APOPTOSIS IS INDUCED BY CHOLINE DEFICIENCY IN FETAL BRAIN AND IN PC12

Treatment of rats with choline during critical periods in brain develo pment results in long-lasting enhancement of spatial memory in their o ffspring. Apoptosis is a normal process during brain development, and, in some tissues, is modulated by the availability of the nutrient cho line. In these studies, we examined whether availability of choline in fluences apoptosis in fetal brain and in the PC12 cell line derived fr om a rat pheochromocytoma. Timed-bred Sprague Dawley rats were fed a c holine-deficient (CD), choline-control, or choline-supplemented (CS) d iet for 6 days and, on embryonic day 18, fetal brain slices were prepa red and apoptosis was assessed using terminal dUTP nucleotide end labe ling (TUNEL) to detect DNA strand breaks and by counting of apoptotic bodies, TUNEL-positive cells were detected in 15.9% (P < 0.01), 8.7% a nd 7.2% of hippocampal cells from fetuses of dams fed the CD, control or CS diets, respectively. A similar inverse relationship between diet ary intake of choline and TUNEL positive cells was detected in an area of cerebral cortex from these fetal brain slices. Counts of apoptotic bodies in fetal brain slices correlated inversely with choline intake of the mothers (6.2% (P < 0.01), 2.5% and 1.9% of hippocampal cells h ad apoptotic bodies in fetuses of dams fed the CD, control and CS diet s, respectively). PC12 cells were grown in DMEM/F12 media supplemented with 70 mu M choline or with 0 mu M choline. The number of apoptotic bodies in PC12 cells increased when cells were grown in 0 mu M choline medium (1.5%; P < 0.05) compared to 70 mu M choline medium (0.55%). I n PC12 cells, TUNEL labeling (DNA strand breaks) increased in choline deficient (13.5%, P < 0.05) compared to sufficient medium (5.0%). In a ddition, cleavage of genomic DNA into 200 bp internucleosomal fragment s was detected in choline-deficient cells. These results show that cho line deficiency induces apoptotic cell death in neuronal-type cells an d in whole brain. We suggest that variations in choline availability t o brain modulate apoptosis rates during development. (C) 1997 Elsevier Science B.V.