Mitochondrial Complex I Activity and Oxidative Damage to Mitochondrial Proteins in the Prefrontal Cortex of Patients with Bipolar Disorder

ORIGINAL ARTICLE Mitochondrial Complex I Activity and Oxidative Damage to Mitochondrial Proteins in the Prefrontal Cortex of Patients With Bipolar Disorder

Ana C. Andreazza, PharmD, PhD; Li Shao, PhD; Jun-Feng Wang, PhD; L. Trevor Young, MD, PhD

Context: Accumulating evidence suggests that mito- der (15 each with bipolar disorder, schizophrenia, and chondrial dysfunction and oxidative stress contribute to major depressive disorder) and 15 nonpsychiatric con- the pathogenesis of bipolar disorder and schizophrenia. trol subjects were studied. It remains unclear whether mitochondrial dysfunction, specifically complex I impairment, is associated with in- Main Outcome Measures: Oxidative damage to pro- creased oxidative damage and, if so, whether this rela- teins and mitochondrial complex I activity. tionship is specific to bipolar disorder. Results: Levels of NDUFS7 and complex I activity were Objective: To evaluate whether decreased levels of the decreased significantly in patients with bipolar disorder electron transport chain complex I subunit NDUFS7 are but were unchanged in those with depression and schizo- associated with complex I activity and increased oxida- phrenia compared with controls. Protein oxidation, as tive damage to mitochondrial proteins in the prefrontal measured by protein carbonylation, was increased sig- cortex of patients with bipolar disorder, schizophrenia, nificantly in the bipolar group but not in the depressed or major depressive disorder. or schizophrenic groups compared with controls. We observed increased levels of 3-nitrotyrosine in the bipolar Design: Postmortem prefrontal cortex from patients and disorder and schizophrenia groups. controls were assessed using immunoblotting, spectro- photometric, competitive enzyme immunoassay to iden- Conclusions: Impairment of complex I may be associ- tify group differences in expression and activity of com- ated with increased protein oxidation and nitration in plex I, and in oxidative damage in mitochondria. the prefrontal cortex of patients with bipolar disorder. Therefore, complex I activity and mitochondrial dys- Setting: University of British Columbia, Vancouver, function may be potential therapeutic targets for bipolar Canada. disorder.

Patients: Forty-five patients with a psychiatric disor- Arch Gen Psychiatry. 2010;67(4):360-368

IPOLAR DISORDER (BD) IS A matter volumes in male patients with fa- chronic psychiatric illness milial BD type I. Mitochondrial dysfunc- characterized by recurrent tion,8,9 and the consequent oxidative dam- episodes of mania, hypoma- age to lipids, proteins, and DNA,9 might nia, mixed states, and de- be one of the possible mechanisms con- pression.B It has been increasingly recog- tributing to neuronal or glial impairment nized that individuals with BD are at higher in BD. risk for chronic general medical condi- Mitochondria are intracellular organ- tions, such as obesity, diabetes mellitus, elles that have a crucial role in adenosine and cardiovascular disease,1,2 which are di- triphosphate (ATP) production through rectly associated with the increased mor- oxidative phosphorylation, a process bidity and mortality observed in BD. De- performed by the electron transport chain spite decades of extensive investigation, the (ETC) complexes I through V.10 Mito- etiology and pathogenesis of this disor- chondria also serve as calcium buffers, der remain unclear. Several postmortem regulators of apoptosis, and generators of 3-6 10,11 Author Affiliations: studies have reported reduced neuro- reactive oxygen species (ROS). Mito- Department of Psychiatry, nal and glial density in discrete regions of chondrial ATP production occurs through University of British Columbia, the prefrontal cortex of patients with BD. the flow of electrons that are passed along Vancouver, British Columbia, In addition, Davis et al7 found reductions ETC complexes in the inner mitochon- Canada. in cortical gray matter and cerebral white drial membrane. Energy lost by protons

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 reentering the mitochondrial matrix through ATP syn- els of lipid peroxidation in the cingulate cortex of pa- thase is used to produce ATP.12 During transfer along the tients with BD. ETC, single electrons sometimes escape and result in a Sun et al,22 using high-density complementary DNA single-electron reduction of molecular oxygen to form a spot microarrays, reported downregulation of 8 mito- − superoxide anion (O2 ), especially in complex I (also chondrial ETC-related genes: NDUFS7 and NDUFS8 known as nicotinamide adenine dinucleotide [NADH]– (complex I), UQCRC2 (complex III), COX5A and COX6C 13 − ubiquinone oxidoreductase). Mitochondrial O2 reacts (complex IV), and ATP5C1, ATP5J, and ATP5G3 (com- − with superoxide dismutase, which converts the O2 into plex V). Using real-time quantitative polymerase chain hydrogen peroxide (H2O2), which can, in the presence reaction, we further verified that mRNA levels of NDUFS7 3ϩ ϩ 2ϩ of ferrous iron (Fe ) via the Fenton reaction (H2O2 Fe were decreased. Because NDUFS7 levels were decreased →Fe3ϩϩOH−ϩOH•), result in the production of highly and may contribute to decreased complex I activity,41 in reactive hydroxyl radicals (OH•). Another relevant event the present study we examined NDUFS7 protein levels – • is the reaction of O2 with nitric oxide (NO ) to form per- and complex I activity as an indication of mitochondrial oxynitrite (ONOO−). When mitochondrial and cyto- complex I impairment. Complex I is a major source of plasm enzymatic and nonenzymatic antioxidant sys- ROS production, which can cause oxidative damage to tems are overwhelmed by elevated levels of ROS and proteins. We, therefore, also analyzed protein oxidation reactive nitrogen species, oxidative damage can occur to (carbonyl content) and tyrosine nitration (3- DNA, lipids (cell and organelle membranes), and pro- nitrotyrosine levels) as markers of oxidative damage to teins (receptors, transcription factors, and enzymes).14 mitochondrial proteins to improve understanding of the Oxidative damage to proteins may be caused by reac- pathogenesis of BD. tions of amino acid residues with (1) ROS, especially OH•, 2ϩ 2ϩ catalyzed by Fe and cupric (Cu ), which introduce car- METHODS bonyl groups in lysine, proline, arginine, and threonine 15,16 − residues ;or(2)ONOO , which nitrates sulfhydryl and POSTMORTEM BRAIN SAMPLES hydroxyl residues in cysteine, methionine, phenylala- nine, and tyrosine; these modifications could inactivate Prefrontal cortex tissue samples were from Brodmann area 10 17,18 the membrane signaling pathways and key enzymes. (1.0-g blocks). Participants were divided into 4 groups: BD, 3-Nitrotyrosine is produced by the nitration of tyrosine MDD, schizophrenia, and nonpsychiatric control subjects (n=15 residues in protein and serves as a marker for in vivo oxi- per group) matched for age, sex, and postmortem interval (PMI) dative damage induced by ONOO−.18 Oxidative modifi- (Table). Diagnoses were retrospectively established by 2 se- cations can also affect the function of specific proteins, nior psychiatrists using DSM-IV criteria. Detailed clinical in- formation, diagnostic procedures, and demographic informa- such as enzymatic activity, DNA binding activities of 42 transcription factors,15 and susceptibility to proteolytic tion on these individuals have been previously published. The 16 investigators were blinded to the group identity, diagnosis, and degradation. demographic variables of the participants during all experi- Several lines of evidence suggest that mitochondrial ments and measurements. Samples were randomly coded nu- dysfunction has a role in the pathogenesis of BD be- merically, and the code was lifted only during data analyses, cause these individuals have been noted, for example, to after all of the experiments were completed. have altered cerebral energy metabolism19,20 and an in- 21 creased ratio of mitochondrial DNA deletion. Recent MITOCHONDRIAL EXTRACTION DNA microarray analyses in postmortem prefrontal cor- tex22,23 and hippocampus24 revealed that the expression Mitochondria-enriched extracts were prepared as described by of many messenger RNAs (mRNAs) coding for ETC com- Smith,43 with minor modifications. Briefly, prefrontal cortex tis- plexes I to V subunits was decreased in patients with BD. sues were homogenized in buffer 1 (0.25M sucrose, 2mM EDTA, In addition, evidence from at least some genotyping stud- and 10mM Tris hydrochloride; pH 7.2) at a ratio of 20 µL/mg ies25,26 suggest that polymorphisms of the complex I sub- of tissue. Homogenized samples were centrifuged (at 5600g for unit NDUFV2 may be associated with BD. Several stud- 3 minutes). The supernatant was kept on ice, and the pellet was ies27-30 have also suggested altered activity and expression resuspended in buffer 1, homogenized, and recentrifuged (at 5600g for 3 minutes). The combined supernatants were cen- of mitochondrial ETC components in postmortem brain trifuged (at 37 500g for 20 minutes). Mitochondria-enriched samples from patients with schizophrenia. Further- pellets were dissolved in buffer 2 (2M aminocaproic acid, 150mM more, mitochondrial ATP production measured in a Bis-Tris hydrochloride, and 500mM EDTA; pH 7.0) at a ratio muscle biopsy specimen was reported to be decreased in of 4 µL/mg of tissue. These homogenates were centrifuged (at patients with major depressive disorder (MDD).31 Com- 12 000g for 15 minutes). The supernatants were collected and plex I is one of the main sites in which electrons are re- used for complex I enzymatic assay and immunoblotting analy- leased and react with oxygen, resulting in ROS produc- sis. Protein concentrations in the supernatants were deter- tion, thus causing oxidative stress. Indeed, recent studies mined using the Bradford assay. To verify that this method is have demonstrated alterations in a diverse set of oxida- reliable with frozen samples, we compared the protein con- tive stress parameters in patients with BD. For example, centrations and the complex I activity in fresh and frozen rat brain. The cerebral cortex was removed (n=4) and submersed studies conducted with peripheral blood cells have dem- in ice-cold calcium (Ca2ϩ)- and magnesium (Mg2ϩ)-free onstrated that BD is associated with alterations in anti- Hanks balanced saline solution. Mean (SD) protein concen- 32-34 33,34 oxidant enzymes, increased lipid peroxidation, in- trations in fresh (2.15 [0.30] µg/µL) and frozen (1.91 [0.21] creased levels of nitric oxide,35-37 and increased DNA µg/µL) mitochondrial fractions were not significantly differ- fragmentation.38,39 Moreover, we40 reported increased lev- ent (t=1.99; P=.13). No significant difference was found in

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 Table. Demographic and Clinical Data for Postmortem Brain Tissue in Patients and Controls

Group P Control BD MDD Schizophrenia F Value Age, mean (SEM) [range], y 48 (2.7) [29-68] 42 (2.9) [25-61] 47 (2.3) [30-65] 45 (3.3) [25-62] 0.728 .54 Sex, M/F, No. 9/6 9/6 9/6 9/6 NA NA PMI, mean (SEM) [range], h 23.7 (2.4) [8-42] 32.5 (4.02) [13-62] 27.5 (2.68) [7-47] 33.7 (3.65) [12-61] 1.857 .15 pH, mean (SEM) [range] 6.3 (0.1) [5.8-6.6] 6.2 (0.1) [5.8-6.5] 6.2 (0.1) [5.8-6.5] 6.2 (0.1) [5.8-6.6] 0.611 .61 Cause of death, No. NA NA Suicide 0974 Cardiopulmonary 13 5 7 8 Accident 2002 Other 0111 Current alcohol/drug abuse or dependence, No. 0433NANA Past alcohol/drug abuse or dependence, No. 2313NANA Medication, No. NA NA Lithium carbonate 0422 Antidepressant 0 8 10 5 Antipsychotic 0 8 0 12

Abbreviations: BD, bipolar disorder; MDD, major depressive disorder; NA, not applicable; PMI, postmortem interval.

mean (SD) complex I activity comparing fresh (2.37 [0.22] PROTEIN OXIDATION µmol/min) and frozen (2.15 [0.21] µmol/min) rat brain samples (t=1.82; P=.16). Protein oxidation was assessed by measuring the levels of carbonyl groups using the OxyBlot Protein Oxidation Detection NDUFS7 LEVELS Kit (catalog No. S7150; Chemicon, Kankakee, Illinois). The carbonyl groups in the protein side chains are derivatized from Protein levels of NDUFS7 were measured by means of immu- 2,4-dinitrophenylhydrazone by reaction with 2,4-dinitrophe- noblotting. Thirty micrograms of mitochondrial extract was nylhydrazine. The 2,4-dinitrophenylhydrazone–derivatized loaded on 12% acrylamide sodium dodecyl sulfate– protein samples were separated by means of polyacrylamide polyacrylamide gel electrophoresis gel and were subsequently gel electrophoresis and Western blotting following the OxyBlot transferred onto polyvinylidene difluoride membranes. Mem- kit instructions. Protein bands were analyzed densitometrically branes were blocked with 0.05% polysorbate 20 in 0.01M phos- and were normalized against the intensity of porin using phate-buffered solution containing 5% nonfat milk (PBS-T) for VersaDoc. 2 hours at room temperature. The blots were independently probed using either mouse anti–human NDUFS7 antibody (No- TYROSINE NITRATION–INDUCED DAMAGE vus Biologicals, Littleton, Colorado), 1:1000, or antiporin antibody (Abcam Inc, Cambridge, Massachusetts), 1:3000, as mi- Tyrosine nitration–induced damage was assessed by measur- tochondria-loading control in PBS-T at 4°C overnight with gentle ing 3-nitrotyrosine levels using the OxiSelect Nitrotyrosine shaking. Membranes were washed with PBS-T 4 times (10 min- ELISA [enzyme-linked immunosorbent assay] Kit (catalog No. utes each time) and then were incubated with rabbit anti– STA-305; Cell Biolabs Inc, San Diego, California). The un- mouse horseradish peroxidase conjunct secondary antibody (Ab- known protein nitrotyrosine sample or nitrated bovine serum cam Inc), 1:3000, in PBS-T for 2 hours at room temperature. albumin standards were first added to a nitrated bovine serum Finally, membranes were washed with PBS-T 4 times (10 min- albumin–preabsorbed enzyme immunoassay plate. After a brief utes each time) before applying electrochemiluminescence re- incubation, the anti–nitrotyrosine antibody was added, fol- agents (GE Co, Piscataway, New Jersey). Protein bands were lowed by the horseradish peroxidase–conjugated secondary an- analyzed densitometrically and were normalized to the porin tibody. The protein nitrotyrosine content in the unknown samples signal using a molecular imaging system (VersaDoc; Bio-Rad was determined by comparison with a standard curve prepared Laboratories, Hercules, California). using predetermined nitrated bovine serum albumin standards.

COMPLEX I ACTIVITY STATISTICAL METHODS

Complex I activity was performed as described by Estornell et Statistical analyses were performed using a computer software al.44 Briefly, mitochondria-enriched pellets were diluted to 10 program (SPSS for Windows version 16.0; SPSS Inc, Chicago, to 15 µg/mL in the assay buffer (50mM potassium chloride, Illinois) software. Normal distribution of data was determined 10mM Tris hydrochloride, 1mM EDTA, and 2mM potassium using the Kolmogorov-Smirnov test. For further analysis, para- cyanide; pH 7.4). The NADH, 75µM, was added. The reaction metric tests were used because most of the data (90%) had a was started by the addition of 50µM coenzyme Q1 and was read normal distribution. Data were analyzed by means of 2-way at 340 nm against the blank containing all the components ex- analysis of variance, followed by the least significant differ- cept the coenzyme Q1 for 5 minutes. The rate after inhibition ence post hoc test. Age, sex, PMI, and brain pH were added as was determined with the same reagents plus the inhibitor ro- covariates, and persistence of the significant difference in main tenone for 5 minutes. Complex I activity was calculated by sub- effect between diagnostic groups was assessed by means of analy- tracting the rate after the addition of rotenone (10µM) from sis of covariance. Correlations were analyzed using the Pear- the overall rate. son correlation test. Data are given as mean (SD).

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2.5 2.0

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Complex I Activity, µmol/min/mg of Protein Complex I Activity, 0.0

5.80 6.00 6.20 6.40 6.60 5.80 6.00 6.20 6.40 6.60 pH pH

C D 4.0 r 2 = 0.008 2000 r 2 = 0.022

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1.0 3-Nitrotyrosine, nM 500 Protein Carbonyl Levels, AU

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5.80 6.00 6.20 6.40 6.60 5.80 6.00 6.20 6.40 6.60 pH pH

Figure 1. Relation between complex I activity (A), NDUFS7 expression (B), protein oxidation (C), and tyrosine nitration–induced damage (D) and pH. Results were assessed using the Pearson correlation test. AU indicates arbitrary units.

RESULTS MITOCHONDRIAL COMPLEX I DYSFUNCTION AND PROTEIN DAMAGE DEMOGRAPHIC VARIABLES We found that NDUFS7 levels were significantly different comparing the 4 groups. The group difference in Demographic and clinical characteristics of patients and NDUFS7 levels (F3,56=5.691, P=.002) was due to a sig- controls are given in the Table. Controls (n=15) were nificant decrease in NDUFS7 levels in the BD group matched with patients with BD (n=15), MDD (n=15), (62.38%, P=.003) compared with controls (Figure 2A). and schizophrenia (n=15) for age and sex; therefore, as Expression of NDUFS7 was not different in patients with expected, no significant differences were noted among MDD (P=.16) or in those with schizophrenia (P=.41) groups on these measures. The PMI (F=1.857, P=.15) compared with controls. To control for potential con- and pH (F=0.611, P=.61) were not significantly differ- founding variables, age, sex, PMI, and brain pH were ent among groups. The PMI was not correlated with added as covariates and were assessed by means of analy- NDUFS7 expression (r2=0.021, P=.10), complex I ac- sis of covariance. The differences between the groups re- tivity (r2 =0.016, P=.11), carbonyl levels (r2 =0.001, mained significant even after these variables were added 2 P=.22), or 3-nitrotyrosine levels (r =0.005, P=.33). The to the analysis (F3=4.798, P=.01). Adding pH to the analy- 2 pH did not correlate with complex I activity (r =0.023, sis had a significant effect (F3=6.271, P=.02) but did not P=.72), carbonyl levels (r2=0.008, P=.29), or 3-nitroty- alter the significant decrease in NDUFS7 levels. rosine levels (r2 =0.022, P=.11) but correlated posi- Complex I activity was markedly decreased (53.12%) 2 Ͻ tively with NDUFS7 levels (r =0.105, P=.01) (Figure 1). in patients with BD (F3,56=12.85, P .001) and was de- To assess whether patients who committed suicide had creased to a lesser extent in those with MDD (17.98%, more mitochondrial dysfunction or protein damage, we P=.003) in relation to controls. There were no significant divided the patients into 2 subgroups: death by suicide differences in complex I activity in patients with schizo- (n=20) and death by other causes (n=25). No signifi- phrenia compared with controls (P=.47) (Figure 2B). Con- cant difference between these groups was observed in trolling for potential confounding variables (age, sex, PMI, complex I activity, NDUFS7 expression, protein carbon- and brain pH) by means of analysis of covariance did not Ͻ ylation, or 3-nitrotyrosine levels. alter the main effect (F3=13.56, P .001).

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 protein oxidation markers. Analysis of covariance did not A Anti-NDUFS7 B demonstrate any effect from the potential confounding factors. Antiporin 3.0 2.5 Next, we analyzed the relationship between complex I activity and NDUFS7 levels, protein oxidation, and ty- 2.0 rosine nitration. As expected, complex I activity was posi- 2.0 2 1.5 tively correlated with NDUFS7 levels (n=60; r =0.185, P=.001). The present results show that complex I activ- 1.0 1.0 ity was correlated negatively with carbonyl levels (n=57;

Complex I Activity, 2 NDUFS7 Levels, AU 0.5 r =0.299, P=.02) and 3-nitrotyrosine levels (n=57; µmol/min/mg of Protein 2 0.0 0.0 r =0.113, P=.01) (Figure 4). CTL BD MDD SCZ CTL BD MDD SCZ Group Group EFFECT OF MEDICATION Figure 2. Levels of NDUFS7 (A) and complex I activity (B) in postmortem prefrontal cortex of control (CTL), bipolar disorder (BD), major depressive Antipsychotic drugs have been reported to be potential in- disorder (MDD), and schizophrenia (SCZ) subjects. Complex I, also known hibitors of complex I activity.27 To assess the potential effect as nicotinamide adenine dinucleotide [NADH]–ubiquinone oxidoreductase, is a multisubunit integral membrane complex of the mitochondrial electron of antipsychotics on the present results, we divided the pa- transport chain that catalyzes electron transfer from NADH to ubiquinone. tients into 2 subgroups: patients with schizophrenia or BD The activity of complex I was measured by following the oxidation of NADH treated with antipsychotics (conventional and second- at 340 nm. The immunocontent of NDUFS7 was analyzed by means of generation drugs) at the time of death (n=20 [12 with Western blotting. Differences among groups were analyzed using 1-way analysis of variance, followed by the least significant difference test schizophrenia and 8 with BD]) (Table) and those who were comparing the patient groups with the control group. Horizontal lines not (n=10 [3 with schizophrenia and 7 with BD]). Be- indicate means; error bars, SE. AU indicates arbitrary units. *PϽ.001; Ͻ cause none of the patients in the other 2 groups were treated †P .005. with this class of drugs at the time of death, they were ex- cluded from the analysis. No significant difference was observed between the 2 subgroups in complex I activity, A B NDUFS7 levels, protein carbonyl content, or 3-nitrotyrosine levels. There was a nonsignificant trend for decreased mean (SD) complex I activity in patients treated with an- CTL BD MDD SCZ tipsychotics (0.81 [0.34] µmol/min) compared with those 8.0 2000 who had not been treated with antipsychotics (1.08 [0.30] µmol/min) (t=−0.456, P=.08). Next, to assess the pos- 6.0 1500 sible effects of antidepressant medications on the results,

4.0 1000 we divided the patients into 2 subgroups: those treated with antidepressant drugs (n=23 [10 with MDD, 5 with schizo- 2.0 500 phrenia, and 8 with BD]) and those not treated with anti- 3-Nitrotyrosine, nM depressant drugs (n=22 [5 with MDD, 10 with schizo- Protein Carbonyl Levels, AU 0.0 0 phrenia, and 7 with BD]). No significant differences were CTL BD MDD SCZ CTL BD MDD SCZ Group Group observed between the groups for any of these measures. Finally, recent studies,45 including ours, have suggested that lithium carbonate has potential antioxidant capacity. We Figure 3. Mitochondrial protein carbonyl immunocontent (A) and 3-nitrotyrosine levels (B) in postmortem prefrontal cortex of control (CTL), divided the patients into groups treated with lithium (n=14 bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia [2 with MDD, 2 with schizophrenia, and 10 with BD]) and (SCZ) subjects. Mitochondrial protein carbonyl levels were analyzed by those who had not received lithium (n=31 [13 with MDD, means of Western blotting. 3-Nitrotyrosine levels were analyzed by means of competitive enzyme-linked immunosorbent assay. Horizontal lines represent 13 with schizophrenia, and 5 with BD]). No significant dif- means; error bars, SE (n=14 CTL; n=15 BD; n=15 MDD; and n=14 SCZ). ferences were observed between these 2 subgroups in com- Differences among groups were analyzed using 1-way analysis of variance, plex I activity, NDUFS7 expression, protein carbonyl con- followed by the least significant difference test comparing the patient groups tent, or 3-nitrotyrosine levels. with the control group. AU indicates arbitrary units. *PϽ.01; †PϽ.05.

To assess oxidative and nitrosative damage to mito- COMMENT chondrial proteins, we analyzed protein carbonylation and 3-nitrotyrosine levels in the postmortem samples. There We report that NDUFS7 levels and complex I activity are were significant differences between groups in carbon- decreased and levels of mitochondrial protein oxidation ylation levels (F3,56=3.01, P=.04) (Figure 3A) and ni- and tyrosine nitration are increased in postmortem pre- tration levels (F3,56=4.56, P=.007) (Figure 3B). Patients frontal cortex of patients with BD compared with age- with BD had increased carbonyl content (P=.01) and 3-ni- and sex-matched nonpsychiatric controls. There were no trotyrosine levels (P=.001) compared with controls. 3-Ni- differences in NDUFS7 levels in patients with either MDD trotyrosine levels were also significantly increased in pa- or schizophrenia, but a small decrease in complex I ac- tients with schizophrenia compared with the control tivity was found in the MDD group compared with con- group (P=.03). Patients with MDD did not differ from trols. Increased 3-nitrotyrosine levels were also found in the control and other patient groups on either of these patients with schizophrenia compared with the control

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0.50 0.50 0.50 Complex I Activity, µmol/min/mg of Protein Complex I Activity, µmol/min/mg of Protein Complex I Activity, µmol/min/mg of Protein Complex I Activity, 0 1.00 2.00 3.00 0.00 1.00 2.00 3.00 0 500 1000 1500 NDUFS7 Levels, AU Carbonyl Levels, AU 3-Nitrotyrosine, nM

Figure 4. Correlations among complex I activity and NDUFS7 levels (A), protein oxidation (carbonyl levels) (B), and tyrosine nitration–induced damage (3-nitrotyrosine) (C). Results were assessed using the Pearson correlation test. AU indicates arbitrary units.

group. We found a negative correlation between com- tion, decreased activity of complex IV was found in the plex I activity and protein carbonylation or 3-nitrotyro- caudate nucleus48 and frontal and temporal cortices,29 and sine levels. These results, together with evidence from succinate dehydrogenase (complex II) activity was in- other neurologic diseases,41 provide evidence that decreased in the putamen and nucleus accumbens of pa- creased NDUFS7 levels contribute to complex I impair- tients with schizophrenia.49 Complex I activity was also ment, in this case, in BD. Decreased complex I activity found to be reduced in the temporal cortex but not in is widely reported to increase superoxide produc- the frontal cortex27 or caudate nucleus in patients with tion,10,11,13,14 and this free radical, in turn, induces oxida- schizophrenia.49 More recently, Karry et al27 observed re- tive15 and nitrosative46 damage to proteins. Neverthe- duced expression of 2 catalytic subunits of mitochon- less, multiple factors regulate complex I activity, including drial complex I, 24 kDa (NDUFV2) and 51 kDa alterations in other complex I subunits, increased neu- (NDUFV1), in the prefrontal cortex of patients with rotoxin levels, glutathione depletion, decreased ATP pro- schizophrenia compared with controls. The present reduction, and increased ONOO− levels.14,41 The present sults did not find differences in NDUFS7 levels and com- study identifies NDUFS7 as a factor that potentially con- plex I activity in the schizophrenia group. The differ- tributes to complex I impairment. There is growing evi- ences in the results may be explained, in part, by the fact dence that mitochondrial impairment, particularly in mi- that Karry et al27 evaluated the RNA expression levels of tochondrial complex I, contributes to the pathogenesis subunits different than those analyzed herein and used of BD.22-24,45 brain tissue from Brodmann area 46/9, whereas the pres- Microarray data have suggested that decreased ex- ent samples were from Brodmann area 10. pression of many mRNAs coding for complexes I through The findings highlighted herein illustrate that down- V subunits are associated with BD.22-24 More specifi- regulation of several complex I subunits occurs in BD, cally, Iwamoto et al47 demonstrated decreased mRNA lev- which may be associated with the susceptibility of BD to els of the complex I subunit NDUFS1, complex III sub- damage through oxidative stress. Human complex I is unit UQCRC2, and complex IV subunit COX15 in the composed of 45 to 46 different subunits and is divided prefrontal cortex of patients with BD. Sun et al22 showed into 3 functional modules. First, the dehydrogenase mod- that 8 genes coding for subunits of ETC complexes I, III, ule is responsible for the oxidation of NADH via flavin IV, and V were downregulated in postmortem prefron- mononucleotide onto a chain of iron-sulfur clusters. Sec- tal cortex of patients with BD: NDUFS7 and NDUFS8 ond, the hydrogenase module guides the released elec- (complex I), UQCRC2 (complex III), COX5A and COX6C trons to electron acceptors. Third, the transporter mod- (complex IV), and ATP5C1, ATP5J, and ATP5G3 (com- ule is responsible for translocation of protons across the plex V). To confirm these data, Sun et al22 evaluated mRNA membrane.50 In BD, decreased expression of NDUFV225,26 levels of NDUFS7, UQCRC2, COX6C, and ATP5G3 using and NDUFS147 in the dehydrogenase module and de- real-time quantitative polymerase chain reaction and creased expression of NDUFS722 and NDUFS822 in the found decreased mRNA levels of NDUFS7 and COX6C. hydrogenase module have been reported. These results Mitochondrial dysfunction has also been reported in suggest that patients with BD may have a reduced abil- schizophrenia. Using transcriptomic, proteomic, and me- ity to oxidize NADH and to transfer electrons to ubiqui- tabolomic analysis, Prabakaran et al30 showed that half none. In that respect, electrons may persist for suffi- of the altered protein expression in patients with schizo- cient time to react with molecular oxygen and, thus, − 51,52 53 phrenia was related to mitochondrial dysfunction and oxi- produce O2 . In addition, Benes et al found that su- dative stress and that these were mirrored by transcrip- peroxide dismutase, catalase, several isoforms of gluta- tional and metabolite perturbations. NDUFS1 was shown thione peroxidase and glutathione S-transferase, and other to be decreased in white and gray matter of the prefron- genes associated with antioxidant reactions were down- tal cortex from patients with schizophrenia.28 In addi- regulated in the hippocampus of patients with BD but

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 not schizophrenia. Together, the organization of com- These results must be interpreted in light of the limi- plex I, the downregulation of complex I subunits, and tations of the samples and methods. First, some of these the decreased levels of the antioxidant system strongly reported changes could be related to lower brain pH, support the susceptibility of mitochondrial proteins to which is commonly associated with antemortem agonal oxidative damage in BD.12-18 states, postmortem delay, and storage of tissue.58 We sug- In this study, patients with BD demonstrated in- gest that such changes in pH may, indeed, be related to creased levels of carbonylated proteins, which were shown the diagnosis and treatment of BD, as suggested by Kato to be negatively correlated with complex I activity. One et al59 and Hamakawa et al.60 We did not find differ- possible mechanism through which impaired complex I ences in pH or PMI between groups (Table). However, activity could be associated with increased protein car- pH correlated positively with NDUFS7 levels but not with bonylation in BD is the overproduction of OH•, by reac- complex I activity, 3-nitrotyrosine levels, and carbonyl − 15,16 • tion of O2 with superoxide dismutase. OH reacts with levels. The covariates pH, PMI, age, and sex did not con- lysine, proline, arginine, and threonine residues of pro- tribute significantly to the main findings, as demon- teins by creating carbonyl groups.15 Carbonylation can strated by the subsequent data analysis. Second, drug treat- alter protein function or can lead to deleterious inter- ment is another important consideration; however, we molecular aggregates that preclude their degradation by did not find significant effects of treatment with lithium, the proteasomal system.15,16 Konradi et al24 demon- antidepressants, or antipsychotics on any of these mea- strated decreased expression of genes involved in the pro- sures. Third, considering that we studied postmortem teasome degradation process in prefrontal cortex from brain, the results also might be limited by a small sample patients with BD, suggesting that in addition to poten- and analysis of a single region, the prefrontal cortex. Re- tial mechanisms leading to increased protein carbonyla- sults of a recent study40 also indicate oxidative stress in tion, the normal process of degradation may also be im- the anterior cingulate cortex of patients with BD. The au- paired, leading to further accumulation. Indeed, thors acknowledge the efforts of many groups to collect accumulation of carbonylated proteins has been impli- postmortem brain samples from larger groups of pa- cated in the etiology or progression of several chronic tients with better control over these important variables central nervous system disorders, including Alzheimer as a future strategy to control for these limitations. There- disease, Parkinson disease, amyotrophic lateral sclero- fore, additional brain regions require further investiga- sis, and multiple sclerosis.54,55 Therefore, future studies tion to understand the specificity of oxidative stress in using redox proteomic techniques will be critical to iden- psychiatric diseases. tify whether specific mitochondrial proteins are targets In conclusion, these results provide evidence of the of protein oxidation and, if so, to define the relationship involvement of mitochondrial complex I dysfunction and between oxidative protein modification (ie, carbonyla- consequent oxidative damage to proteins in BD but not tion) and cellular function in BD. in schizophrenia. Accumulation of oxidative damage to These results also demonstrate increased levels of 3-ni- mitochondrial proteins is thought to lead to neuronal cell trotyrosine in the prefrontal cortex of patients with BD death by apoptosis or as a consequence of aggregation and those with schizophrenia compared with nonpsy- of oxidized protein that may result in neurodegenera- chiatric controls. 3-Nitrotyrosine is a posttranslational tion.61 Complex I deficiency may sensitize neurons to mi- modification in protein tyrosine residue nitrated by tochondria-dependent apoptosis in response to the pro- ONOO− (peroxynitrite).18,46 In the brain, NO• is pro- apoptotic protein Bax, which releases the soluble pool duced by microglia and astrocytes and is subsequently of cytochrome c in the mitochondrial intermembrane transported to neurons, where it may react with super- space, activating the programmed cell death by caspase oxide to yield ONOO−.18 Neuronal nitric oxide synthase 3 and 9.61,62 In addition, Benes et al53 showed that sev- 1, the enzyme that generates NO•, was found to be up- eral apoptotic genes, including FAS, BAK, APAF-1, regulated in the hippocampus of patients with BD,56 in NFkB65, TRAF1, BID, c-Myc, c-jun, and MDM2, are up- addition to increased serum levels of NO• in patients with regulated in the hippocampus of individuals with BD but the same disorder.35-37 Our group57 previously reported not in patients with schizophrenia, suggesting an impor- increased serum levels of 3-nitrotyrosine in patients with tant role of the apoptotic process in BD. Future studies BD early (0-3 years) and late (10-20 years) in the course are needed to identify which specific mitochondrial pro- of the illness. In addition, Murray et al46 showed that teins are targets of carbonylation and nitration in pa- ONOO− reactions with mitochondrial membranes from tients with BD and possibly to identify new targets of neu- bovine heart occur predominantly in complex I sub- roprotective strategies and to help elucidate a better units, resulting in significant inhibition of complex I ac- understanding of the pathogenesis of BD. tivity, suggesting a functional relation between complex I activity and nitration. This is further supported by Submitted for Publication: June 3, 2009; final revision the report of Naoi et al18 of increased 3-nitrotyrosine lev- received August 6, 2009; accepted August 20, 2009. els in the mitochondrial complex I subunits but not in Correspondence: L. Trevor Young, MD, PhD, Depart- other mitochondrial proteins of SH-SY5Y cells incu- ment of Psychiatry, University of British Columbia, 2255 bated with ONOO−. These findings, along with those of Wesbrook Mall, Vancouver, BC V6T 2A1, Canada (trevor the present study, may suggest that mitochondrial com- [email protected]). plex I proteins are susceptible to the nitration process Financial Disclosure: Dr Andreazza has been sup- and that this modification contributes importantly to the ported by CNPq (Brazil) and CAPES (Brazil). Dr Wang mitochondrial dysfunction observed in BD. has received research grants from the Canadian Insti-

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 tutes of Health Research and the National Alliance for dues of glutamine synthetase: both modifications mimic effects of adenylylation. Research on Schizophrenia and Depression. Dr Young Proc Natl Acad Sci U S A. 1998;95(6):2784-2789. 18. Naoi M, Maruyama W, Shamoto-Nagai M, Yi H, Akao Y, Tanaka M. Oxidative stress has received research grants from the Canadian Insti- in mitochondria: decision to survival and death of neurons in neurodegenerative tutes of Health Research and from the Stanley Founda- disorders. Mol Neurobiol. 2005;31(1-3):81-93. tion and is an occasional speaker for Eli Lilly and Astra- 19. Kato T, Takahashi S, Shioiri T, Inubushi T. Alterations in brain phosphorous me- Zeneca. tabolism in bipolar disorder detected by in vivo 31P and 7Li magnetic reso- Funding/Support: This study is supported by grants from nance spectroscopy. J Affect Disord. 1993;27(1):53-59. 20. Frey BN, Stanley JA, Nery FG, Monkul ES, Nicoletti MA, Chen HH, Hatch JP, Cae- the Canadian Institutes of Health Research (Drs Wang tano SC, Ortiz O, Kapczinski F, Soares JC. Abnormal cellular energy and phos- and Young), by the Stanley Medical Research Institute pholipid metabolism in the left dorsolateral prefrontal cortex of medication-free (Dr Young), and by a Young Investigator award from the individuals with bipolar disorder: an in vivo 1H MRS study. Bipolar Disord. 2007; National Alliance for Research on Schizophrenia and De- 9(suppl 1):119-127. 21. Kato T, Stine OC, McMahon FJ, Crowe RR. Increased levels of a mitochondrial pression (Dr Wang). Specimens were donated by the Stan- DNA deletion in the brain of patients with bipolar disorder. Biol Psychiatry. 1997; ley Medical Research Institute Brain Collection cour- 42(10):871-875. tesy of Michael B. Knable, DO; E. Fuller Torrey, MD; 22. Sun X, Wang JF, Tseng M, Young LT. Downregulation in components of mito- Maree J. Webster, PhD; and Robert H. Yolken, MD. chondrial electron transport chain in postmortem frontal cortex from subjects Previous Presentations: This study was presented in part with bipolar disorder. J Psychiatry Neurosci. 2006;31(3):189-196. 23. Iwamoto K, Bundo M, Kato T. Altered expression of mitochondria-related genes in at the 64th Annual Convention of the Society of Biologi- postmortem brains of patients with bipolar disorder or schizophrenia, as revealed cal Psychiatry; May 14, 2009; Vancouver, British Colum- by large-scale DNA microarray analysis. Hum Mol Genet. 2005;14(2):241-253. bia, Canada, and at the 63rd Annual Convention of the 24. Konradi C, Eaton M, MacDonald ML, Walsh J, Benes FM, Heckers S. Molecular Society of Biological Psychiatry; May 1, 2008; Washing- evidence for mitochondrial dysfunction in bipolar disorder. Arch Gen Psychiatry. ton, DC. 2004;61(3):300-308. 25. Washizuka S, Iwamoto K, Kakiuchi C, Bundo M, Kato T. 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Correction

Figure Parts Mislabeled. In the article titled “Mitochon- drial Complex I Activity and Oxidative Damage to Mi- tochondrial Proteins in the Prefrontal Cortex of Pa- tients With Biopolar Disorder,” by Andreazza et al, published in the April issue of the Archives (2010;67[4]: 360-368), the first (A) and second (B) graphs of Figure 4 on page 365 should be switched. The legend is correct.

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