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(The FASEB Journal. 2000;14:2047-2054.)
© 2000 FASEB

GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion

MARIA RITA RIPPO*, FLORENCE MALISAN*, LUIGI RAVAGNAN{dagger}, BARBARA TOMASSINI*, IVANO CONDO*1, PAOLA COSTANTINI{dagger}, SANTOS A. SUSIN{dagger}, ALESSANDRA RUFINI*, MATILDE TODARO{ddagger}, GUIDO KROEMER{dagger} and ROBERTO TESTI*1

* Laboratory of Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, 00133 Rome;
{dagger} Centre National de la Recherche Scientifique, UMR 1599, Institut Gustave Roussy, F94805 Villejuif, France; and
{ddagger} Section of Anatomical Sciences, University of Palermo, Italy

1Correspondence: Laboratory of Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, via di Tor Vergata 135, 00133 Rome. E-mail: tesrob{at}flashnet.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipid and glycolipid diffusible mediators are involved in the intracellular progression and amplification of apoptotic signals. GD3 ganglioside is rapidly synthesized from accumulated ceramide after the clustering of death-inducing receptors and triggers apoptosis. Here we show that GD3 induces dissipation of {Delta}{Psi}m and swelling of isolated mitochondria, which results in the mitochondrial release of cytochrome c, apoptosis inducing factor, and caspase 9. Soluble factors released from GD3-treated mitochondria are sufficient to trigger DNA fragmentation in isolated nuclei. All these effects can be blocked by cyclosporin A, suggesting that GD3 is acting at the level of the permeability transition pore complex. We found that endogenous GD3 accumulates within mitochondria of cells undergoing apoptosis after ceramide exposure. Accordingly, suppression of GD3 synthase (ST8) expression in intact cells substantially prevents ceramide-induced {Delta}{Psi}m dissipation, indicating that endogenously synthesized GD3 induces mitochondrial changes in vivo. Finally, enforced expression of bcl-2 significantly prevents GD3-induced mitochondrial changes, caspase 9 activation, and apoptosis. These results show that mitochondria are a key destination for apoptogenic GD3 ganglioside along the lipid pathway to programmed cell death and indicate that relevant GD3 targets are under bcl-2 control.—Rippo, M. R., Malisan, F., Ravagnan, L., Tomassini, B., Condo, I., Costantini, P., Susin, S. A., Rufini, A., Todaro, M., Kroemer, G., Testi, R. GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion.


Key Words: apoptosis • permeability transition • AIF • cytochrome c • caspase 9


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UNBEARABLE STRESS DUE to internal disfunctions or external stimuli triggers programmed cell death by apoptosis. Whether the initiation of the apoptotic program is inside the cell or at the cell surface, signals are generated and rapidly spread to multiple cellular compartments. Long-distance signal propagation is largely mediated by diffusible hydrolytic products of either protein substrates or phospholipids, mostly generated by the action of caspases and phospholipases.

Ceramide is a diffusible lipid mediator that is generated early during apoptosis from the hydrolysis of membrane sphingomyelin (SM) by sphingomyelinases (1 2 3) . When the apoptotic program is initiated at the cell surface from death receptors, a death domain/FADD/caspases-dependent acidic sphingomyelinase (ASM) is responsible for SM hydrolysis and the transient accumulation of apoptogenic ceramide within 5 to 15 min (4 5 6 7 8 9) . Ceramide is then rapidly converted, through the stepwise addition of sugars and sialic residues, to gangliosides. About 10 to 30 min after CD95 cross-linking, in fact, GD3 ganglioside is neosynthesized and prominently accumulates (10) . The neosynthesis of GD3 ganglioside, mediated by a GD3 synthase ({alpha}2,8-sialyltransferase or ST8) (11 12 13 14) , is critical to signal progression, since GD3 can directly trigger apoptosis and suppression of ST8 expression substantially prevents ceramide- and CD95-induced cell death (10) . In hemopoietic cells, the early accumulation of GD3 requires ASM-derived ceramide, since ASM-deficient cells fail to accumulate GD3 and fail to effectively execute the apoptotic program on CD95 cross-linking, whereas transfer of ASM into ASM-deficient cells reconstitutes GD3 accumulation and efficient apoptosis (15) . Therefore, ASM and ST8 belong to a single pathway that promotes the progression of apoptotic signals by ultimately generating GD3 ganglioside.

GD3 targets, however, remain unclear. Cells undergoing apoptosis on GD3 exposure display early loss of mitochondrial transmembrane potential ({Delta}{Psi}m) (10) . Accordingly, recent evidences indicate that GD3 contributes to the opening of the permeability transition pore complex (PTPC) in isolated mitochondria (16 17 18) . Mitochondria play a central role in the apoptotic program by directing the activation of executioner caspases once irreversible cell damage occurs (19) . Early during the apoptotic process the {Delta}{Psi}m is dissolved, likely due to the opening of the PTPC, causing mitochondrial swelling and rupture of the outer mitochondrial membrane (20) . This is associated with the release of apoptogenic factors, normally confined between the mitochondrial membranes, including cytochrome c (21 , 22) , apoptosis inducing factor (AIF) (23 , 24) , and selected caspases (25) . Cytochrome c and AIF, through different pathways, are both capable of triggering key nuclear events such as chromatin condensation, activation of endonucleases, and DNA fragmentation.

Here, we provide evidence that GD3 ganglioside, which accumulates in cells undergoing apoptosis, can directly interact with mitochondria, causing {Delta}{Psi}m dissipation and the release of cytochrome c, AIF, and caspase 9. These events can be largely prevented by bcl-2. Thus, the lipid pathway recruits mitochondria to the apoptotic program through GD3 ganglioside.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Assessment of mitochondrial parameters in vitro
Mitochondria were purified from rat liver, as described (26) , and resuspended in 250 mM sucrose, 0.1 mM EGTA, 10 mM N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, pH 7.4. For the induction of PT, mitochondria (1 mg protein/ml) were resuspended in PT buffer (200 mM sucrose, 10 mM Tris-MOPS, pH 7.4, 5 mM Tris-succinate, 1 mM Tris-phosphate, 2 µM rotenone, and 10 µM EGTA-Tris) and monitored in an F4500 fluorescence spectrometer (Hitachi, Tokyo, Japan) for the 90° light scattering (545 nm) to determine large amplitude swelling after addition of 5 mM atractyloside, 1 µM cyclosporin A (CsA; Novartis, Basel, Switzerland), and/or 20 µM GD3, GD1a, GM3, GM1, or C2-ceramide (Sigma, St. Louis, Mo.).

Western blotting
Supernatants from mitochondria (6800 g for 15 min; then 20,000 g for 1 h; 4°C) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. Cytochrome c was detected using a monoclonal antibody (clone 7H8.2C12, PharMingen, San Diego, Calif.), AIF by means of a rabbit antiserum directed against the amino acid residues 151–200 (24) , and caspase 9 by a rabbit antiserum directed to the p18 subunit (kindly provided by Dr. D. Nicholson, Merck, Rahway, N.J.). Blots were revealed by ECL (Amersham, Buckinghamshire, U.K.), according to manufacturer instructions.

T cell lymphoma CEM cells (1x106) stably transfected with Bcl-2 cDNA or with the corresponding empty vector as control were treated with GD3 200 µM for 6 h. Cytosolic lysates were subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. The membranes were incubated with a rabbit polyclonal antibody directed to the caspase-9 carboxyl terminus (Santa Cruz, Santa Cruz, Calif.) and revealed by ECL.

Evaluation of nuclear changes in a cell-free system
DNA fragmentation activity in the supernatant of mitochondria was tested on HeLa cell nuclei, as described (27) . Briefly, purified HeLa nuclei were resuspended in 50 mM Tris-HCl, pH 7.2, and supernatants of mitochondria were added (90 min, 37°C). Nuclei were then stained with propidium iodide (Sigma) and analyzed with a FACScan (Becton Dickinson, Rutherford, N.J.) to determine the frequency of hypodiploid nuclei.

Immunoelectron microscopy
Thin sections (80 nm) were prepared from cells treated with C2-ceramide, fixed in 2% paraformaldehyde and 0.5% glutaraldehyde, in 0.1 M Sorensen’s phosphate buffer, embedded in LR white acrylic resin using gelatin embedding capsules (EMS, Ft. Washington, Pa.). Polymerization was accomplished at 50°C for 48 h. Sections mounted on formavar pretreated gold grids were incubated for 10 min with 10% H2O2, rinsed in distilled water, and treated with 1% bovine serum albumin (BSA) for 10 min to minimize nonspecific staining. Sections were then incubated overnight at 4°C with anti-GD3 mAb (clone R24, IgG3, gift from Dr. L. L. Old, Ludwig Institute, New York) and isotype matched control, followed by a 10 nm gold-conjugated goat antiserum to mouse (Aurion BSA-C kit, Aurion Wageningen, The Netherlands). Sections were counterstained with 2% uranyl acetate (5 min) and lead citrate (1 min), then analyzed by electron microscopy (JEOL Jem 1220).

GD3 synthase antisense experiments
T cell lymphoma CEM cells were incubated for 66 h in RPMI 1640 medium containing 10 mM HEPES pH 7.4, 1.0 mM sodium pyruvate, and 10% fetal bovine serum (FBS), with 40 µM of GD3 synthase antisense phosphorothioate oligodeoxynucleotides (5'-CAGTACAGCCATGGCCCCTCT-3') (28) . As control, a scrambled sequence of the same oligodeoxynucleotides (5'-CGACCTACCTATGCGCTACCG-3') or another irrelevant sequence was used at the same concentrations. After GD3 synthase antisense treatment, the cells were viable but unable to synthesize GD3 in response to ceramide (ref 10 and data not shown). Cells were washed once and resuspended in the above medium, then treated with 40 µM C2-ceramide.

Measurement of mitochondrial transmembrane potential in vivo
Dissipation of the mitochondrial transmembrane potential ({Delta}{Psi}m) and generation of reactive oxygen species (ROS) were assessed by staining cells with 16 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6 (2) , Molecular Probes) combined with 4 µM dihydroethidium (HE, Molecular Probes, Eugene, Oreg.) for 20 min at 37°C, followed by FACS analysis (29) .

Transfections of Bcl-2 overexpressing cells
T lymphoma CEM cells stably overexpressing bcl-2 (30) , kindly provided by Dr. R. Kofler (University of Innsbruck), were treated with GD3 (200 µM) in RPMI 1640, 10% FBS. Hypodiploid nuclei were assessed by staining cells with a hypotonic fluorochrome solution (propidium iodide 50 µg/ml (Sigma) in 0.1% sodium citrate plus 0.1% triton X-100) for 4–8 h at 4°C in the dark and analyzed by a FACScan.

The GD3 synthase cDNA was cloned in pEGFP-C3 expression vector (Clontech, Palo Alto, Calif.), fused to the GFP by the amino-terminal portion at the ApaI site. In 0.5 ml of RPMI 1640, 10 x 106 cells were incubated for 10 min on ice with 20 µg of pEGFP-ST8 cDNA or pEGFP empty vector. Cells were then electrophoresed (Gene Pulser, Bio-Rad, Hercules, Calif.) at 290V, 950 µF, left 30 min on ice, and resuspended in 5 ml of RPMI 1640, 10% FBS. After 4 h, live cells were recovered by lymphoprep density gradient centrifugation and replated. After 24 h, apoptotic cells, among green fluorescent cells, were evaluated by fluorescence microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GD3 directly induces dissipation of mitochondrial transmembrane potential ({Delta}{Psi}m)
Treatment of intact cells with exogenous GD3 results in apoptosis preceded by a rapid and dramatic loss of {Delta}{Psi}m (10) . To clarify whether GD3 can be directly responsible for dissipation of {Delta}{Psi}m, isolated rat liver mitochondria were exposed to GD3 and mitochondrial swelling was measured. GD3, but not GD1a (or other commercially available gangliosides such as GM1 and GM3, not shown), was able to induce a dramatic swelling of mitochondria within minutes. On the contrary, ceramide was unable to induce swelling of isolated mitochondria, suggesting that ceramide-induced mitochondrial changes during apoptosis might require GD3 neosynthesis. GD3-induced effects were completely prevented in the presence of CsA, indicating that GD3 is causing the opening of the mitochondrial PTPC (Fig. 1 ).



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  		Figure 1. Mitochondrial large amplitude swelling is induced by GD3. Purified mitochondria (1 mg/ml) were treated with buffer alone, GD3 (20 µM), GD3 (20 µM) + cyclosporine A (CsA,1 µM), GD1a (20 µM), C2-ceramide (20 µM), positive control atractyloside (Atr, 5 mM); diffraction of light was measured at 545 nm with a fluorescence spectrometer. One representative experiment out of five performed is shown.

GD3-induced {Delta}{Psi}m collapse causes the release of apoptogenic factors from mitochondria
Dissipation of {Delta}{Psi}m and swelling is often associated with rupture of the outer mitochondrial membrane and the release of multiple factors from the intermembrane space into the cytosol (19 , 25) . We therefore investigated whether GD3-induced {Delta}{Psi}m loss was causing the release of apoptogenic factors and mitochondrial caspases. Isolated mitochondria exposed for 15 min to GD3, but not to GM3 (or GD1a, not shown), released cytochrome c, AIF (24) , and the p32 cleavage product of caspase 9 (25) (Fig. 2 ). Cytochrome c, AIF, and p32 caspase 9 release was completely prevented by pretreating mitochondria with CsA, indicating that GD3-induced mitochondrial PTPC opening and consequent {Delta}{Psi}m collapse were responsible for the cytosolic release of apoptogenic factors.



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  		Figure 2. GD3 induces the release of cytochrome c, AIF, and caspase 9 (p32 cleavage product) from mitochondria. Supernatants from mitochondria treated as indicated were subjected to immunoblot detection of cytochrome c, AIF, and caspase 9. Supernatants from mitochondria treated with Atr 5 mM and/or Ca2+ (200 µM) were used as control of maximum release of cytochrome c or AIF. One representative experiment out of three performed is shown.

GD3-released mitochondrial factors are sufficient to drive DNA fragmentation
In cells undergoing apoptosis, released cytochrome c and AIF trigger caspase-dependent and independent events that eventually result in the induction of nuclear DNA fragmentation (23 , 24 , 31) . The ability of soluble factors released from mitochondria on GD3 contact to trigger nuclear DNA fragmentation was therefore investigated in a cell-free system. Isolated HeLa nuclei were exposed to supernatants derived from isolated mitochondria forced to {Delta}{Psi}m collapse by in vitro GD3 treatment and DNA content analyzed by flow cytometry. Only supernatants from GD3-treated mitochondria, but not from GM3 or GD1a-treated mitochondria, were effectively inducing DNA fragmentation in isolated nuclei (Fig. 3 ). This effect was completely prevented by cyclosporin A pretreatment of mitochondria. Direct exposure of isolated nuclei to GD3 did not result in DNA fragmentation or nuclear damage (data not shown). Thus, apoptogenic GD3 is sufficient to drive mitochondrial changes, and subsequent nuclear events, which are associated with apoptosis.



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  		Figure 3. The apoptogenic factors released from GD3-treated mitochondria induce nuclear apoptosis. Supernatants from mitochondria treated as indicated in the figure were tested on purified HeLa nuclei for the capacity to induce DNA fragmentation in vitro. DNA content and hypoploidy were determined by propidium iodide (PI) staining and cytofluorometric analysis. Percentage of hypodiploid nuclei is indicated. Fluorescence channels on the x axis, cell counts on the y axis. One representative experiment out of four performed is shown.

Endogenously synthesized GD3 induces mitochondrial changes
To investigate whether endogenous GD3 might target mitochondria in vivo, the possible accumulation of GD3 within mitochondria was investigated by immunoelectron microscopy. After ~30 min exposure to apoptogenic doses of ceramide, GD3 was frequently found associated with membranes of swelling mitochondria (Fig. 4 , black dots). We then investigated whether accumulation of GD3 was causing mitochondrial damage in vivo. CEM cells were pretreated with ST8 antisense oligodeoxynucleotides (28) to suppress the expression of ST8 and prevent the neosynthesis of GD3 on ceramide exposure (10) . Cells were then treated with apoptogenic ceramide and mitochondrial changes evaluated. Pretreatment with ST8-antisense, but not with a scrambled sequence of the same oligodeoxynucleotides, resulted in a substantial reduction of ceramide-induced {Delta}{Psi}m loss and ROS generation (Fig. 5 ). ST8-antisense could not, however, prevent GD3-induced mitochondrial changes (Fig. 5) or GD3-induced apoptosis (not shown). These data strongly suggest that endogenously generated GD3 induces mitochondrial failure during apoptosis.



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  		Figure 4. Ultrastructural analysis of representative mitochondria from unstimulated (A) and stimulated cells with 40 µM C2-ceramide for 30 min (B). Cells were fixed and analyzed by immunoelectron microscopy after treatment with R24 mAb followed by gold-conjugated anti-mouse Ig antibody. GD3 is detectable as black grains on mitochondrial membranes. Original magnification: x30,000. A detailed morphometric analysis of several different images of ceramide-treated cells indicated that the number of the grains associated with mitochondrial membranes was at least 10-fold higher compared to membrane-free cytosolic areas (not shown).



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  		Figure 5. Endogenously synthesized GD3 induces mitochondrial changes. CEM cells were incubated with 40 µM of GD3 synthase antisense (As) or scrambled (Sc) oligonucleotides, then treated with 40 µM C2-ceramide or 200 µM GD3 for 3 h. Mitochondrial changes ({Delta}{Psi}m dissipation and ROS generation) were assayed by FACS analysis (as described in Materials and Methods). Means ± 1 SD of five independent experiments are shown.

Enforced expression of bcl-2 prevents GD3-induced mitochondrial changes, caspase 9 activation, and apoptosis
The above results provide both in vitro and in vivo evidence for a role of GD3 in the induction of mitochondrial {Delta}{Psi}m disruption. Since anti-apoptotic bcl-2 family members directly interfere with PTPC opening and {Delta}{Psi}m loss (32 , 33) , the ability of bcl-2 to protect from GD3-induced mitochondrial damage and apoptosis was investigated. CEM cells stably transfected with bcl-2 or with the corresponding empty vector as control were exposed to GD3, and the induction of permeability transition and ROS generation was measured. Bcl-2 overexpressing CEM cells displayed resistance to GD3-induced mitochondrial changes compared to the control cells (Fig. 6A ).



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  		Figure 6. Bcl-2 prevents GD3-induced mitochondrial changes, caspase 9 activation, and apoptosis. CEM cells stably transfected with bcl-2 or with the empty-vector were incubated with 200 µM GD3 for the indicated time points, and the effects on mitochondrial changes ({Delta}{Psi}m dissipation and ROS generation) (A) and DNA fragmentation (C) were measured by FACS analysis. Means ± 1 SD of four independent experiments are shown. CEM cells stably transfected with bcl-2 or with the empty-vector were incubated with 200 µM GD3 for 6 h and caspase 9 analyzed by immunoblotting of cell lysates (B). One experiment out of two performed with identical results is shown.

Cytochrome c, released concomitantly to {Delta}{Psi}m dissipation, interacts with APAF-1, causing the recruitment and activation of caspase 9, dictating irreversible commitment to apoptosis (34) . This event can be effectively counteracted by bcl-2 (35) . The ability of GD3 to induce caspase 9 activation in vivo and the possible interference by bcl-2 were therefore investigated. As shown in Fig. 6B , exposure of CEM cells to GD3 induced pro-caspase 9 degradation and the appearance of a 32 kDa cleavage product within 6 h, whereas no caspase 9 activation could be detected in CEM cells stably overexpressing bcl-2. Accordingly, GD3-induced apoptosis was substantially delayed in bcl-2 overexpressing CEM cells (Fig. 6C ).

Finally, to assess whether bcl-2 could protect cells from endogenous GD3 overproduction, a GFP-tagged ST8 (GFP-ST8) was transiently expressed in CEM cells stably overexpressing bcl-2. Essentially all cell death induced by GFP-ST8 in control CEM cells could be blocked in CEM overexpressing bcl-2 (Fig. 7 ). Together, these data indicate that GD3-induced damage is mostly confined at sites that can be restrained by bcl-2.



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  		Figure 7. Bcl-2 prevents apoptosis induced by endogenous GD3 accumulation. The effects of the overexpression of pEGFP-ST8 or of the pEGFP empty vector in the bcl-2-transfected cells after 24 h were analyzed by evaluating apoptotic cells among the green fluorescent cells by fluorescence microscopy. Means ± 1 SD of three independent experiments are shown.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented in this paper demonstrate that GD3 ganglioside contributes to the apoptotic program by directly targeting mitochondria in a bcl-2-controlled manner and inducing the mitochondrial release of apoptogenic factors, including cytochrome c, AIF, and caspase 9.

GD3 rapidly accumulates in cells undergoing ceramide-dependent apoptosis by the action of ST8, a polysialyltransferase mostly resident in the endoplasmic reticulum and early Golgi compartment (ER/EG) (11 12 13 14) . ST8 generates GD3 from its immediate precursor GM3 by adding a second sialic acid to the GM3 sialic residue. Although little information is currently available concerning ST8 regulation in vivo, excess ceramide accelerates the rate of GD3 neosynthesis, resulting in GD3 accumulation (10) . In hemopoietic cells, excess ceramide derives initially from the accelerated hydrolysis of sphingomyelin by the action of acidic sphingomyelinase (ASM), boosted by membrane clustering of death receptors and death domain/FADD-dependent caspases activation (4 5 6 7 8 9) . Therefore, excess ceramide generated in acidic compartments by ASM feeds into the ganglioside biosynthetic pathway down to GD3 accumulation (15) , which occurs mostly in the ER/EG compartment. Mitochondria may come into close contact with the ER/EG, which they form a functionally interconnected network with (36 , 37) . GD3 accumulated in the ER/EG could therefore interact with close-by mitochondrial membranes or be physically redistributed to mitochondrial membranes. Further studies will clarify this issue.

GD3 efficiently disrupts {Delta}{Psi}m in intact cells (10) . Moreover, recent reports indicate that GD3 is a potent inducer of mitochondrial permeability transition in isolated mitochondria (16 , 17) . We show here that GD3 is sufficient to cause the release of apoptogenic cytochrome c, AIF, and caspase 9 from isolated mitochondria and, remarkably, that GD3-treated mitochondria can release factors that are sufficient to induce DNA fragmentation in isolated nuclei. Moreover, selective suppression of ST8 expression in vivo, and therefore of endogenous GD3 neosynthesis, significantly prevented ceramide-induced {Delta}{Psi}m dissipation. Although ceramide-dependent pathways might affect mitochondrial function through kinase/phosphatase cascades regulating Akt activity and bad phosphorylation (38 , 39) , the evidence presented indicates that some relevant effects of ceramide on mitochondria may require conversion to GD3.

Endogenously generated GD3 accumulates within mitochondria and causes mitochondrial changes in vivo in cells undergoing apoptosis. This finding allows the enlistment of GD3 ganglioside among the ‘natural’ macromolecular inducers of mitochondrial permeability transition, mobilized to recruit mitochondria to the apoptotic program. They include pro-apoptotic bcl-2 family members such as bax (40 41 42 43) , bak (44) and p15bid (45 46 47) as well as AIF (24) and selected caspases (32) .

Most known mitochondrial permeability transition inducers seem to act at the level of PTPC, a multiprotein complex situated at contact sites between the inner and the outer mitochondrial membranes. Dimeric bax resides in mitochondrial membranes, where it participates to the regulation of the PTPC by directly interacting with the adenine nucleotide translocator (48) and, together with bak, with the voltage-dependent anion channel (44 , 49) , both components of the PTPC. Monomeric bax translocates from the cytosol to the mitochondria in cells undergoing apoptosis (50) , and BH3-mediated homotypic interactions with resident bax might directly affect mitochondrial permeability transition. Similarly, p15bid, resulting from caspase 8-mediated cleavage of bid (45 46 47) , relocates to the mitochondria and very efficiently alters PTPC function (51) . AIF is responsible for both the direct induction of nuclear changes and the cytosolic amplification of the apoptotic response, by acting on nearby mitochondria through yet unknown mechanisms (23 , 24) . Caspases can lower the mitochondrial permeability transition threshold by processing mitochondrial bcl-2 family members known to stabilize the PTPC, such as bcl-2 and bcl-XL (32) . The protective effects of bcl-2 on GD3-induced apoptosis strongly suggest that relevant GD3 targets are controlled by bcl-2.

Different from cytosolic apoptotic effectors of proteic nature, GD3 ganglioside is likely to reach mitochondria via physical continuity between ER/EG and mitochondrial membranes (36 , 37) . Recruitment of available mitochondria through membrane connections is expected to be a slower process compared to diffusion of soluble products within the cytosol. This might explain the generally slower kinetics of apoptosis experimentally induced in vitro by ceramides, or by GD3 itself, compared to death receptor cross-linking agents activating upstream caspases. Accumulation of sphingomyelin-derived ceramides during acute stress responses occurs in simple organisms that lack caspases or bcl-2 family members (52 , 53) . Membrane-directed delivery of death messages may therefore represent an evolutionary ancient mechanism for the recruitment of mitochondria to the cell death program.

Mitochondria appear therefore to represent a critical destination of the lipid pathway. In fact, GD3 is unable to directly affect nuclear membranes and/or cause nuclear events associated with apoptosis, whereas supernatants from GD3-treated mitochondria are entirely competent in inducing DNA fragmentation within isolated nuclei. This suggests that GD3 is not generically perturbing cellular membranes. Pretreatment of isolated mitochondria with cyclosporin A completely suppressed GD3-induced swelling and release of apoptogenic factors, indicating that GD3 is acting at the level of the PTPC. Whether this is due to a direct interaction with any of the PTPC components, some of which are currently unknown, or to a local perturbation of specific mitochondrial membrane microdomains affecting electrical and spatial constraints relevant to the PTPC physiology remains to be established.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. G. Stassi (University of Palermo), R. De Maria (ISS, Rome), L. Lenti, R. Gradini, and F. d’Agostino (University of Rome ‘La Sapienza’) and to Prof. A. Modesti (University of Rome ‘Tor Vergata’) for helpful discussions and advices. We are also indebted to Dr. R Kofler (University of Innsbruch) for providing the bcl-2-transfected CEM cells and to Dr. D. Nicholson (Merck, Rahway, N.J.) for providing the anti-caspase 9 antiserum. This work has been supported by Associazione Italiana Ricerca sul Cancro, Ministero Universita’ e Ricerca Scientifica e Tecnologica, Agenzia Spaziale Italiana, Consiglio Nazionale delle Ricerche Progetto Biotecnologie, Istituto Superiore di Sanita’ Progetto Sclerosi Multipla, European Community Biomed 2 and TMR programs (to R.T), Agence Nationale pour la Recherche sur le SIDA, Association pour la Recherche sur le Cancer, Fondation pour la Recherche Médicale, Ligue Nationale contre le Cancer, and European Commission French Ministry for Science (to G.K.). F.M. and I.C. are AIRC fellowship holders. M.R.R. is a Fondazione Adriano Buzzati-Traverso fellowship holder.

Received for publication December 9, 1999. Revision received April 4, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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