Mitochondrial membrane potential and ischemic neuronal death

doi:10.1016/j.neures.2006.04.005

Neuroscience Research

Volume 55, Issue 3, July 2006, Pages 234-243

https://doi.org/10.1016/j.neures.2006.04.005 Get rights and content

Abstract

Mitochondria are intracellular organelles in which high energy phosphate is produced. Ischemia causes depletion of the materials necessary to produce this phosphate and strongly affects the electron transport chain. Apoptosis commences during and after ischemia. As such, it is likely that a significant relationship exists between inactivation of electron transport and apoptosis. Mitochondrial membrane potential (MMP) reflects performance of the electron transport chain and can indicate a pathological disorder of this system. In an experimental setting, oxygen–glucose depletion (OGD) in neuronal cell culture has been employed to simulate an ischemic condition. The relationship between MMP and subsequent neuronal death during and after OGD has been examined. MMP dissipation and concomitant neuronal death have been reported, but recent studies have demonstrated mitochondrial hyperpolarization preceding neuronal death. The direction of MMP polarization depends on the extent of OGD. Long OGD results in depolarization, while shorter OGD induces hyperpolarization. Neurons are still viable during hyperpolarization, but the process may switch on the apoptotic cascade. Meanwhile, dissipation of MMP seems to be a consequence of severe energy deficit, leading to necrosis. MMP may be a marker of subsequent apoptosis, although a causal relationship remains to be determined.

Introduction

The pathophysiology of ischemic neuronal death has been investigated in a number of laboratories, and investigators have sought to identify a step in the neuronal death pathway that could be blocked through pharmacological intervention. Ischemic neuronal death is roughly categorized to necrosis and apoptosis. Necrosis is an acute cell death occurring immediately after ischemic insult, and apoptosis is a slowly progressing cell death, which appears in the peri-infarct zone or transient global ischemia. Apoptosis may be a target to medically salvage. The mechanism occurring as apoptosis after ischemia is being elucidated. To explore cellular mechanism, neuronal culture is preferentially used. Recently, mitochondria are focused on as a regulator of apoptosis. This review introduces the proposed mechanism involving mitochondria and its membrane potential underling the ischemic neuronal death. Increased glutamate during ischemia opens the NMDA receptor, and allows the abrupt Ca⁺⁺ entry, resulting neuronal death. This glutamate hypothesis disclosed the importance of calcium (Ca⁺⁺) and sodium (Na⁺) ion homeostasis on cell survival and led to the calcium deregulation hypothesis. Calcium deregulation is a term for perturbation of intracellular calcium ion homeostasis (LoPachin and Stys, 1995, Taylor et al., 1999; Chinopoulos et al., 2000a, Chinopoulos et al., 2000b). Following ischemia, there is a sustained increase in intracellular Ca⁺⁺, even after commencement of reoxygenation, which leads to the activation of Ca⁺⁺-dependent enzymes and initiation of the apoptosis cascade. Both calcium buffering and apoptosis are under the control of mitochondria, which are therefore considered a key regulator of cell survival (Kroemer and Reed, 2000, Green and Kroemer, 2004). In light of this, mitochondria are thought to be a good therapeutic target for protection against the apoptotic effects of ischemia, but researchers have not yet confirmed whether pharmaceutical intervention targeting mitochondria can prevent neuronal death. Mitochondrial membrane potential is a key factor in the mechanism by which this organelle affects apoptosis. This review paper describes the behavior of the mitochondrial membrane potential (MMP) in response to ischemic insult, which may provide insight into the factors responsible for neuronal death.

Section snippets

Mitochondrial membrane potential

The mitochondrion is the primary organelle for production of high-energy phosphate. Reducing equivalents, malate and glycerol phosphate, are transported from the cytosol to the mitochondria by the NADH shuttle system. During electron transport, protons are excreted from the matrix into the intermembrane space, generating MMP. Usually the potential is approximately −150 mV, depending on the speed of proton pumping. This potential and the permeability of ions, especially Ca⁺⁺, are determined each

Oxygen–glucose deprivation (OGD) as a model of ischemic cell death

The mitochondrion can be identified under a microscope, and thin-cell culture is generally used for mitochondrial observation. Although ischemic conditions cannot be reproduced in neuronal culture, oxygen–glucose deprivation (OGD) mimics the ischemic condition and provides a model of the depletion of high-energy phosphate. Many differences exist between the in vitro OGD model and the in situ ischemic model. Neuronal swelling in the brain in situ increases intracranial pressure, while the

Mitochondrial membrane potential during OGD and reoxygenation

OGD in cultured cells does not efficiently diminish high-energy phosphate. It sometimes require more than 90 min (Fujii et al., 1994). Changes in the MMP are slow to occur, and it is necessary to monitor continuously. Our approach has been to mount an anaerobic chamber on the stage of an inverted fluorescence microscope and carefully observe the change in MMP. JC-1 is a convenient voltage sensitive probe to monitor MMP (Reers et al., 1991). JC-1 locates in the cytoplasm and mitochondria as long

ATP content in the OGD model

ATP is produced in complex V, the final step in the electron transport chain. Proton excretion during electron transport creates a negative charge in the mitochondrial matrix, establishing the MMP. In order to pump protons out of the mitochondrial matrix FoF1-ATPase must consume ATP. Thus, polarization is an energy-consuming process, and mitochondrial hyperpolarization (MHP) cannot occur if ATP is depleted. In complex V, the protons reenter the matrix, thereby preventing MHP. Dysfunction of

Neuronal validity

After a certain period of OGD, a subset of neurons falls into necrosis or apoptosis. Even in the same culture dish, some neurons remain viable while others do not, and the mode of death varies, with the type of death distinguishable by morphological change. Swollen neurons are necrotic, and a shrunken nucleus indicates apoptosis. However, morphological criteria are sometimes ambiguous, and immunocytochemical techniques are more reliable for gauging viability and identifying the mode of death.

Apoptosis

Apoptosis is characterized as a slowly-progressing death. Basically apoptosis occurs in cells where ATP is conserved, and many studies have confirmed that ATP is conserved in the culture where many neurons become apoptotic.

The terminal deoxynucleotidyl transferase-mediated (dUTP) nick end labeling (TUNEL) method can be used to confirm apoptosis by detecting fragmented ends of DNA. While the TUNEL method is sensitive for detecting apoptosis, it is vulnerable to false-positives in the form of

Pathological hypothesis of hyperpolarization of MMP

MHP has been reported as a pathological step in various experimental settings (Ahmed et al., 2000, Rubi et al., 2004, Perl et al., 2004, Gergely et al., 2002, Kim et al., 2003), and several mechanisms have been proposed for MHP (Fig. 10). MHP is basically induced by the excessive excretion of protons or inhibition of proton reentry. Theoretically an increase in NADH may cause MHP, and increased NADH generation coincident with MHP has been observed in rat pancreatic beta cells overexpressing

Conclusions

Elevation of extracellular glutamate has been considered as one of the events precipitating neuronal death. However, elevation of glutamate also seems to occur after acute ischemic necrosis. Therefore, the specificity of this marker in differentiating survival, apoptosis, and necrosis is very low. Mitochondrial events may be good markers with which to identify the early triggers for cell death. One such indicator, MMP, may turn out to be a useful marker for neuronal death, but additional

Acknowledgement

We would like to thank Dr. Paul Kretchmer ([email protected]) at San Francisco Edit for his assistance in editing this manuscript.

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Review articleMitochondrial membrane potential and ischemic neuronal death

Abstract

Introduction

Section snippets

Mitochondrial membrane potential

Oxygen–glucose deprivation (OGD) as a model of ischemic cell death

Mitochondrial membrane potential during OGD and reoxygenation

ATP content in the OGD model

Neuronal validity

Apoptosis

Pathological hypothesis of hyperpolarization of MMP

Conclusions

Acknowledgement

Neurochem. Int.

Neurochem. Int.

Respir. Physiol.

Brain Res.

Neurochem. Int.

J. Biol. Chem.

Neuroscience

Trends Immunol.

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in culture astrocytes and neurons

J. Neurochem.

Penumbral tissue alkalosis in focal cerebral ischemia: relationship to energy metabolism, blood flow, and steady potential

Ann. Neurol.

Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical call cultures

Proc. Natl. Acad. Sci. U.S.A.

Spontaneous changes in mitochondrial membrane potential in cultured neurons

J. Neurosci.

Exacerbated responses to oxidative stress by an Na+ load in isolated nerve terminals: the role of ATP depletion and rise of Ca2+I

J. Neurosci.

Mitochondrial function as a determinant of recovery or death in cell response to injury

Mol. Cell. Biochem.

Trimetazidine counteracts the hepatic injury associated with ischemia-reperfusion by preserving mitochondiral function

J. Pharmacol. Exp. Ther.

Phosphorylase alpha and labile metabolites during anoxia: correlation to membrane fluxes of K+ and Ca2+

J. Neurochem.

Mitochondrial dysfunction during anoxia/reoxygenation injury of liver sinusoidal endothelial cells

Hepatology

Mitochondrial hyperpolarization and ATP depletion in patients with systemic lupus erythematosus

Arth. Rhemat.

The pathophysiology of mitochondrial cell death

Science

Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning

Circ. Res.

Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function

Am. J. Physiol.

Trifluoperazine and dibucaine-induced inhibition of glutamate-induced mitochondrial depolarization in rat cultured forebrain neurons

Br. J. Pharmacol.

Review article
Mitochondrial membrane potential and ischemic neuronal death

Exacerbated responses to oxidative stress by an Na⁺ load in isolated nerve terminals: the role of ATP depletion and rise of Ca²⁺I

Phosphorylase alpha and labile metabolites during anoxia: correlation to membrane fluxes of K⁺ and Ca²⁺

Sarcolemmal versus mitochondrial ATP-sensitive K⁺ channels and myocardial preconditioning

Mitochondrial ATP-sensitive K⁺ channels modulate cardiac mitochondrial function