Functions of Heparan Sulfate Proteoglycans in Axon Guidance and Degeneration.

描述（由申请人提供）： 神经元连接的精确组织对于处理信息至关重要。神经科学的一个主要挑战是了解这些连接是如何正确建立的，以及这种连接过程中的错误如何导致自闭症或精神分裂症等精神疾病。在发育过程中，神经元延伸轴突，这些轴突在吸引和排斥线索的引导下沿着限定的路径到达它们的大脑目标。除了这个引导过程，涉及修剪或变性的机制纠正了偏离正确路径的轴突，从而确保了回路形成的准确性。有几个因素调节轴突的引导，但它们提供的组合信息不足以塑造整个神经元网络。硫酸乙酰肝素蛋白聚糖（HSPGs）是一种存在于细胞表面和细胞外的核心蛋白，其糖链在轴突寻路过程中起着重要作用，是引发轴突选择性变性的关键 被误导的轴突然而，HSPGs如何控制这些不同的过程仍然是一个谜。HS链经过许多修饰，特别是硫酸化，赋予它们独特的多样性和结构复杂性。这些修改已经被提出来产生一个复杂的“糖代码”，通过调节大脑布线所必需的大多数因素来协调轴突连接的形成。虽然很吸引人，但这种“糖密码”假说仍然是理论上的，尚未在脊椎动物中得到验证。此外，携带这些特定HS模式的核心蛋白的贡献是未知的。使用斑马鱼视网膜顶盖系统作为体内模型，我发现了三种不同的HSPGs（SDC 2，GPC 1a和GPC 1b）调节视网膜轴突导向的不同步骤。在这个项目中，我建议解剖出这些核心蛋白和HS链修饰如何使用体内方法，遗传学和HS生物化学的独特组合来调节轴突导航。本研究的第一个目的是证实SDC 2、GPC 1a和GPC 1b的新功能，并在细胞和分子水平上研究它们的作用方式。它将确定它们的缺失如何影响轴突行为并导致指导错误，它们在哪里起作用，它们的HS链是否是其功能所必需的，以及它们与哪些因素相互作用。该项目的第二个目标是确定特定HS结构基序对轴突寻路和变性的贡献。它将测试6-O和3-O磺基转移酶的作用，修改HS链的硫酸化在特定的位置，并研究如何合成HS寡糖与定义的硫酸化基序和大小调节视网膜轴突导航，无论是在体外培养系统和体内发育胚胎。通过研究核心蛋白和HS精细结构的作用，拟议的研究提供了一个独特的机会来测试糖代码是否在发育过程中协调大脑布线。它还可能为神经系统疾病的治疗和再生策略的未来发展提供新的研究方向。

DESCRIPTION (provided by applicant): Precise organization of neuronal connections is crucial for processing information. A major challenge in neuroscience is to understand how these connections are properly established, and how errors in this wiring process can lead to psychiatric disorders like autism or schizophrenia. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. In addition to this guidance process, mechanisms involving pruning or degeneration correct axons that have deviated from the right path, thereby ensuring accuracy of circuit formation. Several factors regulate axon guidance, but the combined information they provide is not sufficient to sculpt the entire neuronal network. Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular core proteins with attached heparan sulfate (HS) sugar chains that play crucial roles in axon pathfinding, and are essential for triggering the selective degeneration of misguided axons. How HSPGs control these different processes remains however a mystery. HS chains undergo many modifications, especially sulfations, that confer on them a unique diversity and structural complexity. These modifications have been proposed to generate a complex "sugar code" orchestrating the formation of axonal connections by regulating most factors essential for brain wiring. While appealing, this "sugar code" hypothesis is still theoretial and has not been tested in vertebrates. Moreover, the contribution of the core proteins carrying these specific HS patterns is not known. Using the zebrafish retinotectal system as an in vivo model, I have discovered that three distinct HSPGs (SDC2, GPC1a and GPC1b) regulate different steps of retinal axon guidance. In this project, I propose to dissect out how these core proteins and HS chain modifications regulate axon navigation using a unique combination of in vivo approaches, genetics and HS biochemistry. The first aim of this proposal will confirm the novel functions of SDC2, GPC1a and GPC1b, and investigate their mode of action at a cellular and molecular level. It will determine how their absence affects axon behavior and leads to guidance errors, where they act, whether their HS chains are required for their function, and what are the factors they interact with. The second aim of this project will determine the contribution of specific HS structural motifs for axon pathfinding and degeneration. It will test te roles of the 6-O and 3-O sulfotransferases that modify HS chains with sulfations at specific positions, and examine how synthetic HS oligosaccharides with defined sulfation motifs and sizes regulate retinal axon navigation, both in vitro in culture systems and in vivo in the developing embryo. By studying the roles of both core proteins and HS fine structure, the proposed studies give a unique opportunity to test whether a sugar code orchestrates brain wiring during development. It might also provide new lines of research for future development of therapeutic and regenerative strategies in the context of neurological disorders.