The apoptosis-inducing toxin Brefeldin A is taken up and secondarily released by neurons

The Golgi toxin Brefeldin A (BFA) is known to induce apoptosis in various c ancer cells and in cultured neurons. The goal of this work is to study whet her BFA is involved in the apoptotic action of the BFA-exposed neurons supe rnatant. Primary rat cortical cultures (cultures A) were exposed to BFA (50 mug/ml). After 5 h exposure and two careful washes, neurons were replaced into the initial medium for 24 h. Afterwards new cultures (cultures B) were incubated for 24 h in the supernatant of culture A. The percentages of apo ptotic neurons evaluated in both cultures A and B with two methods were com parable in controls A (6.6 +/- 0.8) and in controls B (8.1 +/- 1.6) whereas they were increased in neurons exposed either to BFA (20.4 +/- 2.5) or to the BFA-exposed neurons supernatant (19.3 +/- 2.4). BOC (Boc-asp-FMK) a pan -caspase inhibitor, blocked supernatant-induced neuronal apoptosis in cultu res B (9.3 +/- 2.4). The concentrations of BFA in supernatants of cultures A were measured using liquid chromatography - electrospray - mass spectrome try technique and showed a progressive increase. These BFA levels were able to directly induce apoptosis in exposed neuronal cultures and the percenta ges of apoptotic neurons were comparable to those observed after supernatan t exposures. Our study demonstrates that in primary neuronal cultures, BFA can be trapped in neurons and secondarily released in the supernatant, whic h in turn induces apoptosis.