Improving Brain Recovery Through Glycoengineering

项目摘要 该项目基于代谢糖工程（MGE）的最新进展，MGE是一个技术平台， 非天然单糖阻断细胞表面展示聚糖的生物合成途径。作为 结果，碳水化合物中天然没有的化学功能被安装在糖萼中，这可以改变 细胞粘附、受体活性和下游事件（例如，细胞凋亡、分化和运动性）。在 在之前的工作中，我们开发了N-乙酰甘露糖胺（ManNAc）类似物"Ac5ManNTGc"， 在人类胚胎细胞中，唾液酸的种类很多，他们发现，当细胞生长在"高亲和力"的环境中时， 表面（例如，金，它与硫醇形成配位共价键）-Wnt信号转导在 诱导细胞外Wnt蛋白的缺失和神经元分化。在体内翻译这个 然而，这种方法受到对不可降解的金支架的要求的阻碍。我们最近 通过设计新的ManNAc类似物克服了这一障碍，其中硫醇存在于较长的接头上， 使该官能团进一步远离核心单糖并增加类似物效力。 重要的是，新的类似物在不存在支架的情况下提供促神经原活性，从而简化了神经原活性。 活体翻译本项目将在具体目标1中探索hNSCs中的类似机制;该目标将定义 巯基修饰的ManNAc类似物的化学结构、动力学和剂量（沿着有化学惰性的）如何 大小匹配的对照）调节Aim 1a中的细胞聚糖;评价Aim 1a中细胞粘附和运动的变化。 目的1b，并评估人神经干细胞（hNSCs）在目的1c中的分化。下一篇：Specific Aim 2、将优化的模拟处理条件应用于促进大鼠心脏神经再生 通过将MGE修饰的移植到受伤的动物中来建立脑损伤的脑缺血再灌注（CA）模型。我们将比较hNSCs 用我们的新的巯基修饰的类似物治疗，并适当控制CA后的功能恢复， 评估移植的hNSC在大鼠脑中的存活、粘附、分布和迁移。在具体目标3中， 我们将评估生物化学（Wnt信号传导和钙粘蛋白参与）和细胞（组织浸润免疫 细胞）水平的机制，我们提出有助于MGE在脑损伤恢复中的愈合作用（在Aim 3a）。最后，在目标3b中，我们将通过质谱法表征细胞范围内的"糖位点"，并使用 糖生物信息学分析以鉴定MGE的未知生化介质。具体来说，我们预计 鉴定MGE在植入的hNSCs以及反式作用宿主中的有益作用的新介质 proteins.我们假设硫代类似物通过受体-受体的复杂组合调节hNSC的命运。 对细胞信号传导和粘附的特异性作用，提供了一套多效的愈合效果， 通过传统疗法实现。因此，我们的创新方法开辟了一条新的途径， 用我们新硫醇基MGE技术改进干细胞治疗。

Project Summary This project is based on recent advances in metabolic glycoengineering (MGE), a technology platform where non-natural monosaccharides intercept the biosynthetic pathways for cell surface-displayed glycans. As a result, chemical functionalities not naturally found in carbohydrates are installed in the glycalyx, which can alter cell adhesion, receptor activity, and downstream events (e.g., apoptosis, differentiation, and motility). In previous work, we developed the N-acetylmannosamine (ManNAc) analog “Ac5ManNTGc” to install thiol groups into sialic acids in human embryonic cells and found that – when the cells were grown on a “high affinity” surface (e.g., gold, which forms coordinate covalent bonds with thiols) – Wnt signaling was upregulated in the absence of extracellular Wnt proteins and neuronal differentiation was induced. In vivo translation of this approach, however, was hindered by the requirement for a non-degradable gold scaffold. We recently overcame this impediment by designing new ManNAc analogs with thiols presented on longer linkers, which extends this functional group further away from the core monosccharide and increases analog potency. Critically, the new analogs provide pro-neurogenic activity in the absence of a scaffold thereby simplifying in vivo translation. This project will explore analog mechanism in hNSCs in Specific Aim 1; this aim will define how the chemical structure, kinetics, and dose of thiol-modified ManNAc analogs (along with chemically inert size-matched controls) modulate cellular glycans in Aim 1a; evaluate changes to cell adhesion and motility in Aim 1b, and evaluate the differentiation of human neural stem cells (hNSCs) in Aim 1c. Next, in Specific Aim 2, we will apply the optimized analog-treatment conditions to improve neural regeneration in a rat cardiac arrest (CA) model of brain injury by transplanting MGE-modified into injured animals. We will compare hNSCs treated with our new thiol-modified analogs with appropriate controls on functional recovery after CA by evaluating survival, adhesion, distribution, and migration of transplanted hNSCs in rat brain. In Specific Aim 3, we will evaluate biochemical (Wnt signaling and cadherin involvement) and cellular (tissue infiltrating immune cells) level mechanisms we propose contribute to the healing effects of MGE in brain injury recovery (in Aim 3a). Finally, in Aim 3b we will characterize cell-wide “glycosites” by mass spectrometry and use glycobioinformatics analyses to identify unknown biochemical mediators of MGE. Specifically, we anticipate identifying new mediators of the beneficial effects of MGE in the implanted hNSCs as well as trans-acting host proteins. We hypothesize that thio-analogs modulate hNSC fate through a complex combination of receptor- specific effects on cell signaling and adhesion providing a pleiotropic suite of healing effects that cannot be achieved through conventional therapies. Accordingly, our innovative approach opens a new avenue to improve stem cell therapy with our new thiol-based MGE technique.