The Release of Cytochrome c from Mitochondria during Apoptosis of NGF-deprived Sympathetic Neurons Is a Reversible Event

Sympathetic neurons from newborn rat cervical superior ganglia were cultured as previously described (Martinou et al., 1995). For electron microscopy studies, neurons were fixed with 1% glutaraldehyde in PBS for 48 h at 4°C and washed in PBS before pre-embedding.

Sympathetic neurons (2 × 10⁵ in 3.5-cm-diam Petri dish) were harvested in 100 μl of isotonic buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM Hepes, pH 7.5) supplemented with protease inhibitors cocktail Complete (Boehringer Mannheim) and homogenized with a Dounce homogenizer. Samples were transferred to Eppendorf centrifuge tubes, centrifuged at 900 g for 5 min to remove nuclei, and then followed by centrifugation at 10,000 g for 30 min at 4°C to obtain the heavy membrane pellet (HM) enriched in mitochondria. The HM material was resuspended in 20 μl PBS, 0.2% Triton X-100. The protein concentration was determined by the method of Bradford (1976) in both the HM and soluble fractions. 5 μg (HM fraction) and 8 μg (soluble fraction) were used for Western blotting.

Mitochondria were isolated by sucrose density gradient centrifugation as previously described (Eskes et al., 1998). Mitochondria were incubated with 5 μM BaxΔTm for 30 min at 30°C in a buffer containing 125 mM KCl, 4 mM MgCl₂, 5 mM Na₂HPO₄, 5 mM succinate, 5 μM rotenone, 0.5 mM EGTA, 15 mM Hepes-KOH, pH 7.4. For electron microscopy, pellets of mitochondria were fixed in 1.5% glutaraldehyde in Sorensen phosphate buffer for 1 h at 4°C and processed as indicated.

Pellets of glutaraldehyde-fixed neurons and isolated mitochondria were pre-embedded into low viscosity agarose, washed with Sorensen phosphate buffer, and then postfixed in 2% OsO₄ in phosphate buffer for 1 h at room temperature. Then the samples were washed again in phosphate buffer, dehydrated in alcohol and propylene oxide, and then embedded in Epon. Ultrathin sections of comparable thickness were prepared with a Leica Ultracut ultramicrotome and placed on formvar carbon-coated copper grids. The grids were stained with uranyl acetate and lead citrate and observed with a Philips CM10 transmission electron microscope at 80 kV using a 30–40-μm objective aperture.

Neurons were fixed with 4% paraformaldehyde in PBS, permeabilized for 10 min with PBS containing 0.2% Triton X-100, incubated for 2 h with an anti–cytochrome c monoclonal antibody (dilution 1:15 in PBS with 5% normal goat serum; PharMingen), and then revealed with a fluorescein-labeled goat anti–mouse antibody.

We have studied the distribution of cytochrome c in cultured sympathetic neurons from rat superior cervical ganglia (SCG) undergoing apoptosis induced by NGF deprivation. Cytochrome c was analyzed by Western blotting in both soluble cytosolic and the mitochondria enriched heavy membrane (HM) fractions obtained from SCG neurons at 8, 15, and 24 h after NGF. Fig. 1, a and b, highlights a significant decrease of cytochrome c in the HM fraction of neurons deprived of NGF for 8, 15, and 24 h (Fig. 1 b), accompanied by an increase of cytochrome c in the cytosolic fraction (Fig. 1 a). This result was confirmed by immunostaining studies (Fig. 2, a and b). Although all neurons cultured in the presence of NGF displayed a punctate staining (Fig. 2 a), ∼50–75% of those deprived of NGF for 15 h displayed a diffuse cytosolic pattern (Fig. 2 b) and >95% of the latter had a condensed apoptotic nucleus (data not shown). Immunocytochemistry studies revealed that mitochondria present in neurites preserved their cytochrome c content longer than mitochondria present in soma (data not shown).

We also analyzed the cytochrome c distribution in sympathetic neurons rescued from NGF-deprivation with the caspase peptide inhibitor BAF. In the presence of 100 μM BAF, >80% neurons were able to survive after NGF deprivation for 5 d, although they had markedly smaller somas and atrophic neurites (Fig. 3 c). In agreement with previous data (Deshmukh et al., 1996), we found that BAF was able to inhibit caspase activity as assessed by immunoblot analysis of poly-(ADP ribose) polymerase (data not shown). A major decrease in cytochrome c levels was detected in the HM fraction of those neurons (Fig. 1 c), a finding confirmed by cytochrome c immunostaining (Fig. 2 c). These data indicate that, during apoptosis, the release of cytochrome c from mitochondria of sympathetic neurons does not require caspase activity.

The major objective of this article was to test whether mitochondria from BAF-rescued neurons could recover their cytochrome c content following re-exposure of neurons to NGF. Cytochrome c analysis in HM fraction of neurons deprived of NGF in the presence of BAF for 2 d and then re-exposed to NGF for 3 d, revealed a significant increase in the level of mitochondrial cytochrome c compared with neurons cultured in the absence of NGF and the presence of BAF alone for 5 d (Fig. 1 c). A kinetic analysis revealed a progressive recovery of cytochrome c by mitochondria which was almost complete after a 4-d NGF treatment (Fig. 1 d). Consistent with this finding, immunocytochemistry studies showed reappearance of a punctate mitochondrial cytochrome c staining in almost all neurons (>80%, n = 3) re-exposed to NGF for 24 h (Fig. 2 d). The effect of re-exposure to NGF was blocked by cycloheximide (Fig. 1 c and Fig. 2 f), suggesting the requirement of de novo protein synthesis for its action. It is possible that synthesis of cytochrome c precursor apocytochrome c was a prerequisite for cytochrome c recovery by mitochondria as only apocytochrome c has been shown to be imported into mitochondria (Mayer et al., 1995). In contrast, the recovery of normal cytochrome c levels by mitochondria was not prevented by the microtubule disrupting agent colchicine (Fig. 2 e), therefore excluding the possibility that reappearance of a normal cytochrome c staining pattern in the cell body was due to microtubule associated migration of still intact mitochondria from neurites to the soma.

In agreement with previous data (Deshmukh et al., 1996), we observed that addition of NGF back to BAF-protected SCG neurons caused an increase in soma diameter and neurite extension (Fig. 3 d). Altogether, these results indicate that the NGF receptors TRKA (tyrosine kinase receptor) and their signaling components remained functional in neurons deprived of NGF for at least 5 d. Interestingly, addition of NGF alone (without BAF) back to BAF-rescued neurons was sufficient to promote mitochondria recovery, regrowth of neurons, and long-term survival, suggesting that the caspases which had been activated during apoptosis had been irreversibly inhibited by BAF.

The reappearance of cytochrome c in mitochondria of BAF-rescued neurons re-exposed to NGF suggested that the ultrastructure of mitochondria had been preserved. This hypothesis was tested by electron microscopic studies. Fig. 4 a shows that mitochondria from neurons cultured continuously in the presence of NGF appeared elongated or oval-shaped, with sparse cristae (mean cross-sectional area ± SEM was 0.110 ± 0.007 μm², n = 115, Fig. 5 a). 24 h after NGF deprivation (Fig. 4 b) most mitochondria were round in shape, smaller than normal (mean cross-sectional area: 0.082 ± 0.004 μm², mean ± SEM, n = 124, Fig. 5 a) with a hyperdense matrix. No obvious rupture of the outer mitochondrial membrane has been observed. Mitochondria from BAF-rescued neurons (Fig. 4 c) were even smaller (mean cross-sectional area: 0.071 ± 0.005 μm², mean ± SEM, n = 80, Fig. 5 a), often forming aggregates surrounded by lysosomes (data not shown). After addition of NGF back to BAF-rescued neurons (Fig. 4 d), mitochondria recovered the shape and size typical of neurons continuously cultured in the presence of NGF (0.134 ± 0.08 μm², mean ± SEM, n = 121, Fig. 5 a). Lysosomes containing myelin figures, lipid droplets, as well as images of autophagy, have also been observed as previously described by Martin et al. (1988).

Bax is known to play an essential role in neuronal apoptosis. Sympathetic neurons from Bax-deficient mice can survive more than 20 d in the absence of NGF (Deckwerth et al., 1996). Moreover, Bax is known to trigger cytochrome c release when added directly to isolated mitochondria (Eskes et al., 1998; Jürgensmeier et al., 1998). We therefore wondered whether Bax could also be responsible for the mitochondrial morphological change observed in neurons after NGF deprivation. This hypothesis was tested by electron microscopy using isolated mitochondria that had been incubated for 15 min in the presence of 5 μM BaxΔTm or, for comparison, with 100 μM calcium which stimulates PTP opening (Fig. 5 b and Fig. 6). Although, as previously reported (Petronilli et al., 1993), opening of the PTP with calcium led to mitochondrial swelling, Bax, in contrast, triggered mitochondrial shrinkage resulting in a morphology similar to that observed in apoptotic neurons (Fig. 5 b and Fig. 6).

We report that in sympathetic neurons undergoing apoptosis triggered by NGF deprivation, mitochondria are reduced in size, display a hyperdense matrix, and are depleted of cytochrome c. A similar morphology of mitochondria lacking cytochrome c was observed in neurons protected from NGF deprivation by the caspase peptide inhibitor BAF. Interestingly, upon re-exposure to NGF, the mitochondria from BAF-rescued neurons recovered both a normal size and cytochrome c content. Altogether, these data do not support the hypothesis according to which cytochrome c release is the result of opening of the PTP (Marchetti et al., 1996; Susin et al., 1996; Green and Reed, 1998) and subsequent mitochondrial swelling and rupture of the outer mitochondrial membrane (Vander Heiden et al., 1997) since neither of these two features were observed in apoptotic neurons. Instead, our data agree with previous morphological studies of various cell types undergoing apoptosis showing preservation of mitochondrial membrane ultrastructure (Wyllie et al., 1980) and a reduction in total mitochondrial volume (Mannweiler and Bernhard, 1957; Rouiller, 1957; Svoboda and Higginson, 1963; Wachstein and Besen, 1964; Hackenbrock, 1968). More recently, ultracondensation of mitochondria has also been observed in apoptotic nodal myocytes (James et al., 1993), in lymphoblastic leukemic cells undergoing tumor necrosis factor-induced apoptosis (Lia et al., 1997), in a colon carcinoma cell line treated with herbimycin A (Mancini et al., 1997) and in etoposide-treated THP.1 cells (Zhuang et al., 1998). In some of these studies, a condensed conformation of mitochondria associated with hyperdensity of the matrix, as described here, has been postulated to be the result of water and ion loss from the matrix and to correspond to low-energy states of mitochondria (Hackenbrock, 1968).

Bax, a pro-apoptotic member of the Bcl-2 family essential for neuronal apoptosis (Deckwerth et al., 1996), can form ion channels in synthetic lipid membranes (Antonsson et al., 1997; Schlesinger et al., 1997) and therefore could be a likely candidate responsible for these mitochondrial changes during apoptosis. In support of this hypothesis, addition of Bax directly to isolated mitochondria triggers the release of cytochrome c (Eskes et al., 1998; Jürgensmeier et al., 1998) by a mechanism that may involve pore formation. We now show that this effect is accompanied by a reduction in mitochondrial volume. However, the mechanisms of action of Bax are still unclear. It has been recently reported that Bax can interact with the adenine nucleotide translocator (ANT), a component of the mitochondrial PTP (Marzo et al., 1998). Moreover, in yeast, the ANT appears to be required for the Bax killing function (Marzo et al., 1998). However, the importance of the ANT in apoptosis of mammalian cells has not yet been demonstrated.

One of the most striking observations reported here is the ability of mitochondria from BAF-rescued neurons to recover a normal size and cytochrome c content after re-exposure of neurons to NGF. The transition from small to large mitochondria induced by NGF deserves particular attention. This may be the result of mitochondrial fusion, an hypothesis that we are currently testing. We cannot exclude the possibility that proliferation of mitochondria may also take part in the complete recovery of neurons associated with the ability of neurons to grow in size, to extend neurites and to survive over long periods after re-exposure to NGF.

Recovery of function after protection by caspase inhibitors does not apply to all cell types. Indeed, it has previously been reported that inhibition of caspases in diverse types of apoptosis is incompatible with long-term survival, suggesting that in those cells caspases are activated after the cells become committed to apoptosis (McCarthy et al., 1997). Moreover, it has been shown that overexpression of Bax in Jurkat cells leads to mitochondrial dysfunction and caspase-independent apoptosis (Xiang et al., 1996). In the case of sympathetic neurons undergoing apoptosis induced by NGF deprivation, the situation is different as caspase activation appears to represent the point at which cells become committed to die (Deshmukh et al., 1996). Consistent with this, caspase inhibitors can inhibit apoptosis induced by overexpression of Bax (Vekrellis et al., 1997; Martinou et al., 1998) or Bak (Martinou et al., 1998) in these neurons. The difference between sympathetic neurons and other cell types may reside in intrinsic specificities of their apoptotic pathway, in specific properties of their mitochondria or could be related, at least in vitro, to their ability to produce ATP through glycolysis rather than through oxidative phosphorylation. The ability of mitochondria to recover fully their function when homeostatic conditions are restored may be specific for mitochondria from neurons. This could explain why in both caspase 3– and caspase 9–deficient mice, only neurons are protected from apoptosis during development (Kuida et al., 1996, 1998; Kakem et al., 1998).

 
• ANT

  adenine nucleotide translocator

 
• BAF

  Boc-aspartyl(Ome)-fluoromethylketone

 
• HM

  heavy membrane fraction enriched in mitochondria

 
• PTP

  permeability transition pore

 
• SCG

  sympathetic neurons from newborn rat cervical superior ganglia