Cellular regulation, mechanisms of action, and functions of newly identified polysialic acid-modified proteins in microglia

新发现的小胶质细胞中聚唾液酸修饰蛋白的细胞调节、作用机制和功能

• 批准号: 324633948
• 负责人: Professor Dr. Herbert Hildebrandt
• 金额: --
• 依托单位: Institut für Klinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/324633948?language=en
• 关键词: Cellular; regulation; mechanisms; action; functions

Polysialic acid (polySia) is a glycan modification of a small number of proteins. The most prominent polySia carrier is the neural cell adhesion molecule NCAM, but we previously were able to demonstrate that polySia in microglia and macrophages resides on two other proteins, neuropilin-2 and E-selectin ligand-1. This pool of protein-bound polySia accumulates in the Golgi compartment of cultured microglia and is released in response to inflammatory activation. In the first funding period, we could show that this release depends on calcium, is maintained for at least 24h, and causes feedback inhibition by acting as a trans-activating ligand of Siglec-E in LPS-induced murine microglia in vitro. We also could demonstrate polySia accumulation in activated microglia around a cortical lesion in vivo. Demonstrating that Siglec-E-dependent effects of microglial polySia on neuroinflammation can be studied in the mouse model is important, because so far it has been assumed that the inhibitory polySia receptor Siglec-11 on microglia and macrophages in man has no functional equivalent in mice. Further work in the first funding period revealed an impact of the human-specific Siglec-16 on survival of glioblastoma patients. Siglec-16 has the same polySia-binding domain as Siglec-11 but is linked to activating immune signalling and only 40% of the population is able to express functional Siglec-16. Our data imply that interactions of polySia with Siglec-16 on tumor-associated micoglia and macrophages lead to prolonged survival by promoting a proinflammatory immune milieu. Major open questions arising from the data on microglial polySia production in the mouse model will be in the focus of the current renewal proposal. The time course of polySia release by injury-induced microglia will be studied by our established repertoire of analytical methods in brain slice cultures in situ and after cortical lesion in vivo. Possible effects of polySia release on microglial senescence, inflammatory markers, neuronal damage and complement-dependent phagocytosis of synapses by microglia will be addressed using mice unable to produce microglial polySia. Outcomes will be compared to effects of externally applied polySia on Siglec-E-positive oder –negative slice cultures. In addition polySia fractions with defined polymer lengths will be applied, that either are able to activate Siglec-E-dependent microglia responses, or can only induce Siglec-E independent effects. The physiologically active polymer lengths and a work flow for their production have been established in first project period. Together, the aim of the project is to reveal if and how shedding of polysialylated proteins by microglia affects age-related or injury-induced neuroinflammation, neuronal damage and complement-dependent synapse elimination. The approach to modulate pathological microglia states by defined polySia fractions opens perspectives for new therapeutic interventions.

聚唾液酸（polySia）是少数蛋白质的聚糖修饰。最突出的polySia载体是神经细胞粘附分子NCAM，但我们以前能够证明，polySia在小胶质细胞和巨噬细胞驻留在两个其他蛋白质，神经纤毛蛋白-2和E-选择素配体-1。这种蛋白结合的polySia池在培养的小胶质细胞的高尔基体隔室中积累，并响应于炎症激活而释放。在第一个资助期，我们可以证明这种释放依赖于钙，维持至少24小时，并通过在体外LPS诱导的小鼠小胶质细胞中充当Siglec-E的反式激活配体而引起反馈抑制。我们还可以证明polySia在体内皮质病变周围的活化小胶质细胞中的积累。证明可以在小鼠模型中研究小胶质细胞polySia对神经炎症的Siglec-E依赖性作用是重要的，因为到目前为止，已经假设人中小胶质细胞和巨噬细胞上的抑制性polySia受体Siglec-11在小鼠中没有功能等同物。第一个资助期的进一步工作揭示了人类特异性Siglec-16对胶质母细胞瘤患者生存的影响。Siglec-16具有与Siglec-11相同的多聚Sia结合结构域，但与激活免疫信号传导有关，并且只有40%的群体能够表达功能性Siglec-16。我们的数据表明，polySia与Siglec-16在肿瘤相关微胶质细胞和巨噬细胞上的相互作用通过促进促炎免疫环境而导致存活延长。小鼠模型中小胶质细胞polySia生产数据产生的主要未决问题将成为当前更新提案的重点。损伤诱导的小胶质细胞的polySia释放的时间过程将通过我们建立的分析方法库在原位脑切片培养和皮质损伤后在体内进行研究。将使用无法产生小胶质细胞polySia的小鼠来解决polySia释放对小胶质细胞衰老、炎症标志物、神经元损伤和小胶质细胞对突触的补体依赖性吞噬作用的可能影响。将结果与外部应用polySia对Siglec-E阳性或阴性切片培养物的影响进行比较。此外，将应用具有限定聚合物长度的polySia级分，其能够激活Siglec-E依赖性小胶质细胞应答，或仅能诱导Siglec-E非依赖性效应。在第一个项目期间，已经建立了生理活性聚合物长度及其生产的工作流程。总之，该项目的目的是揭示小胶质细胞的聚唾液酸化蛋白的脱落是否以及如何影响年龄相关或损伤诱导的神经炎症，神经元损伤和补体依赖性突触消除。通过定义的polySia组分调节病理性小胶质细胞状态的方法为新的治疗干预开辟了前景。