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

聚唾液酸（polySia）是一种对少量蛋白质进行聚糖修饰的物质。最突出的polySia载体是神经细胞粘附分子NCAM，但我们之前能够证明，小胶质细胞和巨噬细胞中的polySia存在于另外两种蛋白质上，即neuropilin-2和e -选择素配体-1。这种蛋白结合的多sia聚集在培养的小胶质细胞的高尔基腔室中，并在炎症激活的反应中被释放。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作为siglece的反式激活配体引起反馈抑制。我们也可以证明在体内皮层病变周围激活的小胶质细胞中有多sia积聚。证明可以在小鼠模型中研究小胶质细胞多sia对神经炎症的siglece依赖性作用是重要的，因为到目前为止，人们一直认为人类小胶质细胞和巨噬细胞的抑制性多sia受体siglece -11在小鼠中没有功能等同物。在第一个资助期的进一步工作揭示了人类特异性siglece -16对胶质母细胞瘤患者生存的影响。siglece -16与siglece -11具有相同的多聚体结合结构域，但与激活免疫信号有关，只有40%的人群能够表达功能性的siglece -16。我们的数据表明，polySia与siglece -16在肿瘤相关的小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延长了生存期。从小鼠模型中产生的小胶质细胞多sia的数据中产生的主要开放问题将是当前更新建议的重点。损伤诱导的小胶质细胞释放polySia的时间过程将通过我们建立的分析方法在脑切片原位培养和体内皮层损伤后进行研究。用不能产生小胶质细胞polySia的小鼠研究polySia释放对小胶质细胞衰老、炎症标志物、神经元损伤和补体依赖性突触吞噬的可能影响。结果将与外用polySia对siglece阳性或阴性切片培养的影响进行比较。此外，具有确定聚合物长度的polySia分数将被应用，这些分数要么能够激活siglece - e依赖性小胶质细胞反应，要么只能诱导siglece - e非依赖性效应。在第一个项目阶段，建立了具有生理活性的聚合物长度及其生产流程。总之，该项目的目的是揭示小胶质细胞多唾液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。通过定义多sia分数调节病理小胶质细胞状态的方法为新的治疗干预开辟了前景。

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.