The objective of this proposal is to define the mechanisms regulating intracellular transport of lipids and proteins to myelin. One set of experiments characterizes transport of proteolipid protein (PLP) and sulfatide from their sites of synthesis to assembly into myelin or oligodendroglial (OL) plasma membrane. The hypothesis that PLP and sulfatide are co-transported will be investigated by comparing distribution and metabolism of PLP with sulfatide in intracellular pools. Sulfatide will be compared to PLP with regard to (1) sites of accumulation when transport is blocked by ionophores or agents which disrupt cytoskeleton: (2) orientation in intracellular vesicles and OL plasma membrane; and (3) long-term turnover in extra-myelin pools. These studies will utilize in vivo and in vitro labelling with (3H)leucine and (35S)sulfate, and subcellular fractionation of whole tissue and OL isolated from rat brainstem. Immunocytochemical staining of OL and their membrane sheets in culture will directly visualize these intracellular pools. The second set of experiments characterizes intracellular transport of galactocerebroside:Ab complexes in cultured OL and subsequent effects on OL membranes and metabolism. The effects of GalC Ab on OL will be compared to the effects of Ab to other glycolipids and the lectin peanut agglutinin. Ab to GalC in the presence of second Ab causes patching of GalC on OL membranes, and the GalC:Ab complexes are rapidly internalized. Sensitivity of these events to metabolic inhibitors, ionophores and agents which disrupt cytoskeleton will be examined. Metabolism of GalC will be measured by incorporation of (3H)galactose to determine if its synthesis is stimulated by internalization of GalC:Ab complexes. Exposure of OL to GalC Ab for 2-24 hours results in reversible condensation of membrane sheets; the role of cytoskeleton in this change will be investigated by immunocytochemical and biochemical methods. The hypothesis that continued exposure to GalC Ab alters proliferation and maturation of OL will be investigated by comparing incorporation of (3H)thymidine and time of appearance of GalC, 2'3'-CNP, basic protein and PLP in treated and control cultures. These studies are relevant to further understanding and eventual treatment of diseases involving myelin. Characterization of mechanisms controlling myelin assembly and OL function will provide significant information about regulation of myelination in normal development and remyelination following damage.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS013143-15
Application #
3395117
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1977-04-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
15
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Wayne State University
Department
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Benjamins, Joyce A; Nedelkoska, Liljana (2007) Cyclic GMP-dependent pathways protect differentiated oligodendrocytes from multiple types of injury. Neurochem Res 32:321-9
Boullerne, Anne I; Benjamins, Joyce A (2006) Nitric oxide synthase expression and nitric oxide toxicity in oligodendrocytes. Antioxid Redox Signal 8:967-80
Studzinski, Diane M; Benjamins, Joyce A (2005) Expression of P0 glycoprotein in CNS glia: effects of overexpression in N20.1 cells. Glia 52:234-44
Benjamins, Joyce A; Nedelkoska, Liljana; George, Edwin B (2003) Protection of mature oligodendrocytes by inhibitors of caspases and calpains. Neurochem Res 28:143-52
Studzinski, Diane M; Benjamins, Joyce A (2003) Regulation of CNS glial phenotypes in N20.1 cells. J Neurosci Res 73:31-41
Studzinski, D M; Benjamins, J A (2001) Cyclic AMP differentiation of the oligodendroglial cell line N20.1 switches staurosporine-induced cell death from necrosis to apoptosis. J Neurosci Res 66:691-7
Boullerne, A I; Nedelkoska, L; Benjamins, J A (2001) Role of calcium in nitric oxide-induced cytotoxicity: EGTA protects mouse oligodendrocytes. J Neurosci Res 63:124-35
Studzinski, D M; Callahan, R E; Benjamins, J A (1999) Increased intracellular calcium alters myelin gene expression in the N20.1 oligodendroglial cell line. J Neurosci Res 57:633-42
Boullerne, A I; Nedelkoska, L; Benjamins, J A (1999) Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1. J Neurochem 72:1050-60
Studzinski, D M; Ramaswamy, R; Benjamins, J A (1998) Effects of cyclic AMP on expression of myelin genes in the N20.1 oligodendroglial cell line. Neurochem Res 23:435-41

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