Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons

Abstract

In cultured cerebrocortical neurons, mild excitotoxic insults or staurosporine result in apoptosis. We show here that N-methyl-d-aspartate (NMDA) receptor-mediated, but not staurosporine-mediated, apoptosis is preceded by depolarization of the mitochondrial membrane potential (Deltapsi(m)) and ATP loss. Both insults, however, release cytochrome c (Cyt c) into the cytoplasm. What prompts mitochondria to release Cyt c and the mechanism of release are as yet unknown. We examined the effect of inhibition of the adenine nucleotide translocator (ANT), a putative component of the mitochondrial permeability transition pore. Inhibition of the mitochondrial ANT with bongkrekic acid (BA) prevented NMDA receptor-mediated apoptosis of cerebrocortical neurons. Concomitantly, BA prevented Deltapsi(m) depolarization, promoted recovery of cellular ATP content, and blocked caspase-3 activation. However, in the presence of BA, Cyt c was still released. Because BA prevented NMDA-induced caspase-3 activation and apoptosis, the presence of Cyt c in the neuronal cytoplasm is not sufficient for the induction of caspase activity or apoptosis. In contrast to these findings, BA was ineffective in preventing staurosporine-induced activation of caspases or apoptosis. Additionally, staurosporine-induced, but not NMDA-induced, apoptosis was associated with activation of caspase-8. These results indicate that, in cerebrocortical cultures, excessive NMDA receptor activation precipitates neuronal apoptosis by means of mitochondrial dysfunction, whereas staurosporine utilizes a distinct pathway.

Figure 1

Apoptosis in cerebrocortical cultures. Cultures were exposed to either 300 μM NMDA/5 μM glycine ± 1 μM BA, or 0.5 μM staurosporine. (a–c) Representative labeling of apoptotic cells with PI, (d–f) with SYTO-13, or (g–i) by ISEL+. (j) At 18 h post insult, the presence of cells with condensed nuclei was assessed by staining with either PI (open bars) or SYTO-13 (shaded bars). DNA strand breaks within individual cells were evaluated at 18 h by ISEL+ (filled bars). Values are corrected for the basal level of apoptosis in these cultures (11.7 ± 2.1%; mean ± SEM). Statistical significance of BA/NMDA compared with NMDA indicated by asterisks (**, P < 0.001; ***, P < 0.0001). (k) Protection produced by BA is concentration dependent. Cells treated with NMDA/glycine ± BA (●) were analyzed for apoptosis at 18 h using PI. Treatment of cultures with BA alone for 30 min did not result in an increase in apoptosis at 18 h (□). BA (0.1 μM) did not protect from NMDA-induced apoptosis (***, P < 0.001; n = 8).

Figure 2

Determination of caspase activity. (a) Quantification of conformationally active caspase-3 in individual neurons (biotinylated-DEVD). At 6 h, the amount of active caspase-3 after BA/NMDA was not significantly different (n.s.) from the basal level, but was statistically less than that of NMDA (*, P < 0.05). (b and c) Detection of caspase-3 activity in cerebrocortical cell lysates by cleavage of fluorogenic substrates. (b) NMDA-induced activation (□) of caspase-3 peaked after 6 h (*, P < 0.05 from basal; n = 12). Staurosporine-treated samples (●) displayed a large increase in caspase-3 at 6 h (***, P < 0.001; n = 12). (c) BA prevented NMDA-induced caspase-3 activity at 6 h. The BA/NMDA value was not significantly greater than the basal value but was significantly different from NMDA (*, P < 0.05; n = 12). Staurosporine (staurosp.) values were greater compared with basal (***, P < 0.001; n = 6), but were not statistically decreased by BA. (d) Caspase-8 activity 6 h after exposure to NMDA or staurosporine. Under our conditions, NMDA did not significantly stimulate activation of caspase-8 (n = 8), whereas staurosporine markedly increased caspase-8 (***, P < 0.001: n = 3).

Figure 3

BA prevents NMDA-induced depolarization of Δψ_m in cerebrocortical neurons. (a) NMDA receptor activation induced a partial depolarization of Δψ_m (●). Incubation with 1 μM BA (▵) prevented the NMDA receptor-induced reduction in Δψ_m. Depolarization induced by 1 μM FCCP (★) further decreased TMRM fluorescence intensity. (b) Staurosporine did not depolarize mitochondria for at least 3 h, at which time Δψ_m was still sensitive to FCCP.

Figure 4

Effect of BA on isolated brain mitochondria supported by glutamate/malate oxidation. (a) BA (5 μM) and atractylate (20 μM) both inhibited stimulation of respiration on addition of 0.5 mM ADP. (b) Mitochondrial swelling induced by addition of 0.5 μM Ca²⁺ to de-energized mitochondria at time t = 0 was enhanced in the presence of 20 μM atractylate (●) but prevented by 5 μM BA (○). (c) The concentration of cellular ATP in cerebrocortical cells fell within 20 min of exposure to NMDA, but recovered by 6 h in the presence of BA. Staurosporine was not associated with a fall in ATP concentration (*, P < 0.05; **, P < 0.005; n = 3).

Figure 5

Release of Cyt c. (a) Western blot of Cyt c reveals appearance of Cyt c in the cytoplasm of cerebrocortical cells 3 and 6 h after NMDA receptor activation. (b) Cyt c was detected not only in membrane (m) but also in cytosolic (c) fractions 6 h after exposure to NMDA, NMDA plus BA, or staurosporine; however, under control conditions (Left) cytoplasmic Cyt c remained virtually undetectable. Under all conditions, most of the Cyt c remained in the membrane fraction. (c) Inhibition of caspases with 50 μM z-VAD-fmk (z = benzyloxycarbonyl; fmk = fluoromethyl ketone) during NMDA receptor activation did not prevent release of Cyt c. (d) Inhibition of caspase-8 with 20 μM z-IETD-fmk blocked staurosporine-induced release of Cyt c. (e) Model of staurosporine and NMDA activation of caspases and Cyt c release.