The objective of this application is to fill a major gap in the understanding of the formation and maintenance of the myelin membrane that ensheaths axons in the central and peripheral nervous systems. The myelin sheath is essential for sustaining the speed of neuronal transmission and for maintaining neuronal viability. Loss of myelin underlies a number of devastating neuronal diseases such as multiple sclerosis and so understanding how this specialized membrane is formed and maintained will have direct application to developing treatments for these diseases. Myelin is composed of a distinct set of proteins and lipids. While the mechanisms that regulated the production of myelin proteins is becoming well understood, the mechanisms that control the synthesis of the essential lipid components of myelin have not been addressed. The goal of the proposed studies is to fill that gap. The laboratory of one of the PIs guiding this application has pioneered the study of a set of proteins known as the ORMDLs, which are essential regulators of the synthesis of sphingolipids, a class of lipids essential for myelin formation and function. The ORMDLs keep sphingolipid production in check, their deletion results in unregulated production of these lipids. Recently it was reported that a whole-animal knockout of ORMDLs exhibited a neurological phenotype that was accompanied by myelination defects. Here we directly examine the role of ORMDL regulation of sphingolipid biosynthesis in the myelin-producing cells of the central nervous system. We focus on ORMDL3, one of the three ORMDL isoforms. Whole animal deletion of this isoform results in elevated sphingolipids in the brain. To accomplish our goal we will use two novel mouse lines. One mouse line will have ORMDL3 deleted throughout life in oligodendrocytes, the myelin-producing cells in the central nervous system. This line will test ORMDL function during myelin formation. The second mouse line allows us to delete ORMDL3 in oligodendrocytes in the adult animal to test whether ORMDL3 is required to maintain myelin. Animals with an ORMDL3 deletion will be examined for myelin lipid and protein composition by biochemical analysis. The structure of myelin will be examined by electron microscopy as well has by immunohistochemical analysis. Immunohistochemistry will also be used to determine effects on oligodendrocyte differentiation, numbers, and viability. These morphological analyses will be complemented by electrophysiological measurements that will assess changes in the numbers of myelinated axons and effects on myelin function in promoting conduction velocity. A cell culture model of oligodendrocyte differentiation, utilizing siRNA knockdown of ORMDL3 in primary oligodendrocytes will complement whole animal studies of the role of ORMDL3 on oligodendrocyte differentiation and viability. Finally the experimental autoimmune encephalomyelitis model, which mimics aspects of multiple sclerosis, will be used to test the hypothesis that sphingolipid dysregulation exacerbates this disease.
Many nerves are covered by a special structure, called myelin, that wraps around the nerves and speeds up nerve impulses and keeps nerves healthy. Loss of myelin is the basis of a number of neurological diseases such as multiple sclerosis. We are studying how components of myelin are made, to better understand how we can treat these devastating neurological diseases.
