2Myelin is synthesized as a multilamellar membrane, but the mechanisms of membrane turnover are unknown. We find that myelin pieces are gradually released from aging myelin sheaths and are subsequently cleared by microglia. Myelin fragmentation increases with age and leads to the formation of insoluble, lipofuscin-like lysosomal inclusions in microglia. Thus, age-related myelin fragmentation is substantial leading to lysosomal storage and contributing to microglia senescence and immune dysfunction in aging.Myelin is formed by oligodendrocytes as a multilamellar structure that encloses segments of axons in the central nervous systems (CNS) of vertebrates 1 . Once myelin is laid down, it is unknown to what extent the sheaths require maintenance and remodeling. Membrane turnover may pose a problem for oligodendrocytes that form up to 80 different myelin sheaths of tightly stacked membrane, but harbour little cytoplasm and few lysosomes, the organelles responsible for membrane degradation. Myelin membrane components are metabolically relatively stable with half-lives on the order of several weeks to months 2,3 . Nevertheless, protein/lipid turnover is, in general, necessary to replace potentially impaired molecules with new functional copies in order to combat functional decline [4][5][6][7] . How do molecules trapped within the numerous layers of tightly compacted membrane enter the degradative system? We tested the hypothesis that myelin degradation occurs in part via shedding of myelin fragments into the extracellular space.We analyzed the white matter of aging mice (up to 24 months) by electron microscopy to search for myelin breakdown products. We detected multilamellar myelin fragments more frequently in the brain of the older mice, of which some were associated with myelin sheaths, while others were in the extracellular space or inside of cells (Supplementary Fig. 1). As fixation artefacts frequently affect the appearance of myelin in chemically fixed tissue, we used high-pressure freezing to fix tissue and confirmed the progressive accumulation of multilamellar myelin fragments with age ( Fig. 1a,b).Since some of these myelin fragments were found inside cells, we performed immunhistochemistry to determine whether microglia, the brain phagocytes [8][9][10] , were responsible for the uptake of myelin fragments. An increasing number of myelin basic protein (MBP) and proteolipid protein (PLP) immunoreactive puncta co-localized with Iba1-positive microglia with age ( Fig. 1c, Supplementary Fig. 1).Three-dimensional reconstructions demonstrated that immunoreactive puncta were present inside of microglia (Fig. 1c). Since our results suggested that microglia clear away the myelin fragments that accumulate in the 3 aging brain, we compared microglia number and appearance in young and old animals. Not only had the number of microglia increased in the white matter of old animals as reported previously 11,12 , but also microglia in contact with myelin ( Supplementary Fig. 2). Next, the morphology of lysosomes was evalua...

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To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, in a process thought to be error-free, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in CNS myelin during development by combining ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy and confocal time lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia in a phosphatidylserine-dependent manner. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that requires substantial refinement.

Adult neural stem cells with the ability to generate neurons and glia cells are active throughout life in both the dentate gyrus (DG) and the subventricular zone (SVZ). Differentiation of adult neural stem cells is induced by cell fate determinants like the transcription factor Prox1. Evidence has been provided for a function of Prox1 as an inducer of neuronal differentiation within the DG. We now show that within the SVZ Prox1 induces differentiation into oligodendrocytes. Moreover, we find that loss of Prox1 expression in vivo reduces cell migration into the corpus callosum, where the few Prox1 deficient SVZ-derived remaining cells fail to differentiate into oligodendrocytes. Thus, our work uncovers a novel function of Prox1 as a fate determinant for oligodendrocytes in the adult mammalian brain. These data indicate that the neurogenic and oligodendrogliogenic lineages in the two adult neurogenic niches exhibit a distinct requirement for Prox1, being important for neurogenesis in the DG but being indispensable for oligodendrogliogenesis in the SVZ. STEM CELLS 2016;34:2115-2129 SIGNIFICANCE STATEMENTIn the submitted study, we address the function of the homeobox transcription factor Prox1 for the specification of oligodendrocyte cell fate in adult neural stem cells. A function of Prox1 for neurogenesis is well described in Drosophila. Additionally, recently its implication in neuronal differentiation in neural stem cells has been shown. Therefore, the function seemed to be totally conserved from Drosophila to mammals. However, we here show that the function of Prox1 depends on the regional identity of the investigated neural stem cells. In neural stem cells of the hippocampus, Prox1 induces neuronal differentiation. However, in neural stem cell from the subventricular zone Prox1 induces differentiation in oligodendrocytes.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one “eat me” signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

Successful immune responses depend on the spatiotemporal coordination of immune cell migration, interactions, and effector functions in lymphoid and parenchymal tissues. Real-time intravital microscopy has revolutionized our understanding of the dynamic behavior of many immune cell types in the living tissues of several species. Observing immune cells in their native environment has revealed many unanticipated facets of their biology, which were not expected from experiments outside a living organism. Here we highlight both classic and more recent examples of surprising discoveries that critically relied on the use of live in vivo imaging. In particular, we focus on five major cell types of the innate immune response (macrophages, microglia, neutrophils, dendritic cells, and mast cells), and how studying their dynamics in mouse tissues has helped us advance our current knowledge of immune cell–mediated tissue homeostasis, host defense, and inflammation. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 38 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

I hereby declare that this Doctoral thesis entitled "Role of microglia in myelin turnover" has been written independently with no other aids or sources than quoted.