Phospholipase D activity is important in the neurite outgrowth and synaptogenesis of the neuronal stem cells

Abstract

Phospholipase D (PLD) is a ubiquitous enzyme and believed to be involved in lipid mediated signal transduction and membrane formation in cell proliferation. In the present study, we have investigated the change of PLD activity during the synaptogenesis of rat brain neuronal stem cells to confirm that PLD plays a role in neurite outgrowth and synaptogenesis in neuronal differentiation of the stem cells. Neuronal stem cells were extracted from the fetal rat brain at 12 days of gestation and cultured in N2 media with basic fibroblast growth factor (bFGF). Synaptogenesis was induced by removal of bFGF from the media. Neurite outgrowth and PLD activity were monitored simultaneously within 1 to 5 days from induction of the synaptogenesis. Neurite outgrowth was identified by immunostaining of the cultured cells with Tuj1 antibody under fluorescence microscopy and PLD activity was determined by measuring [3H]-phosphatidylethanol produced via PLD-catalyzed transphosphatidylation in [3H]-palmitic acid-labeled cells. Increased neurite outgrowth was observed after removal of bFGF, and PLD activity was increased according to the progression of neurite outgrowth. Synapsin I expression level was checked by Western blotting of the whole protein extract of the cells. Synapsin I expression was increased as increase of neurite outgrowth and PLD activity. PMA, one of the indirect PLD activator, treatment resulted in increased level of Synapsin I expression and increased neurite outgrowth. When PLD activity was blocked with 1-butanol, neurite outgrowth was inhibited. With the data found, we could conclude PLD might be involved in the progression of neurite outgrowth and synaptogenesis of the rat brain neuronal stem cells.