Biosynthesis of phosphatidylcholine during neuronal differentiation

MARCUCCI, H.; GILARDONI P; CLAUDIA ELENA BANCHIO

Rosario

Congreso; SAIB; 2006

Phosphatidylcholine (PtdCho) is the major phospholipid in membranes cells and is made by the Kennnedy pathway were CTP:phosphocholine citidylyltransferase (CCT)  catalyze the slow step. Two genes encode CCT activities: Pcyt1a encodes CCTa (ubiquitously expressed in mouse tissues) and Pcyt1b encodes two transcripts that differ at the 5 end: CCTb2 and CCTb3 (expressed in brain and gonads). In the present work, using the Affymetrix Mouse Chip U74CV2, we analyzed the expression patterns of genes involved in phospholipid metabolism in differentiating Neuro2A cells. We detected many upregulated genes after retinoic acid (RA) treatment (differentiation condition), including those that encode CCTa and CKa choline kinase (the first enzyme of the pathway). The results were confirmed by real time RT-qPCR. By RT-PCR assays, we could also detect increased levels of CCTb mRNA after RA treatment. To identify the mechanisms that regulate the expression of CCTb and CCTa, we transfected Neuro2A cells with reporter construct harboring 5´ deletions of the promoters. We observed that CCTb2 and CCTa  expression increased after RA treatment. To evaluate the transcriptions factors involved in such regulation, we performed Band Shift and ChiP assays. Our results suggest that the expression of CCT could be coordinately regulated to provide the PtdCho required for neurite outgrowth.