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Mitochondrial oxidative stress and cell death in astrocytes — requirement for stored Ca²⁺ and sustained opening of the permeability transition pore

Department of Physiology, University College London, London, WC1E 6BT, UK

View larger version (41K): [in a new window]   Fig. 3. Differentiation of confocal images revealed dye egress from depolarising mitochondria. (A) High magnification images (x63 oil-immersion objective), revealed depolarisations of individual mitochondria (arrows). (B) Differentiated images reveal only those pixels in which the signal changed between successive image frames. `Clouds' of fluorescence can be seen surrounding individual mitochondria as the TMRE moves into the cytosol. Plots of the signal with time from 3A are shown in C. Arrows indicate the depolarisations illustrated in the preceding images. Bar, 5 µm.

View larger version (39K): [in a new window]   Fig. 5. Stored calcium was necessary for the mitochondrial depolarisations. (A) Astrocytes were exposed to a combination of 10 µM BAPTA-AM and 200 nM thapsigargin in order to empty intracellular Ca2+ stores and to chelate released Ca2+. The efficacy of this manipulation was demonstrated by application of 100 µM ATP, which mediates Ins(1,4,5)P3-mediated Ca2+ release (left), failed to raise [Ca2+]c in the treated cells (right). (B) The relative occurrence of mitochondrial depolarisations was assayed by calculation of `integrated activity'; digital images of each cell were differentiated, revealing how the signal changed with time. The peak signals in the differentiated series were then plotted against time and integrated in order to measure the number or amplitude of depolarisations (see Image processing and statistical analysis). The peak fluorescence with time over representative single cells is shown. Emptying and chelation of ER Ca2+ stores significantly reduced integrated activity: 14.6±5.4 (n=117 cells) versus 10.7±2.4 (n=68) (mean±s.d., P<0.0001, Mann-Whitney U test), confirming that stored Ca2+ was necessary for the mitochondrial depolarisations. Line images extracted from TMRE-loaded cells are shown in C. Treatment of cells with thapsigargin and BAPTA-AM effectively emptied ER Ca2+ stores. Cells were then loaded with TMRE and imaged as before. Multiple transient mitochondrial depolarisations were revealed along a line drawn through a single control cell, but very few were seen in cells treated with thapsigargin and BAPTA.

View larger version (51K): [in a new window]   Fig. 1. TMRE localises to mitochondria in response to the m, where the fluorescence is quenched. (A) TMRE signal co-localises with mitochondrial NADH autofluorescence. Confocal imaging of TMRE loaded astrocytes shows the co-localisation of TMRE staining (top, orange emission) and NADH autofluorescence (bottom, blue emission). Bar, 10 µm. A small section from an image is shown at higher magnification in B, emphasising the colocalisation of the two signals. Bar, 1 µm. (C) A rise in TMRE signal reflects a loss of m. Dissipation of m with the protonophore, FCCP (1 µM) induced a rapid rise in TMRE fluorescence (i,ii), as TMRE fluorescence is quenched by concentration of the probe into mitochondria. When the image sequence was divided by the first image, the values of each pixel were normalised to the `resting' level. The resultant images (iii,iv) reveal the proportional change in each pixel value since the first image. After application of FCCP, the mitochondria appear dark against a bright background of cytosol, consistent with fluorescence dequench as the dye moves from mitochondria to cytosol. The TMRE signal measured over the cytosol of an astrocytes increased both (D) in response to the uncoupler FCCP, and (E) following inhibition of mitochondrial respiration by rotenone (2 µM, in the presence 2.5 µg ml-1 oligomycin).

View larger version (20K): [in a new window]   Fig. 2. Imaging of TMRE-loaded astrocytes revealed transient fluctuations in m. (A-D) Imaging of TMRE-loaded cells revealed brief, spontaneous and reversible depolarisations of individual mitochondria. The fluorescence signal over individual mitochondria is plotted as a function of time. Note that the signal over individual organelles frequently returned to baseline, suggesting complete mitochondrial repolarisation following a depolarisation (A) and single organelles could depolarise and repolarise repeatedly (B,C), further suggesting reversible depolarisations of m. The traces in C and D were obtained from two mitochondria in a single cell, demonstrating that the flickers were independent in time and did not reflect a global loss of m. The histogram in E, shows the distribution of the relative amplitudes of individual transient changes in signal. FCCP was applied at the end of each imaging sequence, and the amplitudes are expressed as a percentage of the response to FCCP. In some cells the global fluorescence was essentially stable, so that individual depolarisations were easily discernible (F), and were much smaller in amplitude than the response to FCCP. In other cells, the flickers rapidly summated to produce a global rise in whole-cell fluorescence that overwhelmed the signal produced by individual mitochondria (G), in which case application of FCCP induced no further change in signal, suggesting that mitochondrial depolarisation was complete.

View larger version (17K): [in a new window]   Fig. 4. Attenuation of illumination intensity increased the time to global depolarisation. (A,B) The rate of mitochondrial depolarisation was systematically slowed by attenuation of the illumination intensity by neutral density filters suggesting that ROS formation by fluorophore illumination was sensitising mPTP formation (P<0001, Kruskal-Wallis non-parametric ANOVA test, n=46, 27 and 38 cells imaged using 1%, 3.5% and 5% transmission filters, respectively).

View larger version (34K): [in a new window]   Fig. 6. The depolarisations reflected transient openings of the mitochondrial permeability transition pore. (A) Treatment of cells with N-methyl 4-valine cyclosporin (400 nM) reduced the incidence of transient depolarisations. Line images illustrate that mitochondrial flickering was almost completely prevented in cells treated with meth-v-Cs, which reduced the integrated activity from control values of 12.0±2.6 (n=122) to 9.8±2.5 (n=82) (mean±s.d., P<0.0001, two-tailed, unpaired t test), suggesting that the transient depolarisations were transient openings of the mPTP. (B) 10 µM Trifluoperazine, another inhibitor of mPTP formation, slowed the rate of global mitochondrial depolarisation, reducing the slope from a control of 0.041±0.018 (n=27 cells) to 0.008±0.006 (n=60 cells; P<0.0001).

View larger version (38K): [in a new window]   Fig. 7. Mitochondrial ROS are required for pore opening. (A) Treatment of cells with an array of antioxidants (1mM ascorbic acid, 250 units/ml catalase, 1mM Trolox and 500 µM of the spin trap TEMPO) reduced the incidence of mitochondrial depolarisations, reducing the integrated activity from a control of 10.0±5.6 (n=53 cells) to 6.4±1.9 (n=57) (P<0.001, Mann-Whitney U test). (B) The antioxidants also significantly increased the time to global mitochondrial depolarisation (P<0.0001, Mann-Whitney U test).

View larger version (61K): [in a new window]   Fig. 8. Illumination of rhod-2 loaded astrocytes caused mitochondrial Ca2+-loading. (A) Confocal images of an astrocyte loaded with the Ca2+-indicator rhod-2. During illumination, the rhod-2 signal over the whole cell rose, including that over mitochondria. Excitation of the dye was elicited using the 543 nm line of a HeNe laser set to 4% of total output. The images were taken using a x63 oil immersion lens; bar, 5 µm. The time course of rhod-2 signal change over a single mitochondrion is shown in B, which illustrates the signal measured over the mitochondrion marked by an arrow in A. Addition of antioxidants significantly reduced the rate of rise of rhod-2 signal (C), suggesting a role for ROS in the mitochondrial Ca2+-loading.

View larger version (24K): [in a new window]   Fig. 9. Transient openings of the mPTP were innocuous: prolonged openings caused necrotic cell death. TMRE loaded cells were imaged using either a 1% neutral density filter, sufficient to image reversible mPTP openings, or a 30% filter, to induce global mitochondrial depolarisation. After a period of imaging, cells were returned to the incubator for 4 hours. They were then exposed to propidium iodide (PI) and Hoechst 33342 (A) in order to identify necrotic or apoptotic nuclei. PI stains damaged or necrotic cells (red arrow). Hoechst stains all nuclei (blue arrow), but apoptotic nuclei display a characteristic bright, condensed staining (not shown). Bar, 10 µm. Transient openings of the mPTP caused no increase in cell death, either by apoptosis or necrosis (B); however, global and complete mitochondrial depolarisation caused a significant increase in necrotic staining (P=0.0002). No sign of apoptotic morphology or Hoechst staining was apparent.