system.*** The binding of Hedgehog ligands to Ptch1 triggers their internalization through the endocytic pathway and ultimately to the lysosome, where Ptch1 is degraded. Thus, one way to set the output range of Shh signaling is to control the steady-state levels of Ptch1 through endocytosis. In Drosophila and mammalian cells, it is known that ubiquitin ligases target Ptch1 to degradation and allow for the activation of Shh signaling. However, it is not known how Ptch1 internalization and targeting to endocytic and lysosomal vesicles is regulated. In a screen for novel Ptch1-interacting proteins, we identified proteins implicated in endocytic shuttling that may regulate the targeting of Ptch1 to intracellular vesicles. This is expected to promote Ptch1 degradation and enhance Shh-dependent ventral patterning in the developing nervous system. Our objectives are: (1) validate the role of these putative Ptch1-interacting proteins in endocyctic processing using assays in cell culture model systems; and (2) evaluate whether the over-expression and knockdown of these proteins during neural development can alter ventral patterning in response to Shh signaling. *** The proposed research addresses a fundamental question in neurobiology: how is the graded response to morphogen signaling controlled? We use a combination of in vitro and in vivo approaches in tractable model systems to address this question, providing an engaging training opportunity in basic cellular and developmental biology. Altogether, this research will significantly advance our understanding of the Shh signaling and lead to fundamental insights into how pattern is generated in the developing vertebrate nervous system.**

脊椎动物神经系统的模式化是细胞多样性产生和大脑布线的关键事件。据认为，Sonic Hedgehog（Shh）信号传导充当基于沿着背腹轴的Shh信号传导的浓度阈值来指定不同细胞类型的“形态原”梯度。默认模型指出Shh是一种信号分子，在腹神经系统中以从高到低的梯度表达。它与细胞表面的Patched 1（Ptch 1）受体结合，引发细胞内一系列事件，最终导致转录激活和细胞命运获得。通常情况下，在没有配体的情况下，Ptch 1阻止称为Smoothened的辅助受体的激活，而Smoothened又是通过Gli转录因子激活Shh通路所必需的。因此，Ptch 1作为Shh通路激活的负调节剂，Shh与Ptch 1的结合缓解了这种抑制。然而，目前还不清楚如何在生长组织（如发育中的神经管）中实现对Shh信号的分级响应。尚待确定的是Ptch 1如何帮助在发育中的脊椎动物神经系统中对Shh形态发生域产生分级反应。Hedgehog配体与Ptch 1的结合通过内吞途径触发它们的内化，并最终到达溶酶体，在那里Ptch 1被降解。因此，设置Shh信号传导的输出范围的一种方法是通过胞吞作用控制Ptch 1的稳态水平。在果蝇和哺乳动物细胞中，已知泛素连接酶靶向Ptch 1降解并允许Shh信号的激活。然而，目前尚不清楚Ptch 1内化和靶向内吞和溶酶体囊泡是如何调节的。在一个新的Ptch 1相互作用的蛋白质的筛选中，我们确定了参与内吞穿梭的蛋白质，这些蛋白质可能调节Ptch 1靶向细胞内囊泡。这有望促进Ptch 1降解，并增强发育中神经系统中Shh依赖的腹侧模式。我们的目标是：（1）使用细胞培养模型系统中的测定来验证这些推定的Ptch 1相互作用蛋白在内吞加工中的作用;以及（2）评估这些蛋白在神经发育期间的过表达和敲低是否可以响应于Shh信号而改变腹侧模式。 * 这项拟议中的研究解决了神经生物学中的一个基本问题：对形态发生素信号的分级反应是如何控制的？我们在易处理的模型系统中使用体外和体内方法的组合来解决这个问题，为基础细胞和发育生物学提供了一个引人入胜的培训机会。总而言之，这项研究将大大推进我们对Shh信号的理解，并导致对发育中的脊椎动物神经系统如何产生模式的基本见解。