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β-Site APP-cleaving Enzyme 1 ( BACE1) is a protease that has been linked to schizophrenia, a severe mental illness that is potentially characterized by enhanced dopamine (DA) release in the striatum. Here, we used acute amphetamine administration to stimulate neuronal activity and investigated the neurophysiological and locomotor-activity response in BACE1-deficient ( BACE1−/−) mice. We measured locomotor activity at baseline and after treatment with amphetamine (3.2 and 10 mg/kg). While baseline locomotor activity did not vary between groups, BACE1−/− mice exhibited reduced sensitivity to the locomotor-enhancing effects of amphetamine. Using high-performance liquid chromatography (HPLC) to measure DA and DA metabolites in the striatum, we found no significant differences in BACE1−/− compared with wild-type mice. To determine if DA neuron excitability is altered in BACE1−/− mice, we performed patch-clamp electrophysiology in putative DA neurons from brain slices that contained the substantia nigra. Pacemaker firing rate was slightly increased in slices from BACE1−/− mice. We next measured G protein-coupled potassium currents produced by activation of D2 autoreceptors, which strongly inhibit firing of these neurons. The maximal amplitude and decay times of D2 autoreceptor currents were not altered in BACE1−/− mice, indicating no change in D2 autoreceptor-sensitivity and DA transporter-mediated reuptake. However, amphetamine (30 µ m)-induced potassium currents produced by efflux of DA were enhanced in BACE1−/− mice, perhaps indicating increased vesicular DA content in the midbrain. This suggests a plausible mechanism to explain the decreased sensitivity to amphetamine-induced locomotion, and provides evidence that decreased availability of BACE1 can produce persistent adaptations in the dopaminergic system. [ABSTRACT FROM AUTHOR]

Depression has strong negative impacts on how individuals function, leading to the assumption that there is strong negative selection on this trait that should deplete genetic variation and decrease its prevalence in human populations. Yet, depressive symptoms remain common. While there has been a large body of work trying to resolve this paradox by mapping genetic variation of this complex trait, there have been few direct empirical tests of the core assumption that there is consistent negative selection on depression in human populations. Here, we use a unique long-term dataset from the National Health and Nutrition Examination Survey that spans four generational cohorts (Silent Generation: 1928-1945, Baby Boomers: 1946-1964, Generation X: 1965-1980, and Millenials: 1981-1996) to measure both depression scores and fitness components (lifetime sexual partners, pregnancies, and live births) of women from the United States born between 1938-1994. We not only assess fitness consequences of depression across multiple generations to determine whether the strength and direction of selection on depression has changed over time, but we also pair these fitness measurements with mixed models to assess how several important covariates, including age, body mass, education, race/ethnicity, and income might influence this relationship. We found that, overall, selection on depression was positive and the strength of selection changed over time-women reporting higher depression had relatively more sexual partners, pregnancies, and births except during the Silent Generation when selection coefficients neared zero. We also found that depression scores and fitness components differed among generations-Baby Boomers showed the highest severity of depression and the most sexual partners. These results were not changed by the inclusion of covariates in our models. A limitation of this study is that for the Millenials, reproduction has not completed and data for this generation is interrupted by right censoring. Most importantly, our results undermine the common belief that there is consistent negative selection on depression and demonstrate that the relationship between depression and fitness changes between generations, which may explain its maintenance in human populations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gurguis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Substance use disorder (SUD) significantly increases the risk of neurotoxicity, inflammation, oxidative stress, and impaired neuroplasticity. The activation of inflammatory pathways by substances may lead to glial activation and chronic neuroinflammation, potentially mediated by the release of extracellular particles (EPs), such as extracellular condensates (ECs) and extracellular vesicles (EVs). These particles, which reflect the physiological, pathophysiological, and metabolic states of their cells of origin, might carry molecular signatures indicative of SUD. In particular, our study investigated neuroinflammatory signatures in SUD by isolating EVs from the dorsolateral prefrontal cortex (dlPFC) Brodmann's area 9 (BA9) in postmortem subjects. We isolated BA9-derived EVs from postmortem brain tissues of eight individuals (controls: n=4, SUD: n=4). The EVs were analyzed for physical properties (concentration, size, zeta potential, morphology) and subjected to integrative multi-omics analysis to profile the lipidomic and proteomic characteristics. We assessed the interactions and bioactivity of EVs by evaluating their uptake by glial cells. We further assessed the effects of EVs on complement mRNA expression in glial cells as well as their effects on microglial migration. No significant differences in EV concentration, size, zeta potential, or surface markers were observed between SUD and control groups. However, lipidomic analysis revealed significant enrichment of glycerophosphoinositol bisphosphate (PIP2) in SUD EVs. Proteomic analysis indicates downregulation of SERPINB12, ACYP2, CAMK1D, DSC1, and FLNB, and upregulation of C4A, C3, and ALB in SUD EVs. Gene ontology and protein-protein interactome analyses highlight functions such as cell motility, focal adhesion, and acute phase response signaling that is associated with the identified proteins. Both control and SUD EVs increased C3 and C4 mRNA expression in microglia, but only SUD EVs upregulated these genes in astrocytes. SUD EVs also significantly enhanced microglial migration in a wound healing assay.This study successfully isolated EVs from postmortem brains and used a multi-omics approach to identify EV-associated lipids and proteins in SUD. Elevated C3 and C4 in SUD EVs and the distinct effects of EVs on glial cells suggest a crucial role in acute phase response signaling and neuroinflammation.

Suicide attempt (SA) risk is elevated in individuals with bipolar disorder (BD), and DNA methylation patterns may serve as possible biomarkers of SA. We conducted epigenome-wide association studies (EWAS) of blood DNA methylation associated with BD and SA. DNA methylation was measured at >700,000 positions in a discovery cohort of n = 84 adults with BD with a history of SA (BD/SA), n = 79 adults with BD without history of SA (BD/non-SA), and n = 76 non-psychiatric controls (CON). EWAS revealed six differentially methylated positions (DMPs) and seven differentially methylated regions (DMRs) between BD/SA and BD/non-SA, with multiple immune-related genes implicated. There were no epigenome-wide significant differences when BD/SA and BD/non-SA were each compared to CON, and patterns suggested that epigenetics differentiating BD/SA from BD/non-SA do not differentiate BD/non-SA from CON. Weighted gene co-methylation network analysis and trait enrichment analysis of the BD/SA vs. BD/non-SA contrast further corroborated immune system involvement, while gene ontology analysis implicated calcium signalling. In an independent replication cohort of n = 48 BD/SA and n = 47 BD/non-SA, fold changes at the discovery cohort's significant sites showed moderate correlation across cohorts and agreement on direction. In both cohorts, classification accuracy for SA history among individuals with BD was highest when methylation at the significant CpG sites as well as information from clinical interviews were combined, with an AUC of 88.8% (CI = 83.8-93.8%) and 82.1% (CI = 73.6-90.5%) for the combined epigenetic-clinical classifier in the discovery and replication cohorts, respectively. Our results provide novel insight to the role of immune system functioning in SA and BD and also suggest that integrating information from multiple levels of analysis holds promise to improve risk assessment for SA in adults with BD., (© 2024. The Author(s).)

The investigation of the effects of prenatal cocaine exposure (PCE) on offspring has been inconsistent, with few studies investigating biological outcomes in humans. We profiled genome-wide DNA methylation (DNAm) of umbilical cord blood (UCB) from newborns with (n = 35) and without (n = 47) PCE. We used DNAm data to (1) assess pediatric epigenetic clocks at birth and (2) to estimate epigenetic scores (ES) for lifetime disorders. We generated gestational epigenetic age estimates (DNAmGA) based on Knight and Bohlin epigenetic clocks. We also investigated the association between DNAmGA and UCB serum brain-derived neurotrophic factor (BDNF) levels. Considering the large-scale DNAm data availability and existing evidence regarding PCE as a risk for health problems later in life, we generated ES for tobacco smoking, psychosis, autism, diabetes, and obesity. A gene ontology (GO) analysis on the CpGs included in the ES with group differences was performed. PCE was associated with lower DNAmGA in newborns, and this effect remained significant when controlling for potential confounders, such as blood cell type composition predicted by DNAm and obstetric data. DNAmGA was negatively correlated with BDNF levels in the serum of UCB. Higher tobacco smoking, psychosis, and diabetes ES were found in the PCE group. The GO analysis revealed GABAergic synapses as a potential pathway altered by PCE. Our findings of decelerated DNAmGA and ES for adverse phenotypes associated with PCE, suggest that the effects of gestational cocaine exposure on the epigenetic landscape of human newborns are detectable at birth., (© 2024. The Author(s).)

Introduction: Human-derived induced pluripotent stem cell (iPSC) models of brain promise to advance our understanding of neurotoxic consequences of drug use. However, how well these models recapitulate the actual genomic landscape and cell function, as well as the drug-induced alterations, remains to be established. New in vitro models of drug exposure are needed to advance our understanding of how to protect or reverse molecular changes related to substance use disorders., Methods: We engineered a novel induced pluripotent stem cell-derived model of neural progenitor cells and neurons from cultured postmortem human skin fibroblasts, and directly compared these to isogenic brain tissue from the donor source. We assessed the maturity of the cell models across differentiation from stem cells to neurons using RNA cell type and maturity deconvolution analyses as well as DNA methylation epigenetic clocks trained on adult and fetal human tissue. As proof-of-concept of this model's utility for substance use disorder studies, we compared morphine- and cocaine-treated neurons to gene expression signatures in postmortem Opioid Use Disorder (OUD) and Cocaine Use Disorder (CUD) brains, respectively., Results: Within each human subject (N = 2, 2 clones each), brain frontal cortex epigenetic age parallels that of skin fibroblasts and closely approximates the donor's chronological age; stem cell induction from fibroblast cells effectively sets the epigenetic clock to an embryonic age; and differentiation of stem cells to neural progenitor cells and then to neurons progressively matures the cells via DNA methylation and RNA gene expression readouts. In neurons derived from an individual who died of opioid overdose, morphine treatment induced alterations in gene expression similar to those previously observed in OUD ex-vivo brain tissue, including differential expression of the immediate early gene EGR1, which is known to be dysregulated by opioid use., Discussion: In summary, we introduce an iPSC model generated from human postmortem fibroblasts that can be directly compared to corresponding isogenic brain tissue and can be used to model perturbagen exposure such as that seen in opioid use disorder. Future studies with this and other postmortem-derived brain cellular models, including cerebral organoids, can be an invaluable tool for understanding mechanisms of drug-induced brain alterations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mendez, Grimm, Stertz, Gorski, Movva, Najera, Moriel, Meyer, Fries, Coarfa and Walss-Bass.)

Introduction: To understand mechanisms and identify potential targets for intervention in the current crisis of opioid use disorder (OUD), postmortem brains represent an under-utilized resource. To refine previously reported gene signatures of neurobiological alterations in OUD from the dorsolateral prefrontal cortex (Brodmann Area 9, BA9), we explored the role of microRNAs (miRNA) as powerful epigenetic regulators of gene function., Methods: Building on the growing appreciation that miRNAs can cross the blood-brain barrier, we carried out miRNA profiling in same-subject postmortem samples from BA9 and blood tissues., Results: miRNA-mRNA network analysis showed that even though miRNAs identified in BA9 and blood were fairly distinct, their target genes and corresponding enriched pathways overlapped strongly. Among the dominant enriched biological processes were tissue development and morphogenesis, and MAPK signaling pathways. These findings point to robust, redundant, and systemic opioid-induced miRNA dysregulation with a potential functional impact on transcriptomic changes. Further, using correlation network analysis, we identified cell-type specific miRNA targets, specifically in astrocytes, neurons, and endothelial cells, associated with OUD transcriptomic dysregulation. Finally, leveraging a collection of control brain transcriptomes from the Genotype-Tissue Expression (GTEx) project, we identified a correlation of OUD miRNA targets with TGF beta, hypoxia, angiogenesis, coagulation, immune system, and inflammatory pathways., Discussion: These findings support previous reports of neurovascular and immune system alterations as a consequence of opioid abuse and shed new light on miRNA network regulators of cellular response to opioid drugs., Competing Interests: SS received research support from Flow Neuroscience and contributes as a study investigator for clinical trials by Compass pathways, Relmada, Liva Nova, and Janssen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Grimm, Mendez, Stertz, Meyer, Fries, Gandhi, Kanchi, Selvaraj, Teixeira, Kosten, Gunaratne, Coarfa and Walss-Bass.)

Microtubules are organelles that usually occur only in the cytosol. Walss et al. (1999) discovered the βII isotype of tubulin, complexed with α , in the nuclei of certain cultured cells, in non-microtubule form. When fluorescently labeled tubulins were microinjected into the cells, only αβ II appeared in the nucleus, and only after one cycle of nuclear disassembly and reassembly. It appeared as if αβ II does not cross the nuclear envelope but is trapped in the nucleus by the re-forming nuclear envelope in whose reassembly β II may be involved. β II is present in the cytoplasm and nuclei of many tumor cells. With some exceptions, normal tissues that expressed βII rarely had βII in their nuclei. It is possible that βII is involved in nuclear reassembly and then disappears from the nucleus. Ruksha et al. (2019) observed that patients whose colon cancer cells in the invasive front showed no βII had a median survival of about 5.5 years, which was more than halved if they had cytosolic β II and further lessened if they had nuclear β II, suggesting that the presence and location of β II in biopsies could be a useful prognostic indicator and also that β II may be involved in cancer progression. Yeh and Ludueña. (2004) observed that many tumors were surrounded by non-cancerous cells exhibiting cytosolic and nuclear β II, suggesting a signaling pathway that causes β II to be synthesized in nearby cells and localized to their nuclei. β II could be useful in cancer diagnosis, since the presence of β II in non-cancerous cells could indicate a nearby tumor. Investigation of this pathway might reveal novel targets for chemotherapy. Another possibility would be to combine αβ II with CRISPR-Cas9. This complex would likely enter the nucleus of a cancer cell and, if guided to the appropriate gene, might destroy the cancer cell or make it less aggressive; possible targets will be discussed here. The possibilities raised here about the utility of β II in cancer diagnosis, prognosis, biology and therapy may repay further investigation., Competing Interests: JG was employed by the company Fosun Pharma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ludueña, Walss-Bass, Portyanko, Guo and Yeh.)

(1) Background: Epigenome-wide association studies (EWAS) in peripheral blood have repeatedly found associations between tobacco smoking and aberrant DNA methylation (DNAm), but little is known about DNAm signatures of smoking in the human brain, which may contribute to the pathophysiology of addictive behavior observed in chronic smokers. (2) Methods: We investigated the similarity of DNAm signatures in matched blood and postmortem brain samples ( n = 10). In addition, we performed EWASs in five brain regions belonging to the neurocircuitry of addiction: anterior cingulate cortex (ACC), Brodmann Area 9, caudate nucleus, putamen, and ventral striatum ( n = 38-72). (3) Results: cg15925993 within the LOC339975 gene was epigenome-wide significant in the ACC. Of 16 identified differentially methylated regions, two ( PRSS50 and LINC00612/A2M-AS1 ) overlapped between multiple brain regions. Functional enrichment was detected for biological processes related to neuronal development, inflammatory signaling and immune cell migration. Additionally, our results indicate the association of the well-known AHRR CpG site cg05575921 with smoking in the brain. (4) Conclusion: The present study provides further evidence of the strong relationship between aberrant DNAm and smoking.

Schizophrenia and bipolar disorder, two of the most severe psychiatric illnesses, have historically been regarded as dichotomous entities but share many features of the premorbid course, clinical profile, genetic factors and treatment approaches. Studies focusing on neuroimaging findings have received considerable attention, as they plead for an improved understanding of the brain regions involved in the pathophysiology of schizophrenia and bipolar disorder. In this review, we summarize the main magnetic resonance imaging findings in both disorders, aiming at exploring the neuroanatomical and functional similarities and differences between the two. The findings show that gray and white matter structural changes and functional dysconnectivity predominate in the frontal and limbic areas and the frontotemporal circuitry of the brain areas involved in the integration of executive, cognitive and affective functions, commonly affected in both disorders. Available evidence points to a considerable overlap in the affected regions between the two conditions, therefore possibly placing them at opposite ends of a psychosis continuum.

Background: Suicide represents a major health concern, especially in developing countries. While many demographic risk factors have been proposed, the underlying molecular pathology of suicide remains poorly understood. A body of evidence suggests that aberrant DNA methylation and expression is involved. In this study, we examined DNA methylation profiles and concordant gene expression changes in the prefrontal cortex of Mexicans who died by suicide., Methods: In collaboration with the coroner's office in Mexico City, brain samples of males who died by suicide (n = 35) and age-matched sudden death controls (n = 13) were collected. DNA and RNA were extracted from prefrontal cortex tissue and analyzed with the Infinium Methylation480k and the HumanHT-12 v4 Expression Beadchips, respectively., Results: We report evidence of altered DNA methylation profiles at 4430 genomic regions together with 622 genes characterized by differential expression in cases vs controls. Seventy genes were found to have concordant methylation and expression changes. Metacore-enriched analysis identified 10 genes with biological relevance to psychiatric phenotypes and suicide (ADCY9, CRH, NFATC4, ABCC8, HMGA1, KAT2A, EPHA2, TRRAP, CD22, and CBLN1) and highlighted the association that ADCY9 has with various pathways, including signal transduction regulated by the cAMP-responsive element modulator, neurophysiological process regulated by the corticotrophin-releasing hormone, and synaptic plasticity. We therefore went on to validate the observed hypomethylation of ADCY9 in cases vs control through targeted bisulfite sequencing., Conclusion: Our study represents the first, to our knowledge, analysis of DNA methylation and gene expression associated with suicide in a Mexican population using postmortem brain, providing novel insights for convergent molecular alterations associated with suicide., (© The Author(s) 2021. Published by Oxford University Press on behalf of CINP.)

Background: Opioid use disorder (OUD) affects millions of people, causing nearly 50 000 deaths annually in the United States. While opioid exposure and OUD are known to cause widespread transcriptomic and epigenetic changes, few studies in human samples have been conducted. Understanding how OUD affects the brain at the molecular level could help decipher disease pathogenesis and shed light on OUD treatment., Methods: We generated genome-wide transcriptomic and DNA methylation profiles of 22 OUD subjects and 19 non-psychiatric controls. We applied weighted gene co-expression network analysis to identify genetic markers consistently associated with OUD at both transcriptomic and methylomic levels. We then performed functional enrichment for biological interpretation. We employed cross-omics analysis to uncover OUD-specific regulatory networks., Results: We found 6 OUD-associated co-expression gene modules and 6 co-methylation modules (false discovery rate <0.1). Genes in these modules are involved in astrocyte and glial cell differentiation, gliogenesis, response to organic substance, and response to cytokine (false discovery rate <0.05). Cross-omics analysis revealed immune-related transcription regulators, suggesting the role of transcription factor-targeted regulatory networks in OUD pathogenesis., Conclusions: Our integrative analysis of multi-omics data in OUD postmortem brain samples suggested complex gene regulatory mechanisms involved in OUD-associated expression patterns. Candidate genes and their upstream regulators revealed in astrocyte, and glial cells could provide new insights into OUD treatment development., (© The Author(s) 2021. Published by Oxford University Press on behalf of CINP.)

White matter (WM) abnormalities in patients with cocaine use disorder (CUD) have been studied; however, the reported effects on the human brain are heterogenous and most results have been obtained from male participants. In addition, biological data supporting the imaging findings and revealing possible mechanisms underlying the neurotoxic effects of chronic cocaine use (CU) on WM are largely restricted to animal studies. To evaluate the neurotoxic effects of CU in the WM, we performed an in vivo diffusion tensor imaging assessment of male and female cocaine users (n = 75) and healthy controls (HC) (n = 58). Moreover, we performed an ex vivo large-scale proteomic analysis using liquid chromatography-tandem mass spectrometry in postmortem brains of patients with CUD (n = 8) and HC (n = 12). Compared with the HC, the CUD group showed significant reductions in global fractional anisotropy (FA) (p < 0.001), and an increase in global mean (MD) and radial diffusion (RD) (both p < 0.001). The results revealed that FA, RD, and MD alterations in the CUD group were widespread along the major WM tracts, after analysis using the tract-based special statistics approach. Global FA was negatively associated with years of CU (p = 0.0421) and female sex (p < 0.001), but not with years of alcohol or nicotine use. Concerning the fibers connecting the left to the right prefrontal cortex, Brodmann area 9 (BA9), the CUD group presented lower FA (p = 0.006) and higher RD (p < 0.001) values compared with the HC group. A negative association between the duration of CU in life and FA values in this tract was also observed (p = 0.019). Proteomics analyses in BA9 found 11 proteins differentially expressed between cocaine users and controls. Among these, were proteins related to myelination and neuroinflammation. In summary, we demonstrate convergent evidence from in vivo diffusion tensor imaging and ex vivo proteomics analysis of WM disruption in CUD.

Human-induced pluripotent stem cells (hiPSCs) allow for the establishment of brain cellular models of psychiatric disorders that account for a patient's genetic background. Here, we conducted an RNA-sequencing profiling study of hiPSC-derived cell lines from schizophrenia (SCZ) subjects, most of which are from a multiplex family, from the population isolate of the Central Valley of Costa Rica. hiPSCs, neural precursor cells, and cortical neurons derived from six healthy controls and seven SCZ subjects were generated using standard methodology. Transcriptome from these cells was obtained using Illumina HiSeq 2500, and differential expression analyses were performed using DESeq2 (|fold change|>1.5 and false discovery rate < 0.3), in patients compared to controls. We identified 454 differentially expressed genes in hiPSC-derived neurons, enriched in pathways including phosphoinositide 3-kinase/glycogen synthase kinase 3 (PI3K/GSK3) signaling, with serum-glucocorticoid kinase 1 (SGK1), an inhibitor of glycogen synthase kinase 3β, as part of this pathway. We further found that pharmacological inhibition of downstream effectors of the PI3K/GSK3 pathway, SGK1 and GSK3, induced alterations in levels of neurite markers βIII tubulin and fibroblast growth factor 12, with differential effects in patients compared to controls. While demonstrating the utility of hiPSCs derived from multiplex families to identify significant cell-specific gene network alterations in SCZ, these studies support a role for disruption of PI3K/GSK3 signaling as a risk factor for SCZ.

The field of proteomics is rapidly gaining territory as a promising alternative to genomic approaches in the efforts to unravel the complex molecular mechanisms underlying schizophrenia and other psychiatric disorders. X-aptamer tech-nology has emerged as a novel proteomic approach for high-sensitivity analyses, and we hypothesized that this technology would identify unique molecular signatures in plasma samples from schizophrenia patients ( n = 60) compared to controls ( n = 20). Using a combinatorial library of X-aptamer beads, we developed a two-color flow cytometer-based approach to identify specific X-aptamers that bound with high specificity to each target group. Based on this, we synthesized two unique X-aptamer sequences, and specific proteins pulled down from the patient and control groups by these X-aptamers were identified by mass spectrometry. We identified two protein biomarkers, complement component C4A and ApoB, upregulated in plasma samples from schizophrenia patients. ELISA validation suggested that the observed differences in C4 levels in patients are likely due to the presence of the illness itself, while ApoB may be a marker of antipsychotic-induced alterations. These studies highlight the utility of the X-aptamer technology in the identification of biomarkers for schizophrenia that will advance our understanding of the pathophysiological mechanisms of this disorder.

Background/aim: Although individuals with substance use disorder (SUD) are at high risk of committing suicide, most studies of postmortem gene expression exclude subjects with SUD due to the potential confounding effect of drugs in the transcriptome. Thus, little is known about the gene expression profile in suicides with SUD. The identification of altered biological processes in suicides with SUD is crucial in the comprehension of the interaction between both pathologies., Methods: We evaluated the gene expression profile in the dorsolateral prefrontal area of suicides and nonsuicides with and without SUD by microarrays., Results: We identified 222 differentially expressed genes, predominately enriched in cell proliferation in the comparison between suicides with and without SUD. When comparing the transcriptome of suicides with SUD to nonsuicides with SUD, we identified 550 differentially expressed genes, mainly enriched in oxidative phosphorylation. Differentially expressed genes (1,417) between suicides and nonsuicides without SUD were detected. Most of them were related to mitochondrial function., Conclusion: Interaction between suicide and SUD seems to influence the expression of genes involved in glial proliferation and glutamatergic neurotransmission. These results highlight, for the first time, that suicides with SUD have a gene expression profile distinct from that of subjects with only one of these disorders.

Chronic cocaine and alcohol use impart significant stress on biological and cognitive systems, resulting in changes consistent with an allostatic load model of neurocognitive impairment. The present study measured potential markers of allostatic load in individuals with comorbid cocaine/alcohol use disorders (CUD/AUD) and control subjects. Measures of brain white matter (WM), telomere length, and impulsivity/attentional bias were obtained. WM (CUD/AUD only) was indexed by diffusion tensor imaging metrics, including radial diffusivity (RD) and fractional anisotropy (FA). Telomere length was indexed by the telomere to single copy gene (T/S) ratio. Impulsivity and attentional bias to drug cues were measured via eye-tracking, and were also modeled using the Hierarchical Diffusion Drift Model (HDDM). Average whole-brain RD and FA were associated with years of cocaine use (R2 = 0.56 and 0.51, both p < .005) but not years of alcohol use. CUD/AUD subjects showed more anti-saccade errors (p < .01), greater attentional bias scores (p < .001), and higher HDDM drift rates on cocaine-cue trials (Bayesian probability CUD/AUD > control = p > 0.99). Telomere length was shorter in CUD/AUD, but the difference was not statistically significant. Within the CUD/AUD group, exploratory regression using an elastic-net model determined that more years of cocaine use, older age, larger HDDM drift rate differences and shorter telomere length were all predictive of WM as measured by RD (model R2 = 0.79). Collectively, the results provide modest support linking CUD/AUD to putative markers of allostatic load., Competing Interests: The authors have declared that no competing interests exist.

Treatments for Cocaine Use Disorder (CUD) are variably effective, and there are no FDA-approved medications. One approach to developing new treatments for CUD may be to investigate and target poor prognostic signs. One such sign is anhedonia (i.e. a loss of pleasure or interest in non-drug rewards), which predicts worse outcomes in existing CUD treatments. Inflammation is thought to underlie anhedonia in many other disorders, but the relationship between anhedonia and inflammation has not been investigated in CUD. Therefore, we assessed peripheral genome-wide gene expression in n = 48 individuals with CUD with high (n = 24) vs. low (n = 24) levels of anhedonia, defined by a median split of self-reported anhedonia. Our hypothesis was that individuals with high anhedonia would show differential gene expression in inflammatory pathways. No individual genes were significantly different between the low and high anhedonia groups when using t-tests with a stringent false discovery rate correction (FDR-corrected p < 0.05). However, an exploratory analysis identified 166 loci where t-tests suggested group differences at a nominal p < 0.05. We used DAVID, a bioinformatics tool that provides functional interpretations of complex lists of genes, to examine representation of this gene list in known pathways. It confirmed that mechanisms related to immunity were the top significant associations with anhedonia in the sample. Further, the two top differentially expressed genes in our sample, IRF1 and GBP5, both have primary inflammation and immune functions, and were significantly negatively correlated with total scores on our self-report of anhedonia across all 48 subjects. These results suggest that prioritizing development of anti-inflammatory medications for CUD may pay dividends, particularly in combination with treatment-matching strategies using either phenotypic measures of anhedonia or biomarkers of inflammatory gene expression to individualize treatment., Competing Interests: The authors have declared that no competing interests exist.

Rationale : Given datasets with a large or diverse set of predictors of aggression, machine learning (ML) provides efficient tools for identifying the most salient variables and building a parsimonious statistical model. ML techniques permit efficient exploration of data, have not been widely used in aggression research, and may have utility for those seeking prediction of aggressive behavior. Objectives : The present study examined predictors of aggression and constructed an optimized model using ML techniques. Predictors were derived from a dataset that included demographic, psychometric and genetic predictors, specifically FK506 binding protein 5 (FKBP5) polymorphisms, which have been shown to alter response to threatening stimuli, but have not been tested as predictors of aggressive behavior in adults. Methods : The data analysis approach utilized component-wise gradient boosting and model reduction via backward elimination to: (a) select variables from an initial set of 20 to build a model of trait aggression; and then (b) reduce that model to maximize parsimony and generalizability. Results : From a dataset of N = 47 participants, component-wise gradient boosting selected 8 of 20 possible predictors to model Buss-Perry Aggression Questionnaire (BPAQ) total score, with R 2 = 0.66. This model was simplified using backward elimination, retaining six predictors: smoking status, psychopathy (interpersonal manipulation and callous affect), childhood trauma (physical abuse and neglect), and the FKBP5&#95;13 gene (rs1360780). The six-factor model approximated the initial eight-factor model at 99.4% of R 2 . Conclusions : Using an inductive data science approach, the gradient boosting model identified predictors consistent with previous experimental work in aggression; specifically psychopathy and trauma exposure. Additionally, allelic variants in FKBP5 were identified for the first time, but the relatively small sample size limits generality of results and calls for replication. This approach provides utility for the prediction of aggression behavior, particularly in the context of large multivariate datasets.

Bipolar disorder (BD) has been previously associated with accelerated aging; yet, the mechanisms underlying this association are largely unknown. The epigenetic clock has been increasingly recognized as a valuable aging marker, although its association with other biological clocks in BD patients and high-risk subjects, such as telomere length and mitochondrial DNA (mtDNA) copy number, has never been investigated. We included 22 patients with BD I, 16 siblings of BD patients, and 20 healthy controls in this analysis. DNA was isolated from peripheral blood and interrogated for genome-wide DNA methylation, mtDNA copy number, and telomere length. DNA methylation age (DNAm age) and accelerated aging were calculated using the Horvath age estimation algorithm in blood and in postmortem brain from BD patients and nonpsychiatric controls using publicly available data. Older BD patients presented significantly accelerated epigenetic aging compared to controls, whereas no difference was detected among the younger subjects. Patients showed higher levels of mtDNA copy number, while no difference was found between controls and siblings. mtDNA significantly correlated with epigenetic age acceleration among older subjects, as well and with global functioning in our sample. Telomere length did not show significant differences between groups, nor did it correlate with epigenetic aging or mtDNA copy number. These results suggest that BD may involve an accelerated epigenetic aging, which might represent a novel target for treating BD and subjects at risk. In particular, our results suggest a complex interplay between biological clocks to determine the accelerated aging and its consequences in BD.

Immune dysfunction has been implicated in the pathophysiology of schizophrenia. Leukocyte migration to the site of inflammation is a fundamental step of immune response which involves P-, E-, and L-selectins. Elevated selectin levels have been reported in un-medicated first-episode patients with schizophrenia but not in medicated patients with multi-episode schizophrenia. We measured fasting plasma soluble P-, E-, and L-selectin in 39 medicated patients with multi-episode schizophrenia and 19 healthy controls. In patients, psychotic symptom severity and cognitive function were assessed with the Positive and Negative Syndrome Scale (PANSS) and the NIH Toolbox Cognitive Test Battery respectively. C-reactive protein (CRP) and Body Mass Index (BMI) were measured in patients and controls. Comparison of selectin levels between patients and controls was done with t-tests and linear regression. Pearson correlation coefficients between plasma selectins and PANSS and cognitive measures were calculated. Geometric mean plasma soluble L-selectin level was lower in patients compared to controls from unadjusted (606.7 ± 1.2 ng/ml vs. 937.7 ± 1.15 ng/ml, p < 0.001) and adjusted analyses (β = 0.59; CI 0.41 to 0.88, p = 0.011). There was a trend towards higher plasma soluble P-selectin in patients compared to controls (90.4 ± 1.2ng/ml vs. 71.8 ± 1.2ng/ml, p = 0.059) in the unadjusted analysis. There was no association between the selectins and psychotic symptoms or cognitive function in the patients. In addition, the selectins were not significantly associated with CRP or BMI. The limitations of this study include small sample size and unavailability of information on medications and blood cell counts. The potential utility of soluble L-selectin as a biomarker of antipsychotic exposure in patients with schizophrenia and the concomitant change in immune response with the use of antipsychotics should be further evaluated.

Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined.

Second-generation antipsychotics (SGAs) are commonly used to treat schizophrenia. However, SGAs cause metabolic disturbances that can manifest as metabolic syndrome (MetS) in a subset of patients. The causes for these metabolic disturbances remain unclear. We performed a comprehensive metabolomic profiling of 60 schizophrenia patients undergoing treatment with SGAs that puts them at high (clozapine, olanzapine), medium (quetiapine, risperidone), or low (ziprasidone, aripiprazole) risk for developing MetS, compared to a cohort of 20 healthy controls. Multiplex immunoassays were used to measure 13 metabolic hormones and adipokines in plasma. Mass spectrometry was used to determine levels of lipids and polar metabolites in 29 patients and 10 controls. We found that levels of insulin and tumor necrosis factor alpha (TNF-α) were significantly higher (p < 0.005) in patients at medium and high risk for MetS, compared to controls. These molecules are known to be increased in individuals with high body fat content and obesity. On the other hand, adiponectin, a molecule responsible for control of food intake and body weight, was significantly decreased in patients at medium and high risk for MetS (p < 0.005). Further, levels of dyacylglycerides (DG), tryacylglycerides (TG) and cholestenone were increased, whereas α-Ketoglutarate and malate, important mediators of the tricarboxylic acid (TCA) cycle, were significantly decreased in patients compared to controls. Our studies suggest that high- and medium-risk SGAs are associated with disruption of energy metabolism pathways. These findings may shed light on the molecular underpinnings of antipsychotic-induced MetS and aid in design of novel therapeutic approaches to reduce the side effects associated with these drugs.

Background: Mitochondrial short and long-range movements are necessary to generate the energy needed for synaptic signaling and plasticity. Therefore, an effective mechanism to transport and anchor mitochondria to pre- and post-synaptic terminals is as important as functional mitochondria in neuronal firing. Mitochondrial movement range is regulated by phosphorylation of cytoskeletal and motor proteins in addition to changes in mitochondrial membrane potential. Movement direction is regulated by serotonin and dopamine levels. However, data on mitochondrial movement defects and their involvement in defective signaling and neuroplasticity in relationship with mood disorders is scarce. We have previously reported the effects of lithium, valproate and a new antipsychotic, paliperidone on protein expression levels at the synaptic level., Hypothesis: Mitochondrial function defects have recently been implicated in schizophrenia and bipolar disorder. We postulate that mood stabilizer treatment has a profound effect on mitochondrial function, synaptic plasticity, mitochondrial migration and direction of movement., Methods: Synaptoneurosomal preparations from rat pre-frontal cortex were obtained after 28 daily intraperitoneal injections of lithium, valproate and paliperidone. Phosphorylated proteins were identified using 2D-DIGE and nano LC-ESI tandem mass spectrometry., Results: Lithium, valproate and paliperidone had a substantial and common effect on the phosphorylation state of specific actin, tubulin and myosin isoforms as well as other proteins associated with neurofilaments. Furthermore, different subunits from complex III and V of the electron transfer chain were heavily phosphorylated by treatment with these drugs indicating selective phosphorylation., Conclusions: Mood stabilizers have an effect on mitochondrial function, mitochondrial movement and the direction of this movement. The implications of these findings will contribute to novel insights regarding clinical treatment and the mode of action of these drugs.

Background: Metabolic syndrome (MetS) is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS) and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined., Methodology/principal Findings: Cultured mouse myoblasts (C2C12), adipocytes (3T3-L1), hepatocytes (FL-83B), and monocytes (RAW 264.7) were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line., Conclusions/significance: Clozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated with the development of these diseases in the general population.

Background: Neuregulin 1 (NRG1) is a key candidate susceptibility gene for both schizophrenia (SCZ) and bipolar disorder (BPD). The function of the NRG1 transmembrane proteins is regulated by cleavage. Alteration of membrane bound-NRG1 cleavage has been previously shown to be associated with behavioral impairments in mouse models lacking expression of NRG1-cleavage enzymes such as BACE1 and gamma secretase. We sought to determine whether alterations in NRG1 cleavage and associated enzymes occur in patients with SCZ and BPD., Methodology/principal Findings: Using human postmortem brain, we evaluated protein expression of NRG1 cleavage products and enzymes that cleave at the external (BACE1, ADAM17, ADAM19) and internal (PS1-gamma secretase) sides of the cell membrane. We used three different cohorts (Controls, SCZ and BPD) and two distinct brain regions: BA9-prefrontal cortex (Controls (n = 6), SCZ (n = 6) and BPD (n = 6)) and hippocampus (Controls (n = 5), SCZ (n = 6) and BPD (n = 6)). In BA9, the ratio of the NRG1 N-terminal fragment relative to full length was significantly upregulated in the SCZ cohort (Bonferroni test, p = 0.011). ADAM17 was negatively correlated with full length NRG1 levels in the SCZ cohort (r = -0.926, p = 0.008). In the hippocampus we found significantly lower levels of a soluble 50 kDa NRG1 fragment in the two affected groups compared the control cohort (Bonferroni test, p = 0.0018). We also examined the relationship of specific symptomatology criteria with measures of NRG1 cleavage using the Bipolar Inventory of Signs and Symptoms Scale (BISS) and the Montgomery Åsberg Depression Rating Scale (MADRS). Our results showed a positive correlation between ADAM19 and psychosis (r = 0.595 p = 0.019); PS1 and mania (r = 0.535, p = 0.040); PS1 and depression (r = 0.567, p = 0.027) in BA9, and BACE1 with anxiety (r = 0.608, p = 0.03) in the hippocampus., Conclusion/significance: Our preliminary findings suggest region-specific alterations in NRG1 cleavage in SCZ and BPD patients. These changes may be associated with specific symptoms in these psychiatric disorders.

Neuregulin 1 (NRG1) has been implicated in several disorders including breast cancer, multiple sclerosis, and schizophrenia. Also, recent evidence suggests that NRG1 may play a role in regulation of inflammation and immune system response. We therefore hypothesized that a schizophrenia-associated missense mutation (valine to leucine) we identified within the transmembrane region of NRG1 would also be linked to immune dysregulation. We used plasma samples from families carrying the mutation to measure levels of antibodies to 41 autoimmune markers and six cytokines (IL-1b, IL-6, IL-10, IL-8, IL-12p70, and TNF-α) and used these levels as quantitative traits to evaluate association with the NRG1 mutation, using FBAT. Next, we used Epstein-Barr virus-transformed B cells from heterozygous mutation carriers and wild-type individuals to evaluate protein and mRNA cytokine expression in vitro using quantitative PCR and ELISA assays. In vivo, increased levels of 25 autoimmune markers as well as elevated levels of cytokines were significantly associated with the NRG1 mutation. In vitro, we observed a significant increase in protein secretion levels of IL-6, TNF-α, and IL-8 in mutation carriers compared with controls. At the mRNA level, we observed a significant increase in IL-6 expression, while IL-4 levels appeared to be down-regulated in heterozygous individuals compared with wild-type controls. This is the first report of association of a NRG1 mutation with immune dysregulation. This study could contribute towards understanding the role of NRG1 in the pathogenesis of schizophrenia and other disorders in which inflammation plays an important role.

The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism has been proposed as a possible candidate for involvement in the pathophysiology of bipolar disorder (BD). To determine whether an association exists between the BDNF Val66Met genotype and morphometric abnormalities of the brain regions involved in memory and learning in BD and healthy subjects. Forty-two BD patients and 42 healthy subjects were studied. Interactions between BDNF Val66Met genotype and diagnosis in gray (GM) volumes were analyzed using an optimized voxel-based morphometry technique. Declarative memory function was assessed with the California Verbal Learning Test II. Left and right anterior cingulate GM volumes showed a significant interaction between genotype and diagnosis such that anterior cingulate GM volumes were significantly smaller in the Val/Met BD patients compared with the Val/Val BD patients (left P=0.01, right P=0.01). Within-group comparisons revealed that the Val/Met carriers showed smaller GM volumes of the dorsolateral prefrontal cortex compared with the Val/Val subjects within the BD patient (P=0.01) and healthy groups (left P=0.03, right P=0.03). The Val/Met healthy subjects had smaller GM volumes of the left hippocampus compared with the Val/Val healthy subjects (P<0.01). There was a significant main effect of diagnosis on memory function (P=0.04), but no interaction between diagnosis and genotype was found (P=0.48). The findings support an association between the BDNF Val66Met genotype and differential gray matter content in brain structures, and suggest that the variation in this gene may play a more prominent role in brain structure differences in subjects affected with BD.

Although atypical antipsychotics are widely known to induce alterations in lipid and glucose metabolism, the mechanisms by which these alterations occur remain unknown. Several recent studies have shown that atypical antipsychotics induce oxidative stress and oxidative cell injury by increasing levels of lipid and protein oxidation. In this study, a novel proteomic approach was used to identify specific proteins oxidized after clozapine treatment. Differentiated neuroblastoma SKNSH cells were treated with 0, 5 or 20 mum clozapine for 24 h and protein extracts were labelled with 6-iodoacetamide fluorescein (6-IAF). The lack of incorporation of 6-IAF to cysteine residues is an indicator of protein oxidation. Labelled proteins were exposed to 2D electrophoresis, and differential protein labelling was assessed. Increased oxidation after clozapine treatment was observed in 10 protein spots (p<0.05), although only four of them remained significant after correcting for analysis with two drug concentrations. Five proteins, corresponding to nine of the spots, were identified by HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) as mitochondrial ribosomal protein S22, mitochondrial malate dehydrogenase, calumenin, pyruvate kinase and 3-oxoacid CoA transferase. The latter four proteins play important roles in energy metabolism. These results suggest that oxidative stress may be a mechanism by which antipsychotics increase the risk for metabolic syndrome and diabetes.