Effect of acetylcholine precursors on proliferation and differentiation of astroglial cells in primary cultures

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 50-diphosphocholine (CDP-choline) and alpha-glyceril-phosphorylcholine (alpha-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 lM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 lM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 lM a-GPC 24h treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 lM choline or a-GPC, whereas in 24 h 1 lM CDP-choline and acetylcholinetreated astrocytes TG expression was unaffected. Treatment with 1 lM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 lM a-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture.