Review articleMitochondrial membrane potential and ischemic neuronal death
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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