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, 20 (2), 353-65

Loss of OPA1 Disturbs Cellular Calcium Homeostasis and Sensitizes for Excitotoxicity

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Loss of OPA1 Disturbs Cellular Calcium Homeostasis and Sensitizes for Excitotoxicity

Y E Kushnareva et al. Cell Death Differ.

Abstract

Optic atrophy 1 (OPA1) mutations cause dominant optic atrophy (DOA) with retinal ganglion cell (RGC) and optic nerve degeneration. The mechanism for the selective degeneration of RGCs in DOA remains elusive. To address the mechanism, we reduced OPA1 protein expression in cell lines and RGCs by RNA interference. OPA1 loss results in mitochondrial fragmentation, deficiency in oxidative phosphorylation, decreased ATP levels, decreased mitochondrial Ca(2+) retention capacity, reduced mtDNA copy numbers, and sensitization to apoptotic insults. We demonstrate profound cristae depletion and loss of crista junctions in OPA1 knockdown cells, whereas the remaining crista junctions preserve their normal size. OPA1-depleted cells exhibit decreased agonist-evoked mitochondrial Ca(2+) transients and corresponding reduction of NAD(+) to NADH, but the impairment in NADH oxidation leads to an overall more reduced mitochondrial NADH pool. Although in our model OPA1 loss in RGCs has no apparent impact on mitochondrial morphology, it decreases buffering of cytosolic Ca(2+) and sensitizes RGCs to excitotoxic injury. Exposure to glutamate triggers delayed calcium deregulation (DCD), often in a reversible manner, indicating partial resistance of RGCs to this injury. However, when OPA1 is depleted, DCD becomes irreversible. Thus, our data show that whereas OPA1 is required for mitochondrial fusion, maintenance of crista morphology and oxidative phosphorylation, loss of OPA1 also results in defective Ca(2+) homeostasis.

Figures

Figure 1
Figure 1
OPA1 loss results in mitochondrial fission without loss in cell viability. (a) Mitochondrial structure in scrambled (control) and OPA1 siRNA-transfected HeLa cells at 3 days after transfection. Mitochondria and nuclei were stained with MitoTracker Red and Hoechst 33342 (blue), respectively. Scale bar, 20 μm. (b) Immunoblot of cytosolic (C) and mitochondrial fraction (M) of siRNA-transfected HeLa cells probed with anti-cytochrome c antibodies. (c and d) Immunocytochemistry and confocal microscopy of fixed control (c) and OPA1 siRNA-transfected (d) HeLa cells labeled with anti-cytochrome c antibodies (green) and MitoTracker Red (red). Hoechst 33342 counterstaining (blue) marks nuclei. (e) Cell viability of scrambled (gray bars) and OPA1 siRNA-transfected (black bars) HeLa cells measured using Calcein-AM 3 days after transfection. (f) DEVDase activity and (g) Annexin-V labeling of scrambled (gray bars) and OPA1 siRNA-transfected (black bars) HeLa cells treated with 1 μM STS for 3–5 h. The rate of zDEVD-AMC cleavage is expressed in arbitrary fluorescence units per minute (FU/min). For (e–g), data are means±S.E.M. of three experiments (* and ** denote P<0.05 and P<0.01 respectively). The color reproduction of this figure is available at the Cell Death and Differentiation journal online
Figure 2
Figure 2
OPA1 loss leads to mitochondrial fission and cristae abnormalities. (A) Two 2-nm thick slices through EM tomographic volumes of mitochondria in fixed scrambled (control) and OPA1 siRNA-transfected HeLa cells in situ. Scale bar, 500 nm. White rectangle marks mitochondria chosen for volume segmentation and surface rendering. (B) Surface-rendered volumes of a segmented control mitochondrion and three segmented OPA1 siRNA mitochondria found in close proximity. The OMM is in blue and cristae are in various colors. (a) Top view of control mitochondrion after segmentation of the outer and cristae membranes into separate objects. (b) Side view of the OMM showing no ruptures. (c and d) Top view of a mitochondrial triplet in OPA1 siRNA-transfected HeLa cells. (e and f) View of same triplet from a side orientation. (d and f) Mitochondria are shown with translucent representation of the OMM to visualize the cristae. Scale bar, 200 nm. (C) Structural analysis of mitochondria by EM tomography. For mitochondrial number and volume, n (number of measured mitochondria)=41 (scrambled siRNA, light gray bars) and 60 (OPA1 siRNA, black bars) (**P<0.01). For cristae surface area (per outer membrane surface area) measurements, n=12 (scrambled siRNA) and 23 (OPA1 siRNA) were used; the same samples were used to determine total cristae surface in the reconstituted volume per cell volume. Data are means±S.E.M. In (d), all of the cristae in the reconstructed volume were summed. The color reproduction of this figure is available at the Cell Death and Differentiation journal online
Figure 3
Figure 3
OPA1 loss leads to mitochondrial structural heterogeneity and depletion of crista junctions, but not their dilation. (A) Electron micrograph showing a field inside a control HeLa cell densely populated with mitochondria having uniform structural features. Scale bar, 500 nm. (B) Electron micrograph showing a field inside an OPA1 siRNA cell densely populated with mitochondria displaying structural heterogeneity in the form of greater matrix condensation in a number of organelles. Same scale as (a). (C) Typical mitochondrial structure in control cells showing well-formed and abundant cristae with light matrix, enlarged from the boxed region in (A). (D) An example of the structure of the majority of mitochondria in OPA1 siRNA cells showing well-formed yet less abundant cristae than in the control, but still having a light matrix, enlarged from the upper boxed region in (C). (E) An example of the structure of the minority of mitochondria in OPA1 siRNA cells again showing less abundant cristae, but having a dark (condensed) matrix, enlarged from the lower boxed region in (B). (F) Inner mitochondrial membrane of the segmented tomographic volume from the scrambled siRNA sample and (G) from the OPA1 siRNA sample. By not displaying the outer membrane, the crista junction openings are easily seen (numbered). In all, 19 crista junctions are in view for the control volume, yet only 3 such junctions are in view for the OPA1 siRNA volume. Scale bar, 50 nm. (H) Crista junction measurements from the volumes. The junctional opening in OPA1 siRNA mitochondria remains roughly circular with a diameter essentially the same as the control junction (a). In all, 52 crista junctions were measured for control (gray bar) and 47 crista junctions for OPA1 siRNA (black bar) mitochondria. For 11 control mitochondria (gray bar) and 26 OPA1 siRNA mitochondria (black bar), the number of crista junctions per mitochondrion was measured and then normalized to each inner boundary membrane surface area (measured from the segmented volume) to determine the density of junctions (b). The crista junction density for the OPA1 siRNA mitochondria was less than half for the control mitochondria; P<0.001
Figure 4
Figure 4
OPA1 loss leads to mitochondrial respiration inhibition, ATP drop, and decrease in mtDNA copy number, and compromises mitochondrial Ca2+ retention capacity. (A) Mitochondrial respiration was measured in digitonin-permeabilized HeLa cells. Arrows indicate additions of cells (8 × 106), 200 μM ADP, 1.25 μg/ml oligomycin, and 100 nM FCCP. Oxygen consumption rates are expressed in nmol of O2 per min per 107 cells. (B) Complex I–III–IV enzyme activity is expressed as mean±S.E.M. of four independent experiments. *Significance at P<0.01 by Student's t-test. (C) ATP levels in scrambled and OPA1 siRNA-transfected HeLa cells expressed as arbitrary luminescence units (AU). Data were collected from replicate wells (n=6) and plotted as means±S.E.M.; *Significance at P<0.01 by Student's t-test. Data shown are representative of eight experiments. (D) MtDNA to nDNA ratio of scrambled and OPA1 siRNA-transfected cells (n=6) measured by Q-PCR and plotted as mean±S.E.M. *Significance at P<0.01 by Mann–Whitney test. (E) Mitochondrial Ca2+ retention capacity was determined in digitonin-permeabilized HeLa cells. Sequential Ca2+ additions cause release of accumulated Ca2+ from mitochondria (a), Δψm decline (b), and mitochondrial swelling (as indicated by a decrease in A660) (c), all attributable to MPT induction. Alamethicin (Alm) was added to induce maximal swelling. Arrows indicate timing of additions of 50 μM Ca2+ and 20 μg/ml Alm. Data are representative of four experiments. (F) Mitochondrial Ca2+ retention capacity in scrambled and OPA1 siRNA cells. Data are means±S.E.M. of four independent experiments. *Significance at P<0.05 by Student's t-test
Figure 5
Figure 5
Effect of OPA1 knockdown on histamine-induced mitochondrial and cytoplasmic [Ca2+] transients. Measurements of mitochondrial (A) and cytoplasmic (B) [Ca2+]. siRNA-transfected HeLa cells were loaded with 2 μM Rhod-2-AM and stimulated by repeated application and washout of 100 μM histamine for 20 s, as indicated. (a) Scrambled siRNA and OPA1 siRNA-cells are represented by gray and black traces, respectively. Data points are means±S.E.M. of individual cells; n=37 and n=41 in 6 and 5 separate experiments for scrambled and OPA1 siRNA, respectively. (b) Relative peak amplitude of the initial [Ca2+] transient and the average of the relative peak amplitudes of all following transients. Bars show means±S.E.M of cells corresponding to (a). ***, **, and * significance at P<0.001, P<0.01, and P<0.05, respectively, by Student's t-test. (c) Decay slopes of sequential [Ca2+] transients (from left to right) approximated by linear regression of the trace immediately following the peak; in a 60-s span for mitochondria (Ac) and in a 20-s span for cytosol (Bc). Bars show means±S.E.M. of cells corresponding to (a). **Significance at P<0.01 by two-way ANOVA comparing OPA1 (black) with scrambled (gray) siRNA categories
Figure 6
Figure 6
OPA1 knockdown leads to more reduced mitochondrial pyridine nucleotide pool. (A) Histamine-stimulated autofluorescence (a) and baseline NAD(P)H autofluorescence (b). Scrambled siRNA and OPA1 siRNA-treated HeLa cells are represented by gray and black traces, respectively. Data points show means±S.E.M. of n=44 and n=49 cells in 3 separate experiments for scrambled and OPA1 siRNA, respectively, for baseline traces; n=122 and n=103 in separate experiments for scrambled and OPA1 siRNA, respectively, for histamine-treated cells. To determine the redox status of the mitochondrial NADH/NAD+ pool at the end of each experiment, mitochondrial NADH was fully oxidized to NAD+ with 1 μM FCCP (0% reduction) followed by inhibition of complex I with rotenone (1 μM) plus substrate (β-hydroxy-butyrate; 10 mM) to fully reduce NAD+ to NADH (100% reduction). (B) Bar graph shows the peak amplitude of the initial transient and the average of the relative peak amplitudes of all subsequent transients of NAD(P)H autofluorescence upon histamine stimuli. (C) Bar graph shows average decay half-time of sequential autofluorescence transients approximated by exponential regression of the 60 s span of the trace immediately following the peak. (D) Analysis of mitochondrial redox status after histamine stimuli in experiments shown in (A). Bar graph shows the resulting NADH redox status expressed as percentage. Gray bars, scrambled siRNA; black bars, OPA1 siRNA. (BD) Data are means±S.E.M. of all cells corresponding to (A). *Significant difference between OPA1 and scrambled siRNA transfection at P<0.05 by Student's t-test
Figure 7
Figure 7
Effect of OPA1 knockdown on intracellular [Ca2+] transients in RGCs. RGCs transfected with FAM-conjugated scrambled or OPA1 siRNA were labeled with Fura-FF plus MitoTracker Red and imaged by low-light-level fluorescence microscopy before time-lapse Ca2+ imaging. (a) Mitochondria labeled with MitoTracker Red are visualized in grayscale; the blue color shows the soma marked by Fura-FF fluorescence; and green indicates fluorescence of the siRNA conjugate FAM. Scale bar, 10 μm. Mitochondrial length (Lmito) and the longitudinal fraction of RGC processes occupied by mitochondria (presence) was measured by image segmentation and skeletonization in FAM-positive cells only (n=11 and n=14 view fields for scrambled and OPA1 siRNA). See also Supplementary Figure 3. (b) Cytoplasmic [Ca2+] transients induced by consecutive depolarization of the plasma membrane with 50 mM KCl for 30 s, as indicated. Scrambled siRNA and OPA1 siRNA RGCs are represented by black and red traces, respectively. Data points are means±S.E.M. of individual FAM-positive cells; for scrambled siRNA, n=15 in 4 experiments and for OPA1 siRNA, n=22 in 4 experiments. Ca2+ extrusion rates (ΔCa) were determined by temporal differentiation of the traces in the recovery phase of the K+-evoked [Ca2+]c transients, where [Ca2+]c was ∼0.75 μM in average. ΔCa was significantly different between OPA1 and scrambled siRNA treatments; P<0.01, two-way ANOVA comparing the siRNA treatments as categories. **Significance at P<0.01 by Welch-test; #Significance at P<0.05 by paired t-test. (c) DCD in RGCs. Fura-FF ratio is shown without calibration because of the presence of close to saturation values. Bar below traces indicates timing of perfusion of 100 μM glutamate plus 10 μM glycine with no Mg2+. Each trace corresponds to an individual FAM-positive cell from three independent cultures of OPA1 (n=11) or scrambled siRNA (n=18)-treated RGCs. Of the 18 scrambled siRNA RGCs, 6 showed reversible secondary rise of [Ca2+]i compared with 0 of 11 OPA1 siRNA RGCs, P<0.05 by one-sided Fisher's exact test. The color reproduction of this figure is available at the Cell Death and Differentiation journal online

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