A chemical approach to elucidating the structure-function relationships of chondronitin sulfate glycosaminoglycans

 描述(由申请人提供)：这个项目将集中在硫酸软骨素糖胺聚糖(CS GAGS)，这是一类在发育、病毒入侵、癌症和脊髓损伤中发挥重要作用的多糖。CS GAG在体内表现出不同的硫酸盐化模式，这些模式在时空上是受调节的。然而，由于CS结构的复杂性和缺乏工具，确定特定硫酸盐基序的功能的努力一直受到阻碍。在这笔赠款中，我们将结合有机化学和生物学的力量来克服这些挑战，并确定神经系统中特定基序的新功能。该计划的主要目标是：(1)促进对CS-GAG结构-功能关系的基本了解；(2)了解CS-GAG在神经可塑性和再生中的作用；(3)开发新的方法来研究和操纵GAG介导的生物过程，以刺激突触可塑性和神经元修复。在上一次授予期间，我们开发了一套化学工具来研究特定的硫酸盐化基序，并发现一个特定的基序CS-E抑制脊髓损伤后的轴突再生。用抗CS-E抗体阻断CS-E基序可刺激体内轴突再生。此外，我们发现，这个相同的基序排斥轴突，并在大脑发育过程中的神经回路形成中发挥关键作用。这项工作的一个重要观察结果是，CS-E的活性需要它与细胞表面受体相互作用，并激活神经元中特定的抑制信号通路。在目前的拨款中，我们将开发新的方法来调节CS-E与神经元受体的相互作用，包括病毒介导的单链抗CS-E抗体、小分子磺基转移酶抑制剂和糖基聚合物模拟物(目标1)。我们将研究CS-E是如何调节蛋白质信号复合体的，尤其是信号素-3A/神经粘连蛋白-1/丛蛋白A(Sema3A/Nrp1/PlxnA)和ePhin/Eph受体复合体(EFN/Eph)(目标2a和3a)。最后，我们将研究目标1中开发的药物在促进视皮层神经可塑性(目标2b，c)和脊髓损伤后轴突再生(目标3b)方面的能力。这些研究有望提供新的化学工具来促进对GAG的理解，并从根本上改变人们对CS GAG的看法-从静态的被动分子到积极调节重要信号通路的配体。最后，如果成功，目标1中开发的药物可能会导致在衰老、损伤和疾病的情况下刺激神经元可塑性和修复的新治疗策略。

 DESCRIPTION (provided by applicant): This project will focus on chondroitin sulfate glycosaminoglycans (CS GAGs), a class of polysaccharides that play important roles in development, viral invasion, cancer, and spinal cord injury. CS GAGs display diverse sulfation patterns that are spatiotemporally regulated in vivo. However, efforts to identify functions for specific sulfation motifs have been hampered by the structural complexity of CS and a lack of tools. In this grant, we will combine the power of both organic chemistry and biology to overcome these challenges and identify novel functions for specific motifs in the nervous system. The broad objectives of this program are to: (1) advance a fundamental understanding of the structure-function relationships of CS GAGs, (2) understand the roles of CS GAGs in neuroplasticity and regeneration, and (3) develop new approaches to study and manipulate GAG-mediated biological processes, with the long-term goal of stimulating synaptic plasticity and neuronal repair. In the last granting period, we developed a set of chemical tools to study specific sulfation motifs and discovered that a particular motif, CS-E, inhibits axon regeneration after spinal cord injury. Blocking the CS-E motif using an anti-CS-E antibody stimulated axon regeneration in vivo. Moreover, we found that this same motif repels axons and plays a critical role in neural circuit formation during brain development. An important observation from this work was that the activity of CS-E required its interaction with cell-surface receptors and activation of specific inhibitory signaling pathways in neurons. In the present grant, we will develop new approaches to modulate the interactions of CS-E with neuronal receptors, including the viral-mediated delivery of single-chain anti-CS-E antibodies, small- molecule sulfotransferase inhibitors, and glycopolymer mimetics (Aim 1). We will study how CS-E regulates protein signaling complexes, with a particular focus on semaphorin-3A/neuropilin-1/plexin A (Sema3A/Nrp1/PlxnA) and ephrin/Eph receptor (Efn/Eph) complexes (Aims 2a and 3a). Finally, we will investigate the ability of the agents developed in Aim 1 to promote neuroplasticity in the visual cortex (Aims 2b,c) and axon regeneration after spinal cord injury (Aim 3b). These studies are expected to provide new chemical tools to advance an understanding of GAGs and fundamentally change how CS GAGs are viewed - from being static, passive molecules to ligands that actively regulate important signaling pathways. Finally, if successful, the agents developed in Aim 1 could lead to novel therapeutic strategies for stimulating neuronal plasticity and repair in the case of aging, injury, and disease.