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This dissertation describes the influence of astrocyte-derived factors during CNS myelination using native in vitro and ex vivo myelinating cultures as well as a novel developed computer-vision algorithm for reliable myelin sheath quantification. Miniaturized immunopanned retinal ganglion celloligodendrocyte precursor cell (RGC-OPC) co-cultures in a higher throughput multi-well plate format in vitro, suitable to test small compound libraries, were established. Maintaining the reciprocal interaction of vital axons and OLs ensured compact myelin formation and allowed the rapid study and manipulation of all the steps of myelination. A novel computer-vision algorithm tailored to quantitate myelin sheaths reliably, permit for the first time analysis at the single cell level and with enough sensitivity to catch dose-responses of myelinating compounds. The robustness and efficacy of these combined experimental and technical advances were demonstrated with published promyelinating compounds BQ3020 and XAV939. This method was the starting point for the study of myelin affecting astrocyte-derived factors from primary human and rat astrocytes. A large number of bioactive proteins and lipids was identified from the astrocyte conditioned media (ACM) and their promoting effect on myelination shown in vitro. On the hunt for astrocyte-derived factors, brevican was identified as a promoter of late-stage myelination in both in vitro and ex vivo cultures. Furthermore, brevican emerged to be particularly important during developmental myelin formation. Brevican knockout (BCAN KO) mice showed profoundly decelerated myelin formation in vivo at 14 days of age compared with wild-type (WT) mice, but a normal myelin phenotype in adulthood. At present, it remains uncertain whether brevican acts directly as a ligand of neuronal or glial receptors or attracts molecules to the OL/axon interphase. Nevertheless, new putative OLderived interactors of brevican such as contactin 1 (CNTN1) via tenascin-R (TN-R) linkage, are proposed in this work. In addition, the changes in astrocytic protein and lipid efflux in response to proinflammatory stimuli were investigated, and the conserved expression patterns in rats and humans were identified. The shift toward a reactive, inflamed, and hypertrophic astrocyte phenotype resulted in particular conserved changes in lipid and protein efflux that ultimately hampered myelination. Furthermore, brevican was significantly downregulated by reactive astrocytes, suggesting a possible mechanistic link. However, brevican was not solely responsible for this reduced myelination. In a disease state, the totality of astrocytic expression changes likely causes multiple disruptions simultaneously, some of which aid myelin homeostasis and some of which hinder it; several of these are discussed in this work. Overall, reactive astrocytes play a dual role, and concerning myelination the dominant harmful role could be determined by the interplay of secreted molecules with the surrounding extracellular milieu or the state of OPCs/OLs. In summary, a valuable workflow to study the steps of myelination in a reliable and automated manner in vitro was established. Moreover, this work provides important insights into the conserved spectrum of astrocyte-derived secretion cues as well as the alterations in protein and lipid efflux caused by astrocyte reactivity. Furthermore, brevican was introduced as a new relevant factor that regulates CNS myelination. Future work on the complex crosstalk between the ECM, astrocytes and myelination in the brain will yield a better understanding of the mechanistic cascade connecting brevican to myelin regulation in health and disease. Finally, these results may open new avenues for future relevant astrocyte-specific therapies that directly target remyelination. |
