Department of Molecular Sciences and ARC Centre of Excellence for Nanoscale Biophotonics, Macquarie University, Sydney, NSW, Australia.
Institute for Glycomics, Griffith University, Gold Coast, Queensland, Australia.
Mol Neurobiol. 2020 Feb;57(2):964-975. doi: 10.1007/s12035-019-01791-7. Epub 2019 Oct 24.
Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.
聚唾液酸（polySia）是一种 2,8-连接的唾液酸的长同聚物，在胚胎脑中含量丰富，在成人脑中主要局限于表现出神经发生和结构可塑性的区域。在中枢神经系统（CNS）中，polySia 对于细胞-细胞相互作用、分化、迁移和细胞因子反应非常重要，这些是调节细胞间相互作用的关键神经元功能，而细胞间相互作用是中枢神经系统免疫信号的基础。在最近的报告中，吗啡的一种代谢物，吗啡-3-葡糖苷酸（M3G）已被证明会在中枢神经系统中引起免疫信号。在这项研究中，我们使用免疫细胞化学和 Western blot 分析比较了神经生长因子（NGF）、脂多糖（LPS）和 M3G 暴露对 PC12 细胞中 polySia 表达的影响。还通过内神经氨酸酶消化从刺激细胞蛋白中提取 polySia，并使用荧光标记进行定量，然后通过 HPLC 分析。与未刺激的细胞或暴露于 TLR4 拮抗剂 LPS-RS 的细胞相比，NGF、M3G 或 LPS 刺激后 polySia 的表达明显增加。此外，我们分析了在这些细胞的细胞表面存在或不存在 polySia 表达的情况下，测试剂暴露对细胞迁移和这些细胞的氧化应激反应的影响。我们观察到没有 polySia 的细胞以及 M3G 或 LPS 刺激后氧化应激增加。我们的研究提供了证据，表明神经元样 PC12 细胞中 polySia 的表达受 M3G 和 LPS 暴露的影响，提示 TLR4 在触发这些事件中起作用。
