The Mitochondrial Permeability Transition Pore and Nitric Oxide Synthase Mediate Early Mitochondrial Depolarization in Astrocytes during Oxygen–Glucose Deprivation

Abstract

Recent studies suggest that the degree of mitochondrial dysfunction in cerebral ischemia may be an important determinant of the final extent of tissue injury. Although loss of mitochondrial membrane potential ( m ), one index of mitochondrial dysfunction, has been documented in neurons exposed to ischemic conditions, it is not yet known whether astrocytes, which are relatively resistant to ischemic injury, experience changes in m under similar conditions. To address this, we exposed cortical astrocytes cultured alone or with neurons to oxygen-glucose deprivation (OGD) and monitored m using tetramethylrhodamine ethyl ester. Both neurons and astrocytes exhibited profound loss of m after 45-60 min of OGD. However, although this exposure is lethal to nearly all neurons, it is hours less than that needed to kill astrocytes. Astrocyte m was rescued during OGD by cyclosporin A, a permeability transition pore blocker, and G N-nitro-arginine, a nitric oxide synthase inhibitor. Loss of mitochondrial membrane potential in astrocytes was not accompanied by depolarization of the plasma membrane. Recovery of astrocyte m after reintroduction of O 2 and glucose occurred over a surprisingly long period (Ͼ1 hr), suggesting that OGD caused specific, reversible changes in astrocyte mitochondrial physiology beyond the simple lack of O 2 and glucose. Decreased m was associated with a cyclosporin A-sensitive loss of cytochrome c but not with activation of caspase-3 or caspase-9. Our data suggest that astrocyte mitochondrial depolarization could be a previously unrecognized event early in ischemia and that strategies that target the mitochondrial component of ischemic injury may benefit astrocytes as well as neurons. Key words: tetramethylrhodamine ethyl ester; mitochondrial permeability transition pore; nitric oxide synthase; cyclosporin A; confocal microscopy; cortical cell culturesMitochondrial dysfunction is an early feature in nervous system ischemia. Functional studies on mitochondria isolated from ischemic brain (Sims et al., 1986;Sims, 1991) and metabolic imaging studies of brain during ischemia (Watanabe et al., 1994;Shiino et al., 1998;McCleary et al., 1999;Shadid et al., 1999) indicate that brief periods of ischemia result in transient mitochondrial respiratory defects that normalize rapidly after reperfusion (Schutz et al., 1973;Hillered et al., 1984;Sims et al., 1986). Longer periods of ischemia, however, result in a secondary, irreversible decline in mitochondrial function that occurs minutes to hours later (for review, see Siesjo et al., 1999). Although mitochondrial failure is associated with loss of mitochondrial membrane potential ( m ) in many injury conditions (Green and Reed, 1998), it is not yet known whether impaired mitochondrial respiration during cerebral ischemia is accompanied by loss of m , because of the lack of sensitive and specific probes for m in the intact brain. Suggestive evidence comes from reports by Fujimura et al. (1998), Andreyev et al. (1998), and Perez-Pinzon et al. (1999, who observed releas...

Inhibition of mitochondrial function in astrocytes: implications for neuroprotection

“…In our study the astrocytes were able to maintain mitochondrial membrane potential during FC inhibition for 2 h by ATPase reversal in pure cultures but not in the presence of co-cultured neurons (Fig 2 and 6). This is in agreement with previous studies that report the failure of astrocytes to use the compensatory mechanism of ATPase reversal in mixed neuron/astrocyte co-cultures (Reichert et al 2001).…”

“…The regulatory mechanisms that control preservation of either mitochondrial or plasma membrane potential under the conditions of energy deprivation remain to be investigated. Of note, failure of astrocytes to maintain mitochondrial membrane potential by F 1 F 0 -ATPase reversal when co-cultured with neurons, without changes in astrocytic plasma membrane potential, was also reported by another group in a previous study (Reichert et al 2001). …”

“…The ability of astrocytes to recover from a profound loss of mitochondrial potential without loss of viability, is in sharp contrast with commonly observed astrocyte behavior (Fig. 9) (Dienel and Hertz 2005), though it has been observed previously (Reichert et al 2001). Studies of this phenomenon are still very limited.…”

“…Because glucose depletion is characteristic of ischemia, glucose-free medium might also have physiological relevance to ischemic conditions. A growing body of studies indicates the reciprocal nature of neuron-astrocyte interactions (Magistretti et al 1993;Reichert et al 2001;Chen and Swanson 2003), but the mechanisms by which neurons might influence astrocyte response and survival remain practically unknown. In this study we observed that the addition of neurons to astrocyte cultures influenced the rates of astrocytic mitochondrial depolarization, and identified one mechanism that can be partially responsible for this phenomenon.…”

Manganese Induces the Mitochondrial Permeability Transition in Cultured Astrocytes

“…Employing intact osteosarcoma cells, Minamikawa et al (46) show a complete reversibility of the MPT without any evidence of cell death. Similarly, it has been reported that astrocytes subjected to oxygen-glucose deprivation showed induction of the MPT without evidence of cell death (47). In this regard, it is noteworthy that our preliminary studies indicate that induction of the MPT in astrocytes when exposed to 100 M manganese acetate for 6 h was completely reversed 48 h after removal of the manganese in the culture medium.…”