Influence of cytosolic and mitochondrial Ca2+, ATP, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

doi:10.1016/S0197-0186(02)00229-2

Neurochemistry International

Volume 43, Issue 2, July 2003, Pages 89-99

https://doi.org/10.1016/S0197-0186(02)00229-2 Get rights and content

Abstract

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 μM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 μM glutamate, 3NP administration resulted in a massive elevation in [Ca²⁺]_c and [Ca²⁺]_m, decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca²⁺]_c within the 24 h time period examined, nor increase [Ca²⁺]_m within 1 h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16 h. Calpain and caspase activity were first evident at 24 h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8 h), and elevated [Ca²⁺]_m as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca²⁺]_c, nor with early changes in [Ca²⁺]_m, mitochondrial membrane potential, ATP levels, or calpain activity.

Introduction

Metabolic impairment induced by 3-nitropropionic acid (3NP), an irreversible succinate dehydrogenase inhibitor, is relevant to CNS injuries and neurodegenerative disorders in which bioenergetic defects are implicated. Systemic 3NP administration results in selective damage to the striatum, and occasionally to hippocampus (Geddes et al., 1996, Hamilton and Gould, 1987) resembling aspects of Huntington’s disease, cerebral ischemia, and Alzheimer’s disease (Beal et al., 1993, Geddes et al., 1996, Panov et al., 2002).

In cultured rat hippocampal neurons, 3NP induces rapid necrosis in some neurons and delayed apoptosis in others (Pang and Geddes, 1997). Low levels of extracellular glutamate shift the cell death mechanism to necrosis, consistent with the indirect excitotoxic hypothesis which proposes that the loss of ATP resulting from energy impairment leads to an inability to maintain ion pumps, resulting in membrane depolarization and removal of the voltage-dependent Mg²⁺ block of the N-methyl-d-aspartate (NMDA) receptor (Novelli et al., 1988, Albin and Greenamyre, 1992, Beal et al., 1993, Turski and Turski, 1993). The NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801, dizocilpine maleate) attenuates the necrotic death, but shifts the cell death mechanism to apoptosis resulting in a delay, but not prevention, of neuron death (Pang and Geddes, 1997, Behrens et al., 1995). These results suggest that Ca²⁺ influx through the NMDA receptor may play a critical role in determining the cell death pathway. The goal of this study was to investigate changes in cytosolic ([Ca²⁺]_c) and mitochondrial ([Ca²⁺]_m) Ca²⁺ concentrations, ATP levels, mitochondrial membrane potential, calpain activity, and caspase activity, under conditions associated with 3NP-induced apoptotic and necrotic neuron death.

Section snippets

Materials

Cell culture reagents and glutamate were purchased from Invitrogen Life Technologies (Carlsbad, CA). 3NP, soybean trypsin inhibitor, MK-801, propidium iodide, and tetramethylrhodamine ethyl ester (TMRE) were purchased from Sigma–Aldrich (St. Louis, MO). Indo-1-acetoxymethyl ester (AM) and calcein AM were obtained from Molecular Probes (Eugene, OR) and rhod-2 AM was obtained from Teflabs (Austin, TX). Bovine pancreas trypsin was from Worthington Chemical Corporation (Lakewood, NJ). ATP levels

Cell death

We previously characterized the extent and morphology of cell death in primary hippocampal neurons exposed to 3NP in Neurobasal/B27 medium for up to 48 h (Pang and Geddes, 1997). 3NP induced both necrotic and apoptotic death. MK-801 shifted the cell death mechanism to apoptosis, while the addition of low levels (10 μM) of extracellular glutamate resulted in predominantly necrotic neuron death.

In the present study, the 1, 8, and 24 h time points were chosen for Ca²⁺ measurement since they preceded

Discussion

The purpose of this study was to examine the mechanisms underlying the shift between apoptotic and necrotic death following exposure of rat hippocampal neurons to 3NP in the presence or absence of added glutamate or the NMDA receptor antagonist MK-801. Previously, we demonstrated that exposure of cultured rat hippocampal neurons to 5 mM 3NP resulted in both apoptotic and necrotic neuron death. The addition of MK-801 results in almost exclusive apoptotic neuron death, whereas low levels of

Acknowledgements

This research was supported by NIH grant AG10836 to JWG.

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¹: Present address: Roche Bioscience, Palo Alto, CA 94304, USA.

²: Present address: Department of Anatomy & Neurobiology, University of California at Irvine, Irvine, CA 92697, USA.

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Influence of cytosolic and mitochondrial Ca2+, ATP, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

Abstract

Introduction

Section snippets

Materials

Cell death

Discussion

Acknowledgements

Brain Res. Mol. Brain Res.

Neuron

Trends Neurosci.

Neurosci. Lett.

J. Biol. Chem.

Trends Cell Biol.

Neurotoxicol. Teratol.

J. Biol. Chem.

Neuroscience

Neuron

J. Biol. Chem.

Neuroscience

Neuroscience

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Brain Res.

Exp. Neurol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Cell Calcium

Calpain activation in neurodegenerative diseases: confocal immunofluorescence study with antibodies specifically recognizing the active form of calpain 2

Acta Neuropathol. (Berl.)

Alternative excitotoxic hypotheses

Neurology

Mitochondrial participation in the intracellular Ca2+ network

J. Cell Biol.

3-Nitropropionic acid induces apoptosis in cultured striatal and cortical neurons

NeuroReport

Excitotoxic cell death

J. Neurobiol.

Calcium-activated neutral protease (calpain) system: structure, function, and regulation

Physiol. Rev.

Intramitochondrial [Ca2+] and membrane potential in ventricular myocytes exposed to anoxia-reoxygenation

Am. J. Physiol.

Influence of cytosolic and mitochondrial Ca²⁺, ATP, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

Mitochondrial participation in the intracellular Ca²⁺ network

Intramitochondrial [Ca²⁺] and membrane potential in ventricular myocytes exposed to anoxia-reoxygenation