Spontaneous Changes in Mitochondrial Membrane Potential in Cultured Neurons

Abstract

Using the mitochondrial membrane potential (⌬⌿ m )-sensitive fluorescent dyes 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolocarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM), we have observed spontaneous changes in the ⌬⌿ m of cultured forebrain neurons. These fluctuations in ⌬⌿ m appear to represent partial, transient depolarizations of individual mitochondria. The frequency of these ⌬⌿ m fluctuations can be significantly lowered by exposure to a photo-induced oxidant burden, an ATP synthase inhibitor, or a glutamate-induced sodium load, without changing overall JC-1 fluorescence intensity. These spontaneous fluctuations in JC-1 signal were not inhibited by altering plasma membrane activity with tetrodotoxin or MK-801 or by blocking the mitochondrial permeability transition pore (PTP) with cyclosporin A. Neurons loaded with TMRM showed similar, low-amplitude, spontaneous fluctuations in ⌬⌿ m . We hypothesize that these ⌬⌿ m fluctuations are dependent on the proper functioning of the mitochondria and reflect mitochondria alternating between the active and inactive states of oxidative phosphorylation. Mitochondria have been implicated in excitotoxic injury pathways, as well as injury mechanisms manifested as apoptotic or necrotic death processes. The mitochondrial membrane potential (⌬⌿ m ) has often been used as a marker for mitochondrial activity and neuronal viability during the various cell death cascades (for review, see Kroemer et al., 1998;Nicholls and Ward, 2000). Injurious stimuli, leading to either excitotoxicity or apoptosis, can lead to profound depolarization of ⌬⌿ m resulting from abnormalities in neuronal processes, including alterations in intracellular calcium dynamics and the opening of the mitochondrial permeability transition pore (PTP) (Ankarcrona et al., 1995;Nieminen et al., 1996;Schinder et al., 1996;White and Reynolds, 1996;Vergun et al., 1999;Budd et al., 2000). Although a loss of ⌬⌿ m may be linked to various inducers of cell death, these are observed as large and possibly catastrophic changes in mitochondrial function.Mitochondria under physiological conditions also play active roles in the maintenance of normal cellular functioning. A key feature of mitochondria that allows them to participate in cell survival is proton pumping across the impermeable inner membrane. This generates an electrochemical gradient, composed of ⌬⌿ m and ⌬pH, which is used for ATP synthesis, ADP-ATP exchange, uptake of respiratory substrates and inorganic phosphate, transport of K ϩ , Na ϩ , and anions to regulate volume, and regulation of protons to control heat production (for review, see Bernardi, 1999). Mitochondria also play protective roles by buffering cells against high concentrations of calcium (Budd and Nicholls, 1996;White and Reynolds, 1997;Stout et al., 1998) and sequestering proapoptotic agents, such as cytochrome c (for review, see Green and Reed, 1998;Desagher and Martinou, 2000). Compared with the catastrophic changes in acute injury states, healthy mitochondria...

Widespread depolarization during expiration: A source of respiratory drive?

“…We propose that decreases in oxygen, at the levels measured during expiration, may lead to slight increases in the degree of membrane depolarizations. Previous studies discussed, found that transient mitochondrial membrane hyperpolarizations and depolarizations may indicate active and inactive states of oxidative phosphorylation [63], consistent with our proposed hypothesis, though they did not directly link these processes with oxygen intake. We propose that these mitochondrial membrane potential changes extend to the membrane of the cell, leading to slight hyperpolarizations and depolarizations of the cell membrane.…”

“…Other studies have also shown mitochondrial membrane potential changes in isolated mitochondria [64,65], and in mitochondria within different cell types, such as cardiomyocytes [66], vascular endothelium [65], and pancreatic B-cells [67]. Buckman and Reynolds found that oligomycin, which inhibits ATP-synthase, decreased the number of fluctuations, suggesting that the fluctuations are related to oxidative phosphorylation [63]. The researchers also performed several experiments to eliminate other explanations for these depolarizations and repolarizations.…”

“…The changing levels of oxygen and C0 2 in inhaled and exhaled air suggest that cellular respiration may slow during exhalation due to the decreased presence of oxygen. One study found spontaneous mitochondrial membrane potential changes in cultured neurons [63]. The researchers proposed that these partial, transient depolarizations may reflect the mitochondria alternating between active and inactive oxidative phosphorylation [63].…”

“…One study found spontaneous mitochondrial membrane potential changes in cultured neurons [63]. The researchers proposed that these partial, transient depolarizations may reflect the mitochondria alternating between active and inactive oxidative phosphorylation [63]. Other studies have also shown mitochondrial membrane potential changes in isolated mitochondria [64,65], and in mitochondria within different cell types, such as cardiomyocytes [66], vascular endothelium [65], and pancreatic B-cells [67].…”

Amyloid precursor protein and amyloid β-peptide bind to ATP synthase and regulate its activity at the surface of neural cells

“…63,64 Thus APP and Ab in mitochondria could inhibit the ATP synthase subunit a within the F 1 F 0 -ATP synthase complex of the electron transport chain, resulting in ATP depletion. It is noteworthy in this respect that mitochondria in cultured neurons display spontaneous changes in mitochondrial membrane potential, 65 which are impaired in mitochondria from Alzheimer's disease patients. 66 It is generally believed that one of the first signs of Alzheimer's disease pathology is a disruption of neural circuits involved in memory formation.…”