Studies of phospholipase D1 and phospholipase D2 expression in rat brain during development and aging and identification of a physiological role for phospholipase D2 in providing the precursor choline for acetylcholine synthesis

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid and choline. The enzyme is encoded by two highly homologous genes in mammals designated PLD1 and PLD2. PLD activity in the whole rat brain was determined at ages ranging from embryonic-day (E) 19 to postnatal-day (P) 49. Basal, oleate-, and phosphatidylinositol-4,5-bisphosphate-stimulated PLD activity increased between E19 and P24 by approximately three-fold and remained unaltered thereafter. The expression of PLD1 and PLD2 mRNA in the whole brain was then examined and showed a similar developmental pattern as PLD activity. The development of PLD1 and PLD2 during the first three postnatal weeks correlates with synaptogenesis and myelination, suggesting that the enzyme might have an important role in these processes. The mRNA and protein expression of PLD1 and PLD2 in brainstem and parietal-cortex was next determined in female rats ranging from newborn to aged. Northern and western blots revealed that both PLD1 and PLD2 expression did not decrease in old age relative to adulthood, indicating that PLD expression is well maintained during aging.;Cholinergic neurons are characterized by an intracellular metabolic pathway catalyzed by a PLD enzyme that hydrolyzes PC to generate the precursor choline for the synthesis of the neurotransmitter acetylcholine (ACh). To identify the PLD involved in this process murine cholinergic SN56 cells were used as a model. PLD2, but not PLD1 mRNA and protein, were detected in these cells, and when a PLD2 antisense oligonucleotide was introduced into the cells, PLD2 mRNA and protein expression were reduced by 34% and 28%, respectively. Moreover, basal- and PMA-stimulated PLD activity and ACh synthesis were reduced by 26--32% by the PLD2 antisense. Overexpression of mouse PLD2 in SN56 cells by transient transfection increased basal and PMA-stimulated PLD activity. The PLD2 transfectants had an increased level of intracellular ACh and released more choline into the medium. These data identify PLD2 as the endogenous enzyme that hydrolyzes PC to generate choline for ACh synthesis in SN56 cells, and indicate that this enzyme provides a possible mechanism whereby cholinergic neurons autoregulate the supply of the acetylcholine precursor.