Regulation of AChE Gene Expression in Neuronally Induced Mouse P19 Cells

Abstract

Mouse P19 embryonic carcinoma cells are uncommitted, multipotent cells which can be induced to terminally differentiate along the neuroectodermal lineage. Neurons, glia and astrocytes develop after free-floating aggregates, cultured 4–6 days in the presence of retinoic acid (RA), are plated onto tissue culture dishes. Neuronal cultures up to 90% purity can be obtained by treatment with mitotic inhibitors. We have used this cell line to examine the developmentally regulated expression of acetylcholinesterase (AChE). AChE is not expressed in untreated cells, but can be detected within 24 hr of plating neuronally-induced cultures. The level of AChE activity increases during neuronal differentiation. This correlates with the increase in cells exhibiting neuronal morphology. Histochemical and immunohistochemical results indicate that AChE activity is associated with cells exhibiting neuronal morphology, but not with glia-like cells. AChE mRNA and enzyme activity increase in parallel. Splicing of AChE mRNA is almost entirely to exon 6, consistent with that observed in mouse brain. Run-on transcription assays demonstrate that the AChE gene is transcribed at a similar rate in undifferentiated monolayer cells, RA-treated aggregates, and differentiated P19 neurons. Transcription inhibition experiments suggest that changes in AChE mRNA stability play a role in regulating expression of the enzyme. RA-treated aggregates maintained in culture for more than six days begin to secrete large amounts of tetrameric AChE. However, if the cells are plated in tissue culture dishes to allow for morphological differentiation, approximately half of the G4 enzyme becomes membrane bound. These results demonstrate that AChE is synthesized early in neuronal development but is not localized to the neuronal cell membrane until later in the differentiation process. These results further suggest that the 20 kD hydrophobic subunit, which most likely tethers AChE to the P19 neuronal membrane, is generated concomitant with morphological differentiation. Thus, P19 cells appear to provide a useful model for examining expression of AChE during neuronal development. (Supported by USPHS GM18360, 24437 and American Heart Association Postdoctoral Fellowship No. 93–58).