Search

Gene expression profiling of brain tissue samples applied to DNA microarrays promises to provide novel insights into the neurobiological bases of primate behavior. The strength of the microarray technology lies in the ability to simultaneously measure the expression levels of all genes in defined brain regions that are known to mediate behavior. The application of microarrays presents, however, various limitations and challenges for primate neuroscience research. Low RNA abundance, modest changes in gene expression, heterogeneous distribution of mRNA among cell subpopulations, and individual differences in behavior all mandate great care in the collection, processing, and analysis of brain tissue. A unique problem for nonhuman primate research is the limited availability of species-specific arrays. Arrays designed for humans are often used, but expression level differences are inevitably confounded by gene sequence differences in all cross-species array applications. Tools to deal with this problem are currently being developed. Here we review these methodological issues, and provide examples from our experiences using human arrays to examine brain tissue samples from squirrel monkeys. Until species-specific microarrays become more widely available, great caution must be taken in the assessment and interpretation of microarray data from nonhuman primates. Nevertheless, the application of human microarrays in nonhuman primate neuroscience research recovers useful information from thousands of genes, and represents an important new strategy for understanding the molecular complexity of behavior and mental health.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

Editorial. Discusses the contributions of microarray technology to neuroscience. Description of the technology; Implications on other forms of biological research; Type of probe in microarray platforms used to examine gene expression.

Presents information on a study which described the bioinformatic selection of human complementary DNA arrayed on nylon membranes at high density for neuroscience applications. Methods; Results; Discussion.

Abstract: Gene expression profiles of postmortem brain tissue represent important resources for understanding neuropsychiatric illnesses. The impact(s) of quality covariables on the analysis and results of gene expression studies are important questions. This paper addressed critical variables which might affect gene expression in two brain regions. Four broad groups of quality indicators in gene expression profiling studies (clinical, tissue, RNA, and microarray quality) were identified. These quality control indicators were significantly correlated, however one quality variable did not account for the total variance in microarray gene expression. The data showed that agonal factors and low pH correlated with decreased integrity of extracted RNA in two brain regions. These three parameters also modulated the significance of alterations in mitochondrial-related genes. The average F-ratio summaries across all transcripts showed that RNA degradation from the AffyRNAdeg program accounted for higher variation than all other quality factors. Taken together, these findings confirmed prior studies, which indicated that quality parameters including RNA integrity, agonal factors, and pH are related to differences in gene expression profiles in postmortem brain. Individual candidate genes can be evaluated with these quality parameters in post hoc analysis to help strengthen the relevance to psychiatric disorders. We find that clinical, tissue, RNA, and microarray quality are all useful variables for collection and consideration in study design, analysis, and interpretation of gene expression results in human postmortem studies. [Copyright &y& Elsevier]