Investigation of myelin maintenance and turnover by inducible MBP knock-out in adult mice

The turnover and maintenance of myelin in the adult CNS Myelin is a multilayered membrane structure generated by oligodendrocytes in the CNS that insulates and supports axons. While most of the oligodendrocyte population differentiates early in life and persist for most of the lifetime, myelin proteins are slowly turned over and replaced within weeks to months. However, the mechanisms of this turnover and replacement processes within the myelin sheath are largely unknown. One reason is the absence of a suitable model system. To study myelin turnover in the adult animal a model system is required that allows to investigate myelin maintenance under conditions of ongoing turnover, survival of oligodendrocytes and the avoidance of acute inflammation. In this thesis a new model system was established that meets these requirements and was used to study turnover within the individual myelin sheath. This model system is based on a strategy for the visualization of myelin renewal by the application of an inducible deletion of myelin basic protein (Mbp) expression in the adult. For this purpose, ablation of MBP was induced in young adult mice after completion of developmental myelination at the age of 8 weeks. After the knock-out was induced, any newly synthetized myelin membrane lacked MBP and therefore the essential component for myelin compaction. The absence of compaction served as a marker for newly made myelin and was studied by electron microscopy. Using volume imaging by focused ion beam scanning electron microscopy (FIB-SEM) we observed the emergence of non-compact myelin membranes at the inner tongue of the juxta-paranodal region. The localized emergence of these membranes suggests the juxta-paranode as a site of MBP integration within the myelin sheath. Furthermore, the progressive loss of compact myelin after ablation of MBP synthesis was quantified and a shortening of internodes in the optic nerve was observed. We performed proteome analysis of the optic nerve eight, 16 and 40 weeks after Mbp knock-out induction to investigate systemic responses after loss of de novo MBP synthesis and found reduced abundance for proteins associated to the myelin sheath and cell adhesion molecules while many cytoskeleton-associated proteins were increased in abundance. These results are in accordance with the detected loss of the compact myelin compartment. To identify potential mechanisms for oligodendrocyte intrinsic or extrinsic recycling of myelin we analyzed individual axons in 3D FIB-SEM volumes and detected an increased number of myelin spheres at the inner tongue of the oligodendrocytes. We suggest that these myelin spheres are very likely involved in disposal of aged myelin. Visualization of the observed processes of membrane integration and removal were made possible by combination of high-quality structural preservation, 3D FIB-SEM microscopy and the novel mouse model system. In conclusion, this study introduced and characterized a powerful tool to study myelin maintenance and turnover and used this to shed light on the process of myelin turnover and the half-life of a myelin sheath in CNS of adult mice.2021-06-1