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.2001 Sep 1;21(17):6608-16.
doi: 10.1523/JNEUROSCI.21-17-06608.2001.

The mitochondrial permeability transition pore and nitric oxide synthase mediate early mitochondrial depolarization in astrocytes during oxygen-glucose deprivation

Affiliations

The mitochondrial permeability transition pore and nitric oxide synthase mediate early mitochondrial depolarization in astrocytes during oxygen-glucose deprivation

S A Reichert et al. J Neurosci..

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 (psi(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 psi(m) under similar conditions. To address this, we exposed cortical astrocytes cultured alone or with neurons to oxygen-glucose deprivation (OGD) and monitored psi(m) using tetramethylrhodamine ethyl ester. Both neurons and astrocytes exhibited profound loss of psi(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 psi(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 psi(m) after reintroduction of O(2) and glucose occurred over a surprisingly long period (>1 hr), suggesting that OGD caused specific, reversible changes in astrocyte mitochondrial physiology beyond the simple lack of O(2) and glucose. Decreased psi(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.

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Figures

Fig. 1.
Fig. 1.
Mitochondrial depolarization results in a graded decline in TMRE fluorescence in astrocytes. Astrocytes were loaded with TMRE (100 nm) and exposed to the mitochondrial depolarizing agents rotenone (complex I inhibitor), rotenone plus oligomycin (F0F1-ATPase inhibitor), and FCCP (mitochondrial protonophore). TMRE fluorescence, reflecting mitochondrial membrane potential, was visualized by confocal microscopy. TMRE fluorescence decreased after rapid depolarization by FCCP or gradual depolarization by rotenone or rotenone plus oligomycin.Panels are pseudocolor representations of TMRE fluorescence intensity (scale onright). Scale bar, 20 μm.Olig, Oligomycin;Rot, rotenone.
Fig. 2.
Fig. 2.
Mitochondrial membrane potential in cortical astrocytes exposed to OGD and subsequent reintroduction of oxygen and glucose. Mixed cortical cultures were exposed to OGD in the presence of the potentiometric dye TMRE.A, C, Mitochondrial ψ, as TMRE fluorescence, was evaluated in the astrocyte monolayer by confocal microscopy after 40, 50, or 60 min of OGD (A) or during recovery after reintroduction of oxygen and glucose after 60 min of OGD (C). Control cultures were maintained in 21% oxygen and 5.5 mm glucose for 60 min.B, D, The time course of mitochondrial TMRE fluorescence loss in astrocytes during OGD (B) and the recovery of mitochondrial ψ after readdition of oxygen and glucose (D) are shown.Dashed line inB indicates interpolation of TMRE fluorescence changes between 0 and 40 min. Values are mean TMRE fluorescence intensity (as % of control) ± SEM;n > 100 for all conditions from 3 or more independent experiments. *p< 0.05 by ANOVA and Tukey'spost hoc analysis.E, Astrocytes were exposed to OGD (60 min) or OGD followed by reintroduction of oxygen plus glucose for an additional 60 or 120 min. Alterations in mitochondrial morphology, visualized by TMRE fluorescence, were apparent after OGD, persisted after reintroduction of O2 plus glucose for 60 min, but had resolved by 120 min after addition of O2 plus glucose. Scale bar:A, C, 20 μm.CTRL, Control;Rep, reperfusion.
Fig. 3.
Fig. 3.
Plasma membrane potential in astrocytes exposed to low K+ (1 mEq K), high K+ (30 mEq K), control, and OGD conditions (90–120 min) in the presence of DiBAC4(3) (1 μm), a plasma membrane potentiometric dye. Control cultures were maintained in 21% oxygen and 5.5 mm glucose for 90–120 min.A, DiBAC4(3) fluorescence was visualized by confocal microscopy (excitation λ = 488 nm, emission λ = 515 nm) at the end of OGD.B, There was no change in plasma membrane potential in astrocytes exposed to OGD compared with the control condition.
Fig. 4.
Fig. 4.
Determination of astrocyte and neuronal death after OGD in cortical cultures. Mixed cortical cultures were exposed to OGD or control conditions for 60 min and then returned to normoxic, glucose-containing solution for 24 hr (data not shown) or 48 hr. The number of dead neurons and astrocytes was determined by staining with propidium iodide, followed by confocal microscopy to allow stained cells in the astrocyte layer (left) to be differentiated from the neurons above (right). PI-positive astrocytes per well in a 24-well culture plate were 35 ± 12 astrocytes in OGD-exposed cultures and 33 ± 15 astrocytes in control cultures (from 4 to 8 wells from 3 independent replications), both of which exhibit <0.001% astrocyte death. Most neurons were dead 24 hr after OGD. Scale bar, 50 μm.
Fig. 5.
Fig. 5.
Comparison of OGD-induced mitochondrial depolarization in astrocytes cultured alone or cocultured with neurons.A, TMRE mitochondrial fluorescence was evaluated after 60 min of OGD in astrocytes cocultured with neurons (right) or after 60 and 90 min of OGD in astrocytes grown alone (left). The optical sectioning ability of the confocal allowed astrocyte mitochondrial fluorescence to be differentiated from that of the overlying neurons in mixed cultures. Astrocytes cultured alone showed delayed loss of ψm compared with astrocytes cultured with neurons. Scale bar, 20 μm.B, Quantitative analyses of mitochondrial TMRE fluorescence in the two culture types are shown. Values are mean TMRE fluorescence intensity (% of control) ± SEM;n > 50 cells from 3 independent experiments. InB, thedashed line represents interpolation of TMRE fluorescence in astrocytes between 0 and 60 min.
Fig. 6.
Fig. 6.
Cytochrome c loss from depolarized astrocyte mitochondria after OGD. Astrocytes were exposed to 90–120 min of OGD.A, After mitochondrial depolarization was observed, mitochondrial and cytosolic fractions were prepared and assayed for cytochrome c concentration. Theleft half of the graph shows cytochrome c concentration (per milligram of protein) in the mitochondrial fraction. Theright half shows cytochrome c concentration in the cytosol. Values are the mean ± SEM;n = 4 independent replications for control and OGD only; 3 additional replications included CsA conditions (*p < 0.05, significantly different from control, and **p < 0.05, significantly different from OGD, by ANOVA and Tukey'spost hoc analysis).B, Apoptosis in astrocyte cultures was initiated by staurosporine at 0.2 μm. Cytochrome c was measured as nanograms per milligram of protein as stated above. Mitochondrial cytochrome c levels are lower in staurosporine-treated cells.
Fig. 7.
Fig. 7.
Activity of caspase-3 and caspase-9 in astrocytes exposed to OGD. Astrocytes were exposed to 90–120 min of OGD until mitochondrial depolarization was confirmed, and then astrocytes were harvested and assayed for caspase-9 (A) or caspase-3 (B) activity (% control) using fluorogenic substrates. No activation of either caspase was observed in astrocytes exposed to OGD. In contrast, caspase-3 and caspase-9 activation was easily observed in astrocytes undergoing apoptosis initiated by staurosporine at 0.2 μm. Values are the mean ± SEM. Error bars for astrocytes exposed to staurosporine and Boc-Asp(oMe)-CH2F (BAF) inA andB are not visible.Stauro, staurosporine.
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