Myelin is a multilamellar membrane structure produced by oligodendrocytes and Schwann cells in nervous systems. Preliminary studies by the applicant have demonstrated that oligodendrocytes and Schwann cells in culture exhibit neurotransmitter receptors that regulate intracellular calcium levels. In this proposal, the applicant plans to test the hypothesis that nerve activity and second messenger systems play a role in the formation or maintenance of myelin.
Four specific aims are listed. (1) To test the hypothesis that oligodendroglia exhibit neuroligand receptors that are developmentally regulated and that these cells are pharmacologically heterogeneous. The expression of developmental markers will be correlated with the expression of neuroligand receptors in cultured oligodendroglia isolated from neonatal rats. Developmental markers to be used for oligodendroglia are glycerol phosphate dehydrogenase (GPDH), galactocerebroside (GC) and myelin basic protein (MBP). Developmental stages are arbitrarily divided into 3 groups that range from the least to the most mature: GPDH-/GC- cells, GC+/MBP- cells, and GC+/MBP+ cells. Also, freshly isolated and cultured oligodendrocytes from adult CNS will be examined for the presence of neuroligand receptors which regulate cytosolic calcium levels. Oligodendrocytes isolated from immature and mature CNS will be co-cultured in order to determine whether neurons influence the expression of neuroligand receptors and whether oligodendroglial cells involved in myelin formation exhibit the receptors. Finally, experiments are planned to examine whether receptor expression by oligodendrocytes isolated from different brain regions is heterogeneous. (2) To test the hypothesis that Schwann cells exhibit neuroligand receptors that are dependent upon an association with specific populations of neurons and that the cells are pharmacologically heterogeneous. Cultured Schwann cells isolated from immature and adult sciatic nerve, DRG and sympathetic ganglia will be examined to see whether cells exhibit neuroligand receptors. Sciatic Schwann cells co-cultured with purified DRG or sympathetic neurons are being examined to see whether the expression of neuroligand receptors by Schwann cells is dependent on an association with neurons and whether the class of neurons present will be exposed to factors that influence cell proliferation and differentiation in order to determine whether the factors induce the expression of neuroligand receptors on Schwann cells. Factors to be tested include those that activate protein kinase A (e.g. db-cAMP), protein kinase C (e.g. PDB) and that increase intracellular calcium (e.g. A23187). (3) To test the hypothesis that receptors on oligodendrocytes and Schwann cells increase intracellular calcium levels through the opening of calcium channels and/or the release of calcium from internal stores. Immature and mature myelin producing cells will be loaded with fura-2 and the influence of neuroligands on their calcium levels monitored in the presence of extra-cellular calcium or drugs (e.g. Dantrolene) that block calcium release from internal stores. (4) To test the hypothesis that nerve depolarization and/or receptor-linked second messenger systems are important in the formation of myelin by cultured oligodendrocytes and Schwann cells. DRG cultures will be co-cultured with Schwann cells or oligodendrocytes and then examined for the influence of the treatments on myelin formation/maintenance. Treatments to be examined will include a blocker of neuronal activity (tetrodotoxin), a blocker of L-type calcium channels (nitrendipine), non-selective blockers of calcium channels (EGTA, Mg++), blocking the release of calcium from internal stores (Dantrolene), inhibitors of protein kinase A (psychosine, H-7, PMA), and activators of protein kinase A (db-cAMP).
| He, M; Howe, D G; McCarthy, K D (1996) Oligodendroglial signal transduction systems are regulated by neuronal contact. J Neurochem 67:1491-9 |
| Lyons, S A; Morell, P; McCarthy, K D (1995) Schwann cell ATP-mediated calcium increases in vitro and in situ are dependent on contact with neurons. Glia 13:27-38 |
| Lyons, S A; Morell, P; McCarthy, K D (1994) Schwann cells exhibit P2Y purinergic receptors that regulate intracellular calcium and are up-regulated by cyclic AMP analogues. J Neurochem 63:552-60 |
| He, M; McCarthy, K D (1994) Oligodendroglial signal transduction systems are developmentally regulated. J Neurochem 63:501-8 |
