Mechanisms Underpinning the Polypharmacy Effects of Medications in Psychiatry

Mechanisms Underpinning the Polypharmacy Effects of Medications in Psychiatry Background: Bipolar disorder is a mental health condition with progressive social and cognitive function disturbances. Most patients’ treatments are based on polypharmacy, but with no biological basis and little is known of the drugs’ interactions. The aim of this study was to analyze the effects of lithium, valproate, quetiapine, and lamotrigine, and the interactions between them, Significance Statement Box Sample on markers of inflammation, bioenergetics, mitochondrial function, and oxidative stress in neuron-like cells and microglial cells.  Methods: Neuron-like cells and lipopolysaccharide-stimulated C8-B4 cells were treated with lithium (2.5  mM), valproate (0.5  mM), quetiapine (0.05  mM), and lamotrigine (0.05  mM) individually and in all possible combinations for 24  h. Twenty cytokines were measured in the media from lipopolysaccharide-stimulated C8-B4 cells. Metabolic flux analysis was used to measure bioenergetics, and real-time PCR was used to measure the expression of mitochondrial function genes in neuron- like cells. The production of superoxide in treated cells was also assessed.  Results: The results suggest major inhibitory effects on proinflammatory cytokine release as a therapeutic mechanism of these medications when used in combination. The various combinations of medications also caused overexpression of PGC1α and ATP5A1 in neuron-like cells. Quetiapine appears to have a proinflammatory effect in microglial cells, but this was reversed by the addition of lamotrigine independent of the drug combination. Conclusion: Polypharmacy in bipolar disorder may have antiinflammatory effects on microglial cells as well as effects on mitochondrial biogenesis in neuronal cells. Keywords: psychiatry, bipolar disorder, polypharmacy, inflammation, mitochondrial function Received: July 20, 2017; Revised: January 18, 2018; Accepted: February 16, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 582 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Bortolasci et al. | 583 Significance Statement This original research aimed to expand knowledge on why polypharmacy works better to treat bipolar disorder than individual drugs. The cellular and molecular basis for altered brain function in patients with bipolar disorder remains poorly understood. Accordingly, mechanisms underlying the molecular effects of drugs used to treat this disorder are largely unknown. Using neu- ronal like cells and microglial cells, we accessed the effects of commonly prescribed bipolar drugs alone and in associations to find out possible mechanism of action of this combinations and create new targets for drug discovery in this field. Mitochondrial dysfunction is noted in BD driving impaired brain Introduction energy metabolism. There is evidence of increased basal metabolic Bipolar disorder (BD) is a potentially neuroprogressive, chronic rate in mania (Caliyurt and Altiay, 2009) and decreased bioenerget- mental health condition characterized by alternate episodes ics in depression, concordant with a biphasic model of energy gen- of mania and depression that has secondary social and cogni- eration in BD paralleling symptomatology. There are high rates of tive consequences. This disorder is ranked in the top 10 lead- comorbidity of BD with mitochondrial diseases, and mood stabiliz- ing causes of lifelong disability worldwide according the World ers have documented effects on mitochondria. Mitochondrial dys- Health Organization and affects approximately 2% of the popu- function can increase production of ROS, leading to enhanced OS, lation (Duong et al., 2016Rihmer et  ; al., 2016). The pathophysi- causing deleterious consequences on signal transduction, synaptic ology of BD is poorly understood. Most useful psychotropic plasticity, and cellular resilience (de Sousa et al., 2014). medications produce maximal responses after several weeks, Understanding the actions of medications used to treat BD and this observation indicates that chronic adaptive molecular on these seemingly diverse yet interacting pathways may con- changes are crucial components of successful treatment rather tribute to our understanding of the underlying pathology of the than simple immediate receptor binding effects (Rizig et  al., disease and also allow new, more effective, and targeted treat- 2012). Operant biological pathways may include mitochondrial ments to be identified. The aim of this study was to augment the dysfunction, alterations in circulating levels of proinflamma- understanding of the mechanisms of action of these medica- tory cytokines, neurotrophins, and oxidative stress (OS) (Bhat tions and, especially, the cumulative effects of these drugs in et al., 2015). combination. Therefore, we tested the effects of combinations Polypharmacy can be defined as the concurrent use of of lithium, valproate, quetiapine, and lamotrigine on markers of multiple medications. For individuals with BD, these typically inflammation, bioenergetics, mitochondrial function, and OS in include agents such as mood stabilizers, atypical antipsychot- neuron-like cells and microglial cells. These drugs were chosen ics, antidepressants, and benzodiazepines (Adli et  al., 2005). from diverse pharmacological classes to represent the mecha- The majority of patients treated for BD receive multiple psy- nism of actions of various medications used to treat psychiatric chotropic medications concurrently (Goldberg et al., 2009 Sac ; hs disorders, including BD. et al., 2014). The Systematic Treatment Enhancement Program for Bipolar Disorder showed that monotherapy is applicable to <20% of the patients (Goldberg et al., 2009). Polypharmacy in the Materials And Methods management of BD is common given the characteristics of the disorder (e.g., chronicity, comorbidity, uncertainty associated NT2 Cell Culture with shifting polarity of illness), characteristics of the drugs (delayed time of action, tolerability, selectivity), and the high NTera2/cloneD1  (NT2) human teratocarcinoma cells (ATCC) frequency of inadequate response to monotherapy (Sachs et al., were cultured in media comprising Dulbecco’s modified Eagle’s 2014). Nevertheless, little is known regarding the drug interac- Medium (Life Technologies) including 10% fetal bovine serum tions or potential cumulative effects, and there is no clear bio- (Thermo Fisher Scientific), and 1% antibiotic/antimycotic solu- logical basis for the selection of drug combinations to improve tion (Life Technologies). These cells are known to express a efficacy. Medications prescribed for BD may have plentiful drug- neuron-like phenotype following retinoic acid-induced dif- drug interactions, raising safety concerns about polypharmacy ferentiation (Pleasure et  al., 1992). To achieve that, the cells -5 and reiterating the need for biologically based evidence (Tsai were treated with retinoic acid (Sigma-Aldrich) at 1 x 10 M for et al., 2014). 28 days with media refreshed every 2 to 3 days. Neuronal mark- The therapeutic effects of some medications used in the ers such as NeuroD (neuronal differentiation), GluR (glutamate management of the disease have recently been suggested to receptor), and Tau (cytoskeletal protein) were measured by real- be related to both mitochondrial and inflammatory mecha- time PCR (RT-PCR) to confirm the neuronal-like state of the cells nisms; however, current literature on this topic is scant. (data not shown) (Megiorni et al., 2005). Cells were seeded onto Notwithstanding this, both central and peripheral inflamma- 24-well and 96-well plates coated with 10  μg/mL poly-D-lysine tory processes seem to play a role in the pathophysiology of BD (Sigma-Aldrich) and 10 μg/mL laminin (Sigma-Aldrich) at 2 × 10 (Berk et al., 2011). Suppression of microglia-mediated inflamma- cells/well (24-well plates) and 8  ×  10 cells/well (96-well plates tion has been proposed as a strategy in therapy as prolonged and 24-well XF24 cell culture microplates, Seahorse Bioscience) immune activation can damage neuronal structure through the with further addition of mitotic inhibitors (1  µM cytosine and production of elevated levels of reactive oxygen species (ROS) 10 µM uridine; Sigma-Aldrich) for a total of 7  days with media and neurotoxins (Block et al., 2007Czeh et  ; al., 2011). Activation refreshed every 2 to 3 days to maintain an enriched culture of of inflammatory pathways and alterations in glutamate metab- differentiated neuronal cells (NT2-N) for the drug treatments. olism appear to converge on glial cells and may play a role in mood disorders. In particular, inflammatory mediators might C8-B4 Cell Culture Conditions act through glial cells to regulate extracellular glutamate con- C8-B4 microglial cells (ATCC CRL-2540) were cultured in centrations in both physiological and pathological conditions (Haroon et al., 2017). Dulbecco’s modified Eagle’s Medium (Life Technologies) with Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 584 | International Journal of Neuropsychopharmacology, 2018 10% fetal bovine serum (Life Technologies) and seeded onto The experiments were carried out in a PikoRealTM qPCR 24-well plates at 1.3 x 10 cells/well for the measurement of machine (Thermo Fisher Scientific) using the following protocol: cytokine release. 95ºC for 7 minutes, 4 cycles of 95ºC for 30 seconds and 60ºC for 1 minute and then data acquisition, 60ºC for 30 seconds, 55ºC to 95ºC, data acquisition and 20ºC for 10 seconds. Resultant melt Drug Treatments curves were used as an indicator of amplification specificity. The The NT2-N cells were treated with lithium (2.5 mM), valproate Quant-iT OliGreen ssDNA Assay Kit (Life Technologies) was used (0.5 mM), quetiapine (0.05 mM), and lamotrigine (0.05 mM) indi- to quantify the cDNA concentration in each sample as per the vidually and in all possible combinations. All drugs were pur - manufacturer’s instructions. Gene expression data were quanti- chased from Sigma-Aldrich. C8-B4 cells were stimulated with fied using the 2ΔCT method normalized to the derived cDNA 1  ng/mL lipopolysaccharide (LPS) per well and then received concentration of each sample. the same treatments as for NT2-N cells. Vehicle control cells were treated with an equal volume of MilliQ water for lithium Mitochondrial Function Quantitation or valproate controls, 0.2% DMSO for lamotrigine or quetiapine controls, or the combination of both (for various drug combina- The cellular bioenergetic profile of NT2-N-treated cells was tions as required). Dose response studies were performed to find assessed using a Seahorse XF24 Flux Analyzer (Seahorse optimal doses to balance the effects of each individual drug on Bioscience). Three basal oxygen consumption rate (OCR) meas- the expression of a number of candidate gene targets, such that urements were performed, and measurements were repeated the effect of one drug did not dominate the overall effect and did following injection of oligomycin (1  mM), carbonyl cyanide- not affect viability of the cells (data not shown). Each treatment 4-(trifluoromethoxy)phenylhydrazone (1  mM) and Antimycin had n = 6 replicates. A  (1  mM). Basal extracellular acidification rate (ECAR) was Following the 24-h treatment, the cells were harvested and determined from data collected at basal measurement points. RNA was extracted using RNeasy mini kits (Qiagen) and reverse- Calculations of respiratory parameters of mitochondrial func- transcribed to produce cDNA using Maxima H Minus first strand tion were performed as previously described (Martin et al., 2014). cDNA synthesis kit (Thermo Fisher Scientific) following the The protein concentration from each well was quantified by manufacturer’s instructions. Pierce BCA protein assay (Thermo Fisher Scientific) to account for differences in cell density during data analysis. Each treat- ment had n= 4 to 6 replicates. Inflammatory Biomarkers A panel of 20 cytokines (interleukin [IL]-1a, IL-1b, IL-3, IL-4, IL-5, Mitochondrial Superoxide Production IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17a, Eotaxin, granulo- cyte-colony stimulating factor [GCSF)], granulocyte macrophage The production of superoxide in NT2-N cells was measured colony-stimulating factor, interferon-gamma, keratinocyte che- using MitoSOXTm (Thermo Fisher Scientific) according to the moattractant, chemokine ligand 5, tumor necrosis factor-alpha) manufacturer’s instructions. The increase in fluorescence inten- was measured with a Bioplex Pro mouse cytokine assay kit sity was measured with a FlexStation II Scanning Fluorometer (Bio-Rad) according to the manufacturer’s protocol, using media (Molecular Devices; λex = 510 nm,λ em = 580 nm). Results are pre- from LPS-stimulated C8-B4 cells following 24-h drug treatments sented relative to vehicle treated cells. and normalized by the protein content (PierceTM BCA protein assay kit; Thermo Fisher Scientific). Statistical Analysis Shapiro-Wilk (inflammatory biomarkers) or Kolmogorov- Gene Expression Smirnov (all the remain analysis) tests were used to check data RT-PCR was used to measure the expression of specific genes sets for normality of distribution. In addition, biomarker data relating to mitochondrial function in NT2-N cells following were visually inspected using box-plots for presence of outli- drug treatments: peroxisome proliferator-activated receptor γ ers and influential data. Levene’s test was used to determine coactivator-1α (PGC1α: forward primer, AAC CAC ACC CAC AGG whether equal variances could be assumed between groups. ATC AGA; reverse primer, TCT TCG CTT TAT TGC TCC ATG A; Since there were no outliers in biomarker data, and there was efficiency: 95.30%), NADH:ubiquinone oxidoreductase subunit no alarming heterogeneity, parametric tests were performed for B8 (forward primer, CAG CCT CCC ACA TGA CCA AG; reverse biomarker data analyses. primer, GCC ATC ATC CGG GTA AGG TT; efficiency: 94.52%), For the mitochondrial function and oxidative stress analysis, succinate dehydrogenase subunit B (forward primer, ATG TGG drug treatment groups were compared against their respective CCC CAT GGT ATT GG; reverse primer, TGG TGT CAA TCC TTC controls using independent samples t tests. Differences were GGG TG; efficiency: 96.70%), ubiquinol-cytochrome C reductase considered statistically significant when P ≤ .05. core protein II (forward primer, GGG AAA GTG TTA GCG GGG The cytokine levels and mitochondrial gene expression were AA; reverse primer, TGG CTT TAA CTT TGG GGG CA; efficiency: analyzed using 1-way ANOVA followed by pairwise compari- 97.31%), mitochondrially encoded cytochrome C oxidase II (for - sons. For each ANOVA test the overall F-test significant level of ward primer, CCG TCT GAA CTA TCC TGC CC; reverse primer, 0.05 was considered. The 1-way ANOVA tests have 7 numerator GAG GGA TCG TTG ACC TCG TC; efficiency: 95.94%), ATP syn- degrees of freedom (df); we are therefore entitled to investigate thase, H+ transporting, mitochondrial F1 complex, alpha sub- 6 (i.e., 1-df) orthogonal contrasts of the treatment means with- unit 1 (ATP5A1: forward primer, TCA GTC TAC GCC GCA CTT out the need to consider the so-called “family-wise” Type I error AC; reverse primer, ATG TAC GCG GGC AAT ACC AT; efficiency: rate for these multiple t tests. However, due to a relatively large 89.63%), and hexokinase II (HK2: forward primer, CCA ACC TTA number of pairwise comparisons (4 comparisons) within each GGC TTG CCA TT; reverse primer, CTT GGA CAT GGG ATG GGG ANOVA, the significance level for pairwise between group com- TG; efficiency: 104.16%). parisons was set at P ≤ .01. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Bortolasci et al. | 585 Statistical analysis was performed using Statistical Package Mitochondrial Gene Expression for the Social Sciences version 22 (SPSS) software. Table  2 shows an overview of expression, relative to control groups, of genes related to mitochondrial function in treated Results NT2-N cells. ATP5A1 expression was significantly increased following most treatments (except for lithium, lamotrigine, Inflammation Biomarkers valproate/quetiapine, lamotrigine/quetiapine, and lithium/val- proate/lamotrigine). PGC1α expression was increased by most Microglial cells are the resident macrophages of the central treatments, including a 60% increase in response to treatment nervous system. They perform key signaling and scavenging with the combination of all 4 drugs. HK2, succinate dehydroge- roles, interact with neurotransmission, and mediate neuro- nase subunit B, mitochondrially encoded cytochrome C oxidase plasticity. Microglia are activated by various neuropathological II, NADH:ubiquinone oxidoreductase subunit B8, and ubiquinol- states and can, in response, secrete proinflammatory cytokines cytochrome C reductase core protein II expression had minor (Derecki and Kipnis, 2013). LPS strongly upregulated the secre- changes across the different treatments. The RT-PCR data sug- tion of most cytokines measured from C8-B4 cells (Figure 1). The gest a tendency for reduced expression of genes related to effects of the tested drugs on the release of cytokines from LPS- mitochondrial function in cells treated with lithium relative to stimulated C8-B4 cells are shown in Table  1. The inflammatory control. This is consistent with the aerobic respiration data of role of microglia in the brain justifies the use of this cell line to lithium-treated cells, which appeared to be reduced (Figure 3A). assess the effects of these drugs on inflammation. Quetiapine significantly increased mRNA levels of ATP5A1. This Lithium (alone) had no effect on cytokine release from LPS- result was unexpected given that flux bioanalysis revealed sig- stimulated C8-B4 cells. Valproate and lamotrigine alone had nificantly decreased basal respiratory rate in cells treated with relatively minor effects. Valproate and lamotrigine had no sig- quetiapine. nificant effect on cytokine levels. These data suggest that these drugs when acting alone have little overall effect on cytokine release from activated glial cells. Quetiapine, however, robustly Mitochondrial Function increased the release of all cytokines measured except for GCSF, Figure  2 shows an overview of mitochondrial function and suggesting a proinflammatory effect on glial cells. Lithium com- basal bioenergetics in NT2-N treated cells. Lithium-treated cells bined with either lamotrigine or quetiapine slightly increased (n = 6) exhibited a 27% lower basal respiratory rate (P = .032) and cytokine release, suggesting a proinflammatory effect. Valproate a 25% lower rate of ATP turnover (P = .032) relative to the con- when combined with either lamotrigine or quetiapine also trol group (n = 5) (Figure  3A). The cells also appeared to exhibit increased release of some cytokines, suggesting an overall pro- increased basal and maximal glycolytic rates, which is consist- inflammatory effect. On the other hand, the combination of ent with decreased capacity for aerobic respiration as shown in lamotrigine and quetiapine robustly reduced cytokine release, lithium-treated NT2-N cells (Figure  3B). Cells treated with val- suggesting a strong antiinflammatory effect. proate tended to have the same direction of changes, although When 3 drugs were used in combination, a clear pattern these were not significant. Quetiapine appeared to decrease emerged. The combinations of lithium/valproate/lamotrigine respiratory activity and capacity in NT2-N cells. Quetiapine- and lithium/valproate/quetiapine increased cytokine produc- treated cells (n = 6) exhibited a 20.1% lower basal respiratory rate tion and release, while the combinations of lithium/lamotrig- (P = .026) compared with the control group (n = 4) ( Figure 3A). ine/quetiapine and valproate/lamotrigine/quetiapine robustly Conversely, lamotrigine was the only drug tested that tended reduced cytokine release. The 4 drugs used in combination also to increase respiratory rate and capacity. However, the variabil- robustly lowered cytokine release. ity in the data rendered these increases statistically insignifi- cant. Basal and maximal glycolytic rates appeared to be reduced in cells treated with quetiapine and lamotrigine; however, no significant differences were found compared with control cells. Cells simultaneously treated with lithium and quetiapine exhibited a 31% lower basal respiratory rate (P = .029) and a 32% lower rate of ATP turnover (P = .041) compared with the control group (Figure  3C), with no significant change in basal or maxi- mal glycolytic rate (Figure 3D). No difference was found in the OCR values, basal or maxi- mal glycolytic rates following treatment with the various com- binations of 3 or 4 drugs relative to the control groups (Figure 2). Overall, using the drugs in combinations appeared to suppress any effect on mitochondrial function caused by the treatment with individual drugs in NT2-N cells. Mitochondrial Superoxide Production Figure  1. Effects of lipopolysaccharide (LPS) on C8-B4 cells (fold change rela- Mitochondrial superoxide is the major intracellular source of tive to vehicle treated cells). LPS treatment increased the release of a number ROS. Cells treated with lithium plus quetiapine showed signifi- of proinflammatory cytokines, including interleukin (IL)-1a, IL-3, IL-4, IL-5, IL-6, cantly decreased superoxide production (P = .05), and the com- IL-12, GCSF, KC, RANTES, and tumor necrosis factor (TNF)α. These data confirm bination of lamotrigine and quetiapine increased superoxide that LPS had a proinflammatory effect on C8-B4 cells. n = 3 samples in duplicate. production (P = .027) (Figure 4). *P < .05 compared with vehicle treated cells (descriptive statistical test). Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 586 | International Journal of Neuropsychopharmacology, 2018 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Table 1. Effects of BD Drugs Alone and in Combination on Cytokine Production and Release from C8-B4 Cells IL-1A IL-1B IL-3 IL-4 IL-5 IL-6 IL-10 IL-12(P40) IL-12(P70) Li 99.67 ± 0.12 94.42 ± 0.12 96.77 ± 0.25 93.29 ± 0.16 86.89 ± 0.16 106.85 ± 0.39 95.94 ± 0.16 76.99 ± 0.28 95.57 ± 0.20 Val 78.68 ± 0.04 81.67 ± 0.00 63.14 ± 0.03 74.09 ± 0.04 68.44 ± 0.04 47.46 ± 0.03 71.98 ± 0.04 20.93 ± 0.03 72.38 ± 0.08 Lam 166.07 ± 0.17 87.40 ± 0.05 166.38 ± 0.26 86.43 ± 0.06 84.70 ± 0.07 37.37 ± 0.07 98.02 ± 0.07 55.78 ± 0.18 83.86 ± 0.10 Quet 284.69 ± 0.23 140.47 ± 0.02* (P = .004) 496.43 ± 0.28* (P < .001) 182.73 ± 0.09* (P = .003) 223.20 ± 0.03* (P < .001) 277.90 ± 0.22* (P = .006) 192.63 ± 0.07* (P = .004) 917.69 ± 1.80* (P < .001) 289.46 ± 0.34* (P = .003) Li/Val 96.25 ± 0.02 101.62 ± 0.01 101.19 ± 0.05 98.38 ± 0.03 93.40 ± 0.04 82.07 ± 0.13 97.12 ± 0.02 30.49 ± 0.04 88.83 ± 0.05 Li/Lam 299.46 ± 0.61* (P = .010) 114.37 ± 0.03 313.87 ± 0.41 131.09 ± 0.19 158.86 ± 0.10 382.98 ± 0.46* (P < .001) 14015 ± 0.18 521.52 ± 1.82 224.56 ± 0.54 Li/Quet 50.07 ± 0.02 111.53 ± 0.07 41.26 ± 0.02 126.77 ± 0.15 210.69 ± 0.41 233.86 ± 0.52 123.58 ± 0.05 305.26 ± 0.68 153.00 ± 0.17 Val/Lam 47.41 ± 0.00 115.47 ± 0.07 46.10 ± 0.06 113.89 ± 0.08 191.64 ± 0.03 259.39 ± 0.10 104.89 ± 0.06 125.90 ± 0.19 119.98 ± 0.13 Val/Quet 49.70 ± 0.02 110.76 ± 0.07 37.26 ± 0.02 106.34 ± 0.06 197.85 ± 0.35 220.97 ± 0.55 114.08 ± 0.09 155.10 ± 0.48 155.63 ± 0.24 Lam/Quet 18.17 ± 0.01* (P < .001) 76.13 ± 0.02 66.58 ± 0.21* 70.64 ± 0.17* (P < .001) 57.27 ± 0.04* (P < .001) 15.78 ± 0.03* (P < .001) 58.12 ± 0.02* (P = .002) 31.68 ± 0.03* (P < .001) 37.65 ± 0.02 Li/Val/Lam 94.17 ± 0.14 149.15 ± 0.06* (P = .006) 79.13 ± 0.16* (P = .001) 173.92 ± 0.25* (P = .008) 427.60 ± 0.27* (P < .001) 634.05 ± 0.72* (P < .001) 164.93 ± 0.17 460.83 ± 0.94 287.94 ± 0.45* (P = .010) Li/Val/Quet 50.58 ± 0.07 130.30 ± 0.12 76.15 ± 0.16 163.64 ± 0.29 372.68 ± 0.30* (P < .001) 337.26 ± 0.31 143.90 ± 0.20 331.43 ± 0.76 236.05 ± 0.68 Li/Lam/Quet 15.74 ± 0.01* (P < .001) 68.84 ± 0.05* (P = .002) 45.31 ± 0.07* (P < .001) 49.75 ± 0.07* (P < .001) 55.64 ± 0.02* (P < .001) 16.87 ± 0.02* (P < .001) 54.46 ± 0.06* (P = .001) 23.85 ± 0.06* (P < .001) 27.02 ± 0.07* (P < .001) Val/Lam/Quet 16.39 ± 0.00* (P < .001) 76.92 ± 0.00 47.21 ± 0.07* (P = .003) 53.25 ± 0.04* (P = .001) 61.55 ± 0.03* (P = .001) 12.12 ± 0.01* (P < .001) 59.08 ± 0.02* (P = .003) 15.69 ± 0.02* (P < .001) 39.20 ± 0.02* P = .002) Li/Val/Lam/Quet 17.41 ± 0.02* (P < .001) 77.08 ± 0.06 63.33 ± 0.10 61.99 ± 0.06* (P = .002) 67.37 ± 0.03* (P = .008) 19.32 ± 0.02* (P < .001) 62.17 ± 0.11* (P = .007) 24.68 ± 0.07* (P < .001) 45.98 ± 0.13* (P = .008) IL-13 IL-17A EOTAXIN GCSF GMCSF IFNG KC RANTES TNFΑ Li 101.51 ± 0.15 98.28 ± 0.20 99.28 ± 0.33 84.31 ± 0.27 99.09 ± 0.23 99.54 ± 0.14 104.63 ± 0.25 68.30 ± 0.20 129.32 ± 0.49 Val 80.42 ± 0.07 71.37 ± 0.04 44.92 ± 0.05 30.59 ± 0.01 66.43 ± 0.06 81.09 ± 0.03 62.56 ± 0.05 60.71 ± 0.08 33.58 ± 0.03 Lam 96.10 ± 0.04 80.87 ± 0.09 87.09 ± 0.41 45.79 ± 0.07 117.27 ± 0.30 113.38 ± 0.09 85.67 ± 0.10 72.67 ± 0.25 58.67 ± 0.09 Quet 182.05 ± 0.08* (P = .004) 163.03 ± 0.09 209.88 ± 0.05 120.48 ± 0.07 207.34 ± 0.01 181.82 ± 0.08 297.50 ± 0.23* (P < .001) 1928.86 ± 7.1* (P = .007) 753.62 ± 1.00* (P = .006) Li/Val 103.13 ± 0.01 107.20 ± 0.01 125.82 ± 0.05 48.74 ± 0.03 118.33 ± 0.01 106.99 ± 0.01 101.24 ± 0.03 67.98 ± 0.12 88.49 ± 0.11 Li/Lam 148.23 ± 0.12 109.34 ± 0.11 76.98 ± 0.07 66.26 ± 0.08 118.52 ± 0.14 129.22 ± 0.12 223.62 ± 0.27* (P = .009) 801.71 ± 3.85 750.64 ± 2.58* (P = .006) Li/Quet 143.54 ± 0.06 121.81 ± 0.01 304.55 ± 1.01 52.62 ± 0.06* (P = .002) 204.03 ± 0.20 143.19 ± 0.02 186.98 ± 0.46 253.87 ± 0.68 159.99 ± 0.89 Val/Lam 116.51 ± 0.07 100.25 ± 0.10 308.97 ± 0.55 49.98 ± 0.01* (P = .002) 153.81 ± 0.29 115.64 ± 0.04 103.06 ± 0.04 213.46 ± 0.14 116.15 ± 0.11 Val/Quet 126.97 ± 0.13 121.44 ± 0.11 166.89 ± 0.24 34.93 ± 0.03* (P < .001) 138.71 ± 0.23 130.52 ± 0.23 110.98 ± 0.10 273.76 ± 0.86 42.50 ± 0.23 Lam/Quet 70.77 ± 0.03 47.69 ± 0.02* (P = .001) 122.50 ± 0.25 8.64 ± 0.00* (P < .001) 28.39 ± 0.10* (P < .001) 46.57 ± 0.05* (P = .007) 40.19 ± 0.04* (P < .001) 13.92 ± 0.01* (P = .001) 1.55 ± 0.00* (P < .001) Li/Val/Lam 204.12 ± 0.21 168.25 ± 0.17 347.42 ± 0.58 83.98 ± 0.03 315.84 ± 0.35* (P < .001) 237.39 ± 0.27* (P = .003) 470.21 ± 0.18* (P < .001) 1360.39 ± 5.4 2377.16 ± 6.1* (P < .001) Li/Val/Quet 169.39 ± 0.34 149.17 ± 0.27 312.24 ± 0.28 48.79 ± 0.10* (P = .001) 233.78 ± 0.26 190.51 ± 0.40 307.22 ± 0.39* (P < .001) 984.03 ± 2.67 821.81 ± 2.47 Li/Lam/Quet 52.99 ± 0.02 42.00 ± 0.06* (P < .001) 103.07 ± 0.24 7.36 ± 0.01* (P < .001) 39.84 ± 0.09* (P < .001) 38.54 ± 0.08* (P = .001) 63.56 ± 0.03* (P = .010) 12.38 ± 0.02* (P = .001) 1.36 ± 0.00* (P < .001) Val/Lam/Quet 68.71 ± 0.03 51.72 ± 0.02* (P = .002) 120.55 ± 0.21 6.47 ± 0.00* (P < .001) 44.78 ± 0.02* (P < .001) 53.98 ± 0.05 35.16 ± 0.01* (P < .001) 16.86 ± 0.01* (P = .002) 0.82 ± 0.00* (P < .001) Li/Val/Lam/Quet 73.88 ± 0.13 56.90 ± 0.11* (P = .006) 124.74 ± 0.40 7.06 ± 0.01* (P < .001) 51.91 ± 0.06* (P = .002) 53.76 ± 0.15 64.14 ± 0.06 (P = .010) 26.05 ± 0.08* (P = .007) 2.34 ± 0.01* (P < .001) Abbreviations: BD, bipolar disorder; Lam, lamotrigine; Li, lithium; Quet, quetiapine; Val, valproate. Percent of vehicle treated cells + SEM. =n 3 samples in duplicate e xcept for DMSO vehicle, n = 6. One-way ANOVA and pairwise comparison corrected for multiple testing *P < .01 compared with vehicle treated cells. Bortolasci et al. | 587 Table 2. Effects of BD Drugs Alone and in Combination on Mitochondrial Gene Expression in NT2-N Cells SDHB ATP5A1 MTCO2 NDUFB8 HKII UQCRC2 PGC1α Li 95.28 ± 20.34 111.58 ± 16.03 73.14 ± 17.20 89.63 ± 19.27 92.90 ± 14.42 96.70 ± 10.81 95.04 ± 10.12 Val 130.31 ± 6.22 189.18 ± 36.41* (P = .009) 146.88 ± 6.09* (P = .003) 85.35 ± 12.20 116.52 ± 3.48 60.11 ± 11.49 145.29 ± 11.45* (P = .010) Quet 88.54 ± 4.48 333.82 ± 11.18* (P < .001) 115.59 ± 6.28 92.85 ± 3.50 111.29 ± 3.35 90.31 ± 5.39 97.79 ± 5.63 Lam 84.38 ± 6.11 139.43 ± 18.42 103.98 ± 18.98 103.84 ± 4.24 128.31 ± 9.04 89.21 ± 5.12 130.57 ± 8.73* (P = .010) Li/Val 122.94 ± 14.29 140.81 ± 11.42* (P = .010) 113.40 ± 14.60 97.71 ± 7.20 104.36 ± 9.01 128.54 ± 17.04 142.28 ± 14.90 Li/Quet 91.29 ± 3.68 594.17 ± 74.09* (P = .010) 92.44 ± 15.00 87.45 ± 9.04 93.09 ± 8.00 104.87 ± 6.49 102.27 ± 5.97 Li/Lam 80.55 ± 7.12 163.76 ± 29.11* (P = .010) 107.68 ± 12.61 99.44 ± 8.38 92.41 ± 11.20 101.56 ± 10.76 91.22 ± 8.04 Val/Quet 87.46 ± 8.56 126.54 ± 22.67 99.96 ± 6.87 90.17 ± 7.06 111.91 ± 3.20 84.75 ± 5.57 126.54 ± 2.15 Val/Lam 76.29 ± 14.44 143.55 ± 41.98* (P = .010) 98.44 ± 22.62 95.52 ± 18.96 92.19 ± 6.83 88.65 ± 12.37 105.81 ± 13.26 Lam/Quet 146.37 ± 33.53 442.98 ± 141.97 118.50 ± 9.33 118.53 ± 30.37 123.96 ± 10.45 130.47 ± 36.14 126.96 ± 12.46 Li/Val/Quet 105.50 ± 1.58 454.76 ± 2.10* (P = .005) 95.56 ± 7.61 99.48 ± 1.68 113.09 ± 4.45 86.29 ± 2.85 153.87 ± 6.13* (P = .010) Li/Val/Lam 104.45 ± 18.49 216.69 ± 6.84 115.12 ± 20.09 94.76 ± 18.72 125.50 ± 13.05 95.23 ± 17.46 142.63 ± 19.53 Li/Quet/Lam 95.93 ± 4.62 362.43 ± 23.76* (P = .010) 71.97 ± 15.57 91.38 ± 3.55 95.33 ± 5.89 86.88 ± 6.46 117.39 ± 5.52 Val/Quet/Lam 93.44 ± 3.71 424.39 ± 50.41* (P = .010) 93.41 ± 7.96 88.83 ± 4.06 118.81 ± 5.90 78.79 ± 6.25 148.32 ± 5.87 Li/Val/Quet/Lam 98.26 ± 9.88 575.31 ± 51.74* (P = .002) 124.89 ± 13.45 98.78 ± 4.24 104.03 ± 14.95 98.09 ± 5.31 160.34 ± 9.45* (P < .001) Abbreviations: BD, bipolar disorder; Lam, lamotrigine; Li, lithium; Quet, quetiapine; Val, valproate. Percent of vehicle treated cells + SEM. n = 6 samples in duplicate . *P < .05 compared with vehicle treated cells (descriptive statistical test). Considering every drug separately in BD patients could offer a Discussion clearer picture of medication effect on cytokines, but that will The major finding in this study is the broadly antiinflamma- be limited to small sample sizes. Analyzing these effects in cell tory effects of polypharmacy in BD, as opposed to monotherapy, culture can clarify the possible mechanism of action of these where less consistent patterns were seen. BD is associated with drugs. In addition, it should be pointed out that the effects of the inflammation in the periphery and in the central nervous sys- drugs in the central nervous system may differ from the periph- tem, and there is increasing evidence to suggest that activation eral effects in circulating cells, that way being more informative of microglia is a key element in this process (Block et al., 2007; than the systemic concentrations of these cytokines. Reus et  al., 2015). Microglial proinflammatory cytokine release Quetiapine was the most cytokine-active drug tested in our can lead to oligodendrocyte cell death by apoptosis, blood brain study, showing strong proinflammatory effects that could in barrier destruction, neuronal damage, and mitochondrial impair - theory contribute to the low-level inflammatory state during ment (Naaldijk et al., 2016). Our results demonstrated effects on antipsychotic treatment reported in previous studies (Sarvari proinflammatory cytokine release that may play a role in the et al., 2014). However, when combined, especially with lamotrig- therapeutic mechanisms of some commonly prescribed drugs ine, this effect was suppressed and an antiinflammatory effect when used in combination. To our knowledge, this is the first appeared to emerge. study investigating differences in cytokine production between Multiple drug combinations that include both lamotrigine these drugs alone and in combination in a microglial cell line. and quetiapine appeared to have strong antiinflammatory It should be noted that this study was conducted in a mouse effects on glial cells. The reduction of proinflammatory cytokines microglial cell line, and it would be interesting to see whether observed in this study could reduce the activated state of micro- these results can be replicated in human microglial cells. The glial cells. It has been recently proposed that glial inflammation results are indicative of additive antiinflammatory effects of spe- can lead to increased release of glutamate into the extrasynaptic cific psychopharmacological combinations on cytokine produc- space by decreasing the capacity of glial transporters to buffer tion, which could provide a protective effect in neurons. and clear glutamate (Haroon et al., 2017). The effects of inflam- The literature regarding human cytokine levels after treat- mation on glial cells can further affect glutamate neurotrans- ment for BD is conflicting. Some studies showed a reduction in mission. Our data suggest that the antiinflammatory effects IFN-γ peripherally within 6 weeks of treatment (Kim et al., 2007; from the combination of lamotrigine with quetiapine may be Uyanik et al., 2015). Recently, 2 meta-analyses on the effects of driven by lamotrigine playing a pivotal role in the inhibition of medications on cytokine profile were published. Modabbernia glutamate release in glial cells. et  al. (2013) in a meta-analysis provided evidence for a lack of Considering antiinflammatory effects, in an association of medication effect on plasma concentrations of IL-2, IL-4, sIL-6R, lithium plus valproate, it is important to add lamotrigine and and INF-γ (Modabbernia et al., 2013). In another meta-analysis, quetiapine rather than just one of them. Both drugs together Munkholm et  al. (2013) reviewed 10 studies on the association had the power to shift the direction of the effect, which did not between plasma cytokine levels and medicines; of these, 6 stud- happen when they are added individually to the lithium plus ies did not find any significant correlation between medication valproate combination. status and cytokine levels, and 4 studies demonstrated correla- Our results demonstrate differential effects of these particular tions for individual cytokine parameters, of which none were four drugs, alone and in combinations, on inflammatory processes. replicated (Munkholm et al., 2013Ric ). ken et al. (2018) recently Given that inflammation is thought to play a role in BD patho- published a study showing the treatment with lithium does not physiology, at least in some patients, these data could provide a change cytokine serum levels (IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, foundation for developing a new treatment strategy to improve INF-γ), even after augmentation (Ricken et al., 2018). In addition, outcomes in some BD patients. It should be noted that lithium a systematic review showed 2 studies that report no effect of alone, which does not appear to affect cytokine release, is effica- valproate on cytokine levels (van den Ameele et al., 2016). cious in some patients, suggesting that antiinflammatory activity It is important to note that most studies analyzed a het- is not required in some patients but is likely required for efficacy in erogeneous group of patients regardless of medication status. others. The concept that a component of the mechanism of action Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 588 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Mitochondrial function and basal bioenergetics in NT2-N treated cells. Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of NT2-N cells exposed for 24 h to either vehicle or bipolar disorder (BD) drugs alone and in combinations. All values are reported as perecent of vehicle ± SEM (n = 6/group). Figure  3. Aerobic respiration and glycolysis in differentiated NT2-N cells as determined by oxygen consumption rate flux in response to mitochondrial probes (for oxygen consumption rate [OCR]) and by flux bioanalysis (for extracellular acidification rate [ECAR]). Data represented as mean OCR (pmol/min) or ECAR (pM/min) ± SEM; n = 4 to 6 per group. *P < .05 compared with vehicle treated cells (descriptive statistical test). of combination of these drugs includes an antiinflammatory effect energy production via glycolysis, increasing lactate levels and is supported by a recent meta-analysis showing that nonsteroidal pH and leading to production of ROS, glutamate excitotoxicity, antiinflammatory drugs have a moderate antidepressant effect in and apoptosis (Konradi et al., 2004). There is a body of evidence subjects with bipolar disorder (Rosenblat et al., 2016). showing an essential dysfunction of mitochondria, such as Mitochondrial dysregulation associated with decreased oxi- decreases in mitochondrial respiration, high-energy phosphates dative phosphorylation shifts metabolism toward anaerobic and pH; changes in mitochondrial morphology; increases in Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Bortolasci et al. | 589 Figure 4. Effects of bipolar disorder (BP) drugs alone and in combination on the intracellular reactive oxygen species (ROS) production in NT2-N cells (relative to vehicle treated cells). n = 5 samples. *P < .05 compared with vehicle treated cells (descriptive statistical test). mitochondrial DNA polymorphisms; and downregulation of Quetiapine significantly decreased basal respiratory rate in NT2-N nuclear mRNA molecules and proteins involved in mitochon- cells and tended to reduce ATP turnover. But when both drugs were drial respiration (Scaini et al., 2016). Other findings in BD include used together, we observed a significant further reduction in basal the dysregulation of mitochondria-related genes, increase of respiratory rate and ATP turnover in NT2-N cells. Together with lactate in the brain, decrease of complex I in postmortem brains, this, a significant reduction in mitochondrial superoxide produc- abnormal mitochondrial structure in cells of bipolar patients,tion and an incr ease in ATP5A1 gene expression was observed in and elevation of isocitrate in cerebrospinal fluid associated with NT2-N cells treated with lithium plus quetiapine. In contrast, only impaired function of isocitrate dehydrogenase (Kato, 2017). lamotrigine increased respiratory rate and capacity, which is note- Evidence from animal studies and cellular models on the worthy given that clinically it is the most robust antidepressant molecular pharmacology of mood stabilizing drugs has impli- of the drugs studied here. Following the polarity index of drugs, cated mitochondrial energy metabolism as a potential target. anticonvulsants (particularly lamotrigine) seem more effective for There is evidence that lithium and valproate modulate mito- prevention of depressive episodes, while atypical antipsychotics chondrial function by affecting the expression and activity of and lithium may have a manic polarity index of efficacy (Popovic mitochondrial complexes. A recent study showed that valproate et al., 2012). That observed efficacy in mania may be related to the reversed the methamphetamine-induced inhibition of complexes decreased bioenergetics highlighted in this study. I, II, III, and IV in rat brains (Valvassori et al., 2010). Similarly, lith- Quetiapine has previously been reported to have antioxidant ium has been shown to increase the activities of mitochondrial properties (Shao et al., 2005W ; ang et al., 2005), so it is likely that complexes I, II, and III in the frontal cortex tissue of human brains the decrease in basal oxygen consumption seen in the present (Maurer et al., 2009). The activity of complex IV was unaffected, study was associated with decreased ROS production. However, suggesting that lithium selectively increases the activity of spe- this hypothesis of redox modulation by quetiapine was not sup- cific enzymes, rather than acting as a general stimulator of the ported by the results of the mitochondrial superoxide produc- electron transport chain (Maurer et al., 2009). A more recent study tion analysis. This result could possibly be explained by the fact detected no difference in complex I activity between the lympho- that the cells were not under a redox stress condition when blasts of bipolar patients and healthy control subjects (Huzayyin treated, and the drugs were therefore unable to cause a down- et al., 2014). It is proposed that the effects of lithium on complex ward shift in the baseline ROS levels. Our results showed a trend I may be specific to neurons. Quetiapine has been shown to dif- for reduced glycolysis in cells following treatment with quetia- ferentially affect genes for complex I subunits causing upregula- pine or lamotrigine, indicating a potential to reverse mitochon- tion of NDUFV2 and downregulation of NDUFA10 (Young, 2007). drial dysregulation. When both drugs were used together, the This evidence is consistent with the idea that commonly opposite effect was observed, suggesting an increase in mito- prescribed bipolar disorder drugs influence mitochondrial func- chondrial dysregulation confirmed by the significant increase in tion. However, more data are required to demonstrate the precise mitochondrial superoxide production in NT2-N cells followed by nature of this association. The present findings showed relatively the treatment with lamotrigine plus quetiapine. minor changes in mitochondrial function in NT2-N cells after Gene expression was measured for one subunit of each of the treatment with BD drugs. The major finding is the combination of mitochondrial complexes and also measured for HK2, an important lithium and quetiapine affected mitochondrial function in NT2-N- regulator of glycolysis, and PGC1 , a tr α anscription factor involved treated cells compared with vehicle. Lithium significantly reduced in mitochondrial biogenesis. PGC1α is expressed at high levels in basal respiratory rate and ATP turnover in NT2-N cells and tended mitochondria rich cells with high energy demands, such as neu- to reduce other parameters of mitochondrial function as well. rons, and has been shown to be a master regulator of mitochondrial Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 590 | International Journal of Neuropsychopharmacology, 2018 biogenesis and cellular energy metabolism (Wareski et  al., 2009; Foundation, Australian Rotary Health, Stanley Medical Research Markham et  al., 2014). The PGC1 family of coactivators controls Institute, Deakin University, Brazilian Society Mobility Program, mitochondrial density in primary neurons (Wareski et  al., 2009). Lilly, NHMRC, Australasian Society for Bipolar and Depressive The upregulation of PGC1α by the drug combinations observed Disorders, and Sevier. She has also received in kind support in our study could compensate for neuronal mitochondrial loss. from BioMedica Nutracuticals, NutritionCare and Bioceuticals. Enhancing mitochondrial biogenesis regulates cellular oxidative M.B.  has received grant/research support from the NIH, capacity, and this may be useful for neuronal recovery and sur - Cooperative Research Centre, Simons Autism Foundation, Cancer vival in neurodegenerative disorders (Wareski et al., 2009). It would Council of Victoria, Stanley Medical Research Foundation, MBF, also be expected that an increase in PGC1 e αxpression would lead NHMRC, Beyond Blue, Rotary Health, Geelong Medical Research to augmented respiration as a result of increased mitochondrial Foundation, Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, biogenesis, but this effect may not be prominent within 24  h of Meat and Livestock Board, Organon, Novartis, Mayne Pharma, commencing treatment. Overall, there was a clear effect on mito- Servier, and Woolworths; has been a speaker for Astra Zeneca, chondrial biogenesis, as indicated by PGC1α gene expression, that Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, Janssen Cilag, may contribute to a delayed effect of treatment with these drugs. Lundbeck, Merck, Pfizer, Sanofi Synthelabo, Servier, Solvay, and The use of human NT2-N cells and microglial cells provided Wyeth; and served as a consultant to Astra Zeneca, Bioadvantex, an element of physiological relevance in the study of psychiatric Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, Janssen Cilag, drugs. The doses of the drugs used in this study are not all con- Lundbeck Merck, and Servier. sistent with expected plasma concentrations when the drugs are used therapeutically; however, the main purpose of this study was References to observe potential interactions between the various drug com- binations, so the doses were modified empirically to ensure that Adli M, Whybrow PC, Grof P, Rasgon N, Gyulai L, Baethge C, Glenn any effects were not dominated by any one drug. Some limita- T, Bauer M (2005) Use of polypharmacy and self-reported tions should be considered, including the fact that the duration of mood in outpatients with bipolar disorder. 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Abstract

Background: Bipolar disorder is a mental health condition with progressive social and cognitive function disturbances. Most patients’ treatments are based on polypharmacy, but with no biological basis and little is known of the drugs’ interactions. The aim of this study was to analyze the effects of lithium, valproate, quetiapine, and lamotrigine, and the interactions between them, Significance Statement Box Sample on markers of inflammation, bioenergetics, mitochondrial function, and oxidative stress in neuron-like cells and microglial cells.  Methods: Neuron-like cells and lipopolysaccharide-stimulated C8-B4 cells were treated with lithium (2.5  mM), valproate (0.5  mM), quetiapine (0.05  mM), and lamotrigine (0.05  mM) individually and in all possible combinations for 24  h. Twenty cytokines were measured in the media from lipopolysaccharide-stimulated C8-B4 cells. Metabolic flux analysis was used to measure bioenergetics, and real-time PCR was used to measure the expression of mitochondrial function genes in neuron- like cells. The production of superoxide in treated cells was also assessed.  Results: The results suggest major inhibitory effects on proinflammatory cytokine release as a therapeutic mechanism of these medications when used in combination. The various combinations of medications also caused overexpression of PGC1α and ATP5A1 in neuron-like cells. Quetiapine appears to have a proinflammatory effect in microglial cells, but this was reversed by the addition of lamotrigine independent of the drug combination. Conclusion: Polypharmacy in bipolar disorder may have antiinflammatory effects on microglial cells as well as effects on mitochondrial biogenesis in neuronal cells. Keywords: psychiatry, bipolar disorder, polypharmacy, inflammation, mitochondrial function Received: July 20, 2017; Revised: January 18, 2018; Accepted: February 16, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 582 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Bortolasci et al. | 583 Significance Statement This original research aimed to expand knowledge on why polypharmacy works better to treat bipolar disorder than individual drugs. The cellular and molecular basis for altered brain function in patients with bipolar disorder remains poorly understood. Accordingly, mechanisms underlying the molecular effects of drugs used to treat this disorder are largely unknown. Using neu- ronal like cells and microglial cells, we accessed the effects of commonly prescribed bipolar drugs alone and in associations to find out possible mechanism of action of this combinations and create new targets for drug discovery in this field. Mitochondrial dysfunction is noted in BD driving impaired brain Introduction energy metabolism. There is evidence of increased basal metabolic Bipolar disorder (BD) is a potentially neuroprogressive, chronic rate in mania (Caliyurt and Altiay, 2009) and decreased bioenerget- mental health condition characterized by alternate episodes ics in depression, concordant with a biphasic model of energy gen- of mania and depression that has secondary social and cogni- eration in BD paralleling symptomatology. There are high rates of tive consequences. This disorder is ranked in the top 10 lead- comorbidity of BD with mitochondrial diseases, and mood stabiliz- ing causes of lifelong disability worldwide according the World ers have documented effects on mitochondria. Mitochondrial dys- Health Organization and affects approximately 2% of the popu- function can increase production of ROS, leading to enhanced OS, lation (Duong et al., 2016Rihmer et  ; al., 2016). The pathophysi- causing deleterious consequences on signal transduction, synaptic ology of BD is poorly understood. Most useful psychotropic plasticity, and cellular resilience (de Sousa et al., 2014). medications produce maximal responses after several weeks, Understanding the actions of medications used to treat BD and this observation indicates that chronic adaptive molecular on these seemingly diverse yet interacting pathways may con- changes are crucial components of successful treatment rather tribute to our understanding of the underlying pathology of the than simple immediate receptor binding effects (Rizig et  al., disease and also allow new, more effective, and targeted treat- 2012). Operant biological pathways may include mitochondrial ments to be identified. The aim of this study was to augment the dysfunction, alterations in circulating levels of proinflamma- understanding of the mechanisms of action of these medica- tory cytokines, neurotrophins, and oxidative stress (OS) (Bhat tions and, especially, the cumulative effects of these drugs in et al., 2015). combination. Therefore, we tested the effects of combinations Polypharmacy can be defined as the concurrent use of of lithium, valproate, quetiapine, and lamotrigine on markers of multiple medications. For individuals with BD, these typically inflammation, bioenergetics, mitochondrial function, and OS in include agents such as mood stabilizers, atypical antipsychot- neuron-like cells and microglial cells. These drugs were chosen ics, antidepressants, and benzodiazepines (Adli et  al., 2005). from diverse pharmacological classes to represent the mecha- The majority of patients treated for BD receive multiple psy- nism of actions of various medications used to treat psychiatric chotropic medications concurrently (Goldberg et al., 2009 Sac ; hs disorders, including BD. et al., 2014). The Systematic Treatment Enhancement Program for Bipolar Disorder showed that monotherapy is applicable to <20% of the patients (Goldberg et al., 2009). Polypharmacy in the Materials And Methods management of BD is common given the characteristics of the disorder (e.g., chronicity, comorbidity, uncertainty associated NT2 Cell Culture with shifting polarity of illness), characteristics of the drugs (delayed time of action, tolerability, selectivity), and the high NTera2/cloneD1  (NT2) human teratocarcinoma cells (ATCC) frequency of inadequate response to monotherapy (Sachs et al., were cultured in media comprising Dulbecco’s modified Eagle’s 2014). Nevertheless, little is known regarding the drug interac- Medium (Life Technologies) including 10% fetal bovine serum tions or potential cumulative effects, and there is no clear bio- (Thermo Fisher Scientific), and 1% antibiotic/antimycotic solu- logical basis for the selection of drug combinations to improve tion (Life Technologies). These cells are known to express a efficacy. Medications prescribed for BD may have plentiful drug- neuron-like phenotype following retinoic acid-induced dif- drug interactions, raising safety concerns about polypharmacy ferentiation (Pleasure et  al., 1992). To achieve that, the cells -5 and reiterating the need for biologically based evidence (Tsai were treated with retinoic acid (Sigma-Aldrich) at 1 x 10 M for et al., 2014). 28 days with media refreshed every 2 to 3 days. Neuronal mark- The therapeutic effects of some medications used in the ers such as NeuroD (neuronal differentiation), GluR (glutamate management of the disease have recently been suggested to receptor), and Tau (cytoskeletal protein) were measured by real- be related to both mitochondrial and inflammatory mecha- time PCR (RT-PCR) to confirm the neuronal-like state of the cells nisms; however, current literature on this topic is scant. (data not shown) (Megiorni et al., 2005). Cells were seeded onto Notwithstanding this, both central and peripheral inflamma- 24-well and 96-well plates coated with 10  μg/mL poly-D-lysine tory processes seem to play a role in the pathophysiology of BD (Sigma-Aldrich) and 10 μg/mL laminin (Sigma-Aldrich) at 2 × 10 (Berk et al., 2011). Suppression of microglia-mediated inflamma- cells/well (24-well plates) and 8  ×  10 cells/well (96-well plates tion has been proposed as a strategy in therapy as prolonged and 24-well XF24 cell culture microplates, Seahorse Bioscience) immune activation can damage neuronal structure through the with further addition of mitotic inhibitors (1  µM cytosine and production of elevated levels of reactive oxygen species (ROS) 10 µM uridine; Sigma-Aldrich) for a total of 7  days with media and neurotoxins (Block et al., 2007Czeh et  ; al., 2011). Activation refreshed every 2 to 3 days to maintain an enriched culture of of inflammatory pathways and alterations in glutamate metab- differentiated neuronal cells (NT2-N) for the drug treatments. olism appear to converge on glial cells and may play a role in mood disorders. In particular, inflammatory mediators might C8-B4 Cell Culture Conditions act through glial cells to regulate extracellular glutamate con- C8-B4 microglial cells (ATCC CRL-2540) were cultured in centrations in both physiological and pathological conditions (Haroon et al., 2017). Dulbecco’s modified Eagle’s Medium (Life Technologies) with Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 584 | International Journal of Neuropsychopharmacology, 2018 10% fetal bovine serum (Life Technologies) and seeded onto The experiments were carried out in a PikoRealTM qPCR 24-well plates at 1.3 x 10 cells/well for the measurement of machine (Thermo Fisher Scientific) using the following protocol: cytokine release. 95ºC for 7 minutes, 4 cycles of 95ºC for 30 seconds and 60ºC for 1 minute and then data acquisition, 60ºC for 30 seconds, 55ºC to 95ºC, data acquisition and 20ºC for 10 seconds. Resultant melt Drug Treatments curves were used as an indicator of amplification specificity. The The NT2-N cells were treated with lithium (2.5 mM), valproate Quant-iT OliGreen ssDNA Assay Kit (Life Technologies) was used (0.5 mM), quetiapine (0.05 mM), and lamotrigine (0.05 mM) indi- to quantify the cDNA concentration in each sample as per the vidually and in all possible combinations. All drugs were pur - manufacturer’s instructions. Gene expression data were quanti- chased from Sigma-Aldrich. C8-B4 cells were stimulated with fied using the 2ΔCT method normalized to the derived cDNA 1  ng/mL lipopolysaccharide (LPS) per well and then received concentration of each sample. the same treatments as for NT2-N cells. Vehicle control cells were treated with an equal volume of MilliQ water for lithium Mitochondrial Function Quantitation or valproate controls, 0.2% DMSO for lamotrigine or quetiapine controls, or the combination of both (for various drug combina- The cellular bioenergetic profile of NT2-N-treated cells was tions as required). Dose response studies were performed to find assessed using a Seahorse XF24 Flux Analyzer (Seahorse optimal doses to balance the effects of each individual drug on Bioscience). Three basal oxygen consumption rate (OCR) meas- the expression of a number of candidate gene targets, such that urements were performed, and measurements were repeated the effect of one drug did not dominate the overall effect and did following injection of oligomycin (1  mM), carbonyl cyanide- not affect viability of the cells (data not shown). Each treatment 4-(trifluoromethoxy)phenylhydrazone (1  mM) and Antimycin had n = 6 replicates. A  (1  mM). Basal extracellular acidification rate (ECAR) was Following the 24-h treatment, the cells were harvested and determined from data collected at basal measurement points. RNA was extracted using RNeasy mini kits (Qiagen) and reverse- Calculations of respiratory parameters of mitochondrial func- transcribed to produce cDNA using Maxima H Minus first strand tion were performed as previously described (Martin et al., 2014). cDNA synthesis kit (Thermo Fisher Scientific) following the The protein concentration from each well was quantified by manufacturer’s instructions. Pierce BCA protein assay (Thermo Fisher Scientific) to account for differences in cell density during data analysis. Each treat- ment had n= 4 to 6 replicates. Inflammatory Biomarkers A panel of 20 cytokines (interleukin [IL]-1a, IL-1b, IL-3, IL-4, IL-5, Mitochondrial Superoxide Production IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17a, Eotaxin, granulo- cyte-colony stimulating factor [GCSF)], granulocyte macrophage The production of superoxide in NT2-N cells was measured colony-stimulating factor, interferon-gamma, keratinocyte che- using MitoSOXTm (Thermo Fisher Scientific) according to the moattractant, chemokine ligand 5, tumor necrosis factor-alpha) manufacturer’s instructions. The increase in fluorescence inten- was measured with a Bioplex Pro mouse cytokine assay kit sity was measured with a FlexStation II Scanning Fluorometer (Bio-Rad) according to the manufacturer’s protocol, using media (Molecular Devices; λex = 510 nm,λ em = 580 nm). Results are pre- from LPS-stimulated C8-B4 cells following 24-h drug treatments sented relative to vehicle treated cells. and normalized by the protein content (PierceTM BCA protein assay kit; Thermo Fisher Scientific). Statistical Analysis Shapiro-Wilk (inflammatory biomarkers) or Kolmogorov- Gene Expression Smirnov (all the remain analysis) tests were used to check data RT-PCR was used to measure the expression of specific genes sets for normality of distribution. In addition, biomarker data relating to mitochondrial function in NT2-N cells following were visually inspected using box-plots for presence of outli- drug treatments: peroxisome proliferator-activated receptor γ ers and influential data. Levene’s test was used to determine coactivator-1α (PGC1α: forward primer, AAC CAC ACC CAC AGG whether equal variances could be assumed between groups. ATC AGA; reverse primer, TCT TCG CTT TAT TGC TCC ATG A; Since there were no outliers in biomarker data, and there was efficiency: 95.30%), NADH:ubiquinone oxidoreductase subunit no alarming heterogeneity, parametric tests were performed for B8 (forward primer, CAG CCT CCC ACA TGA CCA AG; reverse biomarker data analyses. primer, GCC ATC ATC CGG GTA AGG TT; efficiency: 94.52%), For the mitochondrial function and oxidative stress analysis, succinate dehydrogenase subunit B (forward primer, ATG TGG drug treatment groups were compared against their respective CCC CAT GGT ATT GG; reverse primer, TGG TGT CAA TCC TTC controls using independent samples t tests. Differences were GGG TG; efficiency: 96.70%), ubiquinol-cytochrome C reductase considered statistically significant when P ≤ .05. core protein II (forward primer, GGG AAA GTG TTA GCG GGG The cytokine levels and mitochondrial gene expression were AA; reverse primer, TGG CTT TAA CTT TGG GGG CA; efficiency: analyzed using 1-way ANOVA followed by pairwise compari- 97.31%), mitochondrially encoded cytochrome C oxidase II (for - sons. For each ANOVA test the overall F-test significant level of ward primer, CCG TCT GAA CTA TCC TGC CC; reverse primer, 0.05 was considered. The 1-way ANOVA tests have 7 numerator GAG GGA TCG TTG ACC TCG TC; efficiency: 95.94%), ATP syn- degrees of freedom (df); we are therefore entitled to investigate thase, H+ transporting, mitochondrial F1 complex, alpha sub- 6 (i.e., 1-df) orthogonal contrasts of the treatment means with- unit 1 (ATP5A1: forward primer, TCA GTC TAC GCC GCA CTT out the need to consider the so-called “family-wise” Type I error AC; reverse primer, ATG TAC GCG GGC AAT ACC AT; efficiency: rate for these multiple t tests. However, due to a relatively large 89.63%), and hexokinase II (HK2: forward primer, CCA ACC TTA number of pairwise comparisons (4 comparisons) within each GGC TTG CCA TT; reverse primer, CTT GGA CAT GGG ATG GGG ANOVA, the significance level for pairwise between group com- TG; efficiency: 104.16%). parisons was set at P ≤ .01. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Bortolasci et al. | 585 Statistical analysis was performed using Statistical Package Mitochondrial Gene Expression for the Social Sciences version 22 (SPSS) software. Table  2 shows an overview of expression, relative to control groups, of genes related to mitochondrial function in treated Results NT2-N cells. ATP5A1 expression was significantly increased following most treatments (except for lithium, lamotrigine, Inflammation Biomarkers valproate/quetiapine, lamotrigine/quetiapine, and lithium/val- proate/lamotrigine). PGC1α expression was increased by most Microglial cells are the resident macrophages of the central treatments, including a 60% increase in response to treatment nervous system. They perform key signaling and scavenging with the combination of all 4 drugs. HK2, succinate dehydroge- roles, interact with neurotransmission, and mediate neuro- nase subunit B, mitochondrially encoded cytochrome C oxidase plasticity. Microglia are activated by various neuropathological II, NADH:ubiquinone oxidoreductase subunit B8, and ubiquinol- states and can, in response, secrete proinflammatory cytokines cytochrome C reductase core protein II expression had minor (Derecki and Kipnis, 2013). LPS strongly upregulated the secre- changes across the different treatments. The RT-PCR data sug- tion of most cytokines measured from C8-B4 cells (Figure 1). The gest a tendency for reduced expression of genes related to effects of the tested drugs on the release of cytokines from LPS- mitochondrial function in cells treated with lithium relative to stimulated C8-B4 cells are shown in Table  1. The inflammatory control. This is consistent with the aerobic respiration data of role of microglia in the brain justifies the use of this cell line to lithium-treated cells, which appeared to be reduced (Figure 3A). assess the effects of these drugs on inflammation. Quetiapine significantly increased mRNA levels of ATP5A1. This Lithium (alone) had no effect on cytokine release from LPS- result was unexpected given that flux bioanalysis revealed sig- stimulated C8-B4 cells. Valproate and lamotrigine alone had nificantly decreased basal respiratory rate in cells treated with relatively minor effects. Valproate and lamotrigine had no sig- quetiapine. nificant effect on cytokine levels. These data suggest that these drugs when acting alone have little overall effect on cytokine release from activated glial cells. Quetiapine, however, robustly Mitochondrial Function increased the release of all cytokines measured except for GCSF, Figure  2 shows an overview of mitochondrial function and suggesting a proinflammatory effect on glial cells. Lithium com- basal bioenergetics in NT2-N treated cells. Lithium-treated cells bined with either lamotrigine or quetiapine slightly increased (n = 6) exhibited a 27% lower basal respiratory rate (P = .032) and cytokine release, suggesting a proinflammatory effect. Valproate a 25% lower rate of ATP turnover (P = .032) relative to the con- when combined with either lamotrigine or quetiapine also trol group (n = 5) (Figure  3A). The cells also appeared to exhibit increased release of some cytokines, suggesting an overall pro- increased basal and maximal glycolytic rates, which is consist- inflammatory effect. On the other hand, the combination of ent with decreased capacity for aerobic respiration as shown in lamotrigine and quetiapine robustly reduced cytokine release, lithium-treated NT2-N cells (Figure  3B). Cells treated with val- suggesting a strong antiinflammatory effect. proate tended to have the same direction of changes, although When 3 drugs were used in combination, a clear pattern these were not significant. Quetiapine appeared to decrease emerged. The combinations of lithium/valproate/lamotrigine respiratory activity and capacity in NT2-N cells. Quetiapine- and lithium/valproate/quetiapine increased cytokine produc- treated cells (n = 6) exhibited a 20.1% lower basal respiratory rate tion and release, while the combinations of lithium/lamotrig- (P = .026) compared with the control group (n = 4) ( Figure 3A). ine/quetiapine and valproate/lamotrigine/quetiapine robustly Conversely, lamotrigine was the only drug tested that tended reduced cytokine release. The 4 drugs used in combination also to increase respiratory rate and capacity. However, the variabil- robustly lowered cytokine release. ity in the data rendered these increases statistically insignifi- cant. Basal and maximal glycolytic rates appeared to be reduced in cells treated with quetiapine and lamotrigine; however, no significant differences were found compared with control cells. Cells simultaneously treated with lithium and quetiapine exhibited a 31% lower basal respiratory rate (P = .029) and a 32% lower rate of ATP turnover (P = .041) compared with the control group (Figure  3C), with no significant change in basal or maxi- mal glycolytic rate (Figure 3D). No difference was found in the OCR values, basal or maxi- mal glycolytic rates following treatment with the various com- binations of 3 or 4 drugs relative to the control groups (Figure 2). Overall, using the drugs in combinations appeared to suppress any effect on mitochondrial function caused by the treatment with individual drugs in NT2-N cells. Mitochondrial Superoxide Production Figure  1. Effects of lipopolysaccharide (LPS) on C8-B4 cells (fold change rela- Mitochondrial superoxide is the major intracellular source of tive to vehicle treated cells). LPS treatment increased the release of a number ROS. Cells treated with lithium plus quetiapine showed signifi- of proinflammatory cytokines, including interleukin (IL)-1a, IL-3, IL-4, IL-5, IL-6, cantly decreased superoxide production (P = .05), and the com- IL-12, GCSF, KC, RANTES, and tumor necrosis factor (TNF)α. These data confirm bination of lamotrigine and quetiapine increased superoxide that LPS had a proinflammatory effect on C8-B4 cells. n = 3 samples in duplicate. production (P = .027) (Figure 4). *P < .05 compared with vehicle treated cells (descriptive statistical test). Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 586 | International Journal of Neuropsychopharmacology, 2018 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Table 1. Effects of BD Drugs Alone and in Combination on Cytokine Production and Release from C8-B4 Cells IL-1A IL-1B IL-3 IL-4 IL-5 IL-6 IL-10 IL-12(P40) IL-12(P70) Li 99.67 ± 0.12 94.42 ± 0.12 96.77 ± 0.25 93.29 ± 0.16 86.89 ± 0.16 106.85 ± 0.39 95.94 ± 0.16 76.99 ± 0.28 95.57 ± 0.20 Val 78.68 ± 0.04 81.67 ± 0.00 63.14 ± 0.03 74.09 ± 0.04 68.44 ± 0.04 47.46 ± 0.03 71.98 ± 0.04 20.93 ± 0.03 72.38 ± 0.08 Lam 166.07 ± 0.17 87.40 ± 0.05 166.38 ± 0.26 86.43 ± 0.06 84.70 ± 0.07 37.37 ± 0.07 98.02 ± 0.07 55.78 ± 0.18 83.86 ± 0.10 Quet 284.69 ± 0.23 140.47 ± 0.02* (P = .004) 496.43 ± 0.28* (P < .001) 182.73 ± 0.09* (P = .003) 223.20 ± 0.03* (P < .001) 277.90 ± 0.22* (P = .006) 192.63 ± 0.07* (P = .004) 917.69 ± 1.80* (P < .001) 289.46 ± 0.34* (P = .003) Li/Val 96.25 ± 0.02 101.62 ± 0.01 101.19 ± 0.05 98.38 ± 0.03 93.40 ± 0.04 82.07 ± 0.13 97.12 ± 0.02 30.49 ± 0.04 88.83 ± 0.05 Li/Lam 299.46 ± 0.61* (P = .010) 114.37 ± 0.03 313.87 ± 0.41 131.09 ± 0.19 158.86 ± 0.10 382.98 ± 0.46* (P < .001) 14015 ± 0.18 521.52 ± 1.82 224.56 ± 0.54 Li/Quet 50.07 ± 0.02 111.53 ± 0.07 41.26 ± 0.02 126.77 ± 0.15 210.69 ± 0.41 233.86 ± 0.52 123.58 ± 0.05 305.26 ± 0.68 153.00 ± 0.17 Val/Lam 47.41 ± 0.00 115.47 ± 0.07 46.10 ± 0.06 113.89 ± 0.08 191.64 ± 0.03 259.39 ± 0.10 104.89 ± 0.06 125.90 ± 0.19 119.98 ± 0.13 Val/Quet 49.70 ± 0.02 110.76 ± 0.07 37.26 ± 0.02 106.34 ± 0.06 197.85 ± 0.35 220.97 ± 0.55 114.08 ± 0.09 155.10 ± 0.48 155.63 ± 0.24 Lam/Quet 18.17 ± 0.01* (P < .001) 76.13 ± 0.02 66.58 ± 0.21* 70.64 ± 0.17* (P < .001) 57.27 ± 0.04* (P < .001) 15.78 ± 0.03* (P < .001) 58.12 ± 0.02* (P = .002) 31.68 ± 0.03* (P < .001) 37.65 ± 0.02 Li/Val/Lam 94.17 ± 0.14 149.15 ± 0.06* (P = .006) 79.13 ± 0.16* (P = .001) 173.92 ± 0.25* (P = .008) 427.60 ± 0.27* (P < .001) 634.05 ± 0.72* (P < .001) 164.93 ± 0.17 460.83 ± 0.94 287.94 ± 0.45* (P = .010) Li/Val/Quet 50.58 ± 0.07 130.30 ± 0.12 76.15 ± 0.16 163.64 ± 0.29 372.68 ± 0.30* (P < .001) 337.26 ± 0.31 143.90 ± 0.20 331.43 ± 0.76 236.05 ± 0.68 Li/Lam/Quet 15.74 ± 0.01* (P < .001) 68.84 ± 0.05* (P = .002) 45.31 ± 0.07* (P < .001) 49.75 ± 0.07* (P < .001) 55.64 ± 0.02* (P < .001) 16.87 ± 0.02* (P < .001) 54.46 ± 0.06* (P = .001) 23.85 ± 0.06* (P < .001) 27.02 ± 0.07* (P < .001) Val/Lam/Quet 16.39 ± 0.00* (P < .001) 76.92 ± 0.00 47.21 ± 0.07* (P = .003) 53.25 ± 0.04* (P = .001) 61.55 ± 0.03* (P = .001) 12.12 ± 0.01* (P < .001) 59.08 ± 0.02* (P = .003) 15.69 ± 0.02* (P < .001) 39.20 ± 0.02* P = .002) Li/Val/Lam/Quet 17.41 ± 0.02* (P < .001) 77.08 ± 0.06 63.33 ± 0.10 61.99 ± 0.06* (P = .002) 67.37 ± 0.03* (P = .008) 19.32 ± 0.02* (P < .001) 62.17 ± 0.11* (P = .007) 24.68 ± 0.07* (P < .001) 45.98 ± 0.13* (P = .008) IL-13 IL-17A EOTAXIN GCSF GMCSF IFNG KC RANTES TNFΑ Li 101.51 ± 0.15 98.28 ± 0.20 99.28 ± 0.33 84.31 ± 0.27 99.09 ± 0.23 99.54 ± 0.14 104.63 ± 0.25 68.30 ± 0.20 129.32 ± 0.49 Val 80.42 ± 0.07 71.37 ± 0.04 44.92 ± 0.05 30.59 ± 0.01 66.43 ± 0.06 81.09 ± 0.03 62.56 ± 0.05 60.71 ± 0.08 33.58 ± 0.03 Lam 96.10 ± 0.04 80.87 ± 0.09 87.09 ± 0.41 45.79 ± 0.07 117.27 ± 0.30 113.38 ± 0.09 85.67 ± 0.10 72.67 ± 0.25 58.67 ± 0.09 Quet 182.05 ± 0.08* (P = .004) 163.03 ± 0.09 209.88 ± 0.05 120.48 ± 0.07 207.34 ± 0.01 181.82 ± 0.08 297.50 ± 0.23* (P < .001) 1928.86 ± 7.1* (P = .007) 753.62 ± 1.00* (P = .006) Li/Val 103.13 ± 0.01 107.20 ± 0.01 125.82 ± 0.05 48.74 ± 0.03 118.33 ± 0.01 106.99 ± 0.01 101.24 ± 0.03 67.98 ± 0.12 88.49 ± 0.11 Li/Lam 148.23 ± 0.12 109.34 ± 0.11 76.98 ± 0.07 66.26 ± 0.08 118.52 ± 0.14 129.22 ± 0.12 223.62 ± 0.27* (P = .009) 801.71 ± 3.85 750.64 ± 2.58* (P = .006) Li/Quet 143.54 ± 0.06 121.81 ± 0.01 304.55 ± 1.01 52.62 ± 0.06* (P = .002) 204.03 ± 0.20 143.19 ± 0.02 186.98 ± 0.46 253.87 ± 0.68 159.99 ± 0.89 Val/Lam 116.51 ± 0.07 100.25 ± 0.10 308.97 ± 0.55 49.98 ± 0.01* (P = .002) 153.81 ± 0.29 115.64 ± 0.04 103.06 ± 0.04 213.46 ± 0.14 116.15 ± 0.11 Val/Quet 126.97 ± 0.13 121.44 ± 0.11 166.89 ± 0.24 34.93 ± 0.03* (P < .001) 138.71 ± 0.23 130.52 ± 0.23 110.98 ± 0.10 273.76 ± 0.86 42.50 ± 0.23 Lam/Quet 70.77 ± 0.03 47.69 ± 0.02* (P = .001) 122.50 ± 0.25 8.64 ± 0.00* (P < .001) 28.39 ± 0.10* (P < .001) 46.57 ± 0.05* (P = .007) 40.19 ± 0.04* (P < .001) 13.92 ± 0.01* (P = .001) 1.55 ± 0.00* (P < .001) Li/Val/Lam 204.12 ± 0.21 168.25 ± 0.17 347.42 ± 0.58 83.98 ± 0.03 315.84 ± 0.35* (P < .001) 237.39 ± 0.27* (P = .003) 470.21 ± 0.18* (P < .001) 1360.39 ± 5.4 2377.16 ± 6.1* (P < .001) Li/Val/Quet 169.39 ± 0.34 149.17 ± 0.27 312.24 ± 0.28 48.79 ± 0.10* (P = .001) 233.78 ± 0.26 190.51 ± 0.40 307.22 ± 0.39* (P < .001) 984.03 ± 2.67 821.81 ± 2.47 Li/Lam/Quet 52.99 ± 0.02 42.00 ± 0.06* (P < .001) 103.07 ± 0.24 7.36 ± 0.01* (P < .001) 39.84 ± 0.09* (P < .001) 38.54 ± 0.08* (P = .001) 63.56 ± 0.03* (P = .010) 12.38 ± 0.02* (P = .001) 1.36 ± 0.00* (P < .001) Val/Lam/Quet 68.71 ± 0.03 51.72 ± 0.02* (P = .002) 120.55 ± 0.21 6.47 ± 0.00* (P < .001) 44.78 ± 0.02* (P < .001) 53.98 ± 0.05 35.16 ± 0.01* (P < .001) 16.86 ± 0.01* (P = .002) 0.82 ± 0.00* (P < .001) Li/Val/Lam/Quet 73.88 ± 0.13 56.90 ± 0.11* (P = .006) 124.74 ± 0.40 7.06 ± 0.01* (P < .001) 51.91 ± 0.06* (P = .002) 53.76 ± 0.15 64.14 ± 0.06 (P = .010) 26.05 ± 0.08* (P = .007) 2.34 ± 0.01* (P < .001) Abbreviations: BD, bipolar disorder; Lam, lamotrigine; Li, lithium; Quet, quetiapine; Val, valproate. Percent of vehicle treated cells + SEM. =n 3 samples in duplicate e xcept for DMSO vehicle, n = 6. One-way ANOVA and pairwise comparison corrected for multiple testing *P < .01 compared with vehicle treated cells. Bortolasci et al. | 587 Table 2. Effects of BD Drugs Alone and in Combination on Mitochondrial Gene Expression in NT2-N Cells SDHB ATP5A1 MTCO2 NDUFB8 HKII UQCRC2 PGC1α Li 95.28 ± 20.34 111.58 ± 16.03 73.14 ± 17.20 89.63 ± 19.27 92.90 ± 14.42 96.70 ± 10.81 95.04 ± 10.12 Val 130.31 ± 6.22 189.18 ± 36.41* (P = .009) 146.88 ± 6.09* (P = .003) 85.35 ± 12.20 116.52 ± 3.48 60.11 ± 11.49 145.29 ± 11.45* (P = .010) Quet 88.54 ± 4.48 333.82 ± 11.18* (P < .001) 115.59 ± 6.28 92.85 ± 3.50 111.29 ± 3.35 90.31 ± 5.39 97.79 ± 5.63 Lam 84.38 ± 6.11 139.43 ± 18.42 103.98 ± 18.98 103.84 ± 4.24 128.31 ± 9.04 89.21 ± 5.12 130.57 ± 8.73* (P = .010) Li/Val 122.94 ± 14.29 140.81 ± 11.42* (P = .010) 113.40 ± 14.60 97.71 ± 7.20 104.36 ± 9.01 128.54 ± 17.04 142.28 ± 14.90 Li/Quet 91.29 ± 3.68 594.17 ± 74.09* (P = .010) 92.44 ± 15.00 87.45 ± 9.04 93.09 ± 8.00 104.87 ± 6.49 102.27 ± 5.97 Li/Lam 80.55 ± 7.12 163.76 ± 29.11* (P = .010) 107.68 ± 12.61 99.44 ± 8.38 92.41 ± 11.20 101.56 ± 10.76 91.22 ± 8.04 Val/Quet 87.46 ± 8.56 126.54 ± 22.67 99.96 ± 6.87 90.17 ± 7.06 111.91 ± 3.20 84.75 ± 5.57 126.54 ± 2.15 Val/Lam 76.29 ± 14.44 143.55 ± 41.98* (P = .010) 98.44 ± 22.62 95.52 ± 18.96 92.19 ± 6.83 88.65 ± 12.37 105.81 ± 13.26 Lam/Quet 146.37 ± 33.53 442.98 ± 141.97 118.50 ± 9.33 118.53 ± 30.37 123.96 ± 10.45 130.47 ± 36.14 126.96 ± 12.46 Li/Val/Quet 105.50 ± 1.58 454.76 ± 2.10* (P = .005) 95.56 ± 7.61 99.48 ± 1.68 113.09 ± 4.45 86.29 ± 2.85 153.87 ± 6.13* (P = .010) Li/Val/Lam 104.45 ± 18.49 216.69 ± 6.84 115.12 ± 20.09 94.76 ± 18.72 125.50 ± 13.05 95.23 ± 17.46 142.63 ± 19.53 Li/Quet/Lam 95.93 ± 4.62 362.43 ± 23.76* (P = .010) 71.97 ± 15.57 91.38 ± 3.55 95.33 ± 5.89 86.88 ± 6.46 117.39 ± 5.52 Val/Quet/Lam 93.44 ± 3.71 424.39 ± 50.41* (P = .010) 93.41 ± 7.96 88.83 ± 4.06 118.81 ± 5.90 78.79 ± 6.25 148.32 ± 5.87 Li/Val/Quet/Lam 98.26 ± 9.88 575.31 ± 51.74* (P = .002) 124.89 ± 13.45 98.78 ± 4.24 104.03 ± 14.95 98.09 ± 5.31 160.34 ± 9.45* (P < .001) Abbreviations: BD, bipolar disorder; Lam, lamotrigine; Li, lithium; Quet, quetiapine; Val, valproate. Percent of vehicle treated cells + SEM. n = 6 samples in duplicate . *P < .05 compared with vehicle treated cells (descriptive statistical test). Considering every drug separately in BD patients could offer a Discussion clearer picture of medication effect on cytokines, but that will The major finding in this study is the broadly antiinflamma- be limited to small sample sizes. Analyzing these effects in cell tory effects of polypharmacy in BD, as opposed to monotherapy, culture can clarify the possible mechanism of action of these where less consistent patterns were seen. BD is associated with drugs. In addition, it should be pointed out that the effects of the inflammation in the periphery and in the central nervous sys- drugs in the central nervous system may differ from the periph- tem, and there is increasing evidence to suggest that activation eral effects in circulating cells, that way being more informative of microglia is a key element in this process (Block et al., 2007; than the systemic concentrations of these cytokines. Reus et  al., 2015). Microglial proinflammatory cytokine release Quetiapine was the most cytokine-active drug tested in our can lead to oligodendrocyte cell death by apoptosis, blood brain study, showing strong proinflammatory effects that could in barrier destruction, neuronal damage, and mitochondrial impair - theory contribute to the low-level inflammatory state during ment (Naaldijk et al., 2016). Our results demonstrated effects on antipsychotic treatment reported in previous studies (Sarvari proinflammatory cytokine release that may play a role in the et al., 2014). However, when combined, especially with lamotrig- therapeutic mechanisms of some commonly prescribed drugs ine, this effect was suppressed and an antiinflammatory effect when used in combination. To our knowledge, this is the first appeared to emerge. study investigating differences in cytokine production between Multiple drug combinations that include both lamotrigine these drugs alone and in combination in a microglial cell line. and quetiapine appeared to have strong antiinflammatory It should be noted that this study was conducted in a mouse effects on glial cells. The reduction of proinflammatory cytokines microglial cell line, and it would be interesting to see whether observed in this study could reduce the activated state of micro- these results can be replicated in human microglial cells. The glial cells. It has been recently proposed that glial inflammation results are indicative of additive antiinflammatory effects of spe- can lead to increased release of glutamate into the extrasynaptic cific psychopharmacological combinations on cytokine produc- space by decreasing the capacity of glial transporters to buffer tion, which could provide a protective effect in neurons. and clear glutamate (Haroon et al., 2017). The effects of inflam- The literature regarding human cytokine levels after treat- mation on glial cells can further affect glutamate neurotrans- ment for BD is conflicting. Some studies showed a reduction in mission. Our data suggest that the antiinflammatory effects IFN-γ peripherally within 6 weeks of treatment (Kim et al., 2007; from the combination of lamotrigine with quetiapine may be Uyanik et al., 2015). Recently, 2 meta-analyses on the effects of driven by lamotrigine playing a pivotal role in the inhibition of medications on cytokine profile were published. Modabbernia glutamate release in glial cells. et  al. (2013) in a meta-analysis provided evidence for a lack of Considering antiinflammatory effects, in an association of medication effect on plasma concentrations of IL-2, IL-4, sIL-6R, lithium plus valproate, it is important to add lamotrigine and and INF-γ (Modabbernia et al., 2013). In another meta-analysis, quetiapine rather than just one of them. Both drugs together Munkholm et  al. (2013) reviewed 10 studies on the association had the power to shift the direction of the effect, which did not between plasma cytokine levels and medicines; of these, 6 stud- happen when they are added individually to the lithium plus ies did not find any significant correlation between medication valproate combination. status and cytokine levels, and 4 studies demonstrated correla- Our results demonstrate differential effects of these particular tions for individual cytokine parameters, of which none were four drugs, alone and in combinations, on inflammatory processes. replicated (Munkholm et al., 2013Ric ). ken et al. (2018) recently Given that inflammation is thought to play a role in BD patho- published a study showing the treatment with lithium does not physiology, at least in some patients, these data could provide a change cytokine serum levels (IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, foundation for developing a new treatment strategy to improve INF-γ), even after augmentation (Ricken et al., 2018). In addition, outcomes in some BD patients. It should be noted that lithium a systematic review showed 2 studies that report no effect of alone, which does not appear to affect cytokine release, is effica- valproate on cytokine levels (van den Ameele et al., 2016). cious in some patients, suggesting that antiinflammatory activity It is important to note that most studies analyzed a het- is not required in some patients but is likely required for efficacy in erogeneous group of patients regardless of medication status. others. The concept that a component of the mechanism of action Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 588 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Mitochondrial function and basal bioenergetics in NT2-N treated cells. Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of NT2-N cells exposed for 24 h to either vehicle or bipolar disorder (BD) drugs alone and in combinations. All values are reported as perecent of vehicle ± SEM (n = 6/group). Figure  3. Aerobic respiration and glycolysis in differentiated NT2-N cells as determined by oxygen consumption rate flux in response to mitochondrial probes (for oxygen consumption rate [OCR]) and by flux bioanalysis (for extracellular acidification rate [ECAR]). Data represented as mean OCR (pmol/min) or ECAR (pM/min) ± SEM; n = 4 to 6 per group. *P < .05 compared with vehicle treated cells (descriptive statistical test). of combination of these drugs includes an antiinflammatory effect energy production via glycolysis, increasing lactate levels and is supported by a recent meta-analysis showing that nonsteroidal pH and leading to production of ROS, glutamate excitotoxicity, antiinflammatory drugs have a moderate antidepressant effect in and apoptosis (Konradi et al., 2004). There is a body of evidence subjects with bipolar disorder (Rosenblat et al., 2016). showing an essential dysfunction of mitochondria, such as Mitochondrial dysregulation associated with decreased oxi- decreases in mitochondrial respiration, high-energy phosphates dative phosphorylation shifts metabolism toward anaerobic and pH; changes in mitochondrial morphology; increases in Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Bortolasci et al. | 589 Figure 4. Effects of bipolar disorder (BP) drugs alone and in combination on the intracellular reactive oxygen species (ROS) production in NT2-N cells (relative to vehicle treated cells). n = 5 samples. *P < .05 compared with vehicle treated cells (descriptive statistical test). mitochondrial DNA polymorphisms; and downregulation of Quetiapine significantly decreased basal respiratory rate in NT2-N nuclear mRNA molecules and proteins involved in mitochon- cells and tended to reduce ATP turnover. But when both drugs were drial respiration (Scaini et al., 2016). Other findings in BD include used together, we observed a significant further reduction in basal the dysregulation of mitochondria-related genes, increase of respiratory rate and ATP turnover in NT2-N cells. Together with lactate in the brain, decrease of complex I in postmortem brains, this, a significant reduction in mitochondrial superoxide produc- abnormal mitochondrial structure in cells of bipolar patients,tion and an incr ease in ATP5A1 gene expression was observed in and elevation of isocitrate in cerebrospinal fluid associated with NT2-N cells treated with lithium plus quetiapine. In contrast, only impaired function of isocitrate dehydrogenase (Kato, 2017). lamotrigine increased respiratory rate and capacity, which is note- Evidence from animal studies and cellular models on the worthy given that clinically it is the most robust antidepressant molecular pharmacology of mood stabilizing drugs has impli- of the drugs studied here. Following the polarity index of drugs, cated mitochondrial energy metabolism as a potential target. anticonvulsants (particularly lamotrigine) seem more effective for There is evidence that lithium and valproate modulate mito- prevention of depressive episodes, while atypical antipsychotics chondrial function by affecting the expression and activity of and lithium may have a manic polarity index of efficacy (Popovic mitochondrial complexes. A recent study showed that valproate et al., 2012). That observed efficacy in mania may be related to the reversed the methamphetamine-induced inhibition of complexes decreased bioenergetics highlighted in this study. I, II, III, and IV in rat brains (Valvassori et al., 2010). Similarly, lith- Quetiapine has previously been reported to have antioxidant ium has been shown to increase the activities of mitochondrial properties (Shao et al., 2005W ; ang et al., 2005), so it is likely that complexes I, II, and III in the frontal cortex tissue of human brains the decrease in basal oxygen consumption seen in the present (Maurer et al., 2009). The activity of complex IV was unaffected, study was associated with decreased ROS production. However, suggesting that lithium selectively increases the activity of spe- this hypothesis of redox modulation by quetiapine was not sup- cific enzymes, rather than acting as a general stimulator of the ported by the results of the mitochondrial superoxide produc- electron transport chain (Maurer et al., 2009). A more recent study tion analysis. This result could possibly be explained by the fact detected no difference in complex I activity between the lympho- that the cells were not under a redox stress condition when blasts of bipolar patients and healthy control subjects (Huzayyin treated, and the drugs were therefore unable to cause a down- et al., 2014). It is proposed that the effects of lithium on complex ward shift in the baseline ROS levels. Our results showed a trend I may be specific to neurons. Quetiapine has been shown to dif- for reduced glycolysis in cells following treatment with quetia- ferentially affect genes for complex I subunits causing upregula- pine or lamotrigine, indicating a potential to reverse mitochon- tion of NDUFV2 and downregulation of NDUFA10 (Young, 2007). drial dysregulation. When both drugs were used together, the This evidence is consistent with the idea that commonly opposite effect was observed, suggesting an increase in mito- prescribed bipolar disorder drugs influence mitochondrial func- chondrial dysregulation confirmed by the significant increase in tion. However, more data are required to demonstrate the precise mitochondrial superoxide production in NT2-N cells followed by nature of this association. The present findings showed relatively the treatment with lamotrigine plus quetiapine. minor changes in mitochondrial function in NT2-N cells after Gene expression was measured for one subunit of each of the treatment with BD drugs. The major finding is the combination of mitochondrial complexes and also measured for HK2, an important lithium and quetiapine affected mitochondrial function in NT2-N- regulator of glycolysis, and PGC1 , a tr α anscription factor involved treated cells compared with vehicle. Lithium significantly reduced in mitochondrial biogenesis. PGC1α is expressed at high levels in basal respiratory rate and ATP turnover in NT2-N cells and tended mitochondria rich cells with high energy demands, such as neu- to reduce other parameters of mitochondrial function as well. rons, and has been shown to be a master regulator of mitochondrial Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/582/4869972 by Ed 'DeepDyve' Gillespie user on 21 June 2018 590 | International Journal of Neuropsychopharmacology, 2018 biogenesis and cellular energy metabolism (Wareski et  al., 2009; Foundation, Australian Rotary Health, Stanley Medical Research Markham et  al., 2014). The PGC1 family of coactivators controls Institute, Deakin University, Brazilian Society Mobility Program, mitochondrial density in primary neurons (Wareski et  al., 2009). Lilly, NHMRC, Australasian Society for Bipolar and Depressive The upregulation of PGC1α by the drug combinations observed Disorders, and Sevier. She has also received in kind support in our study could compensate for neuronal mitochondrial loss. from BioMedica Nutracuticals, NutritionCare and Bioceuticals. Enhancing mitochondrial biogenesis regulates cellular oxidative M.B.  has received grant/research support from the NIH, capacity, and this may be useful for neuronal recovery and sur - Cooperative Research Centre, Simons Autism Foundation, Cancer vival in neurodegenerative disorders (Wareski et al., 2009). It would Council of Victoria, Stanley Medical Research Foundation, MBF, also be expected that an increase in PGC1 e αxpression would lead NHMRC, Beyond Blue, Rotary Health, Geelong Medical Research to augmented respiration as a result of increased mitochondrial Foundation, Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, biogenesis, but this effect may not be prominent within 24  h of Meat and Livestock Board, Organon, Novartis, Mayne Pharma, commencing treatment. Overall, there was a clear effect on mito- Servier, and Woolworths; has been a speaker for Astra Zeneca, chondrial biogenesis, as indicated by PGC1α gene expression, that Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, Janssen Cilag, may contribute to a delayed effect of treatment with these drugs. Lundbeck, Merck, Pfizer, Sanofi Synthelabo, Servier, Solvay, and The use of human NT2-N cells and microglial cells provided Wyeth; and served as a consultant to Astra Zeneca, Bioadvantex, an element of physiological relevance in the study of psychiatric Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, Janssen Cilag, drugs. The doses of the drugs used in this study are not all con- Lundbeck Merck, and Servier. sistent with expected plasma concentrations when the drugs are used therapeutically; however, the main purpose of this study was References to observe potential interactions between the various drug com- binations, so the doses were modified empirically to ensure that Adli M, Whybrow PC, Grof P, Rasgon N, Gyulai L, Baethge C, Glenn any effects were not dominated by any one drug. Some limita- T, Bauer M (2005) Use of polypharmacy and self-reported tions should be considered, including the fact that the duration of mood in outpatients with bipolar disorder. 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International Journal of NeuropsychopharmacologyOxford University Press

Published: Feb 19, 2018

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