Ultimately, we want to understand how the myelin sheath acquires its extraordinary morphology, and so provide the groundwork for understanding a wide variety of de- and dysmyelinating disease states, such as Multiple Sclerosis, Guillain-Barre Syndrome and the Charcot-Marie-Tooth Diseases. The mechanisms by which the myelin sheath is generated remain elusive. We will test during the next grant period the validity of a new model we have developed for the generation of the myelin spiral. This model anticipates that the first step in the myelination program occurs when a protein scaffold is laid down adhesively linking the myelinating cell with the axon, and forming a guide for new membrane addition. In order to test this model we must first accurately define the positions of certain key marker proteins, (some of which are newly discovered) when these molecules are expressed naturally (in terms of temporal and spatial distribution) in the myelin-axon protein scaffold. We also propose to uncover the actual pattern of new membrane addition to forming myelin, and observe the dynamical movements of protein, lipid and cytoplasmic compartments, using selected fluorescently-labeled myelin protein or axonal moieties as """"""""reference markers."""""""" Our approach is to study living, actively myelinating cells directly observed by high resolution confocal and two photon microscopy, and we will define how initially completely segregated intracellular pathways ultimately converge to yield the myelin sheath. Accordingly, (I) we will engineer mice using bacterial artificial chromosomes (BAC cloning) to express, as naturally as possible, certain key neural cell proteins labeled in tandem with innocuous fluorescent moieties. The protein products of these artificial genes will be fluorescent, obviating the need for antibodies visualized by indirect methods. Furthermore, BAC methodologies yield protein products whose expression (temporal and spatial) is identical to the natural gene. These mice will be used as source material for in vitro myelination studies. (II) We will study fluorescent lipid incorporation into surface membranes of myelinating cells, and compare these data to the assembly parameters of the protein scaffold formed at the internodal ends, as ascertained in Aim I. Finally, (III) we will directly observe dynamical movements of the cytoplasmic channels that enclose non-compact myelin through the injection of fluorescent beads directly into pre-myelinating Schwann cells in culture, and examine the movement of these beads over time as myelination progresses. The studies we propose will allow a comprehensive analysis of the short and long term (several hours to weeks) continuous, relative contributions of proteins, membrane lipid and fluid cytoplasmic components to myelin formation.
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