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

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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.