Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation

Glycosphingolipids, including ganglio‐sides, are emerging as signaling intermediates of extracellular stimuli. Because mitochondria play a key role in the orchestration of death signals, we assessed the interaction of GD3 ganglioside (GD3) with mitochondria and the subsequent cascade of events that culminate in cell death. In vitro studies with isolated mitochondria from rat liver demonstrate that GD3 elicited a burst of peroxide production within 15–30 min, which preceded the opening of the mitochondrial permeability transition, followed by cytochrome c (cyt c) release. These effects were mimicked by lactosylceramide and N‐acetyl‐sphingosine but not by sphinganine or sphingosine and were prevented by cyclosporin A and butylated hydroxytoluene (BHT). Reconstitution of mitochondria pre‐exposed to GD3 with cytosol from rat liver in a cell‐free system resulted in the proteolytic processing of procaspase 3 and subsequent caspase 3 activation. Intact hepatocytes or U937 cells selectively depleted of glutathione in mitochondria by 3‐hydroxyl‐4‐pentenoate (HP) with the sparing of cytosol reduced glutathione (GSH) were sensitized to GD3, manifested as an apoptotic death. Inhibition of caspase 3 prevented the apoptotic phenotype of HP‐treated cells caused by GD3 without affecting cell survival; in contrast, BHT fully protected HP‐treated cells to GD3 treatment. Treatment of cells with tumor necrosis factor increased the level of GD3, whereas blockers of mitochondrial respiration at complex I and II protected sensitized cells to GD3 treatment. Thus, the effect of GD3 as a lipid death effector is determined by its interaction with mitochondria leading to oxidant‐dependent caspase activation. Mitochondrial glutathione plays a key role in controlling cell survival through modulation of the oxidative stress induced by glycosphingolipids.—García‐Ruiz, C., Colell, A., París, R., Fernandez‐Checa, J. C. Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation. FASEB J. 14, 847–858 (2000)