Peripheral Transcription of NRG-ErbB Pathway Genes Are Upregulated in Treatment-Resistant Schizophrenia

Investigation of peripheral gene expression patterns of transcripts within the NRG–ErbB signaling pathway, other than neuregulin-1 (NRG1), among patients with schizophrenia and more specifically treatment-resistant schizophrenia (TRS) is limited. The present study built on our previous work demonstrating elevated levels of NRG1 EGFα, EGFβ, and type I(Ig2) containing transcripts in TRS by investigating 11 NRG–ErbB signaling pathway mRNA transcripts (NRG2, ErbB1, ErbB2, ErbB3, ErbB4, PIK3CD, PIK3R3, AKT1, mTOR, P70S6K, eIF4EBP1) in whole blood of TRS patients (N = 71) and healthy controls (N = 57). We also examined the effect of clozapine exposure on transcript levels using cultured peripheral blood mononuclear cells (PBMCs) from 15 healthy individuals. Five transcripts (ErbB3, PIK3CD, AKT1, P70S6K, eIF4EBP1) were significantly elevated in TRS patients compared to healthy controls but only expression of P70S6K (Pcorrected = 0.018), a protein kinase linked to protein synthesis, cell growth, and cell proliferation, survived correction for multiple testing using the Benjamini–Hochberg method. Investigation of clinical factors revealed that ErbB2, PIK3CD, PIK3R3, AKT1, mTOR, and P70S6K expression were negatively correlated with duration of illness. However, no transcript was associated with chlorpromazine equivalent dose or clozapine plasma levels, the latter supported by our in vitro PBMC clozapine exposure experiment. Taken together with previously published NRG1 results, our findings suggest an overall upregulation of transcripts within the NRG–ErbB signaling pathway among individuals with schizophrenia some of which attenuate over duration of illness. Follow-up studies are needed to determine if the observed peripheral upregulation of transcripts within the NRG–ErbB signaling pathway are specific to TRS or are a general blood-based marker of schizophrenia.

The present investigation, therefore, quantiatively compared (i) whole blood mRNA levels of 11 NRG-ErbB signaling receptors and pathway genes (NRG2, ErbB1, ErbB2, ErbB3, ErbB4, PIK3CD, PIK3R3, AKT1, mTOR, P70S6K, eIF4EBP1) among individuals with TRS and healthy controls, (ii) associations between mRNA levels and symptom severity, age of onset, duration of illness, clozapine plasma level, and chlorpromazine equivalent dosage, and (iii) the effect of clozapine exposure on mRNA expression in PBMCs from healthy controls. We expected that there would be multiple molecular changes in TRS compared to controls that may contribute to the amplification of NRG1 signaling in perhi peral blood in support of a widespread gain of function model of NRG1 in the pathophysiology of schizophrenia.

Clinical Samples
Seventyone participants aged 18-65 with schizophrenia who were treated with clozapine were recruited from inpatient and outpatient clinics in Melbourne, Australia. As these individuals failed to respond to two or more previous trials of antipsychot ics, had poor functioning, and persistent symptoms, they were considered "treatmentresistant, " consistent with current criteria  (25). In addition, 57 age, sex, and socioeconomicmatched unrelated healthy controls were recruited from the general com munity. Controls with a firstdegree family history of psychiatric illness, prior or current use of antipsychotic medication, head injury, seizure, neurological disease, impaired thyroid function, and/or substance abuse/dependence were excluded. Detailed demographic characteristics of all participants are presented in Table 1.
Mini International Neuropsychiatric Interview (26) was administered to all participants to confirm the diagnosis of schizophrenia as well as to rule out the presence of psychiatric disorders in healthy controls. The Positive and Negative Syndrome Scale (PANSS) (27) was used to assess the clinical symptoms and the patients were scored in accordance with the consensus fivefactor (i.e., positive, negative, disorganized/concrete, excited, depressed) PANSS model (28). Information on tobacco, alcohol, and illicit drug use in the past 3 months was collected using a substance use questionnaire. Whole blood samples were collected after overnight fasting and processed according to standardized blood collection and processing protocol (see supplementary methods for more details). Plasma levels of clozapine were measured and chlorpromazine equivalent dosage (excluding clozapine) were calculated for the 31% (n = 22) of participants with schizophrenia who were taking concomitant antipsychotic medication in accordance with published guidelines (29,30). All the participants provided written informed consent and the study protocol was approved by the Melbourne Health Human Research Ethics Committee (MHREC ID 2012.069). The study complied with the Declaration of Helsinki and its subsequent revisions (31).

In Vitro Clozapine Exposure Samples
To assess the effect of clozapine exposure on gene expression of our candidate transcripts, fresh frozen PBMCs from 15 healthy individuals (8 males and 7 females) of European ancestry with a mean age of 35 (SD = 13.5; range 20-54 years) were purchased from STEMCELL™ Technologies, Inc. (Vancouver, BC, Canada). A sample size of 15 was sufficient to detect a large effect (Cohen's d = 0.80) between exposed and unexposed conditions at α = 0.05 and power (1 − β) = 0.80. The percentage of current smokers among the donors was 33.3% (n = 5). All the donors were tested for HIV1, HIV2, hepatitis B and hepatitis C prior to blood collection.
Peripheral blood mononuclear cells isolated from whole blood were supplied as vials containing 100 million cells. PBMCs were rapidthawed from liquid nitrogen and seeded in sixwell plates in triplicates at a concentration of 2 million cells per well (1 × 10 6 cells/mL) in RPMI1640 medium (SigmaAldrich; St. Louis, MO, USA) supplemented with lglutamine (0.3 g/L) and sodium bicarbonate (2 g/L), penicillin (100 U/mL), streptomy cin (100 µg/mL), and 10% fetal bovine serum for 24 h. Cells were then exposed to clozapine (SigmaAldrich, St. Louis, MO, USA) for 24 h and 7 days, at a concentration of 1.2 µM (control cells were exposed to vehicle only, see supplementary methods for details) and incubated at 37°C in 5% CO2. Clozapine was initially dissolved in absolute ethanol and media was used for dilution. The final concentration of ethanol on each well was 1 in 8,000. The concentration of clozapine used was determined from the mean plasma concentration of clozapine found in the first 48 recruited clinical samples (1.2 µM or 384 ng/mL). Toxicity assays (CytoTox 96 ® NonRadioactive Cytotoxicity Assay; Promega Corporation, Madison, WI, USA) were performed at baseline, 24 h and 7day time points after clozapine exposure to meas ure the production of lactate dehydrogenase within the media (see Figure S1 in Supplementary Material for more details).

rna extraction, complementary Dna (cDna) synthesis, and Quantitative realtime Pcr
PureLink RNA Mini Kit (ThermoFisher scientific, Waltham, MA, USA) was used to extract total RNA from both clinical and in vitro samples following standard manufacturer's instruc tions. The RNA integrity number (RIN) range was 3.60-9.50 (mean = 8.59, SD = 0.79). Total RNA was reverse transcribed to complementary DNA (cDNA) using SuperScript ® IV First Strand Synthesis System (Invitrogen, Foster city, CA, USA) using random hexamers. cDNA (10.25 ng) was used as a template for realtime PCR (RTqPCR) using mastermix and gene specific validated Taqman assays from Applied Biosystems, Foster City, CA, USA. Inventoried assays (TaqMan ® , Invitrogen, USA) were used for all the genes of interest as well as for four reference genes (betaactin, ACTB; ubiquitin C, UBC; ABL protooncogene 1, . In addi tion, no reverse transcriptase controls and no template controls were included to rule out genomic DNA contamination and reagent contamination, respectively. Adhering to minimum information for publication of RTqPCR (MIQE) guidelines (32), normalized relative quantities (NRQ), i.e., 2 −ΔCt where ΔCt = [Ct(candidate gene) − Ct (geometric mean of reference genes)] of each mRNA isoform was calculated using the geometric mean expression of two reference genes (UBC and ACTB) that did not differ between groups in the clinical cohort. ABL1 and SDHA were not used as reference genes because their expression differed significantly by group in the clinical cohort ( Figures S2-S4 in Supplementary Material). In the in vitro cohort only, ABL1 was stable after 24 h clozapine exposure and ACTB was stable after 7 days clozapine exposure and were used for normalization and subsequent analysis at specific time points.

statistical analysis
Twosided tests were used for all statistical analyses. Shapiro-Wilk test and quantile-quantile (Q-Q) plots were used to assess normality of variable distributions. Student's ttests were used to test differences for continuous variables between schizophrenia patients and healthy controls, while chisquared (χ 2 ) tests were used for categorical variables. The Benjamini and Hochberg (B-H) stepup procedure (33) was used to adjust for multiple comparisons for all analyses. Effect sizes were calculated using the Hedges' g method (34).
Prior to analysis, the NRQ values for all the mRNA transcripts were checked for normality using Q-Q plots ( Figure S5 in Supplementary Material) and as required were log10 transformed for subsequent analysis. In addition, we assessed the following variables as potential confounders: age, sex, RIN, alcohol use, and smoking status. A variable was considered a confounder and included in our statistical models only when it was significantly different between groups (P < 0.05) and was significantly associ ated with gene expression. The logtransformed NRQ values were compared among groups using general or generalized linear models based on their distribution and adjusted for appropriate covariates. Outliers were identified using the Grubbs' test for outliers and removed from further analysis.
Within the schizophrenia group, Pearson or Spearman correlations, depending on data distribution, were calculated between gene transcript levels and symptom severity, age of onset, illness duration, current chlorpromazine equivalent dose, and clozapine plasma levels. In addition, mRNA transcript levels between participants in positive symptom remission and non remission were assessed using a ttest or Mann-Whitney U test. Positive symptom remission was defined as a PANSS score of ≤3 on delusions, hallucinations, grandiosity, and unusual thought content (28).
To assess differences in gene expression between clozapine exposed and unexposed PBMCs at both time points (24 h and 7 days), Wilcoxon matched paired ttest were used, adjusting for age, gender, and RIN.

NRG-ErbB signaling Pathway Transcripts are Upregulated in Trs
Two (ErbB1, ErbB4) of the 11 NRG-ErbB pathway mRNA tran scripts interrogated, were not detectable in more than 80% of the full cohort and so were removed from further analysis. The rates of nondetects were not significantly different between groups (ErbB1: case 95%, control: 97%; ErbB4: case 81%, control 85%). Analysis on the remaining nine transcripts showed significantly elevated levels of five transcripts: ErbB3 (P = 0.046), PIK3CD (Praw = 0.035), AKT1 (Praw = 0.018), P70S6K (Praw = 0.002), and eIF4EBP1 (Praw = 0.013) in TRS patients compared to healthy controls after adjustment for covariates. However, only P70S6K (PB-H = 0.018) remained significant after correction for multiple comparisons (Figure 2). Importantly, transcript levels were not correlated with clozapine plasma levels or chlorpromazine equivalent antipsychotic exposure (excluding clozapine) ( Table  S2 in Supplementary Material). The lack of relationship between mRNA levels and clozapine levels were further corroborated by our in vitro analysis that showed no difference in mRNA levels of detectable transcripts (n = 9) in clozapine exposed compared to unexposed PBMCs, except mTOR mRNA which showed decreased expression levels in clozapine exposed cells at both 24 h (P = 0.001) and 7day (P = 0.

DiscUssiOn
Our findings suggest transcription in the NRG-ErbB signaling pathway is upregulated in the whole blood of individuals with TRS and is negatively correlated with duration of illness. Among the nine detectable NRG-ErbB pathway transcripts we exam ined, five (ErbB3, PIK3CD, AKT1, P70S6K, and eIF4EBP1) were elevated and, of these, P70S6K survived correction for multiple comparisons. Importantly, we could not attribute this upregula tion of peripheral transcription in the NRG-ErbB pathway to age, sex, or medication. In fact, results from our in vitro clozapine exposure experiment suggested clozapine might reduce rather than increase transcription of genes within the NRG-ErbB signal ing pathway, particularly mTOR expression. Overall, our findings support our hypothesis that there is a generalized increase in NRG1 signaling in people with TRS. Previous findings by us and others support the notion of increased transcription of genes within the NRG-ErbB signal ing pathway in schizophrenia. We recently showed in the same cohort used in the current study, an increased expression of three NRG1 transcripts [i.e., NRG1EGFα, NRG1EGFβ, and NRG1typeI(Ig2)] in TRS compared to controls (24). In addition, several studies by others have reported increased expression of specific isoforms of NRG1 (18) and mRNA of downstream sign aling molecules, including PIK3CD, PIK3CB (16,22), and AKT1  (22,23) in schizophrenia patients. Furthermore, other down stream signaling molecules, such as mTOR, P70S6K, and eIF4B, have been shown to be increased in major depressive disorder (35). However, as we are not aware of any human studies that have interrogated P70S6K, in schizophrenia, we are the first to report increased mRNA of P70S6K in TRS. P70S6K encodes for a vital kinase in the mTOR signaling path way (36)(37)(38) that when phosphorylated by mTOR results in phos phorylation and activation of translation elongation factors eIF4B and eEF2K, thereby promoting protein translation (39,40). Our findings suggest upregulation of P70S6K, in part, may result from an increase in transcription of several genes upstream of P70S6K within the NRG-ErbB signaling pathway. However, other genes (i.e., BDNF, DISC1) as well as neurotransmitters (i.e., glutamate, serotonin) and hormones (e.g., insulin) have also been shown to activate the PI3K-AKT-mTOR signaling pathway (41)(42)(43) and as such may contribute or confound the increase in P70S6K expres sion we have observed. However, most studies find decreased BDNF levels in the blood of people with schizophrenia (44) and suggest some degree of insulin resistance in clozapinetreated patients (45). Future investigations should attempt to account for these other signaling factors and the potential confounders of metabolic changes in people with schizophrenia being treated with clozapine, as doing so will further elucidate the suitability of P70S6K as a peripheral biomarker of overactivity in the NRG1 pathway in schizophrenia.
We also detected trendlevel increases in three transcripts (ErbB3, PIK3CD, and AKT1) upstream of mTOR, within the NRG-ErbB signaling pathway among those with TRS. These increases in whole blood expression are, in part, supported by previous studies that have shown an increased AKT1 mRNA expression in PBMCs from individuals with earlyonset (23) and treatmentnaïve schizophrenia (46), suggesting peripheral upregulation of NRG-ErbB pathway transcripts may not be speci fic to the stage of illness and may occur during the first phases of schizophrenia and continue during the chronic phases. However, six of the mRNA transcripts (ErbB2, PIK3CD, PIK3R3, AKT1, mTOR, and P70S6K) we examined were negatively correlated with duration of illness, suggesting that as the illness progresses the upregulation of transcription within the NRG-ErbB signal ing pathway might become less apparent. However, it is not clear whether this correlation represents a potential disease process and/or a compensatory response in an effort to maintain signaling homeostasis. Studies examining patterns of NRG-ErbB signaling pathway transcripts over the course of the illness are required to confirm this notion and determine the underlying mechanism.
We did not find differences in the peripheral expression of NRG2 between TRS patients and controls. To our knowledge, we are the first to examine NRG2 mRNA in the blood in schizophrenia or other psychiatric disorder. However, a recent study showed that ablation of NRG2 in the adult mouse brain mimicked dopaminergic imbalance seen in schizophrenia (i.e., high subcortical dopamine, low cortical dopamine) and resulted in severe behavioral phenotypes relevant to psychiatric disorders (47). Thus, NRG2 may play a role in the pathophysio logy of schizophrenia but based on our results seems less likely to serve as a peripheral marker of neurobiological changes found in schizophrenia. Likewise, ErbB2 mRNA expression seems an unlikely peripheral marker of schizophrenia based on our null findings as well as findings from others that reported no differ ence in ErbB2 mRNA expression in monocytes of firstepisode, drugnaive patients with schizophrenia compared to healthy controls (48). However, this same study suggested that there may be an exaggerated NRG1 stimulated cytokine response from PBMC in people with schizophrenia compared to controls (48), suggesting a link between overactive NRG1 signaling and inflammation.
Our study has notable limitations. First, we were unable to compare affected individuals with and without TRS and as such the specificity of our results to TRS patients remains to be con firmed. Second, we analyzed crosssectional data, which makes it complicated to predict how gene expression patterns might change with disease progression and their possible relation to clinical symptoms. Third, we measured gene expression in whole blood, as this tissue is clinically accessible and commonly used in biomarker research. However, it is unclear how our findings will relate to other peripheral (PBMCs or lymphocytes) or central tissues (e.g., brain) despite some suggestion for their relevance in schizophrenia (49). Fourth, we did not investigate all transcripts within the NRG-ErbB pathway (i.e., PIK3CA-B, PIK3R1-2, eIF4B, eEF2, and eIF4E). We instead, chose transcripts based on evidence from the current literature in schizophrenia. Furthermore, we only interrogated mRNA levels of our candidate genes within the NRG-ErbB pathway and as such cannot rule out the potential that genetic, protein, and/or epigenetic markers in this pathway may differ in those with schizophrenia. Fifth, our sample size was rela tively small and as such requires independent validation. Finally, our in vitro clozapine exposure experiments examined a single clozapine concentration (1.2 µM) that was guided by pilot data from our study population. While this concentration of clozapine does reflect steady state plasma concentrations (50)(51)(52), future work with PBMCs should examine multiple concentrations that reflect the range of clozapine blood levels observed in the clinic together with interrogating a greater number of candidates at both genetic, gene expression and protein levels.
In summary, our results provide the first peripheral gene expression profile of the major NRG-ErbB pathway genes among individuals with TRS. We detected an overall upregulation of NRG-ErbB pathway transcripts among those with TRS, most robustly for P70S6K. We further showed that most of the tran scripts we examined were negatively correlated with duration of illness, suggesting the upregulation of NRG-ErbB pathway transcripts we observed in the current chronic schizophrenia cohort may be more easily detectable among individuals at earlier stages of the illness relative to healthy individuals. If this notion is substantiated by future research, NRG-ErbB pathway gene expression may serve, in part, as a useful peripheral biomarker for staging of the illness and possibly assist in the identification of those at greatest risk for TRS.

eThics sTaTeMenT
All the participants provided written informed consent and the study protocol was approved by the Melbourne Health Human