Methionine Sulfoxide Reductase a Protects Neuronal Cells Against Brief Hypoxia Reoxygenation

Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia͞reoxygenation

Olena Yermolaieva*, Rong Xu†, Carrie Schinstock‡, Nathan Brot§, Herbert Weissbach¶, Stefan H. Heinemannʈ, and Toshinori Hoshi†**

*Department of Internal Medicine, ‡Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242; §Hospital for Special Surgery, Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021; ¶Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, FL 33431; ʈMolecular and Cellular Biophysics, Medical Faculty of the Friedrich Schiller University Jena, Drackendorfer Strasse 1, D-07747 Jena, Germany; and †Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

Contributed by Herbert Weissbach, December 16, 2003 Hypoxia͞reoxygenation induces cellular injury by promoting oxi- The importance of ROS in ischemia͞hypoxia-induced cellular dative stress. Reversible oxidation of methionine in proteins in- injury and the postulated antioxidant potential of MSRA suggest volving the enzyme peptide methionine sulfoxide reductase type that overexpression of MSRA may protect cells from hypoxia͞ A (MSRA) is postulated to serve a general antioxidant role. There- reoxygenation-mediated cell injury. We tested this hypothesis by fore, we examined whether overexpression of MSRA protected inducing hypoxia in PC12 cells overexpressing MSRA. cells from hypoxia͞reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells Materials and Methods

and promoted apoptotic cell death. Adenovirus-mediated overex- PC12 Cells. PC12 cells were cultured at 37°C in 10% CO2 without pression of MSRA signiﬁcantly diminished the hypoxia-induced any added nerve growth factor as described (21). The cell culture increase in ROS and facilitated cell survival. Measurements of the medium contained Dulbecco’s modified Eagle’s medium sup- membrane potentials of intact mitochondria in PC12 cells and of plemented with 10% horse serum and 5% FBS. isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demon- Overexpression of MSRA. Enhanced green fluorescent protein ͞ strate that MSRA plays a protective role against hypoxia reoxy- (EGFP), bovine MSRA (bMSRA) (22), and EGFP–bMSRA, genation-induced cell injury and suggest the therapeutic potential where EGFP is fused to the N terminus of bMSRA (23), were of MSRA in ischemic heart and brain disease. overexpressed by using the adenovirus-mediated gene transfer method (24). The gene coding sequences were inserted by PCR eactive oxygen species (ROS) promote oxidative damage to into the shuttle plasmid vector pacAd5CMV with a cytomega- Rmany cellular constituents, including amino acids, lipids, and lovirus promoter (24). The gene coding sequences were verified. nucleic acids, and play critical roles in aging and senescence- The recombinant virus particles were prepared by the University associated disorders (1–3). ROS are also likely mediators of of Iowa Gene Transfer Vector Core. Functional overexpression ͞ acute cellular injury events caused by ischemia hypoxia (4). of MSRA in PC12 cells was confirmed by using an assay for ͞ Reperfusion after an ischemic hypoxic episode dramatically MSRA based on the reduction of N-[3H]acetyl methionine increases the overall cellular oxidant level (4). In addition, the sulfoxide (25). Controls contained the virus lacking an insert. oxidant level may increase at least transiently during ischemia͞ hypoxia before reperfusion (5, 6). To protect against the oxida- Determination of met-O Levels. PC12 cells were treated with 90% tive insults induced by a variety of causes, including ischemia͞ N2:10% CO2 (hypoxia), 90% O2:10% CO2 (hyperoxia), or 90% reperfusion, cells contain multiple ‘‘antioxidant’’ mechanisms (1, air:10% CO2 in a commercially available gas-control chamber 2). For example, superoxide dismutase, catalase, and glutathione (Billups-Rothenberg, Del Mar, CA) for 10 min. The cells were peroxidase scavenge the superoxide anion and H2O2 to prevent allowed to recover in the standard growth condition for 24 h, and ROS-induced damages. Nonenzymatic ROS scavengers, such as then they were harvested. The cells were then suspended in 20 vitamins E and C, also contribute to the total antioxidant mM Tris⅐Cl, pH 7.4, and disrupted by freezing and thawing three capacity (7). times. The cell suspension was centrifuged at 12,000 ϫ g, and the The amino acid methionine, both free and in peptide linkage, supernatant was removed. An aliquot of the supernatant was is readily oxidized by ROS, leading to the formation of the R and incubated with 4 ␮g of Pronase for 16 h at 37°C. The mixture was S epimers of methionine sulfoxide (met-O) (8). Reduction of the heated at 100°C for 1 min and then centrifuged to remove S form of met-O in proteins is catalyzed by the enzyme peptide insoluble material. The supernatant was analyzed for its amino methionine sulfoxide reductase A (MSRA) (9–11), whereas the acid composition by a Beckman 7300 amino acid analyzer. The R form is reduced by methionine sulfoxide reductase B (MSRB) two epimers of met-O were the first amino acids to emerge from (11–15). At least one major variant of human MSRA is prefer- the column and readily resolve from the other amino acids. The entially localized in mitochondria, and its N terminus is impor- percentage of met-O present in the samples was calculated as pmol tant in this subcellular localization (16). ϩ Oxidation of selected methionine residues in some proteins, met-O/ (pmol methionine met-O). The estimated values including Kϩ channels (17) and calmodulin (18), drastically

alters their function, suggesting that methionine oxidation and Abbreviations: met-O, methionine sulfoxide; MSRA, methionine sulfoxide reductase A; MSRA may have a role in cellular signal transduction (19). MSRB, methionine sulfoxide reductase B; bMSRA, bovine MSRA; EGFP, enhanced green Methionine oxidation in other proteins, such as glutamine ﬂuorescent protein; PI, propidium iodide; ROS, reactive oxygen species; DHR, dihydro- synthetase, however, does not cause any noticeable functional rhodamine; JC-1, 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolocarbocyanine; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; FCCP, carbonyl cyanide p- change (20). This observation led to the speculation that a triﬂuoromethoxyphenylhydrazone. reversible oxidation–reduction cycle of methionine involving **To whom correspondence should be addressed at: Department of Physiology, University MSRA may also act as a general antioxidant mechanism, of Pennsylvania, Richards D100, 3700 Hamilton Walk, Philadelphia, PA 19104. E-mail:

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308215100 PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1159–1164 Downloaded by guest on September 30, 2021 would include any free met-O that might be present in the 15 min (10 ␮g͞ml; JC-1, Molecular Probes). The cells were cytoplasm. treated with the saline solution that had been previously bubbled with air, O2,orN2. Fluorescence imaging was performed at room Trypan Blue Cell Viability Assay. The cells in the culture medium temperature as described (27) by using a Nikon Eclipse TE200, (21) were challenged with hypoxia (90% N2:10% CO2), hyper- Lambda DG-4 filter changer with a 175-W xenon lamp (Sutter ϫ oxia (90% O2:10% CO2), or normoxia (90% air:10% CO2) for Instruments, Novato, CA), a 100 super fluorescence objective 5 min. The cell culture medium was removed 24 h later, and 30 (Nikon, Melville, NY), dichroic mirror Quad Set 84000, and a ␮l of trypan blue solution [0.4% in PBS (Sigma)] was applied to S490͞20ϫ filter for the green signal and a S555͞28ϫ filter for the the cells on glass coverslips for 5 min. PBS (1 ml) was then added, red signal (Chroma Technology, Brattleboro, VT). Fluorescence and the number of stained and nonstained cells in four to five was captured with a SPOT RT camera (Diagnostic Instruments, randomly selected fields on each coverslip was counted. Typi- Sterling Heights, MI), and image collection and analysis was cally, 300–500 cells were counted in each group. Ethanol (1%) performed by using METAMORPH software (Universal Imaging, was dissolved with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1- West Chester, PA). The fluorescent emission signals at 590 and oxyl (TEMPOL, Calbiochem), the solution of which was present 535 nm in response to excitation at 555 and 490 nm, respectively, for 1 h before the start of the experimental treatments and then were recorded, and the bandwidths for the two signals were 10 washed out after the treatments. nm and 20 nm. The fluorescent signal intensity at 590 nm (red) was divided by the intensity at 535 nm (green), and this red͞ ROS Measurements. The fluorescent dye dihydrorhodamine green ratio was used to estimate the mitochondrial membrane ⌬⌿ ͞ (DHR) 123 (Calbiochem) was used to estimate the ROS level potential ( m). Larger JC-1 red green ratio values reflect ⌬⌿ (21). The excitation and emission wavelengths were 500 nm and more negative m. To quantify JC-1 data from individual 530 nm, respectively. PC12 cells harvested from a confluent flask mitochondria, the JC-1 red͞green ratio signals were compiled by were resuspended in the standard saline solution (see above), using the intensity linescan function of METAMORPH. Briefly, and DHR123 (10 mM stock in DMSO) was added to achieve a four 2-pixel-wide lines were randomly drawn through each final concentration of 10 or 20 ␮M. To measure the ROS acquired image, and the peak red͞green ratio signal was calcu- production during normoxia͞hyperoxia͞hypoxia, the cells were lated for each mitochondrion intersected by the lines. loaded with DHR123 for 5 min at room temperature, centrifuged, resuspended in a 3-ml cuvette containing the solution that Mitochondria Isolation. Male Sprague–Dawley rats were killed ͞ had been bubbled with air, O2,orN2, and immediately placed in with i.p. injections of pentobarbital (15 mg kg), and decapitated the spectrofluorometer (FP-750, JASCO, Tokyo). Because mul- according to a protocol approved by the Institutional Animal tiple ROS convert DHR123 to the stable fluorescent derivative Care and Use Committee. Mitochondria were isolated from rat DHR (26), the slope of signal at 530 nm during the first 4 min liver as described (28). The isolation medium contained (in mM) was used to estimate the overall ROS production rate. Cells were 225 mannitol, 75 sucrose, 1 EGTA, and 10 KH2PO4 (pH 7.2). perfused with the standard saline solution [(in mM) 140 NaCl͞5 The mitochondria were resuspended in the recording medium ͞ ͞ ͞ ͞ ͞ ͞ ͞ ͞ KCl 1 MgCl2 1.5 CaCl2 5 glucose 10 Hepes, pH 7.4] that had [(in mM) 120 KCl succinate 5 Na pyruvate 10 Mops 0.1 ͞ ͞ Ϸ ͞ been bubbled with 100% N2,O2, or air for at least 30 min to MgCl2 0.5 Mg ATP 0.1 EGTA, pH 7.2], at 10 mg ml protein. induce hypoxia, hyperoxia, or normoxia, respectively. The O2 The mitochondria were kept on ice until immediately before use. pressure level of the solution bubbled with N2 in the recording chamber was Ϸ25 mmHg (1 mmHg ϭ 133 Pa) as measured by JC-1 Signals from Isolated Mitochondria. The isolated mitochondrial ⌬⌿ an oxygen meter (ISO2, WPI, Sarasota, FL). To measure the potential m was measured by using JC-1 (27). Before each ROS level on reoxygenation after hypoxia, the cells were sub- experiment, mitochondria in the recording medium (see above) jected to hypoxia for 5 min, washed with the normoxia solution, were incubated with the dye for 5 min in the dark in a 3-ml and placed in the spectrofluorometer. The measurements pre- recording cuvette at 35°C. The emission signals at 590 and 527 sented here represent the ROS levels during the first 2–6 min nm elicited by excitation at 485 nm were measured with a after reoxygenation. spectrofluorometer (FP-750, JASCO). The ratio of the signal at 590 nm over that at 527 nm (red͞green ratio) was calculated to ⌬⌿ Apoptosis and Necrosis Assays. Fractions of the cells undergoing estimate m. The recording chamber was magnetically stirred, apoptosis and necrosis were determined by FACS with a com- and the measurements were carried out at 35°C. To induce mercially available kit that uses Annexin-V-FLUOS and pro- hypoxia and hyperoxia, the mitochondria were placed in the pidium iodide (PI) (Roche Applied Science). The cells in the above solution that had been bubbled with 100% N2 and 100% culture medium were challenged with hypoxia (90% N2:10% O2, respectively, for at least 30 min. The normoxia solution was CO2), hyperoxia (90% O2:10% CO2), or normoxia (90% air:10% bubbled with air. The pH values of the solutions after bubbling CO2) for 10 min, allowed to recover, and then harvested 24 h were verified. later. Approximately 106 cells in each condition were washed with PBS and centrifuged for 2 min. The cell pellet was resus- Statistical Analysis. All results are reported as the mean Ϯ SEM. pended in 100 ␮l of Annexin-V-FLUOS- and PI-labeling solu- Comparisons between the control and MSRA overexpression tions and incubated for 10–15 min at 15–20°C. The mixture was groups in the normoxic, hyperoxic, and hypoxic conditions were analyzed by a FACScan flow cytometer (University of Pennsyl- made by using ANOVA followed by the least significant differ- vania Cancer Center Flow Cytometry and Cell Sorter Shared ence post hoc test (DataDesk, DataDescription, Ithaca, NY). Resource) with a 488-nm excitation and a 515-nm (30-nm Statistical significance was assumed at P Յ 0.05. bandwidth) filter for Annexin-V-FLUORS and a 585-nm (42-nm bandwidth) filter for PI. Results Cellular responses to hypoxia have been extensively studied by Intact Mitochondria Imaging. PC12 cells were plated on glass using dopamine-containing PC12 cells (29). These cells can be coverslips coated with poly-L-lysine (Sigma) the day before the efficiently infected with adenovirus particles to induce gene experiments. The culture medium was replaced with the stan- expression. After treatment with EGFP-bMSRA adenovirus dard saline solution containing a potential-dependent mitochon- particles, virtually every cell showed EGFP fluorescence (data dria-targeted J-aggregate-forming fluorescent dye, 5,5Ј,6,6Ј- not shown). This near 100% efficiency by using the viral method tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolocarbocyanine for allowed us to use fluorescence measurements in populations of

1160 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308215100 Yermolaieva et al. Downloaded by guest on September 30, 2021 mean DHR123 signal (P Ͻ 0.00001), increasing it by Ϸ100%. In contrast, hyperoxia failed to alter the DHR123 signal (P ϭ 0.82). This observation affirms that hypoxia rapidly increases the overall ROS production before reperfusion in PC12 cells.

MSRA Overexpression Lowers ROS. The reversible oxidation– reduction of methionine͞met-O involving MSRA has been postulated to serve as a ROS sink (20). This idea predicts that overexpression of MSRA to facilitate the reduction process should decrease the overall ROS level. Consistent with this prediction, overexpression of MSRA decreased the ROS level (Fig. 1A). Whether the cells were subjected to normoxia, hyperoxia, or hypoxia, the mean DHR123 signal was significantly smaller in the cells treated with bMSRA virus particles than in those treated with blank control virus particles (P Ͻ 0.00001). Within the cells treated with bMSRA adenovirus particles, hyperoxia increased the mean DHR123 signal significantly when compared with the normoxia-treated cells (P ϭ 0.035), but the increase by hypoxia was not significant (P ϭ 0.15). The mean DHR123 signals measured during hyperoxia and hypoxia in the MSRA virus-treated cells were significantly smaller than in the control cells receiving the same treatments (P ϭ 0.002 and P Ͻ 0.00001, respectively). The results show that overexpression of MSRA is effective in reducing the overall ROS level irrespective Fig. 1. Hypoxia increases ROS production and promotes cell death. (A) of the oxygen concentration. It should be noted that the most Hypoxia enhances ROS production in PC12 cells. The slope of the DHR123 striking effect of MSRA virus treatment was observed during signal in the first 4 min of the treatment was used to estimate the ROS level. The results obtained from the cells treated with blank control adenovirus hypoxia; the DHR123 signal in the MSRA group was only 25% particles, bMSRA adenovirus particles, TEMPOL (5 mM), and bMSRA particles of that in the control group during hypoxia. The membrane- and TEMPOL (5 mM) together are shown. The cells were treated with the virus permeable ROS scavenger TEMPOL also decreased the ROS particles 24 h before the measurements and then subjected to normoxia (open production (P Ͻ 0.00001). The lowest levels of ROS were bar), hyperoxia (shaded bar), and hypoxia (filled bar) (10 ␮M DHR123; n ϭ observed in the cells treated with both TEMPOL and MSRA 4–10). (B) ROS production during reoxygenation is inhibited by MSRA over- virus particles (Fig. 1A). expression. The cells were treated with hypoxia, and the DHR123 signals were Reoxygenation after hypoxia increased the ROS level to Ϸ9 measured as in A (20 ␮M DHR123; n ϭ 3). (C) MSRA overexpression protects times that found during hypoxia, and this increase was signifi- cells from hypoxia-induced cell death. Data regarding nonviable fractions in cantly lower in the cells treated with bMSRA adenovirus par- the groups treated with no virus, bMSRA adenovirus particles, control EGFP adenovirus particles, and TEMPOL (5 mM) are shown. The cells were chal- ticles (Fig. 1B). The fractional decrease in the ROS level during lenged with normoxia (open bar), hyperoxia (shaded bar), or hypoxia (filled reoxygenation associated with MSRA overexpression (Fig. 1B) bar) for 5 min. The cell viability assay with trypan blue was performed 24 h was smaller than that during hypoxia (filled bars in Fig. 1A). later. The effect of ethanol (1%) on cell viability was indistinguishable from that of control (data not shown). MSRA Prevents Cell Death After Hypoxia. Hypoxia͞reoxygenation promotes cell death (35). We hypothesized that the decrease in the ROS level caused by the overexpression of MSRA might cells as described below. The in vitro enzymatic assay for MSRA protect cells against hypoxia͞reoxygenation-induced cell death. activity involving the formation of N-acetyl methionine (25) Thus, we assayed the viability of PC12 cells 24 h after brief showed that the MSRA virus treatment led to a Ͼ10-fold normoxia, hyperoxia, and hypoxia treatments by using the trypan increase in the enzyme activity. In a typical experiment, the blue exclusion cell viability assay (Fig. 1C). In untreated control reductase activity was Ϸ14–21 pmol of N-acetyl methionine cells or cells treated with EGFP adenovirus particles, hypoxia formed per ␮g of protein in the untreated control cells and in the (10 min) increased the mean nonviable stained fraction by cells treated with empty virus particles and Ϸ250 pmol͞␮g Ϸ100% (P Ͻ 0.0001). In contrast, hypoxia failed to alter the protein in the cells treated with the MSRA virus particles. These nonviable fraction in the MSRA overexpression group (P ϭ results corroborate the fluorescence measurements and show 0.43), confirming that the protective effect was specific to MSRA that the infection with adenovirus leads to an increase in the expression. Unlike hypoxia, hyperoxia did not alter the nonvi- functional level of MSRA. able cell fraction in any of the groups examined (P ϭ 0.60). Consistent with the finding that the ROS scavenger TEMPOL Hypoxia Increases ROS. Oxidative stress is implicated in reperfu- reduced ROS production (Fig. 1A), hypoxia failed to alter the sion after an ischemic episode (30), and hypoxia may enhance nonviable fraction in the presence of TEMPOL (Fig. 1C). production of ROS before reoxygenation (5, 6, 31). We used two groups of PC12 cells, one group treated with control adenovirus MSRA Prevents Apoptotic Cell Death. To examine whether MSRA particles containing no insert and the other treated with MSRA overexpression protects PC12 cells against apoptosis or ne- virus particles, and challenged them with normoxia, hyperoxia, crosis, flow cytometry measurements were performed by using and hypoxia (Fig. 1A). The ROS production was measured by Annexin-V-FLUOS and PI (36, 37). The Annexin-V-FLUOS using the ROS-sensitive dye DHR123 (21), which has been used assay measures the translocation of phosphatidylserine to the as a general indicator of cellular ROS production (32, 33). external surface of the plasma membrane early in apoptosis, Multiple ROS convert DHR123 into a highly fluorescent form whereas the PI assay primarily detects late apoptotic and necrotic (26), such that the rate of the fluorescent signal increase reflects cells (37, 38). Representative flow cytometry dot plots are shown the ROS production rate. DHR123 is reported to be more in Fig. 2A, where the dots in the bottom right quadrant in each

sensitive in detecting ROS than other dyes tested (34). In the plot represent the Annexin-V-positive early apoptotic cells. BIOCHEMISTRY control group, treatment with hypoxia significantly changed the Hypoxia (10 min) increased the number of early apoptotic PC12

Yermolaieva et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1161 Downloaded by guest on September 30, 2021 Hypoxia itself increases the ROS level (Fig. 1), but a burst of ROS is also created on reperfusion (30). To infer whether reoxygenation is required to trigger cell death, the Annexin-V- FLUOS assay was performed immediately after 24-h hypoxia. This prolonged hypoxia treatment preferentially promoted apoptotic cell death similar to what was observed with short hypoxia (Fig. 2D). The fraction of apoptotic cells after 24-h hypoxia was 18.4 Ϯ 4.48% compared with 5.37 Ϯ 2.19% after 24-h normoxia. MSRA overexpression at least partially protected the cells against apoptosis after 24-h hypoxia (Fig. 2E). The necrotic fraction was unchanged by MSRA overexpression (Fig. 2F).

Cellular met-O Contents After Hypoxia. The finding that the mean ROS level was lower in the cells treated with the MSRA virus particles suggests that the met-O level in the MSRA group may also be lower. This hypothesis was tested by analyzing the met-O levels in the proteins of the cells treated with normoxia, hyperoxia, and hypoxia. There was no significant difference in the level of met-O among the three groups when expressed as a percentage of the total methionine (see Materials and Methods). The levels of met-O ranged from Ϸ9.5% to 13%. The increased levels of ROS in the hypoxic cells did not lead to a global detectable increase in the levels of met-O in these cells, nor did the presence of higher levels of MSRA decrease the total amounts of met-O in proteins. The reasons for the high levels of met-O are unclear, but the observation may indicate that selective oxidation of methionine residues in proteins may be critical and that the majority of met-O in these cell preparations may be functionally inaccessible to MSRA.

Depolarization of Mitochondria. Depolarization of the inner mitochondrial membrane potential facilitated by the opening of large mitochondrial permeability transition pores is regarded as one of the signs of cell death (39). To investigate whether MSRA impedes the effect of hypoxia to promote cell death by preserv- ing the mitochondrial membrane potential, we measured inner mitochondrial membrane potentials by using the fluorescent voltage-sensitive dye JC-1, which preferentially localizes across the inner mitochondrial membrane (27, 40). JC-1 was selected Fig. 2. MSRA overexpression preferentially prevents apoptosis. (A) Flow cyto- because its emission signals could be analyzed in a ratiometric metric analysis of PC12 cells double-stained with Annexin-V-FLOUS and PI. Cells manner and interpreted semiquantitatively (41). Depolarization were treated with normoxia, hyperoxia, or hypoxia for 10 min, and the measurements were made 24 h later. In each of the six plots, the bottom left quadrangle of the inner mitochondrial membrane decreases the emission indicates viable cells that are negative for both Annexin–V binding and PI uptake; signal intensity at 595 nm (red) and increases the signal intensity the bottom right quadrangle includes apoptotic cells positive for Annexin–V at 535 nm (green), such that it reduces the red͞green ratio. binding but negative for PI uptake; and the top right quadrangle primarily Representative JC-1 red͞green ratio signals recorded in two includes necrotic cells positive for both Annexin–V binding and PI uptake. (Upper) groups of cells, control cells treated with control virus particles Representative dot plots from the control cells treated with normoxia (Left), and the cells treated with bMSRA virus particles, are shown in hyperoxia (Center), and hypoxia (Right). (Lower) Representative dot plots from Fig. 3A. In the control cells treated with normoxia, robust JC-1 the cells overexpressing bMSRA. (B) The fractional increases in the number of red͞green ratio signals were observed in a punctate manner, apoptotic Annexin-V-positive cells. The results from the control and MSRA over- representing healthy polarized mitochondria. We found a clear expressing cells are shown. In each cell group, the results after normoxia (open ͞ ϭ main effect of hypoxia on the JC-1 red green ratio signal (Fig. bar), hyperoxia (shaded bar), and hypoxia (ﬁlled bar) treatments are shown (n Ͻ 3). The measurements were made as in A, and the average percentage of cells 3 A and B; P 0.0001). In both the control and bMSRA groups, undergoing apoptosis without any treatment (20.8 Ϯ 3.7%) has been subtracted. hypoxia decreased the JC-1 red͞green ratio signal. Importantly, (C) The fractional increases in the number of necrotic PI-positive cells. The average the mean JC-1 red͞green ratio signal in the MSRA group during percentage of necrotic cells (1.7 Ϯ 0.1%) was subtracted (n ϭ 3). (D) Flow hypoxia was greater than that in the control group (Fig. 3 A cytometric cell death analysis of PC12 cells after 24-hr treatments. Cells were Bottom and B; P Ͻ 0.0001). Application of the mitochondrial treated with normoxia or hypoxia and immediately assayed for cell death. (E) uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydra- MSRA overexpression partially inhibits the increase in the number of apoptotic zone (FCCP) (Fig. 3C) reduced the JC-1 red͞green ratio signals, cells after 24-hr hypoxia treatment. The measurements are made as in A and B. confirming that the signals did reflect ⌬⌿ . The magnitude of The average percentage of apoptotic cells in the group treated with normoxia m (8.9%) was subtracted (nϭ3). (F) MSRA overexpression does not alter the number the inhibition of the JC-1 signal by FCCP was markedly greater of necrotic cells. The measurements are made as in A and B. The average per- than that by hypoxia (Fig. 3 B and C). centage of necrotic cells in the group treated with normoxia (2.7%) was sub- The ratio of the JC-1 emission intensity at 590 nm over that ⌬⌿ tracted (n ϭ 3). at 535 nm is commonly used to infer m in a semiquantitative and ratiometric manner (27). In strong oxidizing conditions induced by experimentally applied H2O2, the decreases in the cells in the control group when measured 24 h later but not in JC-1 emission signal at 595 nm may not reflect mitochondrial ϭ ⌬⌿ the MSRA overexpression group (P 0.04; Fig. 2B). The depolarization of m, whereas the signal increases at 535 nm ⌬⌿ number of necrotic cells remained unaltered (Fig. 2C). still report m (42). To guard against this possibility, the

1162 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308215100 Yermolaieva et al. Downloaded by guest on September 30, 2021 Fig. 4. Membrane potentials of isolated rat liver mitochondria measured by JC-1. (A) JC-1 red͞green ratio after the onset of normoxia, hyperoxia, and hypoxia. Isolated mitochondria were placed in a cuvette and challenged with normoxia, hyperoxia, or hypoxia starting at time 0. It took Ϸ15 s to load the cuvettes into the spectroﬂuorometer, and the recording could not be performed during this period (line width indicates SEM; n ϭ 3–4 in each condition). The values shown are slightly different from those in the Fig. 3B, because the measurements here were made by using isolated mitochondria. Similar results were obtained when the emission results at 535 nm only were compared. (B) Mean JC-1 red͞green ratio slope values in the normoxia, hyperoxia, and hypoxia conditions. The slopes were calculated by differentiating the JC-1 ﬂuorescence ratio traces as shown in A.

depolarization of the inner mitochondrial membrane potential. The changes in the JC-1 red͞green ratio signals as determined by the maximal slopes of the signals are summarized in Fig. 4B. Discussion The results of this study show that brief hypoxia followed by reoxygenation increases the ROS level in PC12 cells and that this increase is associated with mitochondrial depolarization and Fig. 3. Membrane potentials of intact mitochondria in PC12 cells estimated more prevalent cell death. Overexpression of MSRA, which by JC-1. (A) Control PC12 cells treated with blank control virus particles (Left) facilitates the reduction of met-O in proteins to methionine, and the cells treated with bMSRA adenovirus particles (Right) were challenged attenuates these hypoxia͞reperfusion-induced changes. with normoxia (Top), hyperoxia (Middle), and hypoxia (Bottom). The images By using PC12 cells, we detected rapid changes in the overall Ϸ ͞ ⌬⌿ were obtained 5 min after the start of the treatment. The JC-1 red green ROS level and m in response to brief hypoxia. These results ratio signals are represented with the color scale shown. The full scale repre- are in line with those of Roy et al. (44), who demonstrated sents the JC-1 red͞green ratios of 0 and 2. Greater JC-1 red͞green ratio values ⌬⌿ ⌬⌿ significant changes in the plasma membrane potential and m indicated by brighter colors reﬂect more polarized m. The size scale bar by hypoxia in Ͻ30 s in rat glomus cells and PC12 cells. However, represents 20 ␮m. (B) Linescan analysis of the JC-1 results. The JC-1 red͞green ratio results from the mitochondria in two groups are shown. The control other cells may not exhibit such rapid and high sensitivity to group received blank control virus particles, and the MSRA group received hypoxia. For example, more severe and prolonged hypoxia bMSRA particles (n ϭ 19–25 in each condition). (C) JC-1 signals are attenuated treatments may be necessary to induce mitochondrial depolar- by the mitochondrial uncoupler FCCP. FCCP (0.2 ␮M) was applied for 20 min, ization in cardiomyocytes (45). Notwithstanding, our results with and the cells were imaged. dopamine-containing PC12 cells confirm the conclusions of ⌬⌿ previous studies in other systems that hypoxia depolarizes m, as demonstrated by using different voltage-sensitive dyes (44, fluorescent signal intensities at 535 nm alone were also com- 45), and that hypoxia increases the overall cellular ROS level pared. As found with the red͞green ratio signals, hypoxia indeed Ͻ before the onset of reperfusion (6, 31, 46). increased the 535-nm signal in the control group (P 0.0001). Many of the surface-exposed methionine residues in the Furthermore, the mean 535-nm signal in the control group enzyme glutamine synthetase can be oxidized without altering its subjected to hypoxia was also greater than that in the MSRA function (20). Based on this observation, it was proposed that group subjected to hypoxia, indicating greater depolarization in cyclic oxidation and reduction of methionine residues involving ϭ the control cells (P 0.02). These results taken together suggest MSRA might function as a cellular sink for ROS (20). The lower that brief hypoxia is associated with depolarization of the inner overall ROS level observed in our study with MSRA overex- mitochondrial membrane and that overexpression of MSRA pression is consistent with this idea. The ROS-scavenging hy- attenuates this depolarization. pothesis also predicts that the total met-O level may be higher We also investigated the effects of hypoxia on the JC-1 with greater oxidative stress. However, the total cellular met-O red͞green ratio signals from isolated mitochondria because the level was not markedly altered by MSRA overexpression or by interference from the cytoplasm sometimes makes interpreta- the oxygen concentration. Thus, there may be a specialized pool tions of JC-1 signals in intact cells difficult (41). The use of of methionine residues that specifically function as a ROS sink. isolated mitochondria should largely remove the contributions The possibility that repair of specific met-O residues in selected from the cytoplasmic redox-dependent signal transduction path- proteins plays a critical role in enhancing the efficacy of other ways (43). At time 0 (t ϭ 0), the mitochondria were challenged ROS scavenging components cannot be totally ruled out. with the normoxic, hyperoxic, or hypoxic solution (Fig. 4A). In The MSRA virus particle construct used in this study contains the normoxia condition, the JC-1 red͞green ratio signal did not a mitochondrial targeting signal in the N terminus (16). There- change with time, indicating that the inner mitochondrial mem- fore, at least some of the proteins were likely targeted toward brane potential was stable. Hyperoxia caused a slight decrease in mitochondria, but this was not directly confirmed. This mito-

the JC-1 red͞green ratio signal. In contrast, hypoxia rapidly chondrial sequence is present in many mammalian MSRA BIOCHEMISTRY decreased the JC-1 red͞green ratio signal, corresponding to orthologs (16). Because mitochondria are likely to contribute to

Yermolaieva et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1163 Downloaded by guest on September 30, 2021 the hypoxia͞reoxygenation-mediated increase in ROS (6, 44, sufficient to handle the oxidative stress produced by hypoxia. 45), preferential localization of at least some MSRA in or near Thus, MSRA and MSRB, which reduce the S and R forms of mitochondria is consistent with this enzyme functioning to repair met-O in proteins, respectively, may be a viable therapeutic any oxidative damage in this organelle. target. Given that oxidative stress is postulated to be involved in Our finding that the lower ROS level conferred by MSRA many disorders, including diabetes, atherosclerosis, and neuro- overexpression in PC12 cells is associated with greater cell degenerative diseases (4), it is plausible that modulation of the ͞ viability after both hypoxia͞reoxygenation is consistent with the MSRA B activity in cells may be beneficial in the treatment of idea that oxidative stress is an important factor in cell injury these diseases. associated with hypoxia and reoxygenation. The relative importance of ROS generation during hypoxia and on reperfusion We thank H. Daggett for the PC12 culture and mitochondria preparation and Dr. R. Wassef for oxygen measurements. This work was supported remains to be investigated. The finding that heterologous ex- in part by grants from the National Institutes of Health (to T.H.) and the pression of MSRA reduces the hypoxia-mediated apoptosis Thu¨ringer Ministeriums fu¨r Wissenschaft, Forschung, und Kunst (Grant suggests that the endogenous antioxidant mechanism is not B311-25 to S.H.H.).

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