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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.
Insights
Mild excitotoxic insults trigger neuronal apoptosis. Inhibiting the adenine nucleotide translocator (ANT) with bongkrekic acid prevents NMDA receptor-mediated apoptosis by preserving mitochondrial function, but not staurosporine-induced apoptosis.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Excitotoxic insults and staurosporine induce apoptosis in cultured cerebrocortical neurons.
- NMDA receptor-mediated apoptosis involves mitochondrial dysfunction, including loss of mitochondrial membrane potential and ATP depletion.
- Cytochrome c release from mitochondria is observed in both NMDA and staurosporine-induced apoptosis.
Purpose of the Study:
- To investigate the role of the adenine nucleotide translocator (ANT) in NMDA receptor-mediated and staurosporine-induced apoptosis.
- To elucidate the mechanisms underlying mitochondrial involvement in neuronal apoptosis.
Main Methods:
- Utilized cultured cerebrocortical neurons subjected to excitotoxic insults or staurosporine.
- Inhibited the mitochondrial ANT using bongkrekic acid (BA).
- Assessed mitochondrial membrane potential, ATP content, cytochrome c release, and caspase activation.
Main Results:
- Bongkrekic acid (BA) prevented NMDA receptor-mediated apoptosis, mitochondrial depolarization, and ATP loss, while blocking caspase-3 activation.
- Cytochrome c was released into the cytoplasm even when BA inhibited apoptosis, indicating its release is insufficient to trigger apoptosis.
- BA did not prevent staurosporine-induced apoptosis or caspase activation, which was associated with caspase-8 activation.
Conclusions:
- NMDA receptor overactivation triggers neuronal apoptosis via mitochondrial dysfunction, which can be blocked by inhibiting the ANT.
- Staurosporine induces apoptosis through a distinct pathway independent of ANT and mitochondrial dysfunction.
- Mitochondrial dysfunction and cytochrome c release are necessary but not sufficient for NMDA-induced neuronal apoptosis.