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 staurosporinemediated, apoptosis is preceded by depolarization of the mitochondrial membrane potential (⌬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 ⌬ 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, staurosporineinduced, 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. Apoptosis is an important mechanism in both the development and degeneration of the nervous system. Evidence suggests that the loss of neurons in many neurologic disorders occurs by apoptosis (1-4). The overstimulation of glutamate receptors can precipitate the death of neurons by either necrosis or apoptosis depending on the severity of the insult (5, 6). Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, in particular, results in an increase in the intracellular free calcium concentration ([Ca 2ϩ ] i ) to levels above the buffering capacity of neurons (7). This increase in [Ca 2ϩ ] i leads to the activation of toxic events, including oxidative and nitrosative stress (8). Mitochondria, which undergo harmful Ca 2ϩ -loading after NMDA receptor activation (9, 10), also have an important signaling function in apoptosis (11-13).Apoptotic cell death is often mediated by a caspase cascade. Although many stimuli exist, the final phases of apoptosis are executed by a few common effector caspases. Mitochondria appear to provide a link between the initiator caspases and the downstream effector caspases. In nonneuronal cells, mitochondria have been shown to accelerate activation of caspases by releasing proapoptotic molecules, such as cytochrome c (Cyt c) (11, 12) and the apoptosis-inducing factor (13). The release of these molecules can be stimulated by some caspases and by Bid and Bax (14-16), whereas Bcl2 prevents their release (11-13).The mechan...

Staurosporine‐induced apoptosis in human cornea epithelial cells in vitro

“…The overall time course and the concentration range of STS induced apoptosis in SV-40 immortalized HCE cells is in good agreement with apoptotic key-characteristics, detected in primary corneal epithelial cells (19), in cerebrocortical cultures (20), or in cultured lymphoma cells (21). In the present model of the apoptotic pathway, the liberation and activation of caspase-3 from the intermembrane space of the mitochondria has been described as an early apoptotic event, leading to successive cleavage of a number of different proteins, including poly(ADP-ribose) polymerase and DNA-dependent protein kinases (22,23).…”

S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding

“…Excitotoxic injury can be in part mediated by caspases. 16,27 It has been shown that NO can S-nitrosylate the catalytic cysteine of most or all caspases. [12][13][14][15][16] S-Nitrosylation inhibits protease activity of caspases and consequently prevents apoptotic death in many cell types.…”

“…23,24 Intense hyperstimulation of excitatory receptors leads to necrotic cell death, but more mild or chronic overstimulation can result in apoptotic or other forms of cell death. [25][26][27] Two large families of glutamate receptors exist in the nervous system, ionotropic receptors (representing ligandgated ion channels) and metabotropic receptors (coupled to G-proteins). Ionotropic glutamate receptors are further divided into three broad classes, NMDA receptors, a-amino-3-hydroxy-5 methyl-4-isoxazole propionic acid (AMPA) receptors, and kainate receptors, which are each named after a synthetic ligand that can selectively activate its receptor.…”

“…In particular, excessive activation of NMDA receptors leads to the production of damaging free radicals (e.g., NO and ROS) and other enzymatic processes, contributing to cell death. 3,6,26,27,30,31 Ca 2 þ Influx and Generation of NO Increased levels of neuronal Ca 2 þ , in conjunction with the Ca 2 þ -binding protein CaM, trigger the activation of nNOS and subsequent generation of NO from the amino acid L-arginine ( Figure 1). 4 NO is a gaseous-free radical (thus highly diffusible) and a key molecule that plays a vital role in normal signal transduction but in excess can lead to neuronal cell damage and death.…”

Potentiation of excitotoxicity in HIV-1-associated Dementia and the significance of glutaminase

“…This influx disrupts cell homeostasis via hypertonic-induced swelling, ATP depletion and ultimately membrane failure and necrotic cell death. The delayed mechanism is independent of cell swelling, and involves Ca 2+ influx that triggers multiple neurotoxic cascades including p38 and JNK MAP kinase activation, release of cytochrome c, caspase activation, lipid peroxidation, and chromatin condensation (40,(100)(101)(102). The massive influx of Ca 2+ triggers release of Ca 2+ from intracellular stores, further flooding the intracellular space with free Ca 2+ (Fig.…”