Mechanisms of Cell Death Induced by the Mitochondrial Toxin 3-Nitropropionic Acid: Acute Excitotoxic Necrosis and Delayed Apoptosis

Abstract

Impaired energy metabolism may play an important role in neuronal cell death after brain ischemia and in late-onset neurodegenerative diseases. Both excitotoxic necrosis and apoptosis have been implicated in cell death induced by metabolic impairment. However, the factors that determine whether cells undergo apoptosis or necrosis are not known. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), a suicide inhibitor of succinate dehydrogenase. Treatment of cultured rat hippocampal neurons with 3-NP resulted in two types of cell death with distinct morphological, pharmacological, and biochemical features. A rapid necrotic cell death, characterized by cell swelling and nuclear shrinkage, could be completely prevented by the NMDA receptor antagonist MK-801 (10 M) and dose-dependently potentiated by low micromolar levels of extracellular glutamate. A slowly evolving apoptotic death, characterized by nuclear fragmentation, was not attenuated by MK-801 but was prevented by cycloheximide (1 g/ml). The combination of MK-801 and cycloheximide resulted in an almost complete protection against 3-NP-induced cell death. DNA fragmentation, detected by the terminal deoxynucleotidyl transferase-mediated dUTP-X 3Ј nick end-labeling technique, was a late event in apoptosis and also occurred after necrotic cell death. ATP depletion was an early event in the 3-NP-induced neuronal degeneration, and the decline in ATP was exacerbated by glutamate. We conclude that 3-NP triggers two separate cell death pathways: an excitotoxic necrosis as a result of NMDA receptor activation and a delayed apoptosis that is NMDA receptor-independent. Mildly elevated levels of extracellular glutamate shift the cell death mechanism from apoptosis to necrosis.Key words: energy metabolism; succinate dehydrogenase; 3-nitropropionic acid; excitotoxicity; apoptosis; necrosis; nuclear fragmentation; TUNEL; ATP Neuronal function and survival depend on a continuous supply of glucose and oxygen, used to generate ATP through glycolysis and mitochondrial respiration. A perturbation in energy metabolism during conditions such as ischemia, stroke, and brain trauma may cause irreversible neuronal injury. An age-related decline in energy metabolism also may contribute to neuronal loss during normal aging, as well as in neurodegenerative diseases (Wallace, 1994;Beal, 1995).There are discrepancies in the findings regarding the mechanisms of neuronal cell death after metabolic impairment. A large body of evidence supports the "secondary excitotoxicity" hypothesis that a loss of ATP leads to membrane depolarization, removal of the voltage-dependent Mg 2ϩ block of the NMDA receptor, and subsequent activation of NMDA receptor (for review, see Beal, 1992). Both in vivo and in vitro studies have shown that metabolic inhibitors potentiate glutamate and NMDA toxicity (Weller and

The impact of mitochondrial energetic dysfunction on apoptosis in outer hair cells of the cochlea following exposure to intense noise

“…Studies have shown that the resulting level of ATP suppression following a 3-NP treatment is dose-and timedependent. For example, Pang, et al (1997) reported that application of a concentration of 3-NP (5 mM) reduces the cellular ATP level in cultured rat hippocampal neurons by about 25% at 2 hrs after the 3-NP treatment. In the current study, we intended to use the highest dose allowed without causing rapid cell death to generate maximal suppression of SDH activity.…”

FK506 prevents mitochondrial-dependent apoptotic cell death induced by 3-nitropropionic acid in rat primary cortical cultures

“…Several in vitro studies have established that 3-NP exposure induces both apoptosis and necrosis in striatal, cortical, and hippocampal cells (Behrens et al, 1995;Pang and Geddes, 1997). The balance between the two types of cell death depends mainly on the severity of ATP depletion (Eguchi et al, 1997;Leist et al, 1997;Ohgoh et al, 2000) which is closely related with the dose and time of exposure to this toxin.…”

“…In primary rat neuronal cultures, 3-NP induces a gradual increase in mitochondrial calcium and reactive oxygen species which are prevented in the presence of caspase inhibitors (Lee et al, 2002a). At higher concentrations, 3-NP administration results in a massive elevation of mitochondrial and cytosolic calcium, a decrease in ATP levels, a rapid mitochondrial membrane depolarization, and the activation of calpains (Lee et al, 2002b;Nasr et al, 2003;Pang and Geddes, 1997). Under these conditions, caspase activity is not affected, which is in agreement with cells undergoing death by necrosis (Nasr et al, 2003).…”

Excitotoxicity is Required for Induction of Oxidative Stress and Apoptosis in Mouse Striatum by the Mitochondrial Toxin, 3-Nitropropionic Acid

“…It has been reported that 3-NP produced a dose dependent selective toxicity to dopamine neurons in mesencephalic culture that could be attenuated by block ade of NMDA receptors (Zeevalk et al, 1995) and that the initial phase of 3-NP poisoning in vivo selectively inhibited the trichloroacetic acid cycle of GABAergic neurons where the trichloroacetic acid cycle of glia re mained uninhibited, as did the trichloroacetic acid cycle associated with the large neuronal pool of glutamate, which includes glutamatergic neurons (Hassel and Son newald, 1995). However, another study reported that MK-801 and the non-NMDA receptor antagonist, 6-cyano-7 -nitroquinoxaline-2,3-dione, could not protect against neuronal death induced by 3-NP in cultured stria tal and cortical neurons and that MK-801 failed to pre vent apoptotic neuronal death induced by 3-NP in rat hippocampal neuronal cultures (Pang and Geddes, 1997). The possible causes for the discrepancy between those studies and ours might be that the previous ones were in vitro experiments in which the cells would have lacked organized glutamatergic input.…”

“…Thus, we speculated that apoptosis induced by 3-NP in the striatum might be re lated to excitotoxicity, but this has not been clearly elu cidated in vivo. Although MK-801 could not prevent ap optosis induced by 3-NP treatment in a cell culture sys tem (Pang and Geddes, 1997), there is a possibility that 3-NP toxicity in vivo, in an intact system, could involve apoptosis induction by means of excitotoxicity. There fore, the involvement of excitotoxicity and oxidative stress in apoptosis induced by treatment with 3-NP in vivo requires comprehensive elucidation.…”