Structural and dynamical investigations of biomimetic myelin membranes

In this project biomimetic membranes with native and multiple sclerosis like lipid composition have been investigated. To simulate multiple sclerosis we relied on the animal model experimental autoimmune encephalomyelitis (EAE). Special focus was put on the interaction of the membranes with myelin basic protein (MBP). It is an essential protein necessary to build compact myelin. In chapter 3 we utilized several different structural methods to investigate both membrane systems. For this we used the two geometries of large unilamellar vesicles (neutron and x-ray small-angle-scattering) and supported bilayers (neutron reflectivity).We could show that the structural properties of the pure membranes do not differ in the resolution of the instruments. However, if MBP is injected to the samples the situation changes. For the vesicles a rapid aggregation is observed for both lipid compositions. This aggregation is followed by an aging process over the next days. This process was measured by cryo-TEM and small-angle-x-ray-scattering. The EAE membranes show almost no change over this time whereas the native membranes seem to change over the time. Strikingly, the EAE membranes seem to be in a locked state due to a strong binding of the membranes with MBP. In contrast, the native membrane seem to have some kind of plasticity and form giant vesicles with a more than 10fold increased diameter in the micrometer range with partially multi-lamellar structures. These structures resemble more the structure of myelin found in living organisms which are multi-layers wrapped in a cylindrical shape around axons with a diameter of some micrometers. Therefore, we conclude that the native lipid composition has the tendency to form structures similar to the ones observed in nature. This phenomenon could be explained with the bending rigidity of those membranes which is a intrinsic parameter of the membranes. We measured the bending rigidity for both membrane systems by neutron spin echo and received a higher value for the native lipid composition. Concluding, the native membrane has a stronger tendency to reduce its curvature and, therefore, form vesicles with a larger diameter. This explains additionally that sharp edges with locally very high curvature which are only observed in EAE membranes. In chapter 4 we focused more on the formation process of multi-layers in the myelin sheath. To simulate this process we used supported bi-layers of native and EAE modified lipid composition that were coated with MBP. On those bi-layers we injected large unilamellar vesicles with the same lipid compositions, respectively, of native and EAE membrane types. For both lipid compositions we were able to resolve the kinetics in a range of several hours. The kinetics could be modelled by the model of random sequential adsorption. We could show that the vesicles adsorb to the surface of the membrane and reshape there to increase their surface area in touch with the membrane. However, the vesicles did not fuse completely to form a double bi-layer. From those measurements, we were able to estimate the adhesion energy per MBP molecule using a geometrical model in combination with a fitted parameters of the specular data and the bending rigidity measured in the neutron spin echo beforehand. This adhesion energy is larger for native membranes than for EAE modified membranes. These results help to identify biophysical parameters responsible for the properties of native and experimental autoimmune encephalomyelitis (EAE) modified membranes. Therefore, they might contribute to understand the molecular interactions which lead to changes in compact myelin in demyelinating diseases like multiple sclerosis that ultimately destroy this membrane system. However, it is still necessary to investigate the impact of other parts of the myelin membrane system in further studies. For example this study only focused on the cytoplasmic leaflet of myelin. But as the real myelin is highly asymmetric the extracellular leaflets have a very different composition. Also other proteins than MBP can be found in myelin and their interactions with the membranes also need to be looked into to get a broader view on the molecular interactions in myelin