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The frontal pole (Brodmann area 10, BA10) is the largest cytoarchitectonic region of the human cortex, performing complex integrative functions. BA10 undergoes intensive adolescent grey matter pruning prior to the age of onset for bipolar disorder (BP) and schizophrenia (SCHIZ), and its dysfunction is likely to underly aspects of their shared symptomology. In this study, we investigated the role of BA10 neurotransmission-related gene expression in BP and SCHIZ. We performed qPCR to measure the expression of 115 neurotransmission-related targets in control, BP, and SCHIZ postmortem samples (n = 72). We chose this method for its high sensitivity to detect low-level expression. We then strengthened our findings by performing a meta-analysis of publicly released BA10 microarray data (n = 101) and identified sources of convergence with our qPCR results. To improve interpretation, we leveraged the unusually large database of clinical metadata accompanying our samples to explore the relationship between BA10 gene expression, therapeutics, substances of abuse, and symptom profiles, and validated these findings with publicly available datasets. Using these convergent sources of evidence, we identified 20 neurotransmission-related genes that were differentially expressed in BP and SCHIZ in BA10. These results included a large diagnosis-related decrease in two important therapeutic targets with low levels of expression, HTR2B and DRD4, as well as other findings related to dopaminergic, GABAergic and astrocytic function. We also observed that therapeutics may produce a differential expression that opposes diagnosis effects. In contrast, substances of abuse showed similar effects on BA10 gene expression as BP and SCHIZ, potentially amplifying diagnosis-related dysregulation.

Clinical and preclinical studies suggest that some of the behavioral alterations observed in schizophrenia (SZ) may be mechanistically linked to synaptic dysfunction of glutamatergic signaling. Recent genetic and proteomic studies suggest alterations of cortical glutamate receptors of the AMPA-type (AMPARs), which are the predominant ligand-gated ionic channels of fast transmission at excitatory synapses. The impact of gene and protein alterations on the electrophysiological activity of AMPARs is not known in SZ. In this proof of principle work, using human postmortem brain synaptic membranes isolated from the dorsolateral prefrontal cortex (DLPFC), we combined electrophysiological analysis from microtransplanted synaptic membranes (MSM) with transcriptomic (RNA-Seq) and label-free proteomics data in 10 control and 10 subjects diagnosed with SZ. We observed in SZ a reduction in the amplitude of AMPARs currents elicited by kainate, an agonist of AMPARs that blocks the desensitization of the receptor. This reduction was not associated with protein abundance but with a reduction in kainates potency to activate AMPARs. Electrophysiologically-anchored dataset analysis (EDA) was used to identify synaptosomal proteins that linearly correlate with the amplitude of the AMPARs responses, gene ontology functional annotations were then used to determine protein-protein interactions. Protein modules associated with positive AMPARs current increases were downregulated in SZ, while protein modules that were upregulated in SZ were associated with decreased AMPARs currents. Our results indicate that transcriptomic and proteomic alterations, frequently observed in the DLPFC in SZ, converge at the synaptic level producing a functional electrophysiological impairment of AMPARs.

Mitochondrial dysfunction is a neurobiological phenomenon implicated in the pathophysiology of schizophrenia and bipolar disorder that can synergistically affect synaptic neurotransmission. We hypothesized that schizophrenia and bipolar disorder share molecular alterations at the mitochondrial and synaptic levels. Mitochondria DNA (mtDNA) copy number (CN), mtDNA common deletion (CD), mtDNA total deletion, complex I activity, synapse number, and synaptic mitochondria number were studied in the postmortem human dorsolateral prefrontal cortex (DLPFC), superior temporal gyrus (STG), primary visual cortex (V1), and nucleus accumbens (NAc) of controls (CON), and subjects with schizophrenia (SZ), and bipolar disorder (BD). The results showed (i) the mtDNA CN is significantly higher in DLPFC of both SZ and BD, decreased in the STG of BD, and unaltered in V1 and NAc of both SZ and BD; (ii) the mtDNA CD is significantly higher in DLPFC of BD while unaltered in STG, V1, and NAc of both SZ and BD; (iii) The total deletion burden is significantly higher in DLPFC in both SZ and BD while unaltered in STG, V1, and NAc of SZ and BD; (iv) Complex I activity is significantly lower in DLPFC of both SZ and BD, which is driven by the presence of medications, with no alteration in STG, V1, and NAc. In addition, complex I protein concentration, by ELISA, was decreased across three cortical regions of SZ and BD subjects; (v) The number of synapses is decreased in DLPFC of both SZ and BD, while the synaptic mitochondria number was significantly lower in female SZ and female BD compared to female controls. Overall, these findings will pave the way to understand better the pathophysiology of schizophrenia and bipolar disorder for therapeutic interventions.

Primary cilia function as cells' antennas to detect and transduce external stimuli and play crucial roles in cell signaling and communication. The vast majority of cilia genes that are causally linked with ciliopathies are also associated with neurological deficits, such as cognitive impairments. Yet, the roles of cilia dysfunctions in the pathogenesis of psychiatric disorders have not been studied. Our aim is to identify patterns of cilia gene dysregulation in the four major psychiatric disorders: schizophrenia (SCZ), autism spectrum disorder (ASD), bipolar disorder (BP), and major depressive disorder (MDD). For this purpose, we acquired differentially expressed genes (DEGs) from the largest and most recent publicly available databases. We found that 42%, 24%, 17%, and 15% of brain-expressed cilia genes were significantly differentially expressed in SCZ, ASD, BP, and MDD, respectively. Several genes exhibited cross-disorder overlap, suggesting that typical cilia signaling pathways' dysfunctions determine susceptibility to more than one psychiatric disorder or may partially underlie their pathophysiology. Our study revealed that genes encoding proteins of almost all sub-cilia structural and functional compartments were dysregulated in the four psychiatric disorders. Strikingly, the genes of 75% of cilia GPCRs and 50% of the transition zone proteins were differentially expressed in SCZ. The present study is the first to draw associations between cilia and major psychiatric disorders, and is the first step toward understanding the role that cilia components play in their pathophysiological processes, which may lead to novel therapeutic targets for these disorders.

Schizophrenia is a common, chronic and debilitating neuropsychiatric syndrome affecting tens of millions of individuals worldwide. While rare genetic variants play a role in the etiology of schizophrenia, most of the currently explained liability is within common variation, suggesting that variation predating the human diaspora out of Africa harbors a large fraction of the common variant attributable heritability. However, common variant association studies in schizophrenia have concentrated mainly on cohorts of European descent. We describe genome-wide association studies of 6152 cases and 3918 controls of admixed African ancestry, and of 1234 cases and 3090 controls of Latino ancestry, representing the largest such study in these populations to date. Combining results from the samples with African ancestry with summary statistics from the Psychiatric Genomics Consortium (PGC) study of schizophrenia yielded seven newly genome-wide significant loci, and we identified an additional eight loci by incorporating the results from samples with Latino ancestry. Leveraging population differences in patterns of linkage disequilibrium, we achieve improved fine-mapping resolution at 22 previously reported and 4 newly significant loci. Polygenic risk score profiling revealed improved prediction based on trans-ancestry meta-analysis results for admixed African (Nagelkerke's R2 = 0.032; liability R2 = 0.017; P

BackgroundEvidence from anatomical, pharmacological, and genetic studies supports a role for the neuropeptide melanin concentrating hormone system in modulating emotional and cognitive functions. Genome-wide association studies revealed a potential association between the melanin concentrating hormone receptor (MCHR1) gene locus and schizophrenia, and the largest genome-wide association study conducted to date shows a credible genome-wide association.MethodsWe analyzed MCHR1 and pro-melanin concentrating hormone RNA-Seq expression in the prefrontal cortex in schizophrenia patients and healthy controls. Disruptions in the melanin concentrating hormone system were modeled in the mouse brain by germline deletion of MCHR1 and by conditional ablation of melanin concentrating hormone expressing neurons using a Cre-inducible diphtheria toxin system.ResultsMCHR1 expression is decreased in the prefrontal cortex of schizophrenia samples (false discovery rate (FDR) P

Mitochondria have been linked to the etiology of schizophrenia (SZ). However, studies of mitochondria in SZ might be confounded by the effects of pharmacological treatment with antipsychotic drugs (APDs) and other common medications. This review summarizes findings on relevant mitochondria mechanisms underlying SZ, and the potential impact of psychoactive drugs including primarily APDs, but also antidepressants and anxiolytics. The summarized data suggest that APDs impair mitochondria function by decreasing Complex I activity and ATP production and dissipation of the mitochondria membrane potential. At the same time, in the brains of patients with SZ, antipsychotic drug treatment normalizes gene expression modules enriched in mitochondrial genes that are decreased in SZ. This indicates that APDs may have both positive and negative effects on mitochondria. The available evidence suggests three conclusions i) alterations in mitochondria functions in SZ exist prior to APD treatment, ii) mitochondria alterations in SZ can be reversed by APD treatment, and iii) APDs directly cause impairment of mitochondria function. Overall, the mechanisms of action of psychiatric drugs on mitochondria are both direct and indirect; we conclude the effects of APDs on mitochondria may contribute to both their therapeutic and metabolic side effects. These studies support the hypothesis that neuronal mitochondria are an etiological factor in SZ. Moreover, APDs and other drugs must be considered in the evaluation of this pathophysiological role of mitochondria in SZ. Considering these effects, pharmacological actions on mitochondria may be a worthwhile target for further APD development.

Evidence from animal and human studies has linked myo-inositol (MI) with the pathophysiology and/or treatment of psychiatric disorders such as schizophrenia and bipolar disorder. However, there is still controversy surrounding the definitive role of MI in these disorders. Given that brain MI is differentially regulated by three transporters - SMIT1, SMIT2 and/or HMIT (encoded by the genes: SLC5A3, SLC5A11, and SLC2A13, respectively) - we used available datasets to describe the distribution in mouse and human brain of the different MI transporters and to examine changes in mRNA expression of these transporters in patients with schizophrenia and bipolar disorder. We found a differential distribution of the mRNA of each of the three MI transporters in both human and mouse brain regions. Interestingly, while individual neurons express SMIT1 and HMIT, non-neuronal cells express SMIT2, thus partially accounting for different uptake levels of MI and concordance to downstream second messenger signaling pathways. We also found that the expression of MI transporters is significantly changed in schizophrenia and bipolar disorder in a diagnostic-, brain region- and subtype-specific manner. We then examined the effects of germline deletion in mice of Slc5a3 on behavioral phenotypes related to schizophrenia and bipolar disorder. This gene deletion produces behavioral deficits that mirror some specific symptoms of schizophrenia and bipolar disorder. Finally, chronic administration of MI was able to reverse particular, but not all, behavioral deficits in Slc5a3 knockout mice; MI itself induced some behavioral deficits. Our data support a strong correlation between the expression of MI transporters and schizophrenia and bipolar disorder, and suggest that brain region-specific aberration of one or more of these transporters determines the partial behavioral phenotypes and/or symptomatic pattern of these disorders.

Deletions in the 16.6 kb mitochondrial genome have been implicated in numerous disorders that often display muscular and/or neurological symptoms due to the high-energy demands of these tissues. We describe a catalogue of 4489 putative mitochondrial DNA (mtDNA) deletions, including their frequency and relative read rate, using a combinatorial approach of mitochondria-targeted PCR, next-generation sequencing, bioinformatics, post-hoc filtering, annotation, and validation steps. Our bioinformatics pipeline uses MapSplice, an RNA-seq splice junction detection algorithm, to detect and quantify mtDNA deletion breakpoints rather than mRNA splices. Analyses of 93 samples from postmortem brain and blood found (i) the 4977 bp 'common deletion' was neither the most frequent deletion nor the most abundant; (ii) brain contained significantly more deletions than blood; (iii) many high frequency deletions were previously reported in MitoBreak, suggesting they are present at low levels in metabolically active tissues and are not exclusive to individuals with diagnosed mitochondrial pathologies; (iv) many individual deletions (and cumulative metrics) had significant and positive correlations with age and (v) the highest deletion burdens were observed in major depressive disorder brain, at levels greater than Kearns-Sayre Syndrome muscle. Collectively, these data suggest the Splice-Break pipeline can detect and quantify mtDNA deletions at a high level of resolution.

The hypothalamus contains neurons that integrate hunger and satiety endocrine signals from the periphery and are implicated in the pathophysiology of obesity. The limited availability of human hypothalamic neurons hampers our understanding of obesity disease mechanisms. To address this, we generated human induced pluripotent stem cells (hiPSCs) from multiple normal body mass index (BMI; BMI ≤ 25) subjects and super-obese (OBS) donors (BMI ≥ 50) with polygenic coding variants in obesity-associated genes. We developed a method to reliably differentiate hiPSCs into hypothalamic-like neurons (iHTNs) capable of secreting orexigenic and anorexigenic neuropeptides. Transcriptomic profiling revealed that, although iHTNs maintain a fetal identity, they respond appropriately to metabolic hormones ghrelin and leptin. Notably, OBS iHTNs retained disease signatures and phenotypes of high BMI, exhibiting dysregulated respiratory function, ghrelin-leptin signaling, axonal guidance, glutamate receptors, and endoplasmic reticulum (ER) stress pathways. Thus, human iHTNs provide a powerful platform to study obesity and gene-environment interactions.

Subjects with schizophrenia (SZ) and bipolar disorder (BD) show decreased protein and transcript levels for mitochondrial complex I. In vitro results suggest antipsychotic and antidepressant drugs may be responsible. We measured complex I activity in BD, SZ, and controls and presence of antipsychotic and antidepressant medications, mitochondrial DNA (mtDNA) copy number, and the mtDNA common deletion in the brain. Complex I activity in the prefrontal cortex was decreased by 45% in SZ compared to controls (p = 0.02), while no significant difference was found in BD. Complex I activity was significantly decreased (p = 0.01) in pooled cases (SZ and BD) that had detectable psychotropic medications and drugs compared to pooled cases with no detectable levels. Subjects with age at onset in their teens and psychotropic medications showed decreased (p < 0.05) complex I activity compared to subjects with an adult age at onset. Both SZ and BD groups displayed significant increases (p < 0.05) in mtDNA copy number compared to controls; however, common deletion burden was not altered. Complex I deficiency is found in SZ brain tissue, and psychotropic medications may play a role in mitochondrial dysfunction. Studies of medication-free first-episode psychosis patients are needed to elucidate whether mitochondrial pathophysiology occurs independent of medication effects.

BackgroundConventional antidepressants usually require several weeks to achieve a full clinical response in patients with major depressive disorder, an illness associated with dysregulated circadian rhythms and a high incidence of suicidality. Two rapid-acting antidepressant strategies, low-dose ketamine (KT) and sleep deprivation (SD) therapies, dramatically reduce depressive symptoms within 24 hours in a subset of major depressive disorder patients. However, it is unknown whether they exert their actions through shared regulatory mechanisms. To address this question, we performed comparative transcriptomics analyses to identify candidate genes and relevant pathways common to KT and SD.MethodsWe used the forced swim test, a standardized behavioral approach to measure antidepressant-like activity of KT and SD. We investigated gene expression changes using high-density microarrays and pathway analyses (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Gene Set Enrichment Analysis) in KT- and SD-treated mice compared with saline-treated control male mice.ResultsWe show that KT and SD elicit common transcriptional responses implicating distinct elements of the circadian clock and processes involved in neuronal plasticity. There is an overlap of 64 genes whose expression is common in KT and SD. Specifically, there is downregulation of clock genes including Ciart, Per2, Npas4, Dbp, and Rorb in both KT- and SD-treated mice.ConclusionsWe demonstrate a potential involvement of the circadian clock in rapid antidepressant responses. These findings could open new research avenues to help design chronopharmacological strategies to treat major depressive disorder.

The NRF2 (also known as NFE2L2) transcription factor is a critical regulator of genes involved in defense against oxidative stress. Previous studies suggest thatNrf2plays a role in adipogenesisin vitro, and deletion of theNrf2gene protects against diet-induced obesity in mice. Here, we demonstrate that resistance to diet-induced obesity inNrf2(-/-)mice is associated with a 20-30% increase in energy expenditure. Analysis of bioenergetics revealed thatNrf2(-/-)white adipose tissues exhibit greater oxygen consumption. White adipose tissue showed a >2-fold increase inUcp1gene expression. Oxygen consumption is also increased nearly 2.5-fold inNrf2-deficient fibroblasts. Oxidative stress induced by glucose oxidase resulted in increasedUcp1expression. Conversely, antioxidant chemicals (such asN-acetylcysteine and Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) and SB203580 (a known suppressor ofUcp1expression) decreasedUcp1and oxygen consumption inNrf2-deficient fibroblasts. These findings suggest that increasing oxidative stress by limitingNrf2function in white adipocytes may be a novel means to modulate energy balance as a treatment of obesity and related clinical disorders.

Genome-wide association studies of schizophrenia encompassing the major histocompatibility locus (MHC) were highly significant following genome-wide correction. This broad region implicates many genes including the MHC complex class II. Within this interval we examined the expression of two MHC II genes (HLA-DPA1 and HLA-DRB1) in brain from individual subjects with schizophrenia (SZ), bipolar disorder (BD), major depressive disorder (MDD), and controls by differential gene expression methods. A third MHC II mRNA, CD74, was studied outside of the MHC II locus, as it interacts within the same immune complex. Exon microarrays were performed in anterior cingulate cortex (ACC) in BD compared to controls, and both HLA-DPA1 and CD74 were decreased in expression in BD. The expression of HLA-DPA1 and CD74 were both reduced in hippocampus, amygdala, and dorsolateral prefrontal cortex regions in SZ and BD compared to controls by specific qPCR assay. We found several novel HLA-DPA1 mRNA variants spanning HLA-DPA1 exons 2-3-4 as suggested by exon microarrays. The intronic rs9277341 SNP was a significant cis expression quantitative trait locus (eQTL) that was associated with the total expression of HLA-DPA1 in five brain regions. A biomarker study of MHC II mRNAs was conducted in SZ, BD, MDD, and control lymphoblastic cell lines (LCL) by qPCR assay of 87 subjects. There was significantly decreased expression of HLA-DPA1 and CD74 in BD, and trends for reductions in SZ in LCLs. The discovery of multiple splicing variants in brain for HLA-DPA1 is important as the HLA-DPA1 gene is highly conserved, there are no reported splicing variants, and the functions in brain are unknown. Future work on the function and localization of MHC Class II proteins in brain will help to understand the role of alterations in neuropsychiatric disorders. The HLA-DPA1 eQTL is located within a large linkage disequilibrium block that has an irrefutable association with schizophrenia. Future tests in a larger cohort are needed to determine the significance of this eQTL association with schizophrenia. Our findings support the long-held hypothesis that alterations in immune function are associated with the pathophysiology of psychiatric disorders.

Genetic evidence has supported the hypothesis that schizophrenia (SZ) is a polygenic disorder caused by the disruption in function of several or many genes. The most common and reproducible cellular phenotype associated with SZ is a reduction in dendritic spines within the neocortex, suggesting alterations in dendritic architecture may cause aberrant cortical circuitry and SZ symptoms. Here, we review evidence supporting a multifactorial model of mitochondrial dysfunction in SZ etiology and discuss how these multiple paths to mitochondrial dysfunction may contribute to dendritic spine loss and/or underdevelopment in some SZ subjects. The pathophysiological role of mitochondrial dysfunction in SZ is based upon genomic analyses of both the mitochondrial genome and nuclear genes involved in mitochondrial function. Previous studies and preliminary data suggest SZ is associated with specific alleles and haplogroups of the mitochondrial genome, and also correlates with a reduction in mitochondrial copy number and an increase in synonymous and nonsynonymous substitutions of mitochondrial DNA. Mitochondrial dysfunction has also been widely implicated in SZ by genome-wide association, exome sequencing, altered gene expression, proteomics, microscopy analyses, and induced pluripotent stem cell studies. Together, these data support the hypothesis that SZ is a polygenic disorder with an enrichment of mitochondrial targets.

A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.

INTRODUCTION: Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition. METHODS AND RESULTS: Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells. CONCLUSIONS: We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

The rs1344706, an intronic SNP within the zinc-finger protein 804A gene (ZNF804A), was identified as one of the most compelling risk SNPs for schizophrenia (SZ) and bipolar disorder (BD). It is however not clear by which molecular mechanisms ZNF804A increases disease risk. We evaluated the role of ZNF804A in SZ and BD by genotyping the originally associated rs1344706 SNP and an exonic SNP (rs12476147) located in exon four of ZNF804A in a sample of 422 SZ, 382 BD, and 507 controls from the isolated population of the Costa Rica Central Valley. We also investigated the rs1344706 SNP for allelic specific expression (ASE) imbalance in the dorsolateral prefrontal cortex (DLPFC) of 46 heterozygous postmortem brains. While no significant association between rs1344706 and SZ or BD was observed in the Costa Rica sample, we observed an increased risk of SZ for the minor allele (A) of the exonic rs12476147 SNP (p=0.026). Our ASE assay detected a significant over-expression of the rs12476147 A allele in DLPFC of rs1344706 heterozygous subjects. Interestingly, cDNA allele ratios were significantly different according to the intronic rs1344706 genotypes (p-value=0.03), with the rs1344706 A allele associated with increased ZNF804A rs12476147 A allele expression (average 1.06, p-value=0.02, for heterozygous subjects vs. genomic DNA). In conclusion, we have demonstrated a significant association of rs12476147 with SZ, and using a powerful within-subject design, an allelic expression imbalance of ZNF804A exonic SNP rs12476147 in the DLPFC. Although this data does not preclude the possibility of other functional variants in ZNF804A, it provides evidence that the rs1344706 SZ risk allele is the cis-regulatory variant directly responsible for this allelic expression imbalance in adult cortex.

Whole-genome and exome sequencing have already proven to be essential and powerful methods to identify genes responsible for simple Mendelian inherited disorders. These methods can be applied to complex disorders as well, and have been adopted as one of the current mainstream approaches in population genetics. These achievements have been made possible by next generation sequencing (NGS) technologies, which require substantial bioinformatics resources to analyze the dense and complex sequence data. The huge analytical burden of data from genome sequencing might be seen as a bottleneck slowing the publication of NGS papers at this time, especially in psychiatric genetics. We review the existing methods for processing NGS data, to place into context the rationale for the design of a computational resource. We describe our method, the Graphical Pipeline for Computational Genomics (GPCG), to perform the computational steps required to analyze NGS data. The GPCG implements flexible workflows for basic sequence alignment, sequence data quality control, single nucleotide polymorphism analysis, copy number variant identification, annotation, and visualization of results. These workflows cover all the analytical steps required for NGS data, from processing the raw reads to variant calling and annotation. The current version of the pipeline is freely available at http://pipeline.loni.ucla.edu. These applications of NGS analysis may gain clinical utility in the near future (e.g., identifying miRNA signatures in diseases) when the bioinformatics approach is made feasible. Taken together, the annotation tools and strategies that have been developed to retrieve information and test hypotheses about the functional role of variants present in the human genome will help to pinpoint the genetic risk factors for psychiatric disorders.

Suicidal behaviors are frequent in mood disorders patients but only a subset of them ever complete suicide. Understanding predisposing factors for suicidal behaviors in high risk populations is of major importance for the prevention and treatment of suicidal behaviors. The objective of this project was to investigate gene expression changes associated with suicide in brains of mood disorder patients by microarrays (Affymetrix HG-U133 Plus2.0) in the dorsolateral prefrontal cortex (DLPFC: 6 Non-suicides, 15 suicides), the anterior cingulate cortex (ACC: 6NS, 9S) and the nucleus accumbens (NAcc: 8NS, 13S). ANCOVA was used to control for age, gender, pH and RNA degradation, with P≤0.01 and fold change±1.25 as criteria for significance. Pathway analysis revealed serotonergic signaling alterations in the DLPFC and glucocorticoid signaling alterations in the ACC and NAcc. The gene with the lowest p-value in the DLPFC was the 5-HT2A gene, previously associated both with suicide and mood disorders. In the ACC 6 metallothionein genes were down-regulated in suicide (MT1E, MT1F, MT1G, MT1H, MT1X, MT2A) and three were down-regulated in the NAcc (MT1F, MT1G, MT1H). Differential expression of selected genes was confirmed by qPCR, we confirmed the 5-HT2A alterations and the global down-regulation of members of the metallothionein subfamilies MT 1 and 2 in suicide completers. MTs 1 and 2 are neuro-protective following stress and glucocorticoid stimulations, suggesting that in suicide victims neuroprotective response to stress and cortisol may be diminished. Our results thus suggest that suicide-specific expression changes in mood disorders involve both glucocorticoids regulated metallothioneins and serotonergic signaling in different regions of the brain.

Mitochondrial deficiencies with unknown causes have been observed in schizophrenia (SZ) and bipolar disorder (BD) in imaging and postmortem studies. Polymorphisms and somatic mutations in mitochondrial DNA (mtDNA) were investigated as potential causes with next generation sequencing of mtDNA (mtDNA-Seq) and genotyping arrays in subjects with SZ, BD, major depressive disorder (MDD), and controls. The common deletion of 4,977 bp in mtDNA was compared between SZ and controls in 11 different vulnerable brain regions and in blood samples, and in dorsolateral prefrontal cortex (DLPFC) of BD, SZ, and controls. In a separate analysis, association of mitochondria SNPs (mtSNPs) with SZ and BD in European ancestry individuals (n = 6,040) was tested using Genetic Association Information Network (GAIN) and Wellcome Trust Case Control Consortium 2 (WTCCC2) datasets. The common deletion levels were highly variable across brain regions, with a 40-fold increase in some regions (nucleus accumbens, caudate nucleus and amygdala), increased with age, and showed little change in blood samples from the same subjects. The common deletion levels were increased in the DLPFC for BD compared to controls, but not in SZ. Full mtDNA genome resequencing of 23 subjects, showed seven novel homoplasmic mutations, five were novel synonymous coding mutations. By logistic regression analysis there were no significant mtSNPs associated with BD or SZ after genome wide correction. However, nominal association of mtSNPs (p

Major mood disorders have been linked to abnormalities in circadian rhythms, leading to disturbances in sleep, mood, temperature, and hormonal levels. We provide evidence that ketamine, a drug with rapid antidepressant effects, influences the function of the circadian molecular machinery. Ketamine modulates CLOCK:BMAL1-mediated transcriptional activation when these regulators are ectopically expressed in NG108-15 neuronal cells. Inhibition occurs in a dose-dependent manner and is attenuated after treatment with the GSK3β antagonist SB21673. We analyzed the effect of ketamine on circadian gene expression and observed a dose-dependent reduction in the amplitude of circadian transcription of the Bmal1, Per2, and Cry1 genes. Finally, chromatin-immunoprecipitation analyses revealed that ketamine altered the recruitment of the CLOCK:BMAL1 complex on circadian promoters in a time-dependent manner. Our results reveal a yet unsuspected molecular mode of action of ketamine and thereby may suggest possible pharmacological antidepressant strategies.

The imaging genetics approach to studying the genetic basis of disease leverages the individual strengths of both neuroimaging and genetic studies by visualizing and quantifying the brain activation patterns in the context of genetic background. Brain imaging as an intermediate phenotype can help clarify the functional link among genes, the molecular networks in which they participate, and brain circuitry and function. Integrating genetic data from a genome-wide association study (GWAS) with brain imaging as a quantitative trait (QT) phenotype can increase the statistical power to identify risk genes. A QT analysis using brain imaging (DLPFC activation during a working memory task) as a quantitative trait has identified unanticipated risk genes for schizophrenia. Several of these genes (RSRC1, ARHGAP18, ROBO1-ROBO2, GPC1, TNIK, and CTXN3-SLC12A2) have functions related to progenitor cell proliferation, migration, and differentiation, cytoskeleton reorganization, axonal connectivity, and development of forebrain structures. These genes, however, do not function in isolation but rather through gene regulatory networks. To obtain a deeper understanding how the GWAS-identified genes participate in larger gene regulatory networks, we measured correlations among transcript levels in the mouse and human postmortem tissue and performed a gene set enrichment analysis (GSEA) that identified several microRNA associated with schizophrenia (448, 218, 137). The results of such computational approaches can be further validated in animal experiments in which the networks are experimentally studied and perturbed with specific compounds. Glypican 1 and FGF17 mouse models for example, can be used to study such gene regulatory networks. The model demonstrates epistatic interactions between FGF and glypican on brain development and may be a useful model of negative symptom schizophrenia.

BackgroundAbnormalities in the circadian clockwork often characterize patients with major depressive and bipolar disorders. Circadian clock genes are targets of interest in these patients. CRY2 is a circadian gene that participates in regulation of the evening oscillator. This is of interest in mood disorders where a lack of switch from evening to morning oscillators has been postulated.Principal FindingsWe observed a marked diurnal variation in human CRY2 mRNA levels from peripheral blood mononuclear cells and a significant up-regulation (P = 0.020) following one-night total sleep deprivation, a known antidepressant. In depressed bipolar patients, levels of CRY2 mRNA were decreased (P = 0.029) and a complete lack of increase was observed following sleep deprivation. To investigate a possible genetic contribution, we undertook SNP genotyping of the CRY2 gene in two independent population-based samples from Sweden (118 cases and 1011 controls) and Finland (86 cases and 1096 controls). The CRY2 gene was significantly associated with winter depression in both samples (haplotype analysis in Swedish and Finnish samples: OR = 1.8, P = 0.0059 and OR = 1.8, P = 0.00044, respectively).ConclusionsWe propose that a CRY2 locus is associated with vulnerability for depression, and that mechanisms of action involve dysregulation of CRY2 expression.

BackgroundMost studies investigating the neurobiology of depression and suicide have focused on the serotonergic system. While it seems clear that serotonergic alterations play a role in the pathogenesis of these major public health problems, dysfunction in additional neurotransmitter systems and other molecular alterations may also be implicated. Microarray expression studies are excellent screening tools to generate hypotheses about additional molecular processes that may be at play. In this study we investigated brain regions that are known to be implicated in the neurobiology of suicide and major depression are likely to represent valid global molecular alterations.Methodology/Principal FindingsWe performed gene expression analysis using the HG-U133AB chipset in 17 cortical and subcortical brain regions from suicides with and without major depression and controls. Total mRNA for microarray analysis was obtained from 663 brain samples isolated from 39 male subjects, including 26 suicide cases and 13 controls diagnosed by means of psychological autopsies. Independent brain samples from 34 subjects and animal studies were used to control for the potential confounding effects of comorbidity with alcohol. Using a Gene Ontology analysis as our starting point, we identified molecular pathways that may be involved in depression and suicide, and performed follow-up analyses on these possible targets. Methodology included gene expression measures from microarrays, Gene Score Resampling for global ontological profiling, and semi-quantitative RT-PCR. We observed the highest number of suicide specific alterations in prefrontal cortical areas and hippocampus. Our results revealed alterations of synaptic neurotransmission and intracellular signaling. Among these, Glutamatergic (GLU) and GABAergic related genes were globally altered. Semi-quantitative RT-PCR results investigating expression of GLU and GABA receptor subunit genes were consistent with microarray data.Conclusions/SignificanceThe observed results represent the first overview of global expression changes in brains of suicide victims with and without major depression and suggest a global brain alteration of GLU and GABA receptor subunit genes in these conditions.

BackgroundMitochondria provide most of the energy for brain cells by the process of oxidative phosphorylation. Mitochondrial abnormalities and deficiencies in oxidative phosphorylation have been reported in individuals with schizophrenia (SZ), bipolar disorder (BD), and major depressive disorder (MDD) in transcriptomic, proteomic, and metabolomic studies. Several mutations in mitochondrial DNA (mtDNA) sequence have been reported in SZ and BD patients.Methodology/Principal FindingsDorsolateral prefrontal cortex (DLPFC) from a cohort of 77 SZ, BD, and MDD subjects and age-matched controls (C) was studied for mtDNA sequence variations and heteroplasmy levels using Affymetrix mtDNA resequencing arrays. Heteroplasmy levels by microarray were compared to levels obtained with SNaPshot and allele specific real-time PCR. This study examined the association between brain pH and mtDNA alleles. The microarray resequencing of mtDNA was 100% concordant with conventional sequencing results for 103 mtDNA variants. The rate of synonymous base pair substitutions in the coding regions of the mtDNA genome was 22% higher (p = 0.0017) in DLPFC of individuals with SZ compared to controls. The association of brain pH and super haplogroup (U, K, UK) was significant (p = 0.004) and independent of postmortem interval time.ConclusionsFocusing on haplogroup and individual susceptibility factors in psychiatric disorders by considering mtDNA variants may lead to innovative treatments to improve mitochondrial health and brain function.

BackgroundRegulation of Transposable Elements (TEs) expression has already been associated with complex human diseases, in particular cancer [1]. Also, complex diseases cannot be explained only by genetic factors and are likely to be the result of gene-environment interactions with the contribution of TEs. The detection of retroviral transcripts in the brains of schizophrenics suggests that activation or upregulation of distinct human endogenous retroviruses (HERVs) may play a role in the etiopatho-genesis of neuropsychiatric diseases [2], with increasing complications if we consider that TE insertions generate somatic mosaicism in neuronal cells [3]. In addition, mobile elements have been heavily involved in tissue-specific promoter activity [4] that makes them good candidates for brain specific activation of genes related to schizophrenia. Eventually, TEs are thought to be important in regulation of methylation and DNA accessibility to transcription factors [5]. They are also an important source of small RNAs, which usually act to silence TEs [6]; a particular family of small RNAs, miRNAs, has already been shown to alter neural receptors' function [7].

Background: The purpose of this study was to examine the effects of glucose reduction stress on lymphoblastic cell line (LCL) gene expression in subjects with schizophrenia compared to non-psychotic relatives. Methods: LCLs were grown under two glucose conditions to measure the effects of glucose reduction stress on exon expression in subjects with schizophrenia compared to unaffected family member controls. A second aim of this project was to identify cis-regulated transcripts associated with diagnosis. Results: There were a total of 122 transcripts with significant diagnosis by probeset interaction effects and 328 transcripts with glucose deprivation by probeset interaction probeset effects after corrections for multiple comparisons. There were 8 transcripts with expression significantly affected by the interaction between diagnosis and glucose deprivation and probeset after correction for multiple comparisons. The overall validation rate by qPCR of 13 diagnosis effect genes identified through microarray was 62%, and all genes tested by qPCR showed concordant up- or down-regulation by qPCR and microarray. We assessed brain gene expression of five genes found to be altered by diagnosis and glucose deprivation in LCLs and found a significant decrease in expression of one gene, glutaminase, in the dorsolateral prefrontal cortex (DLPFC). One SNP with previously identified regulation by a 3' UTR SNP was found to influence IRF5 expression in both brain and lymphocytes. The relationship between the 3' UTR rs10954213 genotype and IRF5 expression was significant in LCLs (p = 0.0001), DLPFC (p = 0.007), and anterior cingulate cortex (p = 0.002). Conclusion: Experimental manipulation of cells lines from subjects with schizophrenia may be a useful approach to explore stress related gene expression alterations in schizophrenia and to identify SNP variants associated with gene expression.

BackgroundGene expression patterns in the brain are strongly influenced by the severity and duration of physiological stress at the time of death. This agonal effect, if not well controlled, can lead to spurious findings and diminished statistical power in case-control comparisons. While some recent studies match samples by tissue pH and clinically recorded agonal conditions, we found that these indicators were sometimes at odds with observed stress-related gene expression patterns, and that matching by these criteria still sometimes results in identifying case-control differences that are primarily driven by residual agonal effects. This problem is analogous to the one encountered in genetic association studies, where self-reported race and ethnicity are often imprecise proxies for an individual's actual genetic ancestry.ResultsWe developed an Agonal Stress Rating (ASR) system that evaluates each sample's degree of stress based on gene expression data, and used ASRs in post hoc sample matching or covariate analysis. While gene expression patterns are generally correlated across different brain regions, we found strong region-region differences in empirical ASRs in many subjects that likely reflect inter-individual variabilities in local structure or function, resulting in region-specific vulnerability to agonal stress.ConclusionVariation of agonal stress across different brain regions differs between individuals, revealing a new level of complexity for gene expression studies of brain tissues. The Agonal Stress Ratings quantitatively assess each sample's extent of regulatory response to agonal stress, and allow a strong control of this important confounder.