ABSTRACT
Neuronal differentiation plays a key role during embryogenesis. However, based on the capacity of neuronal stem cells to either generate or regenerate neurons and because differentiation stops aberrant neuroblasts proliferation, neuronal differentiation is crucial during neuropathological conditions. Although phosphatidylcholine (PtdCho) has been proposed as an important molecule for neurite growth and neuronal regeneration, the identity of the molecular target has remained elusive. This study originally describes that lysophosphatidylcholine (LPtdCho), either exogenously supplied or generated by the imbalance of PtdCho metabolism through the enzymatic action of cytosolic phospholipase A2, acts as a neurotrophic-like factor. We demonstrated that LPtdCho induces neuronal differentiation by activation of the small G protein Ras followed by the Raf/MEK/ERK signaling pathway. Accordingly, LPtdCho redirects neuroblasts gene expression leading to the generation of functional mature neurons expressing ßIII-tubulin and having increased acetylcholinesterase activity and membrane biosynthesis required for neuritogenesis. These findings provide mechanistic details of the role of cytidine-5-diphosphocholine (CDP-choline) and PtdCho as neuroprotectors. Furthermore, as LPtdCho recapitulates the effect of the therapeutic agent retinoic acid, these results open new avenues for drug discovery for the treatment of neuropathological conditions.
Subject(s)
Cell Lineage , Lysophosphatidylcholines/pharmacology , Neurons/cytology , Neurons/metabolism , Animals , Biomarkers/metabolism , Calcium/metabolism , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Lineage/drug effects , Humans , Lysophosphatidylcholines/metabolism , MAP Kinase Signaling System/drug effects , Mice , Models, Biological , Neurons/drug effects , Neurons/enzymology , Phosphatidylcholines/metabolism , Phospholipases A2, Cytosolic/metabolism , Second Messenger Systems , Tretinoin/pharmacology , ras Proteins/metabolismABSTRACT
Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase alpha (CK(alpha)) and choline cytidylyltransferase alpha (CCT(alpha)). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCT(alpha). Later, the transcription of CK(alpha)- and CCT(alpha)-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CK(alpha) or CCTalpha increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCT(alpha) or CK(alpha) was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration.
Subject(s)
Neurons/cytology , Neurons/metabolism , Phosphatidylcholines/biosynthesis , Animals , Blotting, Western , Cell Differentiation/drug effects , Cell Line, Tumor , Choline Kinase/genetics , Choline-Phosphate Cytidylyltransferase/genetics , Fluorescent Antibody Technique , Mice , Neurons/drug effects , Neurons/enzymology , Oligonucleotide Array Sequence Analysis , Phosphatidylcholines/physiology , Signal Transduction/drug effects , Signal Transduction/genetics , Tretinoin/pharmacologyABSTRACT
CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis by the Kennedy pathway. In mammals, there are two genes that encode the enzyme isoforms that catalyze this reaction: Pcyt1a for CCTalpha and Pcyt1b for CCTbeta. In mouse tissues two different CCTbeta variants named CCTbeta2 and CCTbeta3 have been identified. Although little is known about Pcyt1b gene expression, recent data from cell lines propose a distinct role for CCTbeta2 in neuronal differentiation. Also, gonadal dysfunction in the CCTbeta2 knockout mouse suggests a role for this protein in ovary maturation and the maintenance of sperm production. This work defines and characterizes two alternative promoters that drive the expression of the two murine CCTbeta isoforms. The promoter activities were measured in Neuro-2a (mouse neuroblastoma), TM4 (mouse Sertoli) and C3H10T1/2 (mouse embryo fibroblast) cell lines. The transcriptional start points of each transcript and the promoter regions essential for the expression of each isoform were determined. Analysis of the CCTbeta2 promoter sequence suggested the transcription factor AP-1 as a potential regulator of CCTbeta2 expression in neuronal cells. However, CCTbeta3 was not detected in this cell line suggesting a different role or regulation. The activities of alternative promoters provide for greater flexibility in the control of CCTbeta isoform expression.
