[Frontiers in Bioscience 14, 3550-3562, January 1, 2009]
Pharmacological screening and enzymatic assays for apoptosis
Anne-Sophie Belzacq-Casagrande1,2, Cecile Martel1,3, Claire Pertuiset1, Annie Borgne-Sanchez2, Etienne Jacotot2, Catherine Brenner1
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TABLE OF CONTENTS
1. ABSTRACT
Mitochondria play a central role in the intrinsic pathway of apoptosis. In response to many pro-apoptotic stimuli, mitochondria undergo an irreversible process called mitochondrial membrane permeabilization (MMP). The detection of MMP in isolated mitochondria is most often based on assays that monitor either the loss of the inner transmembrane potential (DYm; classically with Rhodamine 123), permeability transition (PT, cyclosporin A-sensitive matrix swelling), or the release of critical pro-apoptotic intermembrane space effectors. To gain complementary information on MMP mechanisms, we have systematically used three additional assays optimized for the 96-well microplate format: (1) inner membrane permeability, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity. We report that ad hoc combinations of ANT and VDAC ligands, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mastoparan and Vpr52-96 peptide and PT inhibitors, permit to explore relationships between enzymatic functions of sessile mitochondrial proteins (i.e. ANT, VDAC) and MMP. These assays should be useful tools to investigate mitochondrial apoptosis, decipher the implication of inner and outer membrane permeabilization and provide a multi-parametric approach for drug discovery.
2. INTRODUCTION
Apoptosis or programmed cell death has been implicated in metazoan development, as well as during the whole adult life to control cell size population and/or eliminate mutated and surnumerary cells (1). In addition, apoptosis deregulation may lead to diverse severe pathological conditions. For instance, an excess of cell death is observed in neurodegenerative diseases, in ischemia-reperfusion and acute intoxication. In contrast, a defect in apoptosis has been associated with autoimmune diseases and cancer, which are characterized by an uncontrolled cell multiplication (2). In viral infection, host cell apoptosis can be down-regulated in the early phase of infection to promote virus multiplication, whereas apoptosis can be up-regulated at the end of viral cycle to favor virus spreading (3).
Mitochondrial membrane permeabilization (MMP) is a central process in (programmed) cell death pathways and is regulated by Bcl-2 family members via multiple molecular mechanisms (4). Thus, mitochondria collect numerous apoptosis signals, emanating from the extracellular environment (e.g. radiations, growth factors, toxins, death receptors ligands, anoxia) or from the intracellular milieu and other organelles (e.g. calcium (Ca2+), reactive oxygen species (ROS), Bax, p53, caspases, kinases), integrate them and coordinate the caspase-dependent and caspase-independent degradation steps of apoptosis (5). MMP is currently considered as a point of no return in the apoptosis signaling cascade. As a consequence, MMP modulation has been proposed as a therapeutic goal and the development of robust cellular and subcellular assays to identify biomarkers, therapeutic targets and novel therapeutic drugs is currently a major issue for numerous researchers (2, 6-8).
MMP can be divided into several coordinated steps. These steps can occur concomitantly, sequentially, and depend of the cell type and the apoptosis stimulus. Briefly, MMP can affect only the outer membrane or both mitochondrial membranes, i.e. outer and inner membranes (OM and IM), manisfesting by the so-called mitochondrial permeability transition (PT). In most cases, MMP is accompanied by a loss of transmembrane inner potential (DYm loss) (9), an OPA-1-dependent cristae remodeling (10) and a colloidosmotic matrix swelling due to the massive entry of water and solutes (MM<1.5.kDa) trough the irreversible opening of a megachannel, namely the permeability transition pore complex (PTPC) (11, 12). PTPC is a multi-component pore, whose protein composition appeared to vary with the tissues and the condition (e.g. stress, pathology...) (1). Main components would be the voltage-dependent anion channel (VDAC) in the OM and the adenine nucleotide translocase (ANT), in the IM, and cyclophilin D (CypD), whereas other IM components are not excluded (13). Briefly, ANT and VDAC have been proposed as central mediators and/or regulators in MMP in cooperation with pro-apoptotic members of the Bcl-2 family, such as Bax, Bid and Bak (14-17). The striking consequence of membrane permeabilization is the release into the cytosol of a set of proteins/factors that are normally confined into the intermembrane space (18). This applies to the cytochrome c (19), the apoptosis-inducing factor (AIF) (20), caspases (21) and caspase activators such Smac/DIABLO (22). It is noteworthy that some genetic studies in rodents and yeast support an implication of ANT, VDAC or CypD in apoptotic and/or necrotic cell death, whereas other studies argue against their requirement (23-27), suggesting that modalities of cell death in specific models must be carefully considered.
Isolated mitochondria have been instrumental to elucidate the molecular events and the regulation of MMP. Detection of MMP in isolated mitochondria, is most often based on the measurement of the loss of the inner transmembrane potential (DYm; classically with tetramethylrhodamine ethyl ester (TMRM) or Rhodamine 123), the induction of permeability transition (PT) and of matrix volume changes (cyclosporin A-sensitive matrix swelling by optical density changes), or the release of critical pro-apoptotic intermembrane space effectors into the cytosol (e.g. cytochrome c presence into the mitochondrial supernatant) (28-33). Although there is no doubt on the implication of the OM into MMP, the role of the IM is still debated. Therefore, to gain more insights into MMP mechanisms, and study concomitantly apoptotic and modulation of vital functions of VDAC and ANT during MMP induction, we developed a series of coupled enzymatic assays into the 96-well format. Thus, in addition to swelling and depolarization assays, we measured (1) inner membrane permeability to Acetyl CoA, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity in response to various MMP modulators such as calcium, the prototypic PT inducer, carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore, and two pro-apoptotic peptides known to trigger IM permeabilization, mastoparan (MP), a wasp venom peptide (34), and Vpr 52-96, the C-terminal moiety of the viral protein R (Vpr) from HIV-1 (35, 36). Here, we show that these various molecules may have different effects, target various mitochondrial membrane proteins and elicit various MMP mechanisms.
3. MATERIAL AND METHODS
When not indicated, chemicals were from Sigma (St Louis, MO). All assays were performed in 96-well plates (200ml) and were performed at 37�C in a spectrofluorimeter (TECAN genios, TECAN, Austria). For absorbance and optical density measurement, transparent microplates were used and for fluorescent measurement, black microplates were used.
3.1. Isolation of mouse liver mitochondria
Mitochondria were isolated from mouse liver (C57BL/6J, female, 6-12 week old, Charles River, France) by differential centrifugation and purified on Percoll gradient according to (37). Protein concentration was determined using the micro-BCA assay (Pierce, Illinois).
3.2. Swelling and depolarization assays
For swelling and depolarization measurements, mitochondria (25�g of proteins/200�l) were diluted in a hypo-osmotic buffer (10mM Tris-Mops, pH 7.4., 5mM succinate, 200mM sucrose, 1mM Pi, 10�M EGTA, 2�M rotenone) containing or not various doses of agents such as Ca2+, CCCP, Vpr52-96 and MP. The mitochondrial swelling was immediately measured by the decrease in optical density at 540nm for 1800sec at 37�C. Several pharmacological inhibitors, such as cyclosporin A (CsA), which interferes with the ANT-Cyp D binding (38), bongkrekic acid (BA) and carboxyatractyloside (CAT), which inhibits ANT translocase function (39, 40) and 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), which targets VDAC (41), were added to mitochondrial suspension concomitantly with the inducer or not. Similarly, the depolarization of the mitochondria was measured by the rhodamine 123 (1�M) fluorescence dequenching assay (lexc: 485nm, lem: 535nm, Molecular Probes) (42).
3.3. Inner membrane permeabilization assay
In brief, mitochondria (25�g protein/200�l), were treated or not with the agents for 30min at 37�C to stimulate the swelling as described (43). Thereafter, 100�M 5,5'-Dithio-bis 2-nitrobenzoic acid (DTNB), 300�M acetylCoA and 1mM oxaloacetate were added sequentially every 10min and the absorbance at 415nm was recorded for 3000sec. Above cited molecules were used.
3.4. NADH ferricyanide assay of VDAC activity
For NADH ferricyanide activity of VDAC, mitochondria (10�g of proteins/200�l) were equilibrated for 10min at 37�C in the same hypo-osmotic buffer in the presence of molecules or not and then, 250�M NADH were added. After 15min of incubation, addition of 300�M ferricyanide was used to start the measurement of the fluorescence (lexc = 360nm; lem = 465nm). The protocol was adapted from (41). Above cited molecules were used.
3.5. ADP/ATP translocase assay
The ADP/ATP exchange rate was then evaluated on 25�g mitochondrial proteins in 200�l of buffer (20mM Hepes, pH 7.2., 5mM succinate, 300mM sucrose, 10mM KCl, 1mM MgCl2, 1mM Pi, 10�M EGTA, 2�M rotenone) per well of 96-multi-well plate. ATP efflux triggered by externally added ADP was monitored in fluorescence (lexc=360nm, lem=465nm) by following NADP+ reduction occurring in a solution containing 2.5. mM glucose, 1 E.U. hexokinase (E.C. 2.7.1.1.), 0.5. E.U. glucose-6-phosphate-dehydrogenase (Roche/R-Biopharm, France) and 0.2.mM NADPH as described by (24). The choice of fluorescence instead of absorbance was determined by the higher sensitivity of fluorescence and to allow the combination of multiple mode of measurements. Influence of adenylate kinase-dependent ATP synthesis was evaluated after treatment of isolated mitochondria by 10�M of adenylate kinase specific inhibitor Ap5A (P1P5-diadenosine-5'-pentaphosphate). MT21, and above cited agents were used.
3.6. Statistical analysis
IC50 values and standard errors were calculated using Graphpad Prism software.
4. RESULTS AND DISCUSSION
4.1. Ultrastructural effects of Ca2+, MP and Vpr52-96 on mouse liver isolated mitochondria
Mouse liver mitochondria were incubated for 15 or 30min in the absence (Figure 1A) or presence of 25�M Ca2+ (Figure 1B), 2�M MP (Figure 1C), 3�M Vpr52-96 (Figure 1D). Then, their morphology was studied by transmission electronic microscopy. In comparison to control mitochondria, Ca2+, MP and Vpr52-96 induced the matrix swelling and the almost complete loss of internal material. The cristae, which are detected in control mitochondria, cannot be distinguished after treatments, indicating that the IM expanded and came in close contact with the OM due to its large surface. Sites of OM discontinuity, while the IM appeared intact, revealed physical rupture of the OM. Of note, Vpr52-96 , but not MP, preserved the round shape of mitochondria (Figure 1C, D, (36)) suggesting different modalities of mitochondriotoxicity. Global structural changes correspond to previous observations of Jurkat cells mitochondria following cell treatment with anti-Fas antibody (44). Altogether, these results suggest that, in our conditions, Ca2+, MP and Vpr52-96 triggered classical hallmarks of the morphological pro-apoptotic alterations of mitochondria.
4.2. Calcium, MP and Vpr 52-96, but not CCCP promote mitochondrial matrix swelling
Mouse liver isolated mitochondria were treated with various doses of Ca2+ and changes in optical density of the mitochondria suspension was immediately registered. Thus, Ca2+ elicited a dose-dependent matrix swelling, which was reproducibly maximal for 25�M (Figure 2A). This dose was defined as the reference (100%) to estimate the effect of other molecules in percentage for further experiments. Then, we found that MP, and Vpr52-96, but not CCCP, induce a dose-dependent mitochondrial swelling (Figure 2B). The IC50 of MP and Vpr52-96 were respectively 1.86�M and 0.51�M respectively (Figure 2C). Furthermore, the Ca2+ effect was totally inhibited by CsA, and partially by BA and DIDS , as expected from litterature (34, 35, 45). In contrast, MP was only slightly inhibited by DIDS, but was not modulated by CsA and BA, suggesting that MP activity could be mediated via an effect on VDAC, neither ANT nor PTPC. Vpr52-96, also, was not inhibited by CsA, but significantly prevented by BA and DIDS, confirming an effect on both proteins (35, 36). The absence of protective effect of CsA toward both peptides can be explained by differences in mitochondrial origin (rat vs. mouse), in purification protocol (mitochondria purification on Percoll gradient or not) and in pre-incubation conditions in previous studies, but not in our conditions (35, 36). Thus, to elucidate the molecular mechanisms of mitochondrial interaction of an unknown molecule, experimental conditions of the assay may be adapted. For instance, the effects of a molecule on mitochondria can be dependent on the order of addition of the various agents e.g. (inducers vs inhibitors), the temperature, as well as the presence of salts (e.g. MgCl2) (46).
4.3. Calcium, MP and Vpr 52-96, and CCCP induce the mitochondrial depolarization
In parallel with swelling measurement, we analyzed the transmembrane potential using the methodology based on the dequenching of the fluorescent probe, Rhodamine 123 (42). We found that Ca2+ induced a dose-dependent depolarization that is maximal for 25�M (Figure 3A). As expected, CCCP depolarized totally and immediately the mitochondrial membrane (Figure 3B). MP and Vpr 52-96 depolarized also the IM with IC50 of 2�M and 0.4.7�M respectively. According to swelling experiment, CsA, BA and DIDS prevented partially Ca2+-dependent depolarization (Figure 3C). MP-induced depolarization was not prevented by any inhibitor, but was increased by CsA. By contrast and in line with swelling experiment, Vpr52-96 effect was decreased by BA and DIDS, but not by CsA. Of note, CCCP-induced depolarization was partially inhibited by DIDS suggesting an (indirect) implication of VDAC in CCCP activity. For charged molecules such as peptides, we cannot exclude a titration effect due the net negative charge of BA and DIDS.
4.4. Calcium, MP and Vpr 52-96, but not CCCP induce large inner membrane permeability
We used an original enzymatic method to detect changes in IM permeability via the accessibility of a soluble matricial enzyme, citrate synthase (43). This assay can detect IM permeabilization only if exogenous acetyl-coA (MM 809Da) can diffuse into the mitochondrial matrix after opening of a sufficiently large pore, which may be the PTPC, whose exclusion limit is <1.5. kDa (47) (Figure 4A). We decreased the volume of the various constituents of the assay to perform measurement in 96 well-microplate. After the induction of swelling (1800sec), substrates, such as DTNB, acetyl-Coenzyme A and oxaloacetate, were added and the absorbance measured at 415nm. We observed that Ca2+, MP and Vpr52-96 allowed the detection of a citrate synthase activity, suggesting that the molecules stimulated a dose-dependent IM permeabilization (Figure 4B and C). As expected, even at high doses, CCCP did not permeabilize the inner membrane. In line with Korge's conclusion (43), our results support the usefulness of the assay to discriminate compounds acting via the opening of a large pore in the IM or not.
4.5. Vpr 52-96 but not calcium, MP, CCCP modulates NADH reductase activity of VDAC
VDAC has been demonstrated to be a plasma membrane NADH-oxidoreductase (41, 48, 49). Thus, in the presence of NADH and ferricyanide, VDAC generates ferrocyanide and NAD+ as schematized in Figure 5A. This activity is inhibited in a dose-dependent manner by DIDS (Figure 5B). When isolated mitochondria have been treated by our three molecules, we showed that MP and CCCP did not affect the ferricyanide reductase activity, that Vpr52-96 was able to partially inhibit the VDAC activity with an IC50 of 0.86�M (Figure 5C), underscoring again a difference of modality of mitochondriotoxicity of both peptides. Interestingly, a Vpr52-96 effect on the VDAC activity is compatible with a direct peptide-protein interaction as previously suggested (35).
4.6. ADP/ATP translocation activity is impaired by Vpr 52-96, MP, calcium, and CCCP
The physiological function of ANT is to exchange the ADP against ATP in a stoechiometric manner (50, 51, 52). Here, we have developed and used a spectrophotometric assay of ADP/ATP translocation (53, 54) in the microplate format to evaluate the effect of our molecules on the ADP/ATP translocation (Figure 6A). Figure 6B shows a typical analysis based on the use of Ap5A for the inhibition of the adenylate kinase, an enzyme that can interfere with the ADP/ATP translocation assay and of carboxyatractyloside (CAT), as a specific and potent inhibitor of ANT (55). Thus, the specific activity of ANT, after inhibition of adenylate kinase, corresponds to the difference between the Ap5A and Ap5A+CAT curves. In contrast to Passarella et al., ADP/ATP translocation is partially inhibited by Ap5A in our conditions (53). In Figure 6C, we observed that in the presence of CAT, Ca2+, high doses of MP, Vpr52-96, CCCP, and MT21, an ANT inhibitor, described to act independently of permeability transition induction (46, 56), no ADP/ATP translocation activity could be measured. However, when swelling and depolarization have been measured in parallel using the buffer optimized for ATP detection, we noted that for similar concentrations of molecules, (i) CAT did not favored either the swelling and the depolarization (ii) CCCP and MT21 induced the loss of DYm without matrix swelling, and (iii) Ca2+, MP and Vpr52-96 triggered both swelling and depolarization (data not shown). However, these discrepancies can be visualized on kinetics curves by the fact that the induction of swelling allows the ATP release from michondria into the medium and modified the level of the CAT curve in presence of the molecule (arrow, Figure 6D). This demonstrates that the enzymatic assay requires the IM integrity. Therefore, this assay is efficient to determine the effect on the ADP/ATP translocase activity of molecules, such as CAT, which preserves IM permeability barrier, but is not suitable to determine the impact of molecules, which can disrupt the membrane potential.
5. CONCLUDING REMARKS
We described the use of three enzymatic assays based on the measurement of a product apparition or a substrate disparition by spectrophoto- and fluorimetry. These assays can be used to characterize the effects of a panel of different molecules on isolated mitochondria as well as to compare mitochondria of various origins. Ca2+, MP, Vpr52-96 constituted diverse standards for their differences in their chemical structure and their capacity to promote the pro-apoptotic MMP in vitro as well as in cellulo (34, 35, 57). CCCP was a negative control, because as a protonophore, it depolarizes mitochondrial IM, but does not trigger a complete process of MMP (57).
The combination of several assays to the classically used swelling and depolarization assays allowed to deepen the study of the mechanisms of mitochondriotoxicity of candidates molecules and thus, to discriminate molecules, which could seem to act similarly to promote MMP. As a result, Ca2+, MP, Vpr52-96 activated the swelling, the DYm loss, and IM permeabilization via the opening of the PTPC. Moreover, Vpr52-96, but not MP, affects VDAC activity, in line with previous findings revealing that Vpr52-96 requires both ANT and VDAC to kill yeast cells (35). This data was re-enforced by the fact that Vpr52-96 mitochondriotoxic effects are prevented by DIDS and BA, but not those of MP. Moreover, on the basis of their IC50 in the various assays, Vpr52-96 appeared to be more potent than MP. This clearly indicates that the two peptides exert their effect by different molecular mechanisms and presumably target various constitutive mitochondrial proteins.
These assays constitute additional screening assays for drug candidates. Indeed, during the last decade, the development of high throughput screening has increased the need for robust assays using absorbance, fluorescence as well as luminescence (58). Moreover, since the emergence of a concept of apoptosis-based therapy (6), an ever increasing number of companies are interested in the search of apoptosis regulatory molecules and the identification of their target. Here, we propose three microplate assays that can provide complementary information regarding the permeabilization of the mitochondrial IM and the targeting of the two membrane proteins VDAC and ANT, two main actors of the MMP process.
Thus, the results underscore the usefulness of the combination of multiple assays to decipher fine molecular mechanisms of mitochondriotoxicity of candidate molecules (59) and constitute novel tools, which might contribute to the discovery of novel therapeutic molecules.
6. ACKNOWLEDGMENTS
CB is supported by grants funded by ANVAR, ARC (3902) and INCa. ASB and CP have been supported by ANVAR (n�A0505001Z).
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Abbreviations: ANT, adenine nucleotide translocase; Ap5A, P1P5-diadenosine-5'-pentaphosphate; BA, bongrekic acid; Ca2+, calcium; CAT, carboxyatractyloside, CCCP, carbonyl cyanide m-chlorophenylhydrazone; Co., control; CsA, cyclosporine A; cyclophilin D, CypD; IM inner membrane; DTNB, 5,5'-Dithio-bis (2-nitrobenzoic acid; DIDS, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid; DYm, transmembrane inner potential; MP, mastoparan; MMP, mitochondrial membrane permeabilisation; OM, outer membrane; PT, permeability transition; PTPC, permeability transition pore complex; VDAC, voltage-dependent anion channel; Vpr, viral protein
Key Words: Cell Death, Mitochondria, Permeability Transition, VDAC, ANT
Send correspondence to: Catherine Brenner, University of Versailles/SQY, PRES UniverSud Paris, CNRS UMR 8159, LGBC, 45, avenue des Etats-Unis, 78035 Versailles, France, Tel: 33 1 39 25 45 52, Fax: 33 1 39 25 45 72, E-mail:cbrenner@genetique.uvsq.fr