Previous work has shown that potentially important changes in the lipid composition of brain myelin occur during experimental allergic encephalomyelitis (EAE), an established animal model of Multiple Sclerosis. The organization (structure) of normal and EAE lipids are different for both the cytoplasmic and extracellular monolayers. These differences in 'phase behavior' contribute to the differences in the interaction forces between myelin membranes that we have observed using the Surface Forces Apparatus (SFA), which appear to be related to the delamination of the myelin sheath. We hypothesize that the interactions between the cytoplasmic sides of the membranes are due primarily to myelin basic protein (MBP) that couples to the lipid composition, with the anionic lipid and protein isoform content being especially important. The interactions between the extracellular sides of the membrane are due exclusively to the lipids as there are no known adhesive proteins on this side. The adhesion between the extracellular surfaces is therefore likely due to non-specific interactions such as electrostatic and van der Waals forces, which should also be strongly influenced by alterations in the phase behavior and distribution of the lipids in EAE membranes. The SFA will be used to study the complete force vs distance curves of normal and EAE myelin to relate the composition and phase behavior variations to the interaction forces that hold the extracellular and cytoplasmic sides of the myelin sheath together. In addition to variations in lipid composition, the effects of different MBP isoforms (C1, C3, C8) on the membrane adhesion will be determined with the SFA to show how these isoforms couple to the lipid distribution. We will use Langmuir isotherms and fluorescence microscopy of model cytoplasmic and extracellular monolayers and bilayers to determine the relationship between lipid composition and lateral phase separation. We are especially interested in cholesterol, the anionic lipids phosphatdylserine and sphingomyelin, and the neutral lipid phosphatidylcholine, which show the greatest differences between control and EAE myelin. Atomic force microscopy (AFM) will be used in parallel to study the distribution of lipids and MBP and its isoforms at the nanometer scale. We hypothesize that polyethylene glycol (PEG) and poloxamers, non-toxic and FDA-approved polymers, recently used to treat spinal cord injuries in animals, might act to heal the myelin sheath via adding an attractive osmotic 'depletion' force to provide a similar effect that we have recently observed for MBP. This work will provide insights into the role of membrane-composition and organization on the interactions that lead to MS, as well as basic advances in understanding the relationships between lipid phase behavior, protein localization and function, and membrane demyelination. ? ? ?
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