Abstract
Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitro biomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes.
Highlights
From the Section of Mass Spectrometry, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20852
It was observed that the extent of protection or the degree of DNA fragmentation induced by serum starvation differed depending on the cell conditions or lot to lot variations in serum or medium constituents
The protective effect of 22:6n-3 on DNA fragmentation induced by serum starvation was potentiated as the cells were enriched for a longer period of time (Fig. 7A), and this increase paralleled the PS accumulation during the time course of enrichment
Summary
Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. Enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes. We found that enrichment of neuronal cells with 22:6n-3 increased the accumulation of PS and the membrane localization of Raf-1, downregulated caspase-3 activity, and prevented apoptotic cell death under serum-free conditions. Its protective potential was sensitive to the extent of PS accumulation, suggesting that the observed antiapoptotic effect of 22:6n-3 may be mediated at
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