Role of phospholipase D in neurite outgrowth and synaptogenesis of neuronal stem cells

Abstract

Employing neural stem cells from the brain cortex of E14 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neuronal cells during differentiation. PLD activity and expression level were increased as the differentiation of the neuronal stem cells progressed. To further confirm the role of PLD in neurite outgrowth, we constructed the dominant-negative form of rat PLD1 DNA inserted in EGFP plasmid DNA vector (DN-rPLD1). When the DN-rPLD1 was transfected to the neuronal stem cells, the neurite outgrowth was almost completely inhibited under differentiation conditions. Concurrently, the tyrosine phosphorylation of the cells and PLD1 was increased during neuronal differentiation. Since PLD activity and expression level were reduced by the treatment with genistein, tyrosine kinase appeared to be important in the increase of PLD1 expression level and PLD activity. Furthermore, cdc42 was found to be co-localized with PLD1 in neurite, determined by immunostaining, and was revealed to be associated with PLD1 as neuronal differentiation progressed, determined by immunoprecipitation. Overexpression of the dominant negative form of cdc42, N17, resulted in the decrease of PLD activity and neurite outgrowth, and it was restored by overexpression of PLD1. Conversely, expression of the constitutively active form, cdc42 V12, resulted in the increase of PLD activity and neurite outgrowth, suggesting that the interaction of PLD1 and cdc42 may play an important role in neurite outgrowth in neuronal stem cells. Taken together, these results suggest that PLD1 is closely implicated in neurite outgrowth during differentiation of the neuronal stem cells.