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Patterns of cell death triggered in two different cell lines by HypF-N prefibrillar aggregates

Monica Bucciantini^*,1, Stefania Rigacci^*,1, Andrea Berti^*, Laura Pieri^*, Cristina Cecchi^*, Daniele Nosi, Lucia Formigli, Fabrizio Chiti^* and Massimo Stefani^*,,2

^* Department of Biochemical Sciences,
Department of Anatomy, Histology and Forensic Medicine,
Center of Excellence for Molecular and Clinical Studies on Chronic, Inflammatory, Degenerative and Tumoural Diseases for the Development of New Therapies, University of Florence, Florence, Italy

²Correspondence: Viale Morgagni 50, Florence 50134, Italy. E-mail: stefani{at}scibio.unifi.it

SPECIFIC AIMS

We have previously reported that prefibrillar aggregates, but not mature fibrils of a protein unrelated with any disease, the N-terminal domain of the hydrogenase maturation factor HypF (HypF-N), are able to enter into NIH/3T3 cells after incubation in the culture medium. As a consequence, cells display impaired viability due to early modifications of basic biochemical parameters such as intracellular redox status and free Ca²⁺. In this paper, we have investigated the biochemical features of cell death upon exposure to HypF-N toxic prefibrillar aggregates using H-END and NIH/3T3 previously shown to display a different final outcome, apoptosis, or necrosis, respectively, upon exposure to toxic amyloid aggregates. We sought to explain the reasons of the different type of death in either cell line by investigating some biochemical and cellular features, such as extrinsic and intrinsic apoptotic pathways and mitochondria involvement, with respect to controls exposed to the same amounts of the monomeric protein. Our data provide clues to explain the different behavior of the two investigated cell lines taken as examples of the two possible outcomes found in cells experiencing toxic protein aggregates.

PRINCIPAL FINDINGS

1. HypF-N prefibrillar aggregates induce apoptosis in H-END and necrosis in NIH/3T3 cells
NIH/3T3 and H-END cells were exposed for various times to different concentrations of toxic HypF-N prefibrillar aggregates to determine the mode of cell death. NIH/3T3 cells exposed for 24 h to 10 µM prefibrillar aggregates (or less) died with necrotic features, such as cellular and nuclear swelling and cytoplasmic vacuolization. H-END cells displayed typical features of apoptotic death such as nuclear condensation, chromatin aggregation, DNA fragmentation (assayed by nick-end labeling with the Klenow-FragEl^TM method), and plasma membrane blebbing. They also showed annexin V positivity as an annular staining, indicating membrane integrity but phosphatidylserine exposure on the membrane external surface, whereas exposed NIH/3T3 cells showed a cytoplasmic staining, suggesting the presence of plasma membrane ruptures typical of cell necrosis.

2. Caspase activation during H-END and NIH/3T3 cell death
Caspase activation was compared in NIH/3T3 and H-END cells exposed for differing times to 2.0 µM HypF-N prefibrillar aggregates or to the soluble protein. In H-END cells exposed for 5, 16, and 24 h (but not for 8 h) caspase-8 activity was 280, 150, and 350% of that observed in control cells, respectively (Fig. 1 , upper panel), whereas a modest caspase-9 activation was observed only at prolonged times of exposure (16 and 24 h). A higher (150%) caspase-3 activity was detected after 5 and 8 h of cell exposure, possibly deriving from the early caspase-8 activation, while its increase at 24 h could come from the late activities of both caspase-8 and 9 (Fig. 1 , upper panel).

View larger version (26K): [in this window] [in a new window]   Figure 1. Time course of caspase activity in H-END and NIH/3T3 cells after incubation with 2.0 µM toxic HypF-N prefibrillar aggregates. Caspase-9 activity was determined incubating cellular lysates for 2 h at 37°C in the presence of 50 µM Ac-LEHD-AMC. Caspase-3 and -8 activities were determined incubating for 4 h at 37°C in the presence of 200 µM Ac-DEVD-pNA and 200 µM Ac-IETD-pNA, respectively. The values are the mean ± SD of three replicates and are expressed as % with respect to the values found in control cells (100%) treated with 2 µM soluble HypF-N for corresponding intervals of time. These data come from one experiment out of ten, which gave qualitatively identical results.

In exposed NIH/3T3 cells, the pattern of caspase activation was quite different. At 5 h exposure, an 8-fold early increase of caspase-9 activity was observed together with a much lower caspase-8 and caspase-3 activation. At prolonged times of exposure all three caspase activities declined to control levels (Fig. 1 , lower panel).

3. Role of Bid and mitochondria involvement
We investigated whether a proteolysis of Bid did occur in cells exposed to the HypF-N aggregates. A significant decrease of Bid was found both in H-END and NIH/3T3 cells exposed 5 and 8 h to the aggregates, but Bid rose again at higher times of exposure, in agreement with the similar extent of caspase-8 activation observed at 5 h in the two cell lines. These data support the idea that the difference in the behavior of the two cell lines could rely, at least in part, on the early caspase-9 activation found in NIH/3T3 cells exposed to the aggregates. Therefore, the possible involvement of mitochondria in the aggregate-induced biochemical damage was investigated by checking the mitochondrial membrane potential in both cell lines. NIH/3T3 cells displayed a significant early mitochondrial membrane depolarization after 3 h exposure (Fig. 2 E), in agreement with the early caspase-9 activation observed in this cell line; mitochondrial membrane depolarization was maintained even at longer times of exposure (Fig. 2F ). In contrast, most H-END cells displayed significant mitochondrial membrane depolarization only after 24 h exposure (Fig. 2C ), in agreement with the late caspase-9 activation found in this cell line, whereas no significant depolarization was observed at 3 h exposure (Fig. 2B ).

View larger version (31K): [in this window] [in a new window]   Figure 2. JC-1 analysis of mitochondrial membrane potential M in H-END and NIH/3T3 cells. The change in M was detected by a fluorescence-based assay. For experimental details see Experimental procedures. Left panel: H-END cells. Right panel: NIH/3T3 cells. A, D) Control cells exposed 24 h to 2.0 µM soluble HypF-N. B, E) Cells exposed for 3 h to 2.0 µM HypF-N prefibrillar aggregates. C, F) Cells exposed for 24 h to 2.0 µM prefibrillar aggregates of HypF-N.

These data, together with the observed caspase-8-dependent proteolysis of Bid, suggest that mitochondrial injury is not the primary event of the apoptotic process in H-END cells, but could be a late consequence of the activation of the extrinsic pathway. On the other hand, the early mitochondrial injury could support the necrotic pattern observed in NIH/3T3. According to this idea, we measured a 30% decrease in the intracellular ATP levels with respect to controls in exposed NIH/3T3, but not in H-END cells. The reduction of the ATP levels may explain the inability of NIH/3T3 cells to sustain the initially triggered apoptotic program, in agreement with the notion that the latter requires energy to proceed.

Caspase-8 and caspase-9 activities were reduced in H-END cells treated with a cell permeable caspase-8 specific inhibitor prior to the exposure to the aggregates, and Bid cleavage disappeared making it possible to observe an increase in Bid with respect to controls. Such an increase could be due to the up-regulation of the expression of the corresponding gene after p53 activation. We then decided to investigate p53 involvement during H-END cell death.

4. The p53 pathway contributes to the apoptotic program in cells exposed to the aggregates
The p53 levels in cells exposed to the HypF-N aggregates together with the expression levels of two p53 targets, Bax and caspase-6, were investigated through SDS-PAGE and Western blot analysis of cell lysates by using specific antibodies. Sharp increases of all three proteins with respect to controls were found, with apparent maxima at 5 h for p53 and caspase-6, and at 8 h for Bax, suggesting a contribution of the p53 pathway to H-END apoptosis after exposure to HypF-N aggregates.

CONCLUSIONS AND SIGNIFICANCE

It is generally believed that cell death associated with protein aggregates is a result of the stimulation of the apoptotic response, although in some cases cells seem to die by necrosis or secondary necrosis. The pattern of caspase activation and mitochondria injury in exposed H-END and NIH/3T3 cells is consistent with their different fates. In H-END cells, the initial caspase-3 activation by caspase-8 is followed by a reactivation by caspase-8 and caspase-9 at later times of exposure. It is known that the product of Bid cleavage by caspase-8 (tBid) induces oligomerization and insertion of Bax and/or Bak into the outer mitochondrial membrane, with membrane permeabilization, release to the cytosol of death-promoting factors, apoptosome formation, and caspase-9 activation. A significant decrease of Bid was found in the exposed H-END cells, presumably due to the increase of its cleaved form; such a decrease was the highest at 8 h exposure, possibly after the preceding caspase-8 activation and, together with caspase-9 activation, was not found in cells incubated with the cell-permeable caspase-8 inhibitor. These results support the hypothesis of either a cross-talk, in exposed H-END cells, between the extrinsic and intrinsic pathways, and an involvement of the p53 pathway, which could account for the increase in Bid content that can be observed when caspase-8 is inhibited. The sharp increases of the levels of either p53 or two p53 up-regulated gene products (Bax and caspase-6) in exposed H-END cells further support the hypothesis of p53 participation in H-END cell death.

Our data may contribute to an explanation of why NIH/3T3 cells are not able to sustain the initially triggered apoptotic program after their exposure to the toxic aggregates and eventually die by necrosis. The comparison of the patterns of caspase activation in NIH/3T3 and H-END cells shows that in NIH/3T3 cells, caspase-9 undergoes a strong and early (though not sustained) activation, while both cell lines undergo similar caspase-8 and caspase-3 activation and Bid proteolysis after 5 and 8 h of exposure. Caspase-9 activation in NIH/3T3 exposed 3 h to the aggregates agrees with the significant early mitochondrial membrane depolarization found in these cells. At the same time of exposure, no significant mitochondrial membrane depolarization was observed in H-END cells where mitochondrial damage was apparent only at 24 h exposure, in agreement with the late caspase-9 activation.

These data suggest that mitochondrial injury is not the primary event of the apoptotic process in H-END cells; rather, it can follow the activation of the extrinsic pathway possibly elicited by the interaction of prefibrillar aggregates with the plasma membrane, though other triggers of the extrinsic pathway cannot be ruled out. NIH/3T3 cells display early mitochondria impairment and a primary activation of the intrinsic pathway, as indicated by the early onset of caspase-9 activity. The differing mitochondria involvement in cell death in either cell line is confirmed by the energy load in exposed NIH/3T3 and H-END cells. The latter was sharply reduced only in NIH/3T3 cells, confirming the heavier mitochondrial damage in these cells and providing a possible clue to explain their inability to support the initially triggered apoptotic program. Evidence that cells targeted for apoptosis are forced to die by necrosis when intracellular ATP is depleted has previously been reported. The heavy mitochondria impairment in NIH/3T3 cells may be a consequence of the previously reported early increase of free Ca²⁺ and ROS in these cells, after exposure to the same aggregates.

Our data, when added to those previously reported on the biochemical modifications in exposed NIH/3T3 cells, support the existence, at least in most cases, of a common mechanism of aggregate toxicity to cells. This initiates with the interaction of the aggregates with cell membranes and proceeds through early modifications of the intracellular redox status and Ca²⁺ content, followed by the activation of the intrinsic and/or the extrinsic pathway of apoptosis. Our results also indicate that the choice between apoptosis and necrosis could depend on the timing and extent of mitochondria derangement. In NIH/3T3 mitochondrial injury appears to be early and severe, possibly after the massive Ca²⁺ influx, and leads to a substantial reduction of the energy load. Under these conditions, the apoptotic program cannot be sustained and necrosis is the final outcome.

View larger version (44K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3086fje;

¹ These authors contributed equally to this work.

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