Studies on Prostaglandin-Endoperoxide Synthase 1: Lower Levels in Schizophrenia and After Treatment with Antipsychotic Drugs in Conjunction with Aspirin

Studies on Prostaglandin-Endoperoxide Synthase 1: Lower Levels in Schizophrenia and After... Background: Antipsychotic drugs plus aspirin (acetylsalicylic acid), which targets prostaglandin-endoperoxide synthase 1 (PTGS1: COX1), improved therapeutic outcomes when treating schizophrenia. Our microarray data showed higher levels of PTGS1 mRNA in the dorsolateral prefrontal cortex from subjects with schizophrenia of long duration of illness, suggesting aspirin plus antipsychotic drugs could have therapeutic effects by lowering PTGS1 expression in the cortex of subjects with the disorder. Methods: We used Western blotting to measure levels of PTSG1 protein in human postmortem CNS, rat and mouse cortex, and cells in culture. Results: Compared with controls, PTGS1 levels were 41% lower in the dorsolateral prefrontal corte < .01), x (P but not the anterior cingulate or frontal pole, from subjects with schizophrenia. Levels of PTGS1 were not changed in the dorsolateral prefrontal cortex in mood disorders or in the cortex of rats treated with antipsychotic drugs. There was a strong trend (P = .05) to lower cortical PTGS1 10 months after mice were treated postnatally with polyinosinic–polycytidylic acid sodium salt (Poly I:C), consistent with cortical PTGS1 being lower in adult mice after exposure to an immune activator postnatally. In CCF- STTG1 cells, a human-derived astrocytic cell line, aspirin caused a dose-dependent decrease in PTGS1 that was decreased further with the addition of risperidone. Conclusions: Our data suggest low levels of dorsolateral prefrontal cortex PTGS1 could be associated with the pathophysiology of schizophrenia, and improved therapeutic outcome from treating schizophrenia with antipsychotic drugs augmented with aspirin may be because such treatment lowers cortical PTGS1. Keywords: Schizophrenia, aspirin, COX1, PTSG1, postmortem, cortex Received: June 15, 2017; Revised: September 18, 2017; Accepted: October 4, 2017 © The Author(s) 2017. 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 216 medium, 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/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 217 Significance Statement We showed that levels of PTGS1 messenger RNA were higher in the DLPFC from subjects with schizophrenia of long duration of illness. This was significant, because PTGS1 is targeted by aspirin, which increases the ability of antipsychotic drugs to lessen the symptoms of schizophrenia. Here, we report levels of PTGS1 protein is lower in the DLPFC, but not anterior cingulate or frontal pole, from subjects with schizophrenia and that PTGS1 is not altered in subjects with mood disorders or rats treated with antipsychotic drugs. Levels of PTGS1 were lower in human-derived astrocytes treated with aspirin, an effect augmented by the edition of the antipsychotic drug, risperidone. We propose lower levels of PTGS1 protein in the DLPFC are part of the pathophysi- ology of schizophrenia, and treatment with aspirin combined with an antipsychotic drug gives improved therapeutic benefits by lowering PTGS1 in subjects with schizophrenia. Introduction There is a significant body of evidence that suggests inflamma- Our studies showing higher levels of PTGS1 in mRNA in tion is contributing to the pathophysiology of schizophrenia the dorsolateral prefrontal cortex from subjects with schizo- (Na et  al., 2014). This hypothesis is supported by studies that phrenia (Narayan et al., 2008T ; ang et al., 2012b) differed from have shown changes in levels of mRNA for genes involved in findings at the level of protein that suggested PTGS1 was not modulating inflammatory-related pathways in postmortem different in prefrontal cortex, temporal cortex, or occipital cor - CNS tissue from subjects with schizophrenia (Saetre et al., 2007; tex from subjects with the disorder (Maida et al., 2006). Thus, Narayan et  al., 2008; Schmitt et  al., 2011; Hwang et  al., 2013). putting together our data and data on PTGS1 protein, it could Significantly, our study of CNS gene expression showed that, be that there are cortical region-specific changes in PTGS1 compared with controls, levels of prostaglandin-endoperoxide expression in the CNS from subjects with schizophrenia or synthase 1 (PTGS1: alias cyclooxygenase 1 [COX1]) mRNA were changes in PTGS1 expression are not translating into changes significantly higher in the dorsolateral prefrontal cortex from in levels of PTGS1 protein. To address this issue, we decided subjects with schizophrenia who were older than 40 years (av-er to determine if PTGS1 protein was changed in the same cor - age age = 64 ± 7.8  years) (Narayan et al. 2008T ; ang et al., 2012b). tical regions where there were higher levels of mRNA in sub- This finding is of significance with regards to therapeutic inter - jects with schizophrenia. We also decided to measure levels ventions, as it has been shown that acetylsalicylic acid (aspirin), of PTGS1 in the frontal pole and anterior cingulate cortex which targets PTGS1, lessens the severity of some of the symp- from subjects with schizophrenia to determine if changes in toms of schizophrenia when given as an adjunctive treatment PTGS1 could be present throughout the cortex. To address the with antipsychotic drugs compared with what is achieved giv- hypothesis that levels of PTGS1 are altered in the CNS after ing antipsychotic drugs alone (Laan et  al., 2010 W ; eiser et  al., an inflammatory episode, we measured levels of PTGS1 in the 2012). One of these clinical studies noted that acetylsalicylic cortex of neonatal mice treated with polyinosinic-polycytidylic acid adjunctive treatment was more effective in subjects with acid (Poly I:C), which activates inflammatory pathways (Ibi et high levels of blood interferon γ:interleukin 4 ratios, a measure al., 2009). We also measured PTGS1 in the cortex of mice that of increasing inflammatory status (Laan et al., 2010), data sug- had been treated neonatally with phencyclidine hydrochloride gesting that treatment with acetylsalicylic acid was more effect-(PCP), because this model, rather than models that deliver the ive in subjects with schizophrenia who were experiencing an drug to the rat foetus in utero (Jones et al., 2011), is not thought inflammatory episode. The second study (Weiser et  al., 2012) to act through inflammatory or immune pathways (Anastasio showed that acetylsalicylic acid, but not minocycline which is and Johnson, 2008). Significantly, both these animal models a tetracycline antibiotic with antiinflammatory effects that does produce a behavioral phenotype that allows the study of spe- not act on PTGS1, improved symptom severity when given with cific aspects of schizophrenia. We also sought to determine if antipsychotic drugs to subjects with schizophrenia. These data treatment with an antipsychotic drug alone could affect lev- suggest that targeting PTGS1 could be an important mechanism els of PTGS1 in rat cortex. Finally, we addressed the hypoth- of action for a drug that, when given with an antipsychotic drug, esis that treatment with acetylsalicylic acid with antipsychotic improves therapeutic outcomes. drugs affects levels of PTGS1 by measuring levels of the pro- Acetylsalicylic acid acts by targeting PTGS1 and PTGS2 (Vane tein in CCF-STTG1 cells, an eternalized cell line derived from a and Botting, 2003), which both convert arachidonic acid into bio- human astrocytoma (Vik-Mo et al., 2009), which we have estab- active prostanoids. Until recently it was thought that PTGS1 was lished stably express PTGS1, treated with vehicle, acetylsali- responsible for physiological production of prostanoids, whereas cylic acid, risperidone, or acetylsalicylic acid and risperidone. PTGS2 was involved in proinflammatory pathways being indu- cible at sites of inflammation (Aïd and Bosetti, 2011). However, Methods PTGS1 has now been shown to be present in microglia, where it is suggested to have a major role in modulating CNS inflamma- Materials tory processes (Choi et al., 2009). Hence, elevated levels of PTGS1 expression in the CNS could be indicative of generalized inflam- Poly I:C and PCPw ere obtained from Bio-Scientific Pty Ltd. Haloperidol, risperidone, and all general chemicals were matory processes driven by activated microglia in subjects with obtained from Sigma-Aldrich Pty Ltd. CCF-STTG1 cells were schizophrenia (van Berckel et  al., 2008Door ; duin et  al., 2009). obtained from American Type Culture Collection. RPMI 1960 By contrast, PTGS2 is predominantly localized to neurons and media, fetal calf serum (FCS), and antibiotic-antimycotic may therefore be more relevant to inflammatory processes that affect only those cells. (AB-AM) were obtained from ThermoFisher Scientific. Anti-PTGS1 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 218 | International Journal of Neuropsychopharmacology, 2018 antibody was obtained from Abcam (cat no.: AB109025). The goat Studies in Rodent CNS anti-rabbit IgG: horseradish peroxidase complex was obtained Studies in Rats from DAKO. All electrophoresis grade chemicals were from In determining the potential effects of antipsychotic drug treat- BioRad. ment on PTGS1, we decided to use an archetypal, first-genera- tion, antipsychotic drug, because this was the class of drugs used Ethical Approval to treat the cases of subjects with schizophrenia used in this study prior to death. Hence, 6-week-old male Sprague Dawley The Ethics Committee of the Victoria Institute for Forensic rats were exposed to a 12-h-day/-night schedule with free Medicine gave approval to collect human CNS post-mortem, and access to food. The rats (n = 10) received 0.1 mg/kg/d haloperidol all animal experiments were conducted with the approval of the in vehicle or vehicle alone (ethanol 0.1% v/v) in their drinking Florey Institute for Neuroscience and Mental Health Animal water for 28 days. This dose of drug was given on the principle Ethics Committee. that it would give a dopamine-D2 receptor occupancy in a rat equivalent to what is regarded as being optimal when treating subjects with schizophrenia (Kapur et  al., 2003) but adjusted Studies in Human CNS for delivery of haloperidol in drinking water rather than min- CNS tissue was collected from people who had a likely his- ipumps (0.25 mg/kg/d) or by s.c. injection (0.04–0.08 mg/kg s.c./d) tory of a psychiatric disorder and subjects who displayed no over 1 month rather than 7 days. The dose of haloperidol used in obvious symptoms of psychiatric disorders and who had not this study was slightly higher than that used for i.p. injection, as died by suicide (controls). At the time of death, no case had we have shown this gives blood levels of the drug in rats equiva- measurable levels of acetylsalicylic acid in their blood. Tissue lent to what would be expected in humans (Scarr and Dean, was collected from donors after either a witnessed death or 2012). The drug doses were adjusted twice weekly based on the having been seen alive within 5 hours of being found dead. average daily water consumption and increasing weight of each All cadavers were refrigerated soon after discovery, which rat. After the treatment, the rats were left untreated for 48 hours is advantageous as this would aid in slowing the effects of and then sacrificed by decapitation and the brains removed and autolysis (Ferrer et al., 2007). To further optimally preserve o rapidly frozen and stored at -80 C. CNS tissue, the left hemisphere from each donor was pro- cessed in a standardized manner that ensured tissue was fro- Studies in Mice zen to -80 C within 30 minutes of autopsy (Dean et al., 1999). Neonate C57black6J mice were housed with their dams in a Just prior to freezing, a sample of CNS tissue was collected 12-h-light/-dark cycle with free access to food and water. Two from each donor, and CNS pH was measured as described groups of 20 neonate mice received either daily i.p. injections of previously (Kingsbury et al., 1995), because CNS pH is a good pyrogen-free saline or pyrogen-free saline containing 10 mg/kg indicator as to the quality of tissue preservation (Stan et al., Poly I:C between postnatal days (PD) 2 to 6. All mice were imme- 2006). diately returned to their dam after each injection. Two groups of For each case, relevant data from clinical histories 20 mice were injected i.p. on PD 7, 9, and 11 with either pyrogen- and interviews with treating clinicians and relatives were free saline or pyrogen-free saline containing 10 mg/kg PCP and obtained using a standardized instrument, the Diagnostic on each occasion immediately returned to their dam. All mice Instrument for Brain Studies (Hill et al., 1996). On completion were weaned at PD 21, after which the sexes were housed sep- of this review, in cases other than those where it was agreed arately. From each treatment group, 10 mice were killed by cer - the donor had no history of psychiatric illness (controls), a vical dislocation at 1 month of age, which is at a prepubescent diagnosis was made by consensus by 2 psychiatrists and a phase, whilst the remaining 10 mice were killed postpuberty psychologist according to DSM-IV criteria (Roberts et al. 1998). at 10  months of age (Dutta and Sengupta, 2016). Notably, just In addition, the postmortem interval (PMI) was calculated as before being killed, the Poly I:C mice and the PCP mice w ere either the time from witnessed death to autopsy or the time tested with the saccharin preference test. At 1 month old, nei- mid-way between a subject being last seen and being found ther set of treated mice showed altered saccharin preference dead until autopsy. Duration of illness was calculated as the but at 10  months the Poly I:C-treated, but not the PCP-treated time from first presentation with psychiatric symptoms to mice, showed reduced saccharin preference, which is viewed as death. The final recorded doses of different classes of psycho- a behavior analogous to the anhedonia in schizophrenia (Jones tropic drugs were also recorded and standardized to a dose of et al., 2011). Immediately following cervical dislocation, the CNS a specific drug in class (Foster, 1989). was harvested and the frontal cortex was dissected and rapidly For this study, tissue from the dorsolateral prefrontal cortex o frozen in an isopentane bath on dry ice and stored at -80 C until (Brodmann’s [BA] area 46)  was obtained from 20 subjects with needed. Whole brain from one of the control group dams was schizophrenia, 20 subjects with major depressive disorders, 17 used to prepare the internal control used in Western blotting subjects with bipolar disorders, and 20 controls. Tissue was also (see below). obtained from BA 10 and BA 24 from the subjects with schizo- phrenia and the controls. Efforts were made to match age and Studies with CCF-STTG1 Cells sex across diagnoses. Different regions of the cortex were dis- For the study of the impact of acetylsalicylic acid ± risperidone sected guided by cytotectonic markers (Garey, 1994); hence, BA on PTGS1 levels, we chose to use CCF-STTG1 cells, because they 10 was taken as the most rostral portion of the superior frontal express human PTGS1 stably in culture. In these experiments, gyrus and middle frontal gyrus, bounded ventrally by the super - we used risperidone, because that was the antipsychotic drug ior rostral sulcus; BA 24 as anterior cingulate gyrus around the with the least complex neuropsychopharmacology (Goldstein, genu of the corpus callosum; and BA 46 as the lateral surface of 2000) of the drugs used in the study where acetylsalicylic acid the frontal pole, including approximately the middle third of the was used as an adjunctive treatment and caused an improve- middle frontal gyrus. ment in symptom severity (Laan et al., 2010). Thus, CCF-STTG1 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 219 cells were cultured to 70% confluency in RPMI 1960 media con- to control for inter-blot variation, an internal control (IC) of cor - taining 10% heat inactivated FCS and 1x AB-AM at 37 C with 5% tical homogenate was prepared from a single case that was not CO . Cells, at a density of 5 x 10 cells/well, were then subcul- used in the current study. This homogenate was run under the tured (P9) into 6-well cell culture plates and grown for 10 days in exact conditions to be used for the cases of interest in every lane RPMI 1960 media containing 5% FCS and 1x AB-AM. The media on 2 gels and the intensity of the immunogenic band of inter - was then removed, cells washed with 1x phosphate buffered est measured in each lane (30 measurements). These measure- saline and replaced with RPMI 1960 media containing 1x AB-AM, ments were used to establish both intra- and interblot variation no FCS, and either 0, 0.1, 0.5, or 1.0  mM acetylsalicylic acid in for the measurement of PTGS1. This sample was subsequently the absence or presence of risperidone (1 or 10 µM). The pH of run (in duplicate) on every gel, and each gel was exposed so that all media preparations was measured and, where necessary, the sum intensity of the IC fell within the range established adjusted to pH 7.4 prior to commencing the study. Five replicate by calculating the mean ± 2 SD of the initial 30 measurements. wells were prepared for each combination of drugs. The cells The intensity of PTGS1 from all cases were the standardized by were harvested after 24 hours and washed thrice in 1x phos- expressing them as a ratio of IC. For studies in rats and mice, a phate buffered saline. Protein homogenates were prepared by similarly prepared IC from rat and mice cortex was used. sonicating the cells in 10  mM Tris, pH 7.4, 1% SDS, and 1  mM Na VO and stored at -80C until needed. Protein homogenate 3 4 Statistics prepared from CCF-STTG1 cells cultured in RPMI 1960 media Demographic, CNS collection, and psychotropic drug data were containing 10% FCS and 1x AB-AM and harvested at 70% conflu- analyzed using the D’Agostino and Pearson omnibus normal- ency was used as an internal control for Western blotting (see ity test, and nonnormally distributed data were subsequently below). analyzed using nonparametric statistics. Student t tests or 1-way ANOVA were used to compare age, CNS pH, PMI, brain Western Blotting weight, and duration of illness. By contrast, experimental data expressed as ratios are better analyzed with nonparametric Human and rat (frontal cortex) tissue was homogenized into statistics (Siegel, 1957Allison et  ; al., 1995Curr ; an-Everett, 2013; 10 mM Tris containing 1% sodium dodecyl sulphate and 1 mM Dean et  al., 2016). PTGS1 levels in BA 10 and 24 from subjects sodium orthovanadate (pH 7.4) (5 x vol/tissue wet weight) using with schizophrenia and controls were compared using the 20 strokes using a Potter-Elvehjem PTFE pestle and glass tube. Mann-Whitney U test. Suicide and gender ratios across diag- The protein concentration in each homogenate was then meas- noses were compared using the Fisher exact test, and com- ured using the modified BioRad Detergent Compatible assay parisons of levels of PTGS1 across gender and between suicide (Lowry method). Cortical homogenates, containing 20  μg of completers and death by other causes was by Mann-Whitney protein, were then separated in duplicate on a 7.5 % pol yacryl- U test. Comparisons of experimental and with demographic, amide resolving gel. The separated proteins were transferred CNS collection, and pharmacological data were compared using onto nitrocellulose membranes (overnight constant at 40 mA in the Spearman’s rank correlation coefficient. Where there were Towbin transfer buffer). Each membrane was stained with 0.2% strong relationships between experimental and nonexperimen- Ponceau S in 3% TCA to confirm effective transfer of proteins. tal data, comparisons across diagnoses were made using nonex- After destaining, membranes were blocked with 5% non- perimental data as covariates. fat milk powder (NFMP) at room temperature (R/T) for 1 hour The data comparing levels of PTGS1 in the cortex of rats and then incubated with anti-PTGS1 antibody (ABCAM, cat no.: receiving vehicle or treated with haloperidol were compared ab109025 Anti-COX1 / Cyclooxygenase 1 rabbit monoclonal anti- using the Mann-Whitney U test. Levels of PTGS1 at 1 month body, clone no.: EPR5866) at a dilution of 1/200 in Tris-Tween- and 10 months after treatments with Poly I:C or PCP wer e com- Buffer-Saline (TTBS) with 5% NFMP o vernight at 4C. Membranes pared using the Mann-Whitney U test. Data from cell culture were then washed thrice for 10 minutes at R/T in TTBS and then data were analyzed using Kruskal-Wallace test using Dunn’s exposed to a 1/2000 dilution of goat anti-rabbit IgG: horseradish test as a posthoc analysis to compare the effects of risperidone peroxidase in TTBS with 5% NFMPfor 1 hour at R/T . Membranes and acetylsalicylic acid at different doses with the vehicle alone. were washed thrice for 10 minutes at R/T in TTBS and then incu- Statistical analyses were completed using GraphPad Prism v - er bated with Pierce ECL solution for 5 minutes at R/T. Excess ECL sion 6.00 for Windows, GraphPad Software. solution was then removed from the nitrocellulose, an image of chemiluminescence captured using a Kodak 440 CF imaging sys- tem. The intensity of the PTGS1 immunogenic band at ~70 kDa Results was then measured on each nitrocellulose gel. In many studies, Western blot results have been expressed Case History Reviews normalized to a loading control (Aldridge et al., 2008), the notion of the loading control being that the level of the chosen protein There were no significant differences in age or PMI across diag- will not vary as a proportion of total protein between tissues noses (Table  1; supplementary Table 1). There was a significant or between individuals. However, it is becoming increasingly variation in gender ratio between controls and subjects with clear that loading controls and/or reference genes or proteins major depressive disorders as well as subjects with schizophre- are not tenable for studies using human CNS tissue where lev- nia and subjects with major depressive disorders. CNS pH was els of individual proteins vary as a proportion of total protein significantly higher in subjects with major depressive disorders between donors (Eaton et al., 2013). Thus, the best way to control compared with controls, subjects with schizophrenia, and sub- for protein loading, as we have done in this study, is to care- jects with bipolar disorders. There was a significant variation in fully measure the protein concentration of each sample imme- CNS weight across diagnostic cohorts, but no individual diag- diately prior to it being loading onto the gel. This ensures that nosis differed from any other diagnoses. Rates of suicide com- the same protein concentration is loaded for each case (Eaton pletion were higher in subjects with major depressive disorders et al., 2013). Moreover, as described previously (Dean et al., 2006), compared with subjects with schizophrenia and subjects with Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 220 | International Journal of Neuropsychopharmacology, 2018 Table  1. A summary (for numeric: mean ± SEM) of data collected during the case history reviews of subjects with no history of psychiatric illness (controls) as well as for subjects with schizophrenia, major depressive disorders and bipolar disorders. Age Sex pH PMI CNS Weight DI Suicide FRADD LEAP FRAnDD FRMSD (yr) (M / F) (hr) (Kg) (yr) (y / n) CONTROLS 49 ± 4.7 16 / 4 6.28 ± 0.04 41 ± 3.5 1.43 ± 0.34 0 / 20 SCHIZOPHRENIA 48 ± 4.7 16 / 4 6.32 ± 0.05 43 ± 3.1 1.43 ± 0.35 21 ± 4.3 8 / 12 326 ± 76 7.46 ± 2.3 352 ± 115 MAJOR DEPRESSIVE 56 ± 3.6 9 / 11 6.54 ± 0.04 40 ± 3.6 1.30 ± 0.43 16 ± 2.4 17 / 3 303 ± 87 6.8 ± 2.6 862 ± 321 DISORDERS BIPOLAR DISORDERS 56 ± 3.4 9 / 8 6.29 ± 0.04 40 ± 4.0 1.23 ± 0.12 17 ± 3.0 7 / 13 256 ± 57 2.1 ± 0.60 573 ± 318 923 ± 210 F-value 1.01 8.77 0.12 3.03 0.52 0.27 1.56 0.87 d.f. 3,73 3,73 3,73 3,63 2,54 2,21 2,31 2,5 p 0.39 < 0.05 < 0.0001 0.95 < 0.05 0.6 0.002 0.76 0.23 0.47 Cont vs Sz p 1.00 n.s n.s. Cont vs MDD p 0.02 p < 0.001 n.s. Cont vs BD p 0.08 n.s. n.s. Sz vs MDD p 0.02 p < 0.01 n.s. 0.003 Sz vs BD p 0.08 n.s. n.s. 0.700 MDD vs BD p 0.63 p < 0.001 n.s. 0.003 Abbreviations: BD = bipolar disorders, Cont = Controls, DI = duration of illness, F = female, FRADD = final recorded antipsychotic drug does converted to mg chlorpromazine equivalents per day, FRAnD = final recorded antidepressant drug dose converted to mg fluoxetine equivalents per day, FRMSD = final recorded mood stabiliser dose converted to mg lithium equivalents per day, hr = hours, Kg = kilogram, LEAP = lifetime -3 exposure to antipsychotic drugs converted to as chlorpromazine equivalents per year x 10 , M  =  male, MDD  =  major depressive disorder , n = no, Sz = schizophrenia, y = yes, yr = year bipolar disorders. Levels of final recorded antipsychotic drug, (1.15–2.44), P = .73). For those with a psychiatric diagnoses, levels lifetime exposure to antipsychotic drug, final recorded dose of of PTGS1 did not differ between suicide completers and subjects antidepressant drug, or final recorded dose of mood stabilizers who died from other causes in BA 10 ((median± IQR): suicide did not differ significantly between subjects with schizophrenia, completers = 1.18 (0.97–1.53) vs other = 1.16 (0.88–1.56),P = .73), major depressive disorders or bipolar disorders. BA 24 ((median ± IQR): suicide completers= 0.99 (0.67–1.16) vs other = 1.01 (0.65–1.60), P = .61), or BA 46 ((median ± IQR): suicide completers = 1.72 (1.28–2.39) vs other = 1.35 (1.02–2.00),P = .27). Antibody Validation These data suggest that whilst gender ratio and suicide v - ar There is a growing concern about the specificity of antibodies ied with diagnoses (Table 1 ), they were not potential confounds used in various immuno-detection techniques (Manimala et al., when comparing levels of cortical PTGS1 across diagnoses. 2007; Jositsch et  al., 2009); thus, we determined the immuno- There were no significant relationships between levels genic profile of the anti-human PTGS1 antibody used in this of PTGS1 and age, PMI, CNS weight, duration of illness, final study using human cortex and a homogenate of human embry- recorded antipsychotic drug, final recorded dose of antidepres- onic kidney 293T cells that had either been, or not been, trans- sant drug, and final recorded dose of mood stabilizers (Table )2 , fected with the human PTGS1 gene. Using Western blotting, we suggesting these were not potential confounds. showed the presence of a single immunogenic band of molecu- lar weight 70 kDa in 2 samples of human cortex (Figure 1A). An Studies in Rodent Cortex immunogenic band of the same molecular weight was present at high levels in the homogenate from 293T cells transfected Compared with vehicle treatment, levels of PTGS1 did not dif- with the human PTGS1 gene but was absent in nontransfected fer in the cortex of rats treated with 0.1 mg/kg/d haloperidol for 293T cells. These data show the antihuman PTGS1 antibody 28 days (Figure 2A). Similarly, compared with vehicle treatment, used in this study has specificity for PTGS1. levels of PTGS1 did not differ in the cortex of mice 1 month after treatment with 10 mg/kg Poly I:C (Figure 2B), but there was a strong trend to decreased levels of PTGS1 in the cortex of mice Studies in Human Cortex 10 months after such treatment (=P .05). Indeed, a power ana- Compared with controls, levels of PTGS1 were significantly lysis of these data using G*Power Version 3.1.9.2 showed that an lower in BA 46 (P< .01), but not BA 10 (P= .37) or BA 24 (P = .08), addition of 1 animal per treatment group at 10 months w ould from subjects with schizophrenia (Figure 1B). A power analysis be needed to reach significance P < .05. Compared with vehicle of the data from BA 24 using G*Power version 3.1.9.2 showed treatment, levels of PTGS1 did not differ in the cortex of mice 1 that 92 individuals in each diagnostic group would be needed or 10 months after treatment with 10 mg/kg PCP. to reach significance (P < .05). Levels of PTGS1 were measured only in BA 46 from subjects with major depressive disorders and Studies in CCF-STTG1 Cells bipolar disorders and did not differ when compared with con- trols (Figure 1B). Compared with vehicle treatment, levels of PTGS1 did not dif- Levels of PTGS1 did not differ with gender in BA 10 fer in CCF-STTG1 cells after exposure to 1 or 10  μM risperidone (median ± IQR: F = 1.31 (0.88–1.55) vs M = 1.34 (1.02–1.54), P = .74), for 24 hours (Figure 3A). By contrast, acetylsalicylic acid caused BA 24 (median ± IQR: F = 1.12 (0.96–1.51) vs M = 1.09 (0.76–1.84), a dose-dependent decrease in levels of PTGS1 in CCF-STTG1 P = .96), or BA 46 (median ± IQR: F = 1.67 (1.03–2.47) vs M = 1.78 cells after treatment for 24 hours, which was significantly Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 221 Figure 1. Measuring levels of PTSG1 in human and rodent cortex. (A) A Western blot showing levels of a proteins that displayed immunogenic sites that for an anti- prostaglandin synthase 1 (PTGS1) antibody (ABCAM Anti-COX1 / Cyclooxygenase 1 rabbit monoclonal antibody, clone no.: EPR5866) in human, rat, and mouse cortices (20 µg protein) as well as 293T cells that were either transfected or not transfected with the human prostaglandin- endoperoxide synthase 1 (PTGS1) gene. To provide an impression of relative levels of expression 20 µg protein from undiluted 293T or CCF-STTG1 cell lysate (set 3) or cell lysate diluted 1:10 (set 2) or 1:20 (set 1) prior to load- ing onto the polyacrylamide gel are shown. (B) A typical Western blot showing levels of a 70-kDA protein that displayed immunogenic sites for an anti-PTGS1 antibody in homogenates from Brodmann’s areas 10, 24 and 46 from subjects with schizophrenia and age / sex nonpsychotic controls. (B) Levels (median ± interquartile range) of PTGS1 protein in cortical homogenates containing 20 µg total protein from BA 10, 24, and 46 from subjects with schizophrenia and age-/sex-matched controls, as well as BA 46 from subjects with major depressive disorders and bipolar disorders. Table 2. The relationships between levels of prostaglandin-endoperoxide synthase 1 in (A) human BA 10, BA 24 and BA 46 and with (B) case history data in those three cortical regions. Age pH PMI CNS Weight DI FRADD FRAntiD FRMSD ρ p ρ p ρ p ρ p ρ p ρ p ρ p ρ p BA 24 0.19 0.25 0.13 0.42 -0.19 0.25 0.01 0.95 0.28 0.23 -0.10 0.79 n.a n.a BA 46 -0.09 0.46 -0.01 0.92 0.06 0.58 -0.08 0.49 -0.23 0.08 -0.01 0.97 -0.04 0.94 -0.36 0.28 BA 10 0.00 0.99 0.00 0.99 0.03 0.85 -0.16 0.32 0.32 0.17 0.05 0.89 n.a n.a Abbreviations: BA = Brodmann’s area, DI = duration of illness, FRADD = final recorded antipsychotic drug dose expressed as chlorpromazine equivalents per day, FRAntiD = final recorded antidepressant drug dose expressed as fluoxetine equivalents per day, FRMSD = final recorded dose of mood stabiliser drug dose expressed as lithium equivalents per day, PMI = postmortem interval different from vehicle at 1.0 M acetylsalicylic acid (Figure  3B). for levels of mRNA, we do not find that differences between lev- Acetylsalicylic acid decreased levels of PTGS1 in CCF-STTG1 els of PTGS1 protein in BA 46 from subjects with schizophrenia, cells in the presence of both 1  μM and 10 μM risperidone with compared with controls, vary in significance with duration of differences between drug treatment and vehicle reaching sig- illness. Thus, it is possible that lower levels of PTGS1 in BA 46 nificance at 0.5 M acetylsalicylic acid. are present at or close to the onset of schizophrenia and that the higher levels of expression of PTGS1 in BA 46 from subjects with schizophrenia of long duration (Narayan et al., 2008) rep - Discussion resent a slow and unsuccessful response to rectify low protein levels. Alternatively, changes in gene promoter methylation We have shown that levels of PTGS1 are lower in the dorsolateral prefrontal cortex, but not frontal pole or anterior cingulate cor - are known to occur with age (Jung and Pfeifer, 2015) and dur - ation of illness also correlates with age. Therefore, the increased tex, from subjects with schizophrenia and that PTGS1 levels are not altered in the dorsolateral prefrontal cortex from subjects expression of PTGS1 mRNA in schizophrenia may be associated with differential changes in gene promoter methylation with with major depressive disorders or bipolar disorders. Our previ- ous data showed higher levels of PTGS1 mRNA in the dorsolateral age in BA 46 from subjects with schizophrenia and controls (Tang et al., 2012a). However, PTGS1 protein should more closely prefrontal cortex from subjects with schizophrenia of long - dur ation (Narayan et al., 2008; Tang et al., 2012b). By contrast, unlike reflect functional changes in the CNS, and therefore we would Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 222 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Levels of PTSG1 in rodent cortex after treatment with the antipsychotic drug haloperidol, poly I:C or phencyclidine. Levels (median ± interquartile range) of prostaglandin- endoperoxide synthase 1 (PTGS1) protein in the cortex of: (A) rats treated with haloperidol at 0.1 mg/kg/d or vehicle (ethanol 0.1% v/v) in their drinking water for 28 days, (B) mice 1 and 10 months after treatment with 10 mg/kg Poly I:C between postnatal days (PD) 2 to 6, and (C) mice 1 and 10 months after treatment with 10 mg/kg phencyclidine on PD 7, 9, and 11. Figure 3. Levels of PTSB1 in CCF-STTG1 cells after treatment with the antipsychotic drug ripseridone, acetylsalicylic acid or both drugs in combination. Levels (median ± interquartile range) of prostaglandin-endoperoxide synthase 1 (PTGS1) protein in CCF-STTG1 cells after: (A) exposure to vehicle, 1 or 10 µM risperidone for 24 hours or (B) exposure to vehicle and varying doses of acetylsalicylic acid and risperidone for 24 hours. *P < .05 compared with levels of PTGS1 in the absence of acetylsalicylic acid and risperidone. argue that PTGS1 in BA 46 is either involved in or affected by the prefrontal cortex from subjects with major depressive disorders pathophysiology of the disorder. and bipolar disorders, which adds to earlier findings that the Notably, our data suggest that low levels of PTGS1 may be protein is not changed in prefrontal cortex, temporal cortex, or localized to the dorsolateral prefrontal cortex in subjects with occipital cortex from subjects with mood disorders (Maida et al., schizophrenia. This argument is in line with findings from an 2006). There is a report of lower levels of PTGS1 protein and earlier study, which, using immunohistochemistry (Maida et al., mRNA in the frontal cortex from subjects with bipolar disorder 2006) or Western blotting (Rao et  al., 2013), failed to show any (Kim et al., 2012). However, the region of the frontal cortex used change in levels of PTGS1 protein in BA 8, 18, 21, or 22, or hippo- was not defined and may not be one of the cortical regions used campus or PTGS1 mRNA in BA 10 from subjects with schizophre- in this study. nia (Maida et al., 2006; Rao et al., 2013), respectively. In addition, Current data would suggest that changes in PTGS1 levels do we report no change in levels of PTGS1 in the dorsolateral not occur globally in the CNS from subjects with schizophrenia, Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 223 and therefore the changes in PTGS1 may make a unique con- which was further enhanced in the presence of risperidone. Our tribution to the changes in functioning of the dorsolateral data therefore suggest there may be a synergistic interaction prefrontal cortex that have long been associated with schizo- between antipsychotic drugs, such as risperidone, with acetyl- phrenia. Significantly, PTGS1 has recently been proposed salicylic acid to increase its ability to lower levels of PTGS1. Our to form part of a protein-protein interactome that is predicted to data from a human-derived astrocytic-like cell line differs from be affected because of genetic changes that have been shown to that from human monocytic cell lines, which, after exposure to increase the risk for schizophrenia (Ganapathiraju et al., 2016). acetylsalicylic acid, did not change levels of PTGS1 mRNA or pro- Moreover, many of the proteins in this interactome, including tein (Barrios-Rodiles et al., 1996). These data suggest that acetyl- PTGS1, were predicted to be promising drug targets for drugs to salicylic acid may have cell-specific effects on PTGS1 levels treat schizophrenia. Hence, our data would encourage further and that these effects may differ in the CNS and the periphery. study of the PTGS1 containing interactome, as this may reveal Moreover, our data suggest interactions between drugs such as potential sites other than PTGS1 that may have therapeutic acetylsalicylic acid and antipsychotic drugs may be particularly benefits in people with schizophrenia (Laan et al., 2010; Weiser beneficial in the treatment of schizophrenia. This argument is et al., 2012); such drugs may not have the unwanted side effects supported by the finding that the nonselective PTGS inhibitor, associated with the long term use of aspirin (Ridker et al., 2005). naproxen, antagonized haloperidol-induced catalepsy in mice We have begun to explore mechanisms that have the poten- (Naidu and Kulkarni, 2002). Notably, this effect was not observed tial to lower levels of PTGS1 in the CNS of subjects with schizo- with the selective PTGS2 inhibitor, rofecoxib, suggesting the phrenia. We have shown that treating rats or cells in culture interaction between naproxen and haloperidol involved PTGS1. with the antipsychotic drugs, haloperidol, and risperidone, Importantly, it is still not known if the therapeutic response to respectively, does not change levels of PTGS1. Our rat treat- treatment with acetylsalicylic acid and risperidone is due to a ment data agree with an earlier study that reported treatingCNS-wide reduction in levels of PTGS1 in subjects with schizo- rats with haloperidol at 0.15 mg/kg/d for 14 days did not change phrenia or by lowering further levels of PTGS1 in BA 46 from sub- levels of Ptgs1 expression (Wong et al., 2003). By contrast with jects with the disorder. It would seem unlikely that treatment studies in rats using haloperidol, a study treating rats with with acetylsalicylic acid and risperidone would have regionally 6 mg/kg/d olanzapine for 21 days reported no change in PTGS1 selective effects, and therefore at present it would seem most protein, but, compared with vehicle, found higher levels of Ptgs1 likely that the extra benefits obtained by using acetylsalicylic mRNA in the frontal cortex (Cheon et al., 2011). These outcomes acid as an adjunctive treatment with risperidone involves a were the same as a study that reported that, compared with vehi- CNS or cortical-wide reduction in PTGS1. Hence, preclinical and cle, treating rats with clozapine at 10 mg/kg/d for 30 days resulted clinical data would suggest exploring the potential interactions in higher levels of Ptgs1 mRNA without affecting levels of PTGS1 between drugs such as acetylsalicylic acid and antipsychotic protein (Kim et al., 2012). The same study reported that treatment drugs, as this could reveal potential therapeutic benefits asso- with clozapine also reduced the activity of PTGS1. Importantly, ciated with co-administration of such drugs in subjects with whilst data on clozapine and olanzapine raise the interesting schizophrenia (Laan et al., 2010W ; eiser et al., 2012). possibility that some of the therapeutic effects achieved with As with all studies of proteins involved in dynamic processes, these drugs may involve modulating Ptgs1 mRNA levels and/or our data on levels of PTGS1 could be affected by factors such activity, none of the data suggest the lower levels of PTGS1 in BA as the use of antiinflammatory medications, diet, or comorbid 46 are simply due to treatment with antipsychotic drug. inflammatory illness. In addition, although no case used in this Here we report that levels of cortical PTGS1 were very close study had recordable levels of acetylsalicylic acid in their blood to being lower in 10-month-old postpubescent mice (Dutta and at death, it is likely many individuals had taken acetylsalicylic Sengupta, 2016), but not in 1-month-old prepubescent mice who acid during their lifetime. However, although the long-term use had been injected with Poly I:C soon after birth. Such a change of acetylsalicylic acid is used to treat a number of conditions, in cortical PTGS1 was not observed in mice 1 or 10 months after there seems to be no data on how quickly any changes in PTGS1 PCP injections close to birth. These data are significant, because levels revert to normal after cessation of treatment. Unique to only in the 10-month-old Poly I:C-treated mice were we able to all postmortem studies, it is possible a particular cause of death demonstrate a decreased sucrose preference; this change in may affect levels of cortical PTGS1. In addition, there would likely behavior is thought to be analogous to anhedonia in schizophre- be a high level of substance abuse comorbidity in the subjects nia (Jones et al., 2011). Hence, our data raise the possibility that with psychiatric disorders. For example, there are high levels of Poly I:C treatment during the postnatal period causes immune nicotine, cannabis, and cocaine use in subjects with schizophre- activation to bring about a behavioral phenotype in adult mice nia (Winklbaur et al., 2006). It is therefore significant that nico- that models some aspects of the pathophysiology of schizophre- tine has been shown to increase, not decrease, the expression nia (Ibi et al., 2009). In addition, our data also suggest that some of PTGS1 (Schrör et al., 1998). By contrast, tetrahydrocannabinol changes in response to neonatal immune activation do not has been shown not to affect levels of PTGS1 (Chen et al., 2013). become apparent in the CNS until after puberty and that one of We could find no evidence to suggest cocaine affects levels of these changes may result in lower levels of PTGS1. We postulate PTGS1. Thus, available data suggest that the changes in levels of that lower levels of PTGS1 in BA 46 from subjects with schizo- PTGS1 in BA 46 from subjects with schizophrenia are not due to phrenia could be a response to a process involving immune acti- substance abuse. vation some considerable time prior to death and would support In summary, our data suggest there are lower levels of PTGS1 the notion of immunity and inflammation being involved in the in the dorsolateral prefrontal cortex of subjects with schizo- pathophysiology of the disorder. phrenia and that treatment with acetylsalicylic acid acts to We wished to understand the effects of treating with acetyl- lower levels of that protein, at least in a human-derived astro- salicylic acid and risperidone, as was done in subjects with cytic-like cell line, an outcome enhanced by the presence of schizophrenia (Laan et al., 2010W ; eiser et al., 2012) on levels of an antipsychotic drug. The synergistic action of antipsychotic PTGS1. We have now shown a dose-dependent decrease in levels drugs and acetylsalicylic acid on levels of PTGS1 provides a of PTGS1 after exposing CCF-STTG1 cells to acetylsalicylic acid, potential mechanism of action by which treating subjects with Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 224 | International Journal of Neuropsychopharmacology, 2018 schizophrenia with these 2 classes of drugs could improve thera- activity but not mRNA expression in human macrophages. peutic outcomes compared with treatment with antipsychotic Biochem Biophys Res Commun 225:896–900. drugs alone (Laan et al., 2010). However, an observational cohort Chen R, Zhang J, Fan N, Teng Z, Wu Y, Yang H, Tang Y, Sun H, study using data from the Danish nationwide registries suggests Song Y, Chen C (2013) Δ(9)-THC-caused synaptic and mem- there are multiple outcomes linked to the use of antipsychotic ory impairments are mediated through COX-2 signaling. Cell drugs and nonsteroidal antiinflammatory drugs, such as aspirin, 155:1154–1165. in treating schizophrenia (Köhler et al., 2016). One suggested Cheon Y, Park JY, Modi HR, Kim HW, Lee HJ, Chang L, Rao JS, disadvantage of using acetylsalicylic acid or diclofenac was an Rapoport SI (2011) Chronic olanzapine treatment decreases increased risk of relapse and hospitalization. These data would arachidonic acid turnover and prostaglandin E2 concentra- need to be taken into account when considering the use of tion in rat brain. J Neurochem 119:364–376. 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Studies on Prostaglandin-Endoperoxide Synthase 1: Lower Levels in Schizophrenia and After Treatment with Antipsychotic Drugs in Conjunction with Aspirin

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Abstract

Background: Antipsychotic drugs plus aspirin (acetylsalicylic acid), which targets prostaglandin-endoperoxide synthase 1 (PTGS1: COX1), improved therapeutic outcomes when treating schizophrenia. Our microarray data showed higher levels of PTGS1 mRNA in the dorsolateral prefrontal cortex from subjects with schizophrenia of long duration of illness, suggesting aspirin plus antipsychotic drugs could have therapeutic effects by lowering PTGS1 expression in the cortex of subjects with the disorder. Methods: We used Western blotting to measure levels of PTSG1 protein in human postmortem CNS, rat and mouse cortex, and cells in culture. Results: Compared with controls, PTGS1 levels were 41% lower in the dorsolateral prefrontal corte < .01), x (P but not the anterior cingulate or frontal pole, from subjects with schizophrenia. Levels of PTGS1 were not changed in the dorsolateral prefrontal cortex in mood disorders or in the cortex of rats treated with antipsychotic drugs. There was a strong trend (P = .05) to lower cortical PTGS1 10 months after mice were treated postnatally with polyinosinic–polycytidylic acid sodium salt (Poly I:C), consistent with cortical PTGS1 being lower in adult mice after exposure to an immune activator postnatally. In CCF- STTG1 cells, a human-derived astrocytic cell line, aspirin caused a dose-dependent decrease in PTGS1 that was decreased further with the addition of risperidone. Conclusions: Our data suggest low levels of dorsolateral prefrontal cortex PTGS1 could be associated with the pathophysiology of schizophrenia, and improved therapeutic outcome from treating schizophrenia with antipsychotic drugs augmented with aspirin may be because such treatment lowers cortical PTGS1. Keywords: Schizophrenia, aspirin, COX1, PTSG1, postmortem, cortex Received: June 15, 2017; Revised: September 18, 2017; Accepted: October 4, 2017 © The Author(s) 2017. 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 216 medium, 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/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 217 Significance Statement We showed that levels of PTGS1 messenger RNA were higher in the DLPFC from subjects with schizophrenia of long duration of illness. This was significant, because PTGS1 is targeted by aspirin, which increases the ability of antipsychotic drugs to lessen the symptoms of schizophrenia. Here, we report levels of PTGS1 protein is lower in the DLPFC, but not anterior cingulate or frontal pole, from subjects with schizophrenia and that PTGS1 is not altered in subjects with mood disorders or rats treated with antipsychotic drugs. Levels of PTGS1 were lower in human-derived astrocytes treated with aspirin, an effect augmented by the edition of the antipsychotic drug, risperidone. We propose lower levels of PTGS1 protein in the DLPFC are part of the pathophysi- ology of schizophrenia, and treatment with aspirin combined with an antipsychotic drug gives improved therapeutic benefits by lowering PTGS1 in subjects with schizophrenia. Introduction There is a significant body of evidence that suggests inflamma- Our studies showing higher levels of PTGS1 in mRNA in tion is contributing to the pathophysiology of schizophrenia the dorsolateral prefrontal cortex from subjects with schizo- (Na et  al., 2014). This hypothesis is supported by studies that phrenia (Narayan et al., 2008T ; ang et al., 2012b) differed from have shown changes in levels of mRNA for genes involved in findings at the level of protein that suggested PTGS1 was not modulating inflammatory-related pathways in postmortem different in prefrontal cortex, temporal cortex, or occipital cor - CNS tissue from subjects with schizophrenia (Saetre et al., 2007; tex from subjects with the disorder (Maida et al., 2006). Thus, Narayan et  al., 2008; Schmitt et  al., 2011; Hwang et  al., 2013). putting together our data and data on PTGS1 protein, it could Significantly, our study of CNS gene expression showed that, be that there are cortical region-specific changes in PTGS1 compared with controls, levels of prostaglandin-endoperoxide expression in the CNS from subjects with schizophrenia or synthase 1 (PTGS1: alias cyclooxygenase 1 [COX1]) mRNA were changes in PTGS1 expression are not translating into changes significantly higher in the dorsolateral prefrontal cortex from in levels of PTGS1 protein. To address this issue, we decided subjects with schizophrenia who were older than 40 years (av-er to determine if PTGS1 protein was changed in the same cor - age age = 64 ± 7.8  years) (Narayan et al. 2008T ; ang et al., 2012b). tical regions where there were higher levels of mRNA in sub- This finding is of significance with regards to therapeutic inter - jects with schizophrenia. We also decided to measure levels ventions, as it has been shown that acetylsalicylic acid (aspirin), of PTGS1 in the frontal pole and anterior cingulate cortex which targets PTGS1, lessens the severity of some of the symp- from subjects with schizophrenia to determine if changes in toms of schizophrenia when given as an adjunctive treatment PTGS1 could be present throughout the cortex. To address the with antipsychotic drugs compared with what is achieved giv- hypothesis that levels of PTGS1 are altered in the CNS after ing antipsychotic drugs alone (Laan et  al., 2010 W ; eiser et  al., an inflammatory episode, we measured levels of PTGS1 in the 2012). One of these clinical studies noted that acetylsalicylic cortex of neonatal mice treated with polyinosinic-polycytidylic acid adjunctive treatment was more effective in subjects with acid (Poly I:C), which activates inflammatory pathways (Ibi et high levels of blood interferon γ:interleukin 4 ratios, a measure al., 2009). We also measured PTGS1 in the cortex of mice that of increasing inflammatory status (Laan et al., 2010), data sug- had been treated neonatally with phencyclidine hydrochloride gesting that treatment with acetylsalicylic acid was more effect-(PCP), because this model, rather than models that deliver the ive in subjects with schizophrenia who were experiencing an drug to the rat foetus in utero (Jones et al., 2011), is not thought inflammatory episode. The second study (Weiser et  al., 2012) to act through inflammatory or immune pathways (Anastasio showed that acetylsalicylic acid, but not minocycline which is and Johnson, 2008). Significantly, both these animal models a tetracycline antibiotic with antiinflammatory effects that does produce a behavioral phenotype that allows the study of spe- not act on PTGS1, improved symptom severity when given with cific aspects of schizophrenia. We also sought to determine if antipsychotic drugs to subjects with schizophrenia. These data treatment with an antipsychotic drug alone could affect lev- suggest that targeting PTGS1 could be an important mechanism els of PTGS1 in rat cortex. Finally, we addressed the hypoth- of action for a drug that, when given with an antipsychotic drug, esis that treatment with acetylsalicylic acid with antipsychotic improves therapeutic outcomes. drugs affects levels of PTGS1 by measuring levels of the pro- Acetylsalicylic acid acts by targeting PTGS1 and PTGS2 (Vane tein in CCF-STTG1 cells, an eternalized cell line derived from a and Botting, 2003), which both convert arachidonic acid into bio- human astrocytoma (Vik-Mo et al., 2009), which we have estab- active prostanoids. Until recently it was thought that PTGS1 was lished stably express PTGS1, treated with vehicle, acetylsali- responsible for physiological production of prostanoids, whereas cylic acid, risperidone, or acetylsalicylic acid and risperidone. PTGS2 was involved in proinflammatory pathways being indu- cible at sites of inflammation (Aïd and Bosetti, 2011). However, Methods PTGS1 has now been shown to be present in microglia, where it is suggested to have a major role in modulating CNS inflamma- Materials tory processes (Choi et al., 2009). Hence, elevated levels of PTGS1 expression in the CNS could be indicative of generalized inflam- Poly I:C and PCPw ere obtained from Bio-Scientific Pty Ltd. Haloperidol, risperidone, and all general chemicals were matory processes driven by activated microglia in subjects with obtained from Sigma-Aldrich Pty Ltd. CCF-STTG1 cells were schizophrenia (van Berckel et  al., 2008Door ; duin et  al., 2009). obtained from American Type Culture Collection. RPMI 1960 By contrast, PTGS2 is predominantly localized to neurons and media, fetal calf serum (FCS), and antibiotic-antimycotic may therefore be more relevant to inflammatory processes that affect only those cells. (AB-AM) were obtained from ThermoFisher Scientific. Anti-PTGS1 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 218 | International Journal of Neuropsychopharmacology, 2018 antibody was obtained from Abcam (cat no.: AB109025). The goat Studies in Rodent CNS anti-rabbit IgG: horseradish peroxidase complex was obtained Studies in Rats from DAKO. All electrophoresis grade chemicals were from In determining the potential effects of antipsychotic drug treat- BioRad. ment on PTGS1, we decided to use an archetypal, first-genera- tion, antipsychotic drug, because this was the class of drugs used Ethical Approval to treat the cases of subjects with schizophrenia used in this study prior to death. Hence, 6-week-old male Sprague Dawley The Ethics Committee of the Victoria Institute for Forensic rats were exposed to a 12-h-day/-night schedule with free Medicine gave approval to collect human CNS post-mortem, and access to food. The rats (n = 10) received 0.1 mg/kg/d haloperidol all animal experiments were conducted with the approval of the in vehicle or vehicle alone (ethanol 0.1% v/v) in their drinking Florey Institute for Neuroscience and Mental Health Animal water for 28 days. This dose of drug was given on the principle Ethics Committee. that it would give a dopamine-D2 receptor occupancy in a rat equivalent to what is regarded as being optimal when treating subjects with schizophrenia (Kapur et  al., 2003) but adjusted Studies in Human CNS for delivery of haloperidol in drinking water rather than min- CNS tissue was collected from people who had a likely his- ipumps (0.25 mg/kg/d) or by s.c. injection (0.04–0.08 mg/kg s.c./d) tory of a psychiatric disorder and subjects who displayed no over 1 month rather than 7 days. The dose of haloperidol used in obvious symptoms of psychiatric disorders and who had not this study was slightly higher than that used for i.p. injection, as died by suicide (controls). At the time of death, no case had we have shown this gives blood levels of the drug in rats equiva- measurable levels of acetylsalicylic acid in their blood. Tissue lent to what would be expected in humans (Scarr and Dean, was collected from donors after either a witnessed death or 2012). The drug doses were adjusted twice weekly based on the having been seen alive within 5 hours of being found dead. average daily water consumption and increasing weight of each All cadavers were refrigerated soon after discovery, which rat. After the treatment, the rats were left untreated for 48 hours is advantageous as this would aid in slowing the effects of and then sacrificed by decapitation and the brains removed and autolysis (Ferrer et al., 2007). To further optimally preserve o rapidly frozen and stored at -80 C. CNS tissue, the left hemisphere from each donor was pro- cessed in a standardized manner that ensured tissue was fro- Studies in Mice zen to -80 C within 30 minutes of autopsy (Dean et al., 1999). Neonate C57black6J mice were housed with their dams in a Just prior to freezing, a sample of CNS tissue was collected 12-h-light/-dark cycle with free access to food and water. Two from each donor, and CNS pH was measured as described groups of 20 neonate mice received either daily i.p. injections of previously (Kingsbury et al., 1995), because CNS pH is a good pyrogen-free saline or pyrogen-free saline containing 10 mg/kg indicator as to the quality of tissue preservation (Stan et al., Poly I:C between postnatal days (PD) 2 to 6. All mice were imme- 2006). diately returned to their dam after each injection. Two groups of For each case, relevant data from clinical histories 20 mice were injected i.p. on PD 7, 9, and 11 with either pyrogen- and interviews with treating clinicians and relatives were free saline or pyrogen-free saline containing 10 mg/kg PCP and obtained using a standardized instrument, the Diagnostic on each occasion immediately returned to their dam. All mice Instrument for Brain Studies (Hill et al., 1996). On completion were weaned at PD 21, after which the sexes were housed sep- of this review, in cases other than those where it was agreed arately. From each treatment group, 10 mice were killed by cer - the donor had no history of psychiatric illness (controls), a vical dislocation at 1 month of age, which is at a prepubescent diagnosis was made by consensus by 2 psychiatrists and a phase, whilst the remaining 10 mice were killed postpuberty psychologist according to DSM-IV criteria (Roberts et al. 1998). at 10  months of age (Dutta and Sengupta, 2016). Notably, just In addition, the postmortem interval (PMI) was calculated as before being killed, the Poly I:C mice and the PCP mice w ere either the time from witnessed death to autopsy or the time tested with the saccharin preference test. At 1 month old, nei- mid-way between a subject being last seen and being found ther set of treated mice showed altered saccharin preference dead until autopsy. Duration of illness was calculated as the but at 10  months the Poly I:C-treated, but not the PCP-treated time from first presentation with psychiatric symptoms to mice, showed reduced saccharin preference, which is viewed as death. The final recorded doses of different classes of psycho- a behavior analogous to the anhedonia in schizophrenia (Jones tropic drugs were also recorded and standardized to a dose of et al., 2011). Immediately following cervical dislocation, the CNS a specific drug in class (Foster, 1989). was harvested and the frontal cortex was dissected and rapidly For this study, tissue from the dorsolateral prefrontal cortex o frozen in an isopentane bath on dry ice and stored at -80 C until (Brodmann’s [BA] area 46)  was obtained from 20 subjects with needed. Whole brain from one of the control group dams was schizophrenia, 20 subjects with major depressive disorders, 17 used to prepare the internal control used in Western blotting subjects with bipolar disorders, and 20 controls. Tissue was also (see below). obtained from BA 10 and BA 24 from the subjects with schizo- phrenia and the controls. Efforts were made to match age and Studies with CCF-STTG1 Cells sex across diagnoses. Different regions of the cortex were dis- For the study of the impact of acetylsalicylic acid ± risperidone sected guided by cytotectonic markers (Garey, 1994); hence, BA on PTGS1 levels, we chose to use CCF-STTG1 cells, because they 10 was taken as the most rostral portion of the superior frontal express human PTGS1 stably in culture. In these experiments, gyrus and middle frontal gyrus, bounded ventrally by the super - we used risperidone, because that was the antipsychotic drug ior rostral sulcus; BA 24 as anterior cingulate gyrus around the with the least complex neuropsychopharmacology (Goldstein, genu of the corpus callosum; and BA 46 as the lateral surface of 2000) of the drugs used in the study where acetylsalicylic acid the frontal pole, including approximately the middle third of the was used as an adjunctive treatment and caused an improve- middle frontal gyrus. ment in symptom severity (Laan et al., 2010). Thus, CCF-STTG1 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 219 cells were cultured to 70% confluency in RPMI 1960 media con- to control for inter-blot variation, an internal control (IC) of cor - taining 10% heat inactivated FCS and 1x AB-AM at 37 C with 5% tical homogenate was prepared from a single case that was not CO . Cells, at a density of 5 x 10 cells/well, were then subcul- used in the current study. This homogenate was run under the tured (P9) into 6-well cell culture plates and grown for 10 days in exact conditions to be used for the cases of interest in every lane RPMI 1960 media containing 5% FCS and 1x AB-AM. The media on 2 gels and the intensity of the immunogenic band of inter - was then removed, cells washed with 1x phosphate buffered est measured in each lane (30 measurements). These measure- saline and replaced with RPMI 1960 media containing 1x AB-AM, ments were used to establish both intra- and interblot variation no FCS, and either 0, 0.1, 0.5, or 1.0  mM acetylsalicylic acid in for the measurement of PTGS1. This sample was subsequently the absence or presence of risperidone (1 or 10 µM). The pH of run (in duplicate) on every gel, and each gel was exposed so that all media preparations was measured and, where necessary, the sum intensity of the IC fell within the range established adjusted to pH 7.4 prior to commencing the study. Five replicate by calculating the mean ± 2 SD of the initial 30 measurements. wells were prepared for each combination of drugs. The cells The intensity of PTGS1 from all cases were the standardized by were harvested after 24 hours and washed thrice in 1x phos- expressing them as a ratio of IC. For studies in rats and mice, a phate buffered saline. Protein homogenates were prepared by similarly prepared IC from rat and mice cortex was used. sonicating the cells in 10  mM Tris, pH 7.4, 1% SDS, and 1  mM Na VO and stored at -80C until needed. Protein homogenate 3 4 Statistics prepared from CCF-STTG1 cells cultured in RPMI 1960 media Demographic, CNS collection, and psychotropic drug data were containing 10% FCS and 1x AB-AM and harvested at 70% conflu- analyzed using the D’Agostino and Pearson omnibus normal- ency was used as an internal control for Western blotting (see ity test, and nonnormally distributed data were subsequently below). analyzed using nonparametric statistics. Student t tests or 1-way ANOVA were used to compare age, CNS pH, PMI, brain Western Blotting weight, and duration of illness. By contrast, experimental data expressed as ratios are better analyzed with nonparametric Human and rat (frontal cortex) tissue was homogenized into statistics (Siegel, 1957Allison et  ; al., 1995Curr ; an-Everett, 2013; 10 mM Tris containing 1% sodium dodecyl sulphate and 1 mM Dean et  al., 2016). PTGS1 levels in BA 10 and 24 from subjects sodium orthovanadate (pH 7.4) (5 x vol/tissue wet weight) using with schizophrenia and controls were compared using the 20 strokes using a Potter-Elvehjem PTFE pestle and glass tube. Mann-Whitney U test. Suicide and gender ratios across diag- The protein concentration in each homogenate was then meas- noses were compared using the Fisher exact test, and com- ured using the modified BioRad Detergent Compatible assay parisons of levels of PTGS1 across gender and between suicide (Lowry method). Cortical homogenates, containing 20  μg of completers and death by other causes was by Mann-Whitney protein, were then separated in duplicate on a 7.5 % pol yacryl- U test. Comparisons of experimental and with demographic, amide resolving gel. The separated proteins were transferred CNS collection, and pharmacological data were compared using onto nitrocellulose membranes (overnight constant at 40 mA in the Spearman’s rank correlation coefficient. Where there were Towbin transfer buffer). Each membrane was stained with 0.2% strong relationships between experimental and nonexperimen- Ponceau S in 3% TCA to confirm effective transfer of proteins. tal data, comparisons across diagnoses were made using nonex- After destaining, membranes were blocked with 5% non- perimental data as covariates. fat milk powder (NFMP) at room temperature (R/T) for 1 hour The data comparing levels of PTGS1 in the cortex of rats and then incubated with anti-PTGS1 antibody (ABCAM, cat no.: receiving vehicle or treated with haloperidol were compared ab109025 Anti-COX1 / Cyclooxygenase 1 rabbit monoclonal anti- using the Mann-Whitney U test. Levels of PTGS1 at 1 month body, clone no.: EPR5866) at a dilution of 1/200 in Tris-Tween- and 10 months after treatments with Poly I:C or PCP wer e com- Buffer-Saline (TTBS) with 5% NFMP o vernight at 4C. Membranes pared using the Mann-Whitney U test. Data from cell culture were then washed thrice for 10 minutes at R/T in TTBS and then data were analyzed using Kruskal-Wallace test using Dunn’s exposed to a 1/2000 dilution of goat anti-rabbit IgG: horseradish test as a posthoc analysis to compare the effects of risperidone peroxidase in TTBS with 5% NFMPfor 1 hour at R/T . Membranes and acetylsalicylic acid at different doses with the vehicle alone. were washed thrice for 10 minutes at R/T in TTBS and then incu- Statistical analyses were completed using GraphPad Prism v - er bated with Pierce ECL solution for 5 minutes at R/T. Excess ECL sion 6.00 for Windows, GraphPad Software. solution was then removed from the nitrocellulose, an image of chemiluminescence captured using a Kodak 440 CF imaging sys- tem. The intensity of the PTGS1 immunogenic band at ~70 kDa Results was then measured on each nitrocellulose gel. In many studies, Western blot results have been expressed Case History Reviews normalized to a loading control (Aldridge et al., 2008), the notion of the loading control being that the level of the chosen protein There were no significant differences in age or PMI across diag- will not vary as a proportion of total protein between tissues noses (Table  1; supplementary Table 1). There was a significant or between individuals. However, it is becoming increasingly variation in gender ratio between controls and subjects with clear that loading controls and/or reference genes or proteins major depressive disorders as well as subjects with schizophre- are not tenable for studies using human CNS tissue where lev- nia and subjects with major depressive disorders. CNS pH was els of individual proteins vary as a proportion of total protein significantly higher in subjects with major depressive disorders between donors (Eaton et al., 2013). Thus, the best way to control compared with controls, subjects with schizophrenia, and sub- for protein loading, as we have done in this study, is to care- jects with bipolar disorders. There was a significant variation in fully measure the protein concentration of each sample imme- CNS weight across diagnostic cohorts, but no individual diag- diately prior to it being loading onto the gel. This ensures that nosis differed from any other diagnoses. Rates of suicide com- the same protein concentration is loaded for each case (Eaton pletion were higher in subjects with major depressive disorders et al., 2013). Moreover, as described previously (Dean et al., 2006), compared with subjects with schizophrenia and subjects with Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 220 | International Journal of Neuropsychopharmacology, 2018 Table  1. A summary (for numeric: mean ± SEM) of data collected during the case history reviews of subjects with no history of psychiatric illness (controls) as well as for subjects with schizophrenia, major depressive disorders and bipolar disorders. Age Sex pH PMI CNS Weight DI Suicide FRADD LEAP FRAnDD FRMSD (yr) (M / F) (hr) (Kg) (yr) (y / n) CONTROLS 49 ± 4.7 16 / 4 6.28 ± 0.04 41 ± 3.5 1.43 ± 0.34 0 / 20 SCHIZOPHRENIA 48 ± 4.7 16 / 4 6.32 ± 0.05 43 ± 3.1 1.43 ± 0.35 21 ± 4.3 8 / 12 326 ± 76 7.46 ± 2.3 352 ± 115 MAJOR DEPRESSIVE 56 ± 3.6 9 / 11 6.54 ± 0.04 40 ± 3.6 1.30 ± 0.43 16 ± 2.4 17 / 3 303 ± 87 6.8 ± 2.6 862 ± 321 DISORDERS BIPOLAR DISORDERS 56 ± 3.4 9 / 8 6.29 ± 0.04 40 ± 4.0 1.23 ± 0.12 17 ± 3.0 7 / 13 256 ± 57 2.1 ± 0.60 573 ± 318 923 ± 210 F-value 1.01 8.77 0.12 3.03 0.52 0.27 1.56 0.87 d.f. 3,73 3,73 3,73 3,63 2,54 2,21 2,31 2,5 p 0.39 < 0.05 < 0.0001 0.95 < 0.05 0.6 0.002 0.76 0.23 0.47 Cont vs Sz p 1.00 n.s n.s. Cont vs MDD p 0.02 p < 0.001 n.s. Cont vs BD p 0.08 n.s. n.s. Sz vs MDD p 0.02 p < 0.01 n.s. 0.003 Sz vs BD p 0.08 n.s. n.s. 0.700 MDD vs BD p 0.63 p < 0.001 n.s. 0.003 Abbreviations: BD = bipolar disorders, Cont = Controls, DI = duration of illness, F = female, FRADD = final recorded antipsychotic drug does converted to mg chlorpromazine equivalents per day, FRAnD = final recorded antidepressant drug dose converted to mg fluoxetine equivalents per day, FRMSD = final recorded mood stabiliser dose converted to mg lithium equivalents per day, hr = hours, Kg = kilogram, LEAP = lifetime -3 exposure to antipsychotic drugs converted to as chlorpromazine equivalents per year x 10 , M  =  male, MDD  =  major depressive disorder , n = no, Sz = schizophrenia, y = yes, yr = year bipolar disorders. Levels of final recorded antipsychotic drug, (1.15–2.44), P = .73). For those with a psychiatric diagnoses, levels lifetime exposure to antipsychotic drug, final recorded dose of of PTGS1 did not differ between suicide completers and subjects antidepressant drug, or final recorded dose of mood stabilizers who died from other causes in BA 10 ((median± IQR): suicide did not differ significantly between subjects with schizophrenia, completers = 1.18 (0.97–1.53) vs other = 1.16 (0.88–1.56),P = .73), major depressive disorders or bipolar disorders. BA 24 ((median ± IQR): suicide completers= 0.99 (0.67–1.16) vs other = 1.01 (0.65–1.60), P = .61), or BA 46 ((median ± IQR): suicide completers = 1.72 (1.28–2.39) vs other = 1.35 (1.02–2.00),P = .27). Antibody Validation These data suggest that whilst gender ratio and suicide v - ar There is a growing concern about the specificity of antibodies ied with diagnoses (Table 1 ), they were not potential confounds used in various immuno-detection techniques (Manimala et al., when comparing levels of cortical PTGS1 across diagnoses. 2007; Jositsch et  al., 2009); thus, we determined the immuno- There were no significant relationships between levels genic profile of the anti-human PTGS1 antibody used in this of PTGS1 and age, PMI, CNS weight, duration of illness, final study using human cortex and a homogenate of human embry- recorded antipsychotic drug, final recorded dose of antidepres- onic kidney 293T cells that had either been, or not been, trans- sant drug, and final recorded dose of mood stabilizers (Table )2 , fected with the human PTGS1 gene. Using Western blotting, we suggesting these were not potential confounds. showed the presence of a single immunogenic band of molecu- lar weight 70 kDa in 2 samples of human cortex (Figure 1A). An Studies in Rodent Cortex immunogenic band of the same molecular weight was present at high levels in the homogenate from 293T cells transfected Compared with vehicle treatment, levels of PTGS1 did not dif- with the human PTGS1 gene but was absent in nontransfected fer in the cortex of rats treated with 0.1 mg/kg/d haloperidol for 293T cells. These data show the antihuman PTGS1 antibody 28 days (Figure 2A). Similarly, compared with vehicle treatment, used in this study has specificity for PTGS1. levels of PTGS1 did not differ in the cortex of mice 1 month after treatment with 10 mg/kg Poly I:C (Figure 2B), but there was a strong trend to decreased levels of PTGS1 in the cortex of mice Studies in Human Cortex 10 months after such treatment (=P .05). Indeed, a power ana- Compared with controls, levels of PTGS1 were significantly lysis of these data using G*Power Version 3.1.9.2 showed that an lower in BA 46 (P< .01), but not BA 10 (P= .37) or BA 24 (P = .08), addition of 1 animal per treatment group at 10 months w ould from subjects with schizophrenia (Figure 1B). A power analysis be needed to reach significance P < .05. Compared with vehicle of the data from BA 24 using G*Power version 3.1.9.2 showed treatment, levels of PTGS1 did not differ in the cortex of mice 1 that 92 individuals in each diagnostic group would be needed or 10 months after treatment with 10 mg/kg PCP. to reach significance (P < .05). Levels of PTGS1 were measured only in BA 46 from subjects with major depressive disorders and Studies in CCF-STTG1 Cells bipolar disorders and did not differ when compared with con- trols (Figure 1B). Compared with vehicle treatment, levels of PTGS1 did not dif- Levels of PTGS1 did not differ with gender in BA 10 fer in CCF-STTG1 cells after exposure to 1 or 10  μM risperidone (median ± IQR: F = 1.31 (0.88–1.55) vs M = 1.34 (1.02–1.54), P = .74), for 24 hours (Figure 3A). By contrast, acetylsalicylic acid caused BA 24 (median ± IQR: F = 1.12 (0.96–1.51) vs M = 1.09 (0.76–1.84), a dose-dependent decrease in levels of PTGS1 in CCF-STTG1 P = .96), or BA 46 (median ± IQR: F = 1.67 (1.03–2.47) vs M = 1.78 cells after treatment for 24 hours, which was significantly Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 221 Figure 1. Measuring levels of PTSG1 in human and rodent cortex. (A) A Western blot showing levels of a proteins that displayed immunogenic sites that for an anti- prostaglandin synthase 1 (PTGS1) antibody (ABCAM Anti-COX1 / Cyclooxygenase 1 rabbit monoclonal antibody, clone no.: EPR5866) in human, rat, and mouse cortices (20 µg protein) as well as 293T cells that were either transfected or not transfected with the human prostaglandin- endoperoxide synthase 1 (PTGS1) gene. To provide an impression of relative levels of expression 20 µg protein from undiluted 293T or CCF-STTG1 cell lysate (set 3) or cell lysate diluted 1:10 (set 2) or 1:20 (set 1) prior to load- ing onto the polyacrylamide gel are shown. (B) A typical Western blot showing levels of a 70-kDA protein that displayed immunogenic sites for an anti-PTGS1 antibody in homogenates from Brodmann’s areas 10, 24 and 46 from subjects with schizophrenia and age / sex nonpsychotic controls. (B) Levels (median ± interquartile range) of PTGS1 protein in cortical homogenates containing 20 µg total protein from BA 10, 24, and 46 from subjects with schizophrenia and age-/sex-matched controls, as well as BA 46 from subjects with major depressive disorders and bipolar disorders. Table 2. The relationships between levels of prostaglandin-endoperoxide synthase 1 in (A) human BA 10, BA 24 and BA 46 and with (B) case history data in those three cortical regions. Age pH PMI CNS Weight DI FRADD FRAntiD FRMSD ρ p ρ p ρ p ρ p ρ p ρ p ρ p ρ p BA 24 0.19 0.25 0.13 0.42 -0.19 0.25 0.01 0.95 0.28 0.23 -0.10 0.79 n.a n.a BA 46 -0.09 0.46 -0.01 0.92 0.06 0.58 -0.08 0.49 -0.23 0.08 -0.01 0.97 -0.04 0.94 -0.36 0.28 BA 10 0.00 0.99 0.00 0.99 0.03 0.85 -0.16 0.32 0.32 0.17 0.05 0.89 n.a n.a Abbreviations: BA = Brodmann’s area, DI = duration of illness, FRADD = final recorded antipsychotic drug dose expressed as chlorpromazine equivalents per day, FRAntiD = final recorded antidepressant drug dose expressed as fluoxetine equivalents per day, FRMSD = final recorded dose of mood stabiliser drug dose expressed as lithium equivalents per day, PMI = postmortem interval different from vehicle at 1.0 M acetylsalicylic acid (Figure  3B). for levels of mRNA, we do not find that differences between lev- Acetylsalicylic acid decreased levels of PTGS1 in CCF-STTG1 els of PTGS1 protein in BA 46 from subjects with schizophrenia, cells in the presence of both 1  μM and 10 μM risperidone with compared with controls, vary in significance with duration of differences between drug treatment and vehicle reaching sig- illness. Thus, it is possible that lower levels of PTGS1 in BA 46 nificance at 0.5 M acetylsalicylic acid. are present at or close to the onset of schizophrenia and that the higher levels of expression of PTGS1 in BA 46 from subjects with schizophrenia of long duration (Narayan et al., 2008) rep - Discussion resent a slow and unsuccessful response to rectify low protein levels. Alternatively, changes in gene promoter methylation We have shown that levels of PTGS1 are lower in the dorsolateral prefrontal cortex, but not frontal pole or anterior cingulate cor - are known to occur with age (Jung and Pfeifer, 2015) and dur - ation of illness also correlates with age. Therefore, the increased tex, from subjects with schizophrenia and that PTGS1 levels are not altered in the dorsolateral prefrontal cortex from subjects expression of PTGS1 mRNA in schizophrenia may be associated with differential changes in gene promoter methylation with with major depressive disorders or bipolar disorders. Our previ- ous data showed higher levels of PTGS1 mRNA in the dorsolateral age in BA 46 from subjects with schizophrenia and controls (Tang et al., 2012a). However, PTGS1 protein should more closely prefrontal cortex from subjects with schizophrenia of long - dur ation (Narayan et al., 2008; Tang et al., 2012b). By contrast, unlike reflect functional changes in the CNS, and therefore we would Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 222 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Levels of PTSG1 in rodent cortex after treatment with the antipsychotic drug haloperidol, poly I:C or phencyclidine. Levels (median ± interquartile range) of prostaglandin- endoperoxide synthase 1 (PTGS1) protein in the cortex of: (A) rats treated with haloperidol at 0.1 mg/kg/d or vehicle (ethanol 0.1% v/v) in their drinking water for 28 days, (B) mice 1 and 10 months after treatment with 10 mg/kg Poly I:C between postnatal days (PD) 2 to 6, and (C) mice 1 and 10 months after treatment with 10 mg/kg phencyclidine on PD 7, 9, and 11. Figure 3. Levels of PTSB1 in CCF-STTG1 cells after treatment with the antipsychotic drug ripseridone, acetylsalicylic acid or both drugs in combination. Levels (median ± interquartile range) of prostaglandin-endoperoxide synthase 1 (PTGS1) protein in CCF-STTG1 cells after: (A) exposure to vehicle, 1 or 10 µM risperidone for 24 hours or (B) exposure to vehicle and varying doses of acetylsalicylic acid and risperidone for 24 hours. *P < .05 compared with levels of PTGS1 in the absence of acetylsalicylic acid and risperidone. argue that PTGS1 in BA 46 is either involved in or affected by the prefrontal cortex from subjects with major depressive disorders pathophysiology of the disorder. and bipolar disorders, which adds to earlier findings that the Notably, our data suggest that low levels of PTGS1 may be protein is not changed in prefrontal cortex, temporal cortex, or localized to the dorsolateral prefrontal cortex in subjects with occipital cortex from subjects with mood disorders (Maida et al., schizophrenia. This argument is in line with findings from an 2006). There is a report of lower levels of PTGS1 protein and earlier study, which, using immunohistochemistry (Maida et al., mRNA in the frontal cortex from subjects with bipolar disorder 2006) or Western blotting (Rao et  al., 2013), failed to show any (Kim et al., 2012). However, the region of the frontal cortex used change in levels of PTGS1 protein in BA 8, 18, 21, or 22, or hippo- was not defined and may not be one of the cortical regions used campus or PTGS1 mRNA in BA 10 from subjects with schizophre- in this study. nia (Maida et al., 2006; Rao et al., 2013), respectively. In addition, Current data would suggest that changes in PTGS1 levels do we report no change in levels of PTGS1 in the dorsolateral not occur globally in the CNS from subjects with schizophrenia, Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dean et al. | 223 and therefore the changes in PTGS1 may make a unique con- which was further enhanced in the presence of risperidone. Our tribution to the changes in functioning of the dorsolateral data therefore suggest there may be a synergistic interaction prefrontal cortex that have long been associated with schizo- between antipsychotic drugs, such as risperidone, with acetyl- phrenia. Significantly, PTGS1 has recently been proposed salicylic acid to increase its ability to lower levels of PTGS1. Our to form part of a protein-protein interactome that is predicted to data from a human-derived astrocytic-like cell line differs from be affected because of genetic changes that have been shown to that from human monocytic cell lines, which, after exposure to increase the risk for schizophrenia (Ganapathiraju et al., 2016). acetylsalicylic acid, did not change levels of PTGS1 mRNA or pro- Moreover, many of the proteins in this interactome, including tein (Barrios-Rodiles et al., 1996). These data suggest that acetyl- PTGS1, were predicted to be promising drug targets for drugs to salicylic acid may have cell-specific effects on PTGS1 levels treat schizophrenia. Hence, our data would encourage further and that these effects may differ in the CNS and the periphery. study of the PTGS1 containing interactome, as this may reveal Moreover, our data suggest interactions between drugs such as potential sites other than PTGS1 that may have therapeutic acetylsalicylic acid and antipsychotic drugs may be particularly benefits in people with schizophrenia (Laan et al., 2010; Weiser beneficial in the treatment of schizophrenia. This argument is et al., 2012); such drugs may not have the unwanted side effects supported by the finding that the nonselective PTGS inhibitor, associated with the long term use of aspirin (Ridker et al., 2005). naproxen, antagonized haloperidol-induced catalepsy in mice We have begun to explore mechanisms that have the poten- (Naidu and Kulkarni, 2002). Notably, this effect was not observed tial to lower levels of PTGS1 in the CNS of subjects with schizo- with the selective PTGS2 inhibitor, rofecoxib, suggesting the phrenia. We have shown that treating rats or cells in culture interaction between naproxen and haloperidol involved PTGS1. with the antipsychotic drugs, haloperidol, and risperidone, Importantly, it is still not known if the therapeutic response to respectively, does not change levels of PTGS1. Our rat treat- treatment with acetylsalicylic acid and risperidone is due to a ment data agree with an earlier study that reported treatingCNS-wide reduction in levels of PTGS1 in subjects with schizo- rats with haloperidol at 0.15 mg/kg/d for 14 days did not change phrenia or by lowering further levels of PTGS1 in BA 46 from sub- levels of Ptgs1 expression (Wong et al., 2003). By contrast with jects with the disorder. It would seem unlikely that treatment studies in rats using haloperidol, a study treating rats with with acetylsalicylic acid and risperidone would have regionally 6 mg/kg/d olanzapine for 21 days reported no change in PTGS1 selective effects, and therefore at present it would seem most protein, but, compared with vehicle, found higher levels of Ptgs1 likely that the extra benefits obtained by using acetylsalicylic mRNA in the frontal cortex (Cheon et al., 2011). These outcomes acid as an adjunctive treatment with risperidone involves a were the same as a study that reported that, compared with vehi- CNS or cortical-wide reduction in PTGS1. Hence, preclinical and cle, treating rats with clozapine at 10 mg/kg/d for 30 days resulted clinical data would suggest exploring the potential interactions in higher levels of Ptgs1 mRNA without affecting levels of PTGS1 between drugs such as acetylsalicylic acid and antipsychotic protein (Kim et al., 2012). The same study reported that treatment drugs, as this could reveal potential therapeutic benefits asso- with clozapine also reduced the activity of PTGS1. Importantly, ciated with co-administration of such drugs in subjects with whilst data on clozapine and olanzapine raise the interesting schizophrenia (Laan et al., 2010W ; eiser et al., 2012). possibility that some of the therapeutic effects achieved with As with all studies of proteins involved in dynamic processes, these drugs may involve modulating Ptgs1 mRNA levels and/or our data on levels of PTGS1 could be affected by factors such activity, none of the data suggest the lower levels of PTGS1 in BA as the use of antiinflammatory medications, diet, or comorbid 46 are simply due to treatment with antipsychotic drug. inflammatory illness. In addition, although no case used in this Here we report that levels of cortical PTGS1 were very close study had recordable levels of acetylsalicylic acid in their blood to being lower in 10-month-old postpubescent mice (Dutta and at death, it is likely many individuals had taken acetylsalicylic Sengupta, 2016), but not in 1-month-old prepubescent mice who acid during their lifetime. However, although the long-term use had been injected with Poly I:C soon after birth. Such a change of acetylsalicylic acid is used to treat a number of conditions, in cortical PTGS1 was not observed in mice 1 or 10 months after there seems to be no data on how quickly any changes in PTGS1 PCP injections close to birth. These data are significant, because levels revert to normal after cessation of treatment. Unique to only in the 10-month-old Poly I:C-treated mice were we able to all postmortem studies, it is possible a particular cause of death demonstrate a decreased sucrose preference; this change in may affect levels of cortical PTGS1. In addition, there would likely behavior is thought to be analogous to anhedonia in schizophre- be a high level of substance abuse comorbidity in the subjects nia (Jones et al., 2011). Hence, our data raise the possibility that with psychiatric disorders. For example, there are high levels of Poly I:C treatment during the postnatal period causes immune nicotine, cannabis, and cocaine use in subjects with schizophre- activation to bring about a behavioral phenotype in adult mice nia (Winklbaur et al., 2006). It is therefore significant that nico- that models some aspects of the pathophysiology of schizophre- tine has been shown to increase, not decrease, the expression nia (Ibi et al., 2009). In addition, our data also suggest that some of PTGS1 (Schrör et al., 1998). By contrast, tetrahydrocannabinol changes in response to neonatal immune activation do not has been shown not to affect levels of PTGS1 (Chen et al., 2013). become apparent in the CNS until after puberty and that one of We could find no evidence to suggest cocaine affects levels of these changes may result in lower levels of PTGS1. We postulate PTGS1. Thus, available data suggest that the changes in levels of that lower levels of PTGS1 in BA 46 from subjects with schizo- PTGS1 in BA 46 from subjects with schizophrenia are not due to phrenia could be a response to a process involving immune acti- substance abuse. vation some considerable time prior to death and would support In summary, our data suggest there are lower levels of PTGS1 the notion of immunity and inflammation being involved in the in the dorsolateral prefrontal cortex of subjects with schizo- pathophysiology of the disorder. phrenia and that treatment with acetylsalicylic acid acts to We wished to understand the effects of treating with acetyl- lower levels of that protein, at least in a human-derived astro- salicylic acid and risperidone, as was done in subjects with cytic-like cell line, an outcome enhanced by the presence of schizophrenia (Laan et al., 2010W ; eiser et al., 2012) on levels of an antipsychotic drug. The synergistic action of antipsychotic PTGS1. We have now shown a dose-dependent decrease in levels drugs and acetylsalicylic acid on levels of PTGS1 provides a of PTGS1 after exposing CCF-STTG1 cells to acetylsalicylic acid, potential mechanism of action by which treating subjects with Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/216/4401462 by Ed 'DeepDyve' Gillespie user on 16 March 2018 224 | International Journal of Neuropsychopharmacology, 2018 schizophrenia with these 2 classes of drugs could improve thera- activity but not mRNA expression in human macrophages. peutic outcomes compared with treatment with antipsychotic Biochem Biophys Res Commun 225:896–900. drugs alone (Laan et al., 2010). However, an observational cohort Chen R, Zhang J, Fan N, Teng Z, Wu Y, Yang H, Tang Y, Sun H, study using data from the Danish nationwide registries suggests Song Y, Chen C (2013) Δ(9)-THC-caused synaptic and mem- there are multiple outcomes linked to the use of antipsychotic ory impairments are mediated through COX-2 signaling. Cell drugs and nonsteroidal antiinflammatory drugs, such as aspirin, 155:1154–1165. in treating schizophrenia (Köhler et al., 2016). One suggested Cheon Y, Park JY, Modi HR, Kim HW, Lee HJ, Chang L, Rao JS, disadvantage of using acetylsalicylic acid or diclofenac was an Rapoport SI (2011) Chronic olanzapine treatment decreases increased risk of relapse and hospitalization. These data would arachidonic acid turnover and prostaglandin E2 concentra- need to be taken into account when considering the use of tion in rat brain. J Neurochem 119:364–376. 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It could also be argued that the therapeutic bene- brain using in situ radioligand binding and autoradiography. fit from augmenting antipsychotic drug treatment with acetyl- In: Using CNS tissue in psychiatric research: A practical guide salicylic acid is because this decreases levels of PTGS1 in regions (Dean B, Kleinman JE, Hyde TM, eds), pp 67–83. Amsterdam: of the CNS other than BA 46. This study cannot resolve this issue Harwood Academic Press. but does suggest that, given the important role for PTGS1 in CNS Dean B, Gray L, Scarr E (2006) Regionally specific changes in lev- inflammation (Choi et al., 2009), lower levels of PTGS1 in the CNS els of cortical S100beta in bipolar 1 disorder but not schizo- from subjects with schizophrenia should be protective against phrenia. Aust NZ J Psychiatry 40:217–224. neuroinflammation (Fillman et al., 2013). 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International Journal of NeuropsychopharmacologyOxford University Press

Published: Mar 1, 2018

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