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In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

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Generation of choline for acetylcholine synthesis by phospholipase D isoforms.

D Zhao¹, M A Frohman, J K Blusztajn

¹Department of Pathology, Boston University, School of Medicine, Boston, MA, USA. dizhao@bu.edu

BMC Neuroscience

|December 6, 2001

Summary

This summary is machine-generated.

Phospholipase D2 (PLD2) generates choline for acetylcholine (ACh) synthesis in cholinergic neurons. This study identified PLD2 as the primary enzyme responsible, though PLD1 may also contribute in certain conditions.

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Area of Science:

Neuroscience
Biochemistry
Cell Biology

Background:

Cholinergic neurons synthesize acetylcholine (ACh) using choline derived from phosphatidylcholine (PC) hydrolysis.
Phospholipase D (PLD) enzymes are implicated in this choline generation pathway.

Purpose of the Study:

To identify the specific PLD isoform responsible for choline production for ACh synthesis in cholinergic neurons.
To investigate the roles of PLD1 and PLD2 in this process using a cellular model.

Main Methods:

Utilized murine basal forebrain cholinergic SN56 cells with experimentally modified PLD1 and PLD2 expression and activity.
Assessed ACh levels in cells cultured in choline-free medium to track de novo synthesis.
Employed antisense oligonucleotides to reduce PLD2 expression and transient transfection for PLD1 and PLD2 overexpression.

Main Results:

PLD2, but not PLD1, was detected in SN56 cells; its activity and ACh synthesis were stimulated by PMA.
Reducing PLD2 expression with antisense oligonucleotides decreased PLD activity and ACh levels.
Overexpressing PLD2 significantly increased both basal and PMA-stimulated PLD activity and ACh synthesis.
Overexpressing PLD1 also enhanced PLD activity and ACh synthesis, particularly with PMA stimulation.

Conclusions:

PLD2 is identified as the primary endogenous enzyme generating choline for ACh synthesis in cholinergic cells.
Choline generated by PLD1 can also be utilized for ACh synthesis, particularly in a model system or under specific stimulation.