Postmortem Dorsolateral Prefrontal Cortex Research Articles

Pathological alterations of chondroitin sulfate moiety in postmortem hippocampus of patients with schizophrenia

Perineuronal nets comprise chondroitin sulfate moieties and their core proteins, and their neuropathological alterations have been implicated in schizophrenia. To explore the molecular mechanism of the perineuronal net impairments in schizophrenia, we measured the immunoreactivity of chondroitin sulfate moieties, major components of perineuronal nets, in three brain regions (postmortem dorsolateral prefrontal cortex, caudate nucleus, and hippocampus) of schizophrenia patients and control subjects. Immunoblotting for chondroitin 4-sulfate and chondroitin 6-sulfate moieties revealed a significant increase in intensity of a 180 kD band of chondroitin 4-sulfate immunoreactivity in the hippocampus of patients, although we detected no significant alteration in their immunoreactivities with any other molecular sizes or in other brain regions. The levels of immunoreactivity were not correlated with postmortem interval, age, or storage time. We failed to find such an increase in a similar molecular range of the chondroitin 4-sulfate immunoreactivity in the hippocampus of the rats chronically treated with haloperidol. These results suggest that the level alteration of the chondroitin 4-sulfate moiety might contribute to the perineuronal net abnormality found in patients with schizophrenia.

Expression of dopamine signaling genes in the post-mortem brain of individuals with mental illnesses is moderated by body mass index and mediated by insulin signaling genes

Preclinical studies implicate insulin signaling as a modulator of dopamine transmission, but human data is currently limited. We hypothesize that changes in the expression of insulin receptor-related genes in the post-mortem brain tissue of patients with mood and psychotic disorders mediate the expression of dopamine regulation-related genes. From a database containing microarray data from the post-mortem dorsolateral prefrontal cortex (dlPFC) (healthy controls [HC]: n = 209; patients: n = 321) and hippocampus (HC: n = 180; patients: n = 196), we conducted a hypothesis-driven analysis through the a priori selection of 12 dopamine- and 3 insulin-related genes. Mediation and moderated mediation models, accounting for the role of body mass index (BMI), were used. In the dlPFC, expressions of insulin receptor- and dopamine regulation-related genes were moderated by BMI, with significantly lower expression in high BMI patients. In the hippocampus, there were significantly lower expressions of these genes, which were not moderated by BMI. Illnesses by BMI effects on expression of dopamine genes were fully mediated by expression of insulin receptor gene (INSR). Analysis of conditional indirect effects showed interactions between INSR and BMI, indicating significantly stronger indirect effects at higher BMI values. In the hippocampus we observed that expression of insulin receptor substrate 1 and 2 fully mediated the effects of illnesses on expression of dopamine genes. In conclusion, differential expression of dopamine-related genes was related to altered expression of insulin signaling genes. BMI had region-specific effects, supporting the hypothesis that metabolic systems are critical mediators of dopaminergic function.

An Interleukin-1 beta (IL1B) haplotype linked with psychosis transition is associated with IL1B gene expression and brain structure

We investigated IL1B genetic variation previously associated with risk for transition to psychosis for its association with gene expression in human post-mortem dorsolateral prefrontal cortex (DLPFC) from 74 (37 schizophrenia, 37 control) individuals and brain structure in 92 (44 schizophrenia, 48 control) living individuals. The IL1B A-G-T ‘risk for psychosis transition’ haplotype (rs16944|rs4848306|rs12621220) was associated with upregulation of IL1B mRNA expression in the DLPFC as well as reduced total grey matter and left middle frontal volumes and enlarged left lateral ventricular volume. Our results suggest IL1B genetic variation may confer psychosis risk via elevated mRNA expression and/or brain structure abnormalities.

MGluR5 hypofunction is integral to glutamatergic dysregulation in schizophrenia

Multiple lines of evidence point to glutamatergic signaling in the postsynaptic density (PSD) as a pathophysiologic mechanism in schizophrenia. Integral to PSD glutamatergic signaling is reciprocal interplay between GluN and mGluR5 signaling. We examined agonist-induced mGluR5 signaling in the postmortem dorsolateral prefrontal cortex (DLPFC) derived from 17 patients and age- and sex- matched controls. The patient group showed a striking reduction in mGluR5 signaling, manifested by decreases in Gq/11 coupling and association with PI3K and Homer compared to controls (P<0.01 for all). This was accompanied by increases in serine and tyrosine phosphorylation of mGluR5, which can decrease mGluR5 activity via desensitization (P<0.01). In addition, we find altered protein – protein interaction (PPI) of mGluR5 with RGS4, norbin, Preso 1 and tamalin, which can also attenuate mGluR5 activity. We previously reported molecular underpinnings of GluN hypofunction (decreased GluN2 phosphorylation) and here show those of reduced mGluR5 signaling in schizophrenia. We find that reduced GluN2 phosphorylation can be precipitated by attenuated mGluR5 activity and that increased mGluR5 phosphorylation can result from decreased GluN function, suggesting a reciprocal interplay between the two pathways in schizophrenia. Interestingly, the patient group showed decreased mGluR5 – GluN association (P<0.01), a mechanistic basis for the reciprocal facilitation. In sum, we present the first direct evidence for mGluR5 hypoactivity, propose a reciprocal interplay between GluN and mGluR5 pathways as integral to glutamatergic dysregulation and suggest protein - protein interactions in mGluR5 – GluN complexes as potential targets for intervention in schizophrenia.

Intracellular inflammatory and antioxidant pathways in postmortem frontal cortex of subjects with major depression: effect of antidepressants

BackgroundStudies show that Toll-like receptors (TLRs), members of the innate immune system, might participate in the pathogenesis of the major depressive disorder (MDD). However, evidence of this participation in the brain of patients with MDD has been elusive.MethodsThis work explores whether the protein expression by immunodetection assays (Western blot) of elements of TLR-4 pathways controlling inflammation and the oxidative/nitrosative stress are altered in postmortem dorsolateral prefrontal cortex of subjects with MDD. The potential modulation induced by the antidepressant treatment on these parameters was also assessed. Thirty MDD subjects (15 antidepressant-free and 15 under antidepressant treatment) were matched for gender and age to 30 controls in a paired design.ResultsNo significant changes in TLR-4 expression were detected. An increased expression of the TLR-4 endogenous ligand Hsp70 (+ 33%), but not of Hsp60, and the activated forms of mitogen-activated protein kinases (MAPKs) p38 (+ 47%) and JNK (+ 56%) was observed in MDD. Concomitantly, MDD subjects present a 45% decreased expression of DUSP2 (a regulator of MAPKs) and reduced (− 21%) expression of the antioxidant nuclear factor Nrf2. Antidepressant treatment did not modify the changes detected in the group with MDD and actually increased (+ 25%) the expression of p11, a protein linked with the transport of neurotransmitters and depression.ConclusionData indicate an altered TLR-4 immune response in the brain of subjects with MDD. Additional research focused on the mechanisms contributing to the antidepressant-induced TLR-4 pathway modulation is warranted and could help to develop new treatment strategies for MDD.

Reproductive period and epigenetic modifications of the oxidative phosphorylation pathway in the human prefrontal cortex.

PurposeHuman females have a unique duration of post-reproductive longevity, during which sex-specific mechanisms ma influence later-life mechanisms of neuronal resilience and vulnerability. The maintenance of energy metabolism, through the oxidative phosphorylation (OXPHOS) apparatus, is essential for brain health. Given the known association between reproductive period (years from menarche to menopause) and cognitive aging, we examined the hypothesis that cumulative estrogen exposure across the lifetime may be associated with differential methylation of genes in the OXPHOS pathway.MethodsUsing DNA methylation patterns in the post-mortem dorsolateral prefrontal cortex (DLPFC) of 426 women prospectively followed until death in the Religious Orders Study and Rush Memory and Aging Project, we examined the relationship between reproductive period (subtracting age at menarche from age at menopause) and DNA methylation of a published set of autosomal OXPHOS genes previously implicated in stroke susceptibility. We then performed an unsupervised analysis of methylation levels across the Hallmark pathways from the Molecular Signatures Database.ResultsWe observed a strong association between reproductive period and DNA methylation status across OXPHOS CpGs. We replicated this association between reproductive period and DNA methylation in a much larger set of OXPHOS genes in our unsupervised analysis. Here, reproductive period also showed associations with methylation in genes related to E2F, MYC and MTORC1 signaling, fatty acid metabolism and DNA repair.ConclusionThis study provides evidence from both a supervised and unsupervised analyses, that lifetime cumulative endogenous steroid exposures may play a role in maintenance of post-menopausal cellular balance, including in brain tissue.

12.1 CELL-SUBTYPE SPECIFIC BIOENERGETIC DEFECTS IN SCHIZOPHRENIA

BackgroundNovel insights into the pathophysiology of schizophrenia are needed to move the field forward by providing the conceptual framework to facilitate development of new treatment strategies. It is well established that glutamatergic systems are disrupted in schizophrenia, which are intimately linked to metabolic function. While there are many promising new directions, accumulating evidence suggests that bioenergetic function is impaired in the brain in schizophrenia. There are multiple mechanisms in the brain to meet neuronal energy demands, including glycolysis, lactate uptake, and oxidative phosphorylation. In normal brain, neurons and astrocytes are coupled through the astrocyte-neuron lactate shuttle, where astrocytes metabolize glucose to lactate and pyruvate, primary energy substrates that are transported to neurons via monocarboxylate transporters (MCTs). Lactate generated by glycolysis in glial cells constitutively supports synaptic transmission even under conditions in which a sufficient supply of glucose and intracellular adenosine triphosphate (ATP) are present. Interestingly, working memory and other cognitive domains are dependent on the shuttling of lactate from astrocytes to neurons. This process highlights the bioenergetic coupling between astrocytes and neurons that develops as the brain matures, forming a critical biological process in the mature adult brain. We assessed elements of these systems in postmortem brain, testing the hypothesis that there are cell-subtype defects in bioenergetics function in the frontal cortex in schizophrenia.MethodsWell-validated assays were used to assess the activity of three glycolytic enzymes in postmortem dorsolateral prefrontal cortex (DLPFC) samples (n=16/group): lactate dehydrogenase (LDH), hexokinase (HXK), and phosphofructokinase (PFK). Each sample was assayed with and without a specific inhibitor (in duplicate) and normalized to protein loaded into the assay. We also probed for differences in protein expression using western blot analysis. Western blot analyses were run in duplicate using the following antibodies optimized for postmortem brain: MCT1, LDH, LDHA, LDHB, HXK1, glucose transporter 3 (GLUT3). We performed real time quantitative polymerase chain reaction (RT-qPCR) using TaqMan PCR assays (MCT1, MCT4, HXK1, HXK2, LDHA, LDHB, PFK1, GLUT1, and GLUT3) in duplicate on cDNA samples in 96-well optical plates on a Stratagene MX3000P (Stratagene, La Jolla, California). We also coupled laser capture microdissection (LCM) with RT-qPCR from superficial and deep layers of DLPFC using the Veritas Microdissection instrument and CapSure Macro LCM caps (Life Technologies, formerly Arcturus, Mountain View, CA, USA). Similar studies were performed in haloperidol-decanoate or vehicle (sesame oil) treated rats (intramuscular injection every 3 weeks for 9 months).ResultsWe found a 24% decrease in PFK1 mRNA expression in the dorsolateral prefrontal cortex in schizophrenia (p=0.039). We also found decreases in HXK (26%, p=0.002) and PFK (16%, p<0.001) activity in the dorsolateral prefrontal cortex. These changes were not present in haloperidol treated rats. At the cell-level, in pyramidal neurons we found an increase in MCT1 mRNA expression (22%, p= 0.038), and decreases in HXK1 (19%, p= 0.023), PFK1 (22%, p=0.003), GLUT1 (20%, p=0.008), and GLUT3 (20%, p=0.023) mRNA expression. We found increases in MCT1 (17%, p<0.05) and GLUT3 (20%, p<0.05), but not HXK1, PFK1, or GLUT1, mRNA expression in enriched pyramidal neuron samples of antipsychotic treated rats.DiscussionAs the brain develops, bioenergetic organization and the formation of synapses occur simultaneously, creating a fundamentally interdependent system. There is accumulating evidence of implicating a number of abnormalities associated with glucose metabolism, the lactate shuttle, and bioenergetic coupling in schizophrenia, suggesting energy storage and usage deficits in the brain. Bioenergetic deficits and genetic risk for synaptic dysfunction in schizophrenia could contribute to the pathophysiology of this illness. In normal brain, glucose enters cells through GLUTs and is processed by glycolytic enzymes resulting in bioenergetic substrates such as pyruvate. Pyruvate can then be converted to lactate and transported between cells or intracellularly by MCTs to be oxidized in the TCA cycle when neuronal energy demand is high. Our findings of decreased glycolytic enzyme and lactate transporter mRNA expression suggests a decrease in the capacity of pyramidal neurons to generate bioenergetic substrates from glucose via glycolytic pathways. Additionally, if neurons were unable to take up adequate amounts of glucose for glycolysis, the intracellular pool of available pyruvate/lactate for transport into mitochondria may be diminished, ultimately impacting energy supply. It is also possible that there is attenuated glycolysis in pyramidal neurons, with a shift towards pathways that boost protection from oxidative stress (pentose phosphate pathway). Other studies also report region and cell-subtype specific changes in the expression of genes encoding proteins involved in metabolism in this illness. Importantly, the above changes were not attributable to antipsychotic treatment. Both synaptic function and meeting of energetic demands are essential for cognition, and failure of either could contribute to the cognitive symptoms seen in schizophrenia. Augmenting affected systems such as glucose utilization pathways could offer a novel approach to restoring cognitive function in schizophrenia. This could include targeting pro-metabolic substrates pharmacologically.

Loneliness 5 years ante-mortem is associated with disease-related differential gene expression in postmortem dorsolateral prefrontal cortex

Subjective social isolation, loneliness, is associated with poor mental and physical health, but the underlying molecular mechanisms are poorly understood. Here we analyzed loneliness data collected on average 5 years ante-mortem and RNA gene expression at death in postmortem dorsolateral prefrontal cortex (DLPFC) from 181 participants in the Rush Memory and Aging Project (MAP), a longitudinal, prospective cohort study of common chronic conditions of aging. Our analytic protocol controlled for biographical variables (age, sex, education), psychological and health variables (depressive symptoms, interval between assessment and autopsy, slope of cognitive decline, AD pathology, presence of infarcts) and RNA integrity. Our results are based on a pre-ranked Gene Set Enrichment Analysis (GSEA) at FDR-corrected q-values <0.05, using these collections from the Molecular Signatures Database (v6.0 MSigDB): (1) Hallmarks, (2) Canonical, (3) Gene Ontology (GO), (4) Chemical and Genetic Perturbations, (5) Immunologic Signatures, (6) Oncogenic Signatures, and (7) Cancer Modules. We now report on 337 up-regulated and 43 down-regulated gene sets, among which the most significant ones were associated with Alzheimer’s disease, psychiatric illness, immune dysfunction, and cancer. These gene sets constitute attractive targets for future studies into the molecular mechanisms by which loneliness exacerbates a wide range of neurodegenerative, psychiatric, and somatic illnesses.

Analysis of LINE-1 Elements in DNA from Postmortem Brains of Individuals with Schizophrenia.

Whereas some rare genetic variants convey high risk for schizophrenia (SZ), common alleles conveying even moderate risk remain elusive. Long interspersed element-1s (L1) are mobile retrotransposons comprising ~17% of the human genome. L1 retrotransposition can cause somatic mosaicism during neurodevelopment by insertional mutagenesis. We hypothesized that, compared to controls, patients diagnosed with schizophrenia (PDS) may have increased numbers of deleterious L1 insertions, perhaps occurring de novo, in brain-expressed genes of dorsolateral prefrontal cortex (DLPFC) neurons. Neuronal and non-neuronal nuclei were separated by fluorescence-activated cell sorting from postmortem DLPFC of 36 PDS and 26 age-matched controls. Genomic sequences flanking the 3'-side of L1s were amplified from neuronal DNA, and neuronal L1 libraries were sequenced. Aligned sequences were analyzed for L1 insertions using custom bioinformatics programs. Ontology and pathway analyses were done on lists of genes putatively disrupted by L1s in PDS and controls. Cellular or population allele frequencies of L1s were assessed by droplet digital PCR or Taqman genotyping. We observed a statistically significant increase in the proportion of intragenic novel L1s in DLPFC of PDS. We found over-representation of L1 insertions within the gene ontologies 'cell projection' and 'postsynaptic membrane' in the gene lists derived from PDS samples, but not from controls. Cellular allele frequencies of examined L1 insertions indicated heterozygosity in genomes of DLPFC cells. An L1 within ERI1 exoribonuclease family member 3 (ERI3) was found to associate with SZ. These results extend prior work documenting increased L1 genetic burden in the brains of PDS and also identify unique genes that may provide new insight into the pathophysiology of schizophrenia.

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.

Altered expression of histamine signaling genes in autism spectrum disorder

The histaminergic system (HS) has a critical role in cognition, sleep and other behaviors. Although not well studied in autism spectrum disorder (ASD), the HS is implicated in many neurological disorders, some of which share comorbidity with ASD, including Tourette syndrome (TS). Preliminary studies suggest that antagonism of histamine receptors 1–3 reduces symptoms and specific behaviors in ASD patients and relevant animal models. In addition, the HS mediates neuroinflammation, which may be heightened in ASD. Together, this suggests that the HS may also be altered in ASD. Using RNA sequencing (RNA-seq), we investigated genome-wide expression, as well as a focused gene set analysis of key HS genes (HDC, HNMT, HRH1, HRH2, HRH3 and HRH4) in postmortem dorsolateral prefrontal cortex (DLPFC) initially in 13 subjects with ASD and 39 matched controls. At the genome level, eight transcripts were differentially expressed (false discovery rate <0.05), six of which were small nucleolar RNAs (snoRNAs). There was no significant diagnosis effect on any of the individual HS genes but expression of the gene set of HNMT, HRH1, HRH2 and HRH3 was significantly altered. Curated HS gene sets were also significantly differentially expressed. Differential expression analysis of these gene sets in an independent RNA-seq ASD data set from DLPFC of 47 additional subjects confirmed these findings. Understanding the physiological relevance of an altered HS may suggest new therapeutic options for the treatment of ASD.

Decreased free d-aspartate levels are linked to enhanced d-aspartate oxidase activity in the dorsolateral prefrontal cortex of schizophrenia patients

It is long acknowledged that the N-methyl d-aspartate receptor co-agonist, d-serine, plays a crucial role in several N-methyl d-aspartate receptor-mediated physiological and pathological processes, including schizophrenia. Besides d-serine, another free d-amino acid, d-aspartate, is involved in the activation of N-methyl d-aspartate receptors acting as an agonist of this receptor subclass, and is abundantly detected in the developing human brain. Based on the hypothesis of N-methyl d-aspartate receptor hypofunction in the pathophysiology of schizophrenia and considering the ability of d-aspartate and d-serine to stimulate N-methyl d-aspartate receptor-dependent transmission, in the present work we assessed the concentration of these two d-amino acids in the post-mortem dorsolateral prefrontal cortex and hippocampus of patients with schizophrenia and healthy subjects. Moreover, in this cohort of post-mortem brain samples we investigated the spatiotemporal variations of d-aspartate and d-serine. Consistent with previous work, we found that d-aspartate content was selectively decreased by around 30% in the dorsolateral prefrontal cortex, but not in the hippocampus, of schizophrenia-affected patients, compared to healthy subjects. Interestingly, such selective reduction was associated to greater (around 25%) cortical activity of the enzyme responsible for d-aspartate catabolism, d-aspartate oxidase. Conversely, no significant changes were found in the methylation state and transcription of DDO gene in patients with schizophrenia, compared to control individuals, as well as in the expression levels of serine racemase, the major enzyme responsible for d-serine biosynthesis, which also catalyzes aspartate racemization. These results reveal the potential involvement of altered d-aspartate metabolism in the dorsolateral prefrontal cortex as a factor contributing to dysfunctional N-methyl d-aspartate receptor-mediated transmission in schizophrenia.

A Study of TNF Pathway Activation in Schizophrenia and Bipolar Disorder in Plasma and Brain Tissue.

A proinflammatory imbalance in the tumor necrosis factor (TNF) system may contribute to the pathogenesis of schizophrenia (SCZ) and bipolar disorders (BDs) and related comorbidities. We investigated the relative distribution of TNF-related molecules in blood and dorsolateral prefrontal cortex (DLPFC) in these disorders. We measured plasma levels of TNF, soluble TNF receptor 1 (sTNFR1), soluble TNF receptor 2 (sTNFR2), and a disintegrin and metalloprotease-17 (ADAM17) using enzyme immunoassays and calculated the TNF/sTNFRs ratio (TNF/sTNFR1+sTNFR2) in a sample of 816 SCZ and BD spectrum patients and 624 healthy controls (HCs). TNF, TNFRSF1A (TNFR1), TNFRSF1B (TNFR2), and ADAM17 mRNA levels were determined in whole blood, and postmortem DLPFC obtained from an independent cohort (n = 80 SCZ, n = 44 BD, and n = 86 HC). In peripheral blood, we show increased TNF-related measures in patients compared to HC, with an increased TNF/sTNFRs ratio (p = 6.00 × 10-5), but decreased TNF mRNA expression (p = 1 × 10-4), with no differences between SCZ and BD. Whole blood ADAM17 mRNA expression was markedly higher in BD vs SCZ patients (p = 1.40 × 10-14) and vs HC (p = 1.22 × 10-8). In postmortem DLPFC, we found no significant differences in mRNA expression of TNF pathway genes between any groups. SCZ and BD patients have increased plasma TNF pathway markers without corresponding increase in blood cell gene expression. ADAM17 expression in leukocytes is markedly different between the two disorders, while alterations in TNF-related gene expression in DLPFC are uncertain. Further studies are necessary to elucidate the aberrant regulation of the TNF pathway in severe mental disorders.

Neurobehavioral Phenotype Analysis in Molecular Intervention Models of Mir-137 Expression

Small non-coding microRNA (miRNA) coordinate mRNA translation and stability, and their dysregulation is thought to have significant consequences in psychiatric disorders. This hypothesis is clearly supported by strong genome-wide association of the MIR137 containing locus with schizophrenia. In accordance with previous studies, we show that the common risk variant is associated with reduced expression of the mature miRNA in postmortem dorsolateral prefrontal cortex, whereas no difference was observed on a case-control basis in schizophrenia. To try and understand the functional significance of developmental and acute alteration of this molecule in the brain, we used both chemical and transgenic approaches in the zebrafish. While overexpression produced no noticeable phenotype, miR-137 suppression by morpholino antisense and transgene directed miR-sponge expression, produced a significant sensorimotor phenotype in response to touch stimuli. The absence of anatomical or histological pathology in the nervous system of these animals, suggested that the behavioral phenotype was more likely due to a change in synaptic function and neural connectivity. More recently, we observed a significant alteration of neurobehavioural phenotype relevant to schizophrenia in miR-137 overexpressing lentiviral transformed rats. This research suggests that the risk variant is a miR-eQTL responsible for neurodevelopmentally sensitive haploinsufficiency in carriers that manifest as neurocognitive deficits mediated by dysregulation in several pathways involved with schizophrenia and other psychiatric disorders.

Temporal, Diagnostic, and Tissue-Specific Regulation of NRG3 Isoform Expression in Human Brain Development and Affective Disorders.

Genes implicated in schizophrenia are enriched in networks differentially regulated during human CNS development. Neuregulin 3 (NRG3), a brain-enriched neurotrophin, undergoes alternative splicing and is implicated in several neurological disorders with developmental origins. Isoform-specific increases in NRG3 are observed in schizophrenia and associated with rs10748842, a NRG3 risk polymorphism, suggesting NRG3 transcriptional dysregulation as a molecular mechanism of risk. The authors quantitatively mapped the temporal trajectories of NRG3 isoforms (classes I-IV) in the neocortex throughout the human lifespan, examined whether tissue-specific regulation of NRG3 occurs in humans, and determined if abnormalities in NRG3 transcriptomics occur in mood disorders and are genetically determined. NRG3 isoform classes I-IV were quantified using quantitative real-time polymerase chain reaction in human postmortem dorsolateral prefrontal cortex from 286 nonpsychiatric control individuals, from gestational week 14 to 85 years old, and individuals diagnosed with either bipolar disorder (N=34) or major depressive disorder (N=69). Tissue-specific mapping was investigated in several human tissues. rs10748842 was genotyped in individuals with mood disorders, and association with NRG3 isoform expression examined. NRG3 classes displayed individually specific expression trajectories across human neocortical development and aging; classes I, II, and IV were significantly associated with developmental stage. NRG3 class I was increased in bipolar and major depressive disorder, consistent with observations in schizophrenia. NRG3 class II was increased in bipolar disorder, and class III was increased in major depression. The rs10748842 risk genotype predicted elevated class II and III expression, consistent with previous reports in the brain, with tissue-specific analyses suggesting that classes II and III are brain-specific isoforms of NRG3. Mapping the temporal expression of genes during human brain development provides vital insight into gene function and identifies critical sensitive periods whereby genetic factors may influence risk for psychiatric disease. Here the authors provide comprehensive insight into the transcriptional landscape of the psychiatric risk gene, NRG3, in human neocortical development and expand on previous findings in schizophrenia to identify increased expression of developmentally and genetically regulated isoforms in the brain of patients with mood disorders. Principally, the finding that NRG3 classes II and III are brain-specific isoforms predicted by rs10748842 risk genotype and are increased in mood disorders further implicates a molecular mechanism of psychiatric risk at the NRG3 locus and identifies a potential developmental role for NRG3 in bipolar disorder and major depression. These observations encourage investigation of the neurobiology of NRG3 isoforms and highlight inhibition of NRG3 signaling as a potential target for psychiatric treatment development.

Altered CSNK1E, FABP4 and NEFH protein levels in the dorsolateral prefrontal cortex in schizophrenia

Schizophrenia constitutes a complex disease. Negative and cognitive symptoms are enduring and debilitating components of the disorder, highly associated to disability and burden. Disrupted neurotransmission circuits in dorsolateral prefrontal cortex (DLPFC) have been related to these symptoms. To identify candidates altered in schizophrenia, we performed a pilot proteomic analysis on postmortem human DLPFC tissue from patients with schizophrenia (n=4) and control (n=4) subjects in a pool design using differential isotope peptide labelling followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). We quantified 1315 proteins with two or more unique peptides, 116 of which showed altered changes. Of these altered proteins, we selected four with potential roles on cell signaling, neuronal development and synapse functioning for further validation: casein kinase I isoform epsilon (CSNK1E), fatty acid-binding protein 4 (FABP4), neurofilament triplet H protein (NEFH), and retinal dehydrogenase 1 (ALDH1A1). Immunoblot validation confirmed our proteomic findings of these proteins being decreased in abundance in the schizophrenia samples. Additionally, we conducted immunoblot validation of these candidates on an independent sample cohort comprising 23 patients with chronic schizophrenia and 23 matched controls. In this second cohort, CSNK1E, FABP4 and NEFH were reduced in the schizophrenia group while ALDH1A1 did not significantly change. This study provides evidence indicating these proteins are decreased in schizophrenia: CSNK1E, involved in circadian molecular clock signaling, FABP4 with possible implication in synapse functioning, and NEFH, important for cytoarchitecture organization. Hence, these findings suggest the possible implication of these proteins in the cognitive and/or negative symptoms in schizophrenia.

The glial phosphorylase of glycogen isoform is reduced in the dorsolateral prefrontal cortex in chronic schizophrenia

Reduced glutamatergic activity and energy metabolism in the dorsolateral prefrontal cortex (DLPFC) have been described in schizophrenia. Glycogenolysis in astrocytes is responsible for providing neurons with lactate as a transient energy supply helping to couple glutamatergic neurotransmission and glucose utilization in the brain. This mechanism could be disrupted in schizophrenia. The aim of this study was to explore whether the protein levels of the astrocyte isoform of glycogen phosphorylase (PYGM), key enzyme of glycogenolysis, and the isoform A of Ras-related C3 botulinum toxin substrate 1 (RAC1), a kinase that regulates PYGM activity, are altered in the postmortem DLPFC of chronic schizophrenia patients (n=23) and matched controls (n=23). We also aimed to test NMDAR blockade effect on these proteins in the mouse cortex and cortical astrocytes and antipsychotic treatments in rats. Here we report a reduction in PYGM and RAC1 protein levels in the DLPFC in schizophrenia. We found that treatment with the NMDAR antagonist dizocilpine in mice as a model of psychosis increased PYGM and reduced RAC1 protein levels. The same result was observed in rat cortical astroglial-enriched cultures. 21-day haloperidol treatment increased PYGM levels in rats. These results show that PYGM and RAC1 are altered in the DLPFC in chronic schizophrenia and are controlled by NMDA signalling in the rodent cortex and cortical astrocytes suggesting an altered NMDA-dependent glycogenolysis in astrocytes in schizophrenia. Together, this study provides evidence of a NMDA-dependent transient local energy deficit in neuron-glia crosstalk in schizophrenia, contributing to energy deficits of the disorder.

Expression of the Longest RGS4 Splice Variant in the Prefrontal Cortex Is Associated with Single Nucleotide Polymorphisms in Schizophrenia Patients.

The Regulator of G protein signaling 4 (RGS4) gene is a candidate susceptibility gene for schizophrenia (SCZ). Previous studies showed that the mRNA level of the longest splice variant RGS4-1 was decreased in the dorsolateral prefrontal cortex (DLPFC) of SCZ patients compared with healthy controls. In this pilot study, we examined the possible mechanisms of RGS4-1 mRNA reduction in SCZ. We genotyped SNP1 (rs10917670), rs2661347, SNP4 (rs951436), SNP7 (rs951439), SNP18 (rs2661319), and rs10799897 (SNP9897) and tested the methylation status of CpG islands of the RGS4 gene in the postmortem DLPFC samples obtained from subjects with SCZ and bipolar disorder as well as healthy controls. RGS4-1 mRNA level was associated with five SNPs (SNP1, rs2661347, SNP4, SNP7, and SNP18) and their haplotypes but not with SNP9897. In addition, this study revealed that RGS4-1 mRNA was low in subjects with specific genotypes of SNP1, rs2661347, SNP4, SNP7, and SNP18. Lower RGS4-1 mRNA expression in the DLPFC of SCZ is associated with SNPs in the 5′ regulatory region of the RGS4 gene but not with the methylation status of its CpG islands.

CHRNA7 and CHRFAM7A mRNAs: co-localized and their expression levels altered in the postmortem dorsolateral prefrontal cortex in major psychiatric disorders.

CHRNA7, coding α-7 nicotinic acetylcholine receptor (α7 nAChR), is involved in cognition through interneuron modulation of dopamine and glutamate signaling. CHRNA7 and its partially duplicated chimeric gene CHRFAM7A have been implicated in schizophrenia through linkage and association studies. Expression of CHRNA7 and CHRFAM7A mRNA was measured in the postmortem prefrontal cortex in more than 700 subjects, including patients with schizophrenia, bipolar disorder, major depression, and normal comparison subjects. The effects of antipsychotics and nicotine, as well as associations of CHRNA7 SNPs with gene expression, were explored. Fluorescent in-situ hybridization was used to examine coexpression of both transcripts in the human cortex. CHRFAM7A expression and CHRFAM7A/CHRNA7 ratios were higher in fetal compared with postnatal life, whereas CHRNA7 expression was relatively stable. CHRFAM7A expression was significantly elevated in all diagnostic groups, while CHRNA7 expression was reduced in the schizophrenia group and increased in the major depression group compared with the comparison group. CHRFAM7A/CHRNA7 ratios were significantly increased in the schizophrenia and bipolar disorder groups compared with the comparison group. There was no effect of nicotine or antipsychotics and no association of SNPs in CHRNA7 with expression. CHRNA7 and CHRFAM7A mRNAs were expressed in the same neuronal nuclei of the human neocortex. These data show preferential fetal CHRFAM7A expression in the human prefrontal cortex and suggest abnormalities in the CHRFAM7A/CHRNA7 ratios in schizophrenia and bipolar disorder, due mainly to overexpression of CHRFAM7A. Given that these transcripts are coexpressed in a subset of human cortical neurons and can interact to alter function of nAChRs, these results support the concept of aberrant function of nAChRs in mental illness.

Src kinase as a mediator of convergent molecular abnormalities leading to NMDAR hypoactivity in schizophrenia.

Numerous investigations support decreased glutamatergic signaling as a pathogenic mechanism of schizophrenia: yet molecular underpinnings for such dysregulation are largely unknown. In the postmortem dorsolateral prefrontal cortex, we found striking decreases in tyrosine phosphorylation of N-methyl-D aspartate (NMDA) receptor subunit 2 (GluN2), which is critical for neuroplasticity. The decreased GluN2 activity in schizophrenia may not be due to downregulation of NMDA receptors since MK-801 binding and NMDA receptor complexes in the PSD were in fact increased in schizophrenia cases. At the post-receptor level, however, we found striking reductions in the protein kinase C, Pyk 2 and Src kinase activity, which in tandem can decrease GluN2 activation. Given that Src serves as a hub of various signaling mechanisms impacting GluN2 phosphorylation, we postulated that Src hypoactivity may result from convergent alterations of various schizophrenia susceptibility pathways and thus mediate their impacts on NMDA receptor signaling. Indeed, the DLPFC of schizophrenia cases exhibit increased PSD-95 and erbB4 and decreased RPTPa and dysbindin-1, each of which reduces Src activity via protein interaction with Src. To test genomic underpinnings for Src hypoactivity, we examined genome wide association study results, incorporating 13,394 cases and 34,676 controls, which yielded no significant association of individual variants of Src and its direct regulators with schizophrenia. However, a wider protein-protein interaction based network centered on Src, showed significant enrichment of gene-level associations with schizophrenia compared to other psychiatric illnesses. Our results together demonstrate striking decreases in NMDA receptor signaling at the post-receptor level and propose Src as a nodal point of convergent dysregulations impacting NMDA receptor pathway via protein-protein associations.