Investigating the neuroimmunogenic architecture of schizophrenia.

The role of the immune system in schizophrenia remains controversial despite numerous studies to date. Most studies have profiled expression of select genes or proteins in peripheral blood, but none have focused on the expression of canonical pathways that mediate overall immune response. The current study used a systematic genetic approach to investigate the role of the immune system in a large sample of post-mortem brain of patients with schizophrenia: RNA sequencing was performed to assess the differential expression of 561 immune genes and 20 immune pathways in dorsolateral prefrontal cortex (DLPFC) (144 schizophrenia and 196 control subjects) and hippocampus (83 schizophrenia and 187 control subjects). The effect of RNA quality on gene expression was found to be highly correlated with the effect of diagnosis even after adjustment for observable RNA quality parameters (i.e. RNA integrity), thus this confounding relationship was statistically controlled using principal components derived from the gene expression matrix. In DLPFC, 23 immune genes were found to be differentially expressed (false discovery rate <0.05), of which seven genes replicated in both directionality and at nominal significance (P<0.05) in an independent post-mortem DLPFC data set (182 schizophrenia and 212 control subjects), although notably at least five of these genes have prominent roles in pathways other than immune function and overall the effect sizes were minimal (fold change <1.1). In the hippocampus, no individual immune genes were identified to be differentially expressed, and in both DLPFC and hippocampus none of the individual immune pathways were relatively differentially expressed. Further, genomic schizophrenia risk profiles scores were not correlated with the expression of individual immune pathways or differentially expressed genes. Overall, past reports claiming a primary pathogenic role of the immune system intrinsic to the brain in schizophrenia could not be confirmed.

GAD1 alternative transcripts and DNA methylation in human prefrontal cortex and hippocampus in brain development, schizophrenia.

Genetic variations and adverse environmental events in utero or shortly after birth can lead to abnormal brain development and increased risk of schizophrenia. γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, plays a vital role in normal brain development. GABA synthesis is controlled by enzymes derived from two glutamic acid decarboxylase (GAD) genes, GAD1 and GAD2, both of which produce transcript isoforms. While the full-length GAD1 transcript (GAD67) has been implicated in the neuropathology of schizophrenia, the transcript structure of GAD1 in the human brain has not been fully characterized. In this study, with the use of RNA sequencing and PCR technologies, we report the discovery of 10 novel transcripts of GAD1 in the human brain. Expression levels of four novel GAD1 transcripts (8A, 8B, I80 and I86) showed a lifespan trajectory expression pattern that is anticorrelated with the expression of the full-length GAD1 transcript. In addition, methylation levels of two CpG loci within the putative GAD1 promoter were significantly associated with the schizophrenia-risk SNP rs3749034 and with the expression of GAD25 in dorsolateral prefrontal cortex (DLPFC). Moreover, schizophrenia patients who had completed suicide and/or were positive for nicotine exposure had significantly higher full-length GAD1 expression in the DLPFC. Alternative splicing of GAD1 and epigenetic state appear to play roles in the developmental profile of GAD1 expression and may contribute to GABA dysfunction in the PFC and hippocampus of patients with schizophrenia.

Longitudinal analyses of the DNA methylome in deployed military servicemen identify susceptibility loci for post-traumatic stress disorder.

In order to determine the impact of the epigenetic response to traumatic stress on post-traumatic stress disorder (PTSD), this study examined longitudinal changes of genome-wide blood DNA methylation profiles in relation to the development of PTSD symptoms in two prospective military cohorts (one discovery and one replication data set). In the first cohort consisting of male Dutch military servicemen (n=93), the emergence of PTSD symptoms over a deployment period to a combat zone was significantly associated with alterations in DNA methylation levels at 17 genomic positions and 12 genomic regions. Evidence for mediation of the relation between combat trauma and PTSD symptoms by longitudinal changes in DNA methylation was observed at several positions and regions. Bioinformatic analyses of the reported associations identified significant enrichment in several pathways relevant for symptoms of PTSD. Targeted analyses of the significant findings from the discovery sample in an independent prospective cohort of male US marines (n=98) replicated the observed relation between decreases in DNA methylation levels and PTSD symptoms at genomic regions in ZFP57, RNF39 and HIST1H2APS2. Together, our study pinpoints three novel genomic regions where longitudinal decreases in DNA methylation across the period of exposure to combat trauma marks susceptibility for PTSD.

Increasing BMI is associated with reduced expression of P-glycoprotein (ABCB1 gene) in the human brain with a stronger association in African Americans than Caucasians.

The efflux pump, p-glycoprotein, controls bioavailability and excretion of pharmaceutical compounds. In the blood-brain barrier, p-glycoprotein regulates the delivery of pharmaceutical substances to the brain, influencing efficacy and side effects for some drugs notably antipsychotics. Common side effects to antipsychotics include obesity and metabolic disease. Polymorphisms in the ABCB1 gene coding for p-glycoprotein are associated with more severe side effects to neuro-pharmaceuticals as well as weight gain, indicating a potential link between p-glycoprotein function and metabolic regulation. Using microarray data analysis from 145 neurologically sound adults, this study investigated the association between body mass index (BMI) and ABCB1 expression in the frontal cortex. Increasing BMI values were associated with a statistically significantly reduced expression of ABCB1. Investigation of DNA methylation patterns in a subgroup of 52 individuals found that the methylation/expression ratios of ABCB1 were unaffected by increasing BMI values. Interestingly, the effect of BMI on ABCB1 expression appeared stronger in African Americans than in Caucasians.

Neurexin 1 (NRXN1) splice isoform expression during human neocortical development and aging.

Neurexin 1 (NRXN1), a presynaptic cell adhesion molecule, is implicated in several neurodevelopmental disorders characterized by synaptic dysfunction including autism, intellectual disability and schizophrenia. To gain insight into NRXN1's involvement in human cortical development we used quantitative real-time PCR to examine the expression trajectories of NRXN1, and its predominant isoforms, NRXN1-α and NRXN1-ß, in prefrontal cortex from fetal stages to aging. In addition, we investigated whether prefrontal cortical expression levels of NRXN1 transcripts are altered in schizophrenia or bipolar disorder in comparison with non-psychiatric control subjects. We observed that all three NRXN1 transcripts were highly expressed during human fetal cortical development, markedly increasing with gestational age. In the postnatal dorsolateral prefrontal cortex, expression levels were negatively correlated with age, peaking at birth until ~3 years of age, after which levels declined markedly to be stable across the lifespan. NRXN1-ß expression was modestly but significantly elevated in the brains of patients with schizophrenia compared with non-psychiatric controls, whereas NRXN1-α expression was increased in bipolar disorder. These data provide novel evidence that NRXN1 expression is highest in human dorsolateral prefrontal cortex during critical developmental windows relevant to the onset and diagnosis of a range of neurodevelopmental disorders, and that NRXN1 expression may be differentially altered in neuropsychiatric disorders.

Sex differences in glutamate receptor gene expression in major depression and suicide.

Accumulating data indicate that the glutamate system is disrupted in major depressive disorder (MDD), and recent clinical research suggests that ketamine, an antagonist of the N-methyl-d-aspartate (NMDA) glutamate receptor (GluR), has rapid antidepressant efficacy. Here we report findings from gene expression studies of a large cohort of postmortem subjects, including subjects with MDD and controls. Our data reveal higher expression levels of the majority of glutamatergic genes tested in the dorsolateral prefrontal cortex (DLPFC) in MDD (F21,59=2.32, P=0.006). Posthoc data indicate that these gene expression differences occurred mostly in the female subjects. Higher expression levels of GRIN1, GRIN2A-D, GRIA2-4, GRIK1-2, GRM1, GRM4, GRM5 and GRM7 were detected in the female patients with MDD. In contrast, GRM5 expression was lower in male MDD patients relative to male controls. When MDD suicides were compared with MDD non-suicides, GRIN2B, GRIK3 and GRM2 were expressed at higher levels in the suicides. Higher expression levels were detected for several additional genes, but these were not statistically significant after correction for multiple comparisons. In summary, our analyses indicate a generalized disruption of the regulation of the GluRs in the DLPFC of females with MDD, with more specific GluR alterations in the suicides and in the male groups. These data reveal further evidence that, in addition to the NMDA receptor, the AMPA, kainate and the metabotropic GluRs may be targets for the development of rapidly acting antidepressant drugs.

Revisiting DARPP-32 in postmortem human brain: changes in schizophrenia and bipolar disorder and genetic associations with t-DARPP-32 expression.

Dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32 or PPP1R1B) has been of interest in schizophrenia owing to its critical function in integrating dopaminergic and glutaminergic signaling. In a previous study, we identified single-nucleotide polymorphisms (SNPs) and a frequent haplotype associated with cognitive and imaging phenotypes that have been linked with schizophrenia, as well as with expression of prefrontal cortical DARPP-32 messenger RNA (mRNA) in a relatively small sample of postmortem brains. In this study, we examined the association of expression of two major DARPP-32 transcripts, full-length (FL-DARPP-32) and truncated (t-DARPP-32), with genetic variants of DARPP-32 in three brain regions receiving dopaminergic input and implicated in schizophrenia (the dorsolateral prefrontal cortex (DLPFC), hippocampus and caudate) in a much larger set of postmortem samples from patients with schizophrenia, bipolar disorder, major depression and normal controls (>700 subjects). We found that the expression of t-DARPP-32 was increased in the DLPFC of patients with schizophrenia and bipolar disorder, and was strongly associated with genotypes at SNPs (rs879606, rs90974 and rs3764352), as well as the previously identified 7-SNP haplotype related to cognitive functioning. The genetic variants that predicted worse cognitive performance were associated with higher t-DARPP-32 expression. Our results suggest that variation in PPP1R1B affects the abundance of the splice variant t-DARPP-32 mRNA and may reflect potential molecular mechanisms implicated in schizophrenia and affective disorders.

APOE2 enhances neuroprotection against Alzheimer's disease through multiple molecular mechanisms.

The common APOE2 gene variant is neuroprotective against Alzheimer's disease (AD) and reduces risk by nearly 50%. However, the mechanisms by which APOE2 confers neuroprotection are largely unknown. Here we showed that ApoE protein abundance in human postmortem cortex follows an isoform-dependent pattern (E2>E3>E4). We also identified a unique downstream transcriptional profile determined by microarray and characterized by downregulation of long-term potentiation (LTP) related transcripts and upregulation of extracellular matrix (ECM)/integrin-related transcripts in E2 cases and corroborated this finding at the protein level by demonstrating increases in ECM collagens and laminins. In vivo studies of healthy older individuals demonstrated a unique and advantageous biomarker signature in E2 carriers. APOE2 also reduced the risk of mild cognitive impairment to AD conversion by half. Our findings suggest that ApoE2 protein abundance, coupled with its inability to bind to LDLRs, may act to increase amyloid-beta (Ab) clearance. In addition, increased ECM and reduced LTP-related expression results in diminished activity-dependent Ab secretion and/or excitotoxicity, and thus also promotes neuroprotection.

Contrasting changes in DRD1 and DRD2 splice variant expression in schizophrenia and affective disorders, and associations with SNPs in postmortem brain.

Dopamine 2 receptor (DRD2) is of major interest to the pathophysiology of schizophrenia (SCZ) both as a target for antipsychotic drug action as well as a SCZ-associated risk gene. The dopamine 1 receptor (DRD1) is thought to mediate some of the cognitive deficits in SCZ, including impairment of working memory that relies on normal dorsolateral prefrontal cortex (DLPFC) function. To better understand the association of dopamine receptors with SCZ, we studied the expression of three DRD2 splice variants and the DRD1 transcript in DLPFC, hippocampus and caudate nucleus in a large cohort of subjects (~700), including patients with SCZ, affective disorders and nonpsychiatric controls (from 14th gestational week to 85 years of age), and examined genotype-expression associations of 278 single-nucleotide polymorphisms (SNPs) located in or near DRD2 and DRD1 genes. Expression of D2S mRNA and D2S/D2-long (D2L) ratio were significantly increased in DLPFC of patients with SCZ relative to controls (P<0.0001 and P<0.0001, respectively), whereas D2L, D2Longer and DRD1 were decreased (P<0.0001). Patients with affective disorders showed an opposite pattern: reduced expression of D2S (major depressive disorder, P<0.0001) and increased expression of D2L and DRD1 (bipolar disorder, P<0.0001). Moreover, SCZ-associated risk alleles at rs1079727, rs1076560 and rs2283265 predicted increased D2S/D2L expression ratio (P<0.05) in control individuals. Our data suggest that altered splicing of DRD2 and expression of DRD1 may constitute a pathophysiological mechanism in risk for SCZ and affective disorders. The association between SCZ risk-associated polymorphism and the ratio of D2S/D2L is consistent with this possibility.

Altered gene expression in the dorsolateral prefrontal cortex of individuals with schizophrenia.

The underlying pathology of schizophrenia (SZ) is likely as heterogeneous as its symptomatology. A variety of cortical and subcortical regions, including the prefrontal cortex, have been implicated in its pathology, and a number of genes have been identified as risk factors for disease development. We used in situ hybridization (ISH) to examine the expression of 58 genes in the dorsolateral prefrontal cortex (DLPFC, comprised of Brodmann areas 9 and 46) from 19 individuals with a premorbid diagnosis of SZ and 33 control individuals. Genes were selected based on: (1) previous identification as risk factors for SZ; (2) cell type markers or (3) laminar markers. Cell density and staining intensity were compared in the DLPFC, as well as separately in Brodmann areas 9 and 46. The expression patterns of a variety of genes, many of which are associated with the GABAergic system, were altered in SZ when compared with controls. Additional genes, including C8orf79 and NR4A2, showed alterations in cell density or staining intensity between the groups, highlighting the need for additional studies. Alterations were, with only a few exceptions, limited to Brodmann area 9, suggesting regional specificity of pathology in the DLPFC. Our results agree with previous studies on the GABAergic involvement in SZ, and suggest that areas 9 and 46 may be differentially affected in the disease. This study also highlights additional genes that may be altered in SZ, and indicates that these potentially interesting genes can be identified by ISH and high-throughput image analysis techniques.

RNA-sequencing of the brain transcriptome implicates dysregulation of neuroplasticity, circadian rhythms and GTPase binding in bipolar disorder.

RNA-sequencing (RNA-seq) is a powerful technique to investigate the complexity of gene expression in the human brain. We used RNA-seq to survey the brain transcriptome in high-quality postmortem dorsolateral prefrontal cortex from 11 individuals diagnosed with bipolar disorder (BD) and from 11 age- and gender-matched controls. Deep sequencing was performed, with over 350 million reads per specimen. At a false discovery rate of <5%, we detected five differentially expressed (DE) genes and 12 DE transcripts, most of which have not been previously implicated in BD. Among these, Prominin 1/CD133 and ATP-binding cassette-sub-family G-member2 (ABCG2) have important roles in neuroplasticity. We also show for the first time differential expression of long noncoding RNAs (lncRNAs) in BD. DE transcripts include those of serine/arginine-rich splicing factor 5 (SRSF5) and regulatory factor X4 (RFX4), which along with lncRNAs have a role in mammalian circadian rhythms. The DE genes were significantly enriched for several Gene Ontology categories. Of these, genes involved with GTPase binding were also enriched for BD-associated SNPs from previous genome-wide association studies, suggesting that differential expression of these genes is not simply a consequence of BD or its treatment. Many of these findings were replicated by microarray in an independent sample of 60 cases and controls. These results highlight common pathways for inherited and non-inherited influences on disease risk that may constitute good targets for novel therapies.

Molecular signatures in post-mortem brain tissue of younger individuals at high risk for Alzheimer's disease as based on APOE genotype.

Alzheimer's disease (AD) is a neurodegenerative condition characterized histopathologically by neuritic plaques and neurofibrillary tangles. The objective of this transcriptional profiling study was to identify both neurosusceptibility and intrinsic neuroprotective factors at the molecular level, not confounded by the downstream consequences of pathology. We thus studied post-mortem cortical tissue in 28 cases that were non-APOE4 carriers (called the APOE3 group) and 13 cases that were APOE4 carriers. As APOE genotype is the major genetic risk factor for late-onset AD, the former group was at low risk for development of the disease and the latter group was at high risk for the disease. Mean age at death was 42 years and none of the brains had histopathology diagnostic of AD at the time of death. We first derived interregional difference scores in expression between cortical tissue from a region relatively invulnerable to AD (primary somatosensory cortex, BA 1/2/3) and an area known to be susceptible to AD pathology (middle temporal gyrus, BA 21). We then contrasted the magnitude of these interregional differences in between-group comparisons of the APOE3 (low risk) and APOE4 (high risk) genotype groups. We identified 70 transcripts that differed significantly between the groups. These included EGFR, CNTFR, CASP6, GRIA2, CTNNB1, FKBPL, LGALS1 and PSMC5. Using real-time quantitative PCR, we validated these findings. In addition, we found regional differences in the expression of APOE itself. We also identified multiple Kyoto pathways that were disrupted in the APOE4 group, including those involved in mitochondrial function, calcium regulation and cell-cycle reentry. To determine the functional significance of our transcriptional findings, we used bioinformatics pathway analyses to demonstrate that the molecules listed above comprised a network of connections with each other, APOE, and APP and MAPT. Overall, our results indicated that the abnormalities that we observed in single transcripts and in signaling pathways were not the consequences of diagnostic plaque and tangle pathology, but preceded it and thus may be a causative link in the long molecular prodrome that results in clinical AD.

Coupling of dopamine D1 recognition sites with adenylate cyclase in nuclei accumbens and caudatus of schizophrenics.

Sodium fluoride, guanylimidodiphosphate, and the D1 dopamine receptor agonist SKF 38393 elicited a greater activation of adenylate cyclase in homogenates of caudate nucleus in schizophrenic than in nonschizophrenic subjects used as controls. Similarly, a greater activation of adenylate cyclase by sodium fluoride was observed in the nucleus accumbens of schizophrenics. These findings suggest that the coupling of dopamine D1 recognition sites with adenylate cyclase is more efficient in the brain of the schizophrenic, presumably because of an increased affinity of the G/F protein for guanosine 5'-triphosphate.

A correlation between platelet monoamine oxidase activity and plasma prolactin concentrations in man.

Increases in plasma prolactin concentrations produced by alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, varied inversely with baseline platelet monoamine oxidase activity in 12 patients with chronic schizophrenia. In normal volunteers with low monoamine oxidase activity and in unmedicated patients with chronic schizophrenia, plasma prolactin concentrations varied directly with platelet monoamine oxidase activity. No such relationship was found in normal subjects with high platelet monoamine oxidase activity. These data suggest that platelet monoamine oxidase activity reflects monoaminergic activity in the tubero-infundibular system, which in turn affects plasma prolactin concentrations. This relationship may be important in patients with low platelet monoamine oxidase activity, such as some chronic schizophrenics.

Intravenous naloxone administration in schizophrenia and affective illness.

Fourteen schizophrenic patients and five patients with affective disorders were given naloxone (0.4 to 10 milligrams) or placebo intravenously in a double-blind fashion. Physicians' ratings of hallucinations, mannerisms and posturing, conceptual disorganization, psychosis, and mood did not change significantly. A single item, unusual thought content, improved significantly on the naloxone day compared to the placebo day. There was no improvement in mood in affectively ill patients rated either by themselves or by physicians. Naloxone did not markedly improve any patient studied, which suggests that the acute blockade of opiate receptors is not associated with global improvement in psychotic symptomatology.

Genetic vulnerability to DUSP22 promoter hypermethylation is involved in the relation between in utero famine exposure and schizophrenia.

Epigenetic changes may account for the doubled risk to develop schizophrenia in individuals exposed to famine in utero. We therefore investigated DNA methylation in a unique sample of patients and healthy individuals conceived during the great famine in China. Subsequently, we examined two case-control samples without famine exposure in whole blood and brain tissue. To shed light on the causality of the relation between famine exposure and DNA methylation, we exposed human fibroblasts to nutritional deprivation. In the famine-exposed schizophrenia patients, we found significant hypermethylation of the dual specificity phosphatase 22 (DUSP22) gene promoter (Chr6:291687-293285) (N = 153, p = 0.01). In this sample, DUSP22 methylation was also significantly higher in patients independent of famine exposure (p = 0.025), suggesting that hypermethylation of DUSP22 is also more generally involved in schizophrenia risk. Similarly, DUSP22 methylation was also higher in two separate case-control samples not exposed to famine using DNA from whole blood (N = 64, p = 0.03) and postmortem brains (N = 214, p = 0.007). DUSP22 methylation showed strong genetic regulation across chromosomes by a region on chromosome 16 which was consistent with new 3D genome interaction data. The presence of a direct link between famine and DUSP22 transcription was supported by data from cultured human fibroblasts that showed increased methylation (p = 0.048) and expression (p = 0.019) in response to nutritional deprivation (N = 10). These results highlight an epigenetic locus that is genetically regulated across chromosomes and that is involved in the response to early-life exposure to famine and that is relevant for a major psychiatric disorder.

Postnatal alterations in dopaminergic markers in the human prefrontal cortex.

Dopamine in the prefrontal cortex plays a critical role in normal cognition throughout the lifespan and has been implicated in the pathophysiology of neuropsychiatric disorders such as schizophrenia and attention deficit disorder. Little is known, however, about the postnatal development of the dopaminergic system in the human prefrontal cortex. In this study, we examined pre- and post-synaptic markers of the dopaminergic system in postmortem tissue specimens from 37 individuals ranging in age from 2 months to 86 years. We measured the levels of tyrosine hydroxylase, the rate limiting enzyme in dopamine biosynthesis, using Western immunoblotting. We also examined the gene expression of the three most abundant dopamine receptors (DARs) in the human prefrontal cortex: DAR1, DAR2 and DAR4, by in situ hybridization. We found that tyrosine hydroxylase concentrations and DAR2 mRNA levels were highest in the cortex of neonates. In contrast, the gene expression of DAR1 was highest in adolescents and young adults. No significant changes across age groups were detected in mRNA levels of DAR4. Both DAR1 and DAR2 mRNA were significantly lower in the aged cortex. Taken together, our data suggest dynamic changes in markers of the dopamine system in the human frontal cortex during postnatal development at both pre-and post-synaptic sites. The peak in DAR1 mRNA levels around adolescence/early adulthood may be of particular relevance to neuropsychiatric disorders such as schizophrenia in which symptoms manifest during the same developmental period.

High BMI levels associate with reduced mRNA expression of IL10 and increased mRNA expression of iNOS (NOS2) in human frontal cortex.

Several studies link increasing body mass index (BMI) to cognitive decline both as a consequence of obesity per se and as a sequela of obesity-induced type 2 diabetes. Obese individuals are prone to a chronic low-grade inflammation as the metabolically active visceral fat produces proinflammatory cytokines. Animal studies indicate that these cytokines can cross the blood-brain barrier. Such crossover could potentially affect the immune system in the brain by inducing gene expression of proinflammatory genes. The relationship between obesity and neuroinflammation in the human brain is currently unknown. Therefore we aim to examine the relationship between BMI and gene expression of central inflammatory markers in the human frontal cortex. Microarray data of 141 neurologically and psychiatrically healthy individuals were obtained through the BrainCloud database. A simple linear regression analysis was performed with BMI as variable on data on IL10, IL1ß, IL6, PTGS2 (COX2) and NOS2 (iNOS). Increasing BMI is associated with a decrease in the mRNA expression of IL10 (P=0.014) and an increase in the expression of NOS2 (iNOS; P=0.040). Expressions of IL10 and NOS2 (iNOS) were negatively correlated (P<0.001). The expression of IL10 was mostly affected by individuals with BMI ⩾40. Multiple linear regression analyses with BMI, age, sex and race as variables were performed in order to identify potential confounders. In conclusion, increasing BMI could affect the IL10-mediated anti-inflammatory defense in the brain and induce iNOS-mediated inflammatory activity.