We will study events involved in peripheral nervous system (PNS) demyelination - and the remyelination which occurs under some conditions - with the long range goal of manipulating these events. Experimental models used are rats in which Wallerian degeneration is induced surgically by nerve crush, or in which primary demyelination is induced by tellurium feeding. Our first specific aim is to ascertain the sources of lipids used for remyelination of the PNS following demyelinating insult. With respect to cholesterol, possible sources include: (a) de novo synthesis in Schwann cells, (b) that """"""""salvaged"""""""" by macrophages during the demyelination phase, and (c) that derived from the circulation. Experiments involve measurement of incorporation of label from 3H2O into cholesterol of sciatic nerve myelin. That cholesterol coming from the circulation will be assessed by altering specific radioactivity of circulating cholesterol by dietary manipulations, and then examining dependence of specific radioactivity of PNS myelin cholesterol on specific radioactivity of circulating cholesterol. Variations of this methodology will be used to check whether phospholipids and cerebrosides may be salvaged for reutilization and/or, in the case of phospholipids, there is utilization of circulating stores. Any utilization of circulating lipids will be correlated with blood-nerve barrier integrity. In another set of studies involving rats, the role of macrophages in specific steps of demyelination and remyelination, including storage of lipids to be used for remyelination, will be examined. We will attempt to define which steps, if any, in downregulation of expression of""""""""myelin- specific"""""""" proteins and lipid synthetic enzymes are influenced by presence of macrophages. Experiments will involve use of monoclonal antibodies or Clodronate treatment to block entry of macrophages into damaged sciatic nerve, and then determining which steps in myelin breakdown are dependent on the presence of macrophages. The third specific aim involves transfection of primary rat Schwann cells with constructs to enable study of control of cholesterol metabolism at the gene level. Cholesterol synthesis and synthesis of other myelin components are coordinately regulated in Schwann cells during development and during demyelination. We will test the hypothesis that the promoter region for the rate-limiting enzyme in cholesterol biosynthesis, hydroxymethylglutaryl-CoA reductase, is activated in a tissue-specific (Schwann cell) manner by a signaling cascade inducing myelination - and that this is different from the well-characterized upregulation of this gene in the cholesterol-deficient state.
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