George H. DeVries - US grants

The molecular events which occur when an axolemma-enriched membrane fraction or a myelin membrane fraction stimulate a mitotically quiescent population of Schwann cells (SC) to divide will be investigated. In particular, the following metabolic events in cultured SC will be evaluated in the presence and absence of membrane mitogens: increased turnover of phosphoinositides (as revealed by radioactivity labeled lipid turnover studies), increased calcium flux and/or intracellular mobilization of calcium as revealed by radioactive calcium flux measurements and Quin-2 measurements of intracellular calcium, increased sodium influx and hydrogen ion efflux as revealed by radioactive sodium flux experiments and fluorescent dye measurements of intracelular pH; increased phosphorylation of proteins as revealed by protein labeling via P32-phosphorus. The molecules required for specific interaction of each membrane fraction with SC will be investigated by evaluating how these membrane stimulated metabolic-sequalae may be compromised as a consequence of specific perturbation of the stimulation membrane. Phorbol esters and calcium ionophores will be used to stimulate both "branches" of the phosphoinositide effect to bring about mitosis. The relative efficiency and timing for the stimulation of each branch reactive to that of the membrane mitogens will be investigated as well as the nature of the proteins phosphorylation via each pathway. Finally, myelin which has been radioactively prelabeled with lipid precursors will be given to SC and the nature and timing of the lysosomal breakdown of that myelin will be evaluated relative to the onset of mitosis in order to identify potential mitogens which may be released from the myelin membrane. It is anticipated that these studies will given new information concerning the molecular events which must occur both for normal myelination to occur and for remyelination after Wallerian degeneration.

This is a proposal to investigate the molecular components of the axonal plasma membrane particularly as they relate to the ensheathing glial cell. The molecular properties of premyelinated axolemma (isolated from newborn optic nerve), unmyelinated axolemma (isolated from the parallel fibers in the molecular layer of cerebellum and post myelinated axolemma (isolated from uniformly well-myelinated fibers in the pons, cerebellar peduncle or white matter of the cervical region of spinal cord) will be determined by HPLC separation and quantitative analysis of lipids and two dimensional separaton of proteins by polyacrylamide electrophoresis. A procedure will be developed to isolate the nodal axolemma from myelinated axons and its protein and lipid composition will be determined. Polyvalent antisera will be raised in rabbits to each isolated membrane fraction. If the antisera shows axonal plasma membrane specificity in situ the antisera will be used in conjunction with goat anti-rabbit coated polyacrylamide beads to specifically isolate and further purify an axonal plasma membrane fraction from each starting source. The molecular basis for the mitogenic effect of axolemma enriched fractions on cultured Schwann cells will be further investigated by solubilization and purification of the active molecule; the role of c-AMP in the mitogenic effect will also be investigated. It is anticipated that these studies will give new insights into specific axonal molecules required for neuron-glial interaction.

The long term objective of this research is to characterize axolemmnal and myelin molecules which cause Schwann cells (SC) to proliferate and to understand the mechanism by which these signals are transduced across the Schwann cell plasma membrane. Axolemma- enriched fractions (AEF) from myelinated, non-myelinated and unmyelinated sources will be incubated with cultured SC. After stimulation of SC with AEF, fluorescent dyes will be used to study calcium mobilization and activation of the Na+/H+ antiporter; the pattern of cytoplasmic and membrane phosphorylation by 32P will also be examined. Using fluorescent labelled AEF, we will quantitate the amount of AEF per SC to test the hypothesis that a threshold amount of AEF-SC interaction is required to "trigger" mitosis. The myelin-related mitogens will be solubilized and purified by high performance liquid chromatography (HPLC) followed by biochemical characterization. The role of macrophage versus SC processing of exogenous myelin membrane to produce a SC mitogen will be evaluated via the development of macrophage-free cultures. A transformed SC line will be produced utilizing a new DNA construct containing a metallothionein promoter linked to the large T antigen of SV-40 virus in the absence of divalent cation the SC line should undergo normal differentiation when presented with a myelin-competent neurite. Soluble neuronrelated SC mitogen will be produced from cultured neurons by severing them from their neurites and inhibiting the synthesis of a class of molecules to which they are normally bound. The nature of axonal molecules responsible for three molecular manifestations of SC differentiation will be examined; secretion of Type IV procollogen, aggregation of SC surface laminin into linear arrays and possible increase of SC laminin receptors and increase of myelin specific lipids (galactocerebroside) as visualized immunologically and protein (Po, the major myelin glycoprotein) as determined by increased levels of Po mRNA or increased translation of Po mRNA. It is anticipated that these studies will profice a baseline from which to evaluate how glial cells normally respond to neuronal signals os that we can assess how such signalling may be compromised in cases of uncontrolled SC proliferation as in neurofibromatosis. In addition, this research will clarify the relative roles of macrophages verus SC in the proliferative response which follows the Wallerian degeneration characteristic of peripheral nerve injury.