Postmortem Prefrontal Cortex Research Articles

Opposite alterations of 5\xadHT2A receptor brain density in subjects with schizophrenia: relevance of radiotracers pharmacological profile

The status of serotonin 5­HT2A receptors (5­HT2ARs) in schizophrenia has been controversial. In vivo positron emission tomography neuroimaging and in vitro post-mortem binding studies have reported conflicting results about 5­HT2AR density. Radiotracers bind different receptor conformations depending on their agonist, antagonist or inverse agonist properties. This study investigates 5­HT2AR density in the post-mortem prefrontal cortex from subjects with schizophrenia and controls using three radiotracers with a different pharmacological profile. The specific binding parameters of the inverse agonist [18F]altanserin, the agonist [3H]lysergic acid diethylamide (LSD) and the antagonist [3H]MDL100907 to brain cortex membranes from 20 subjects with schizophrenia and 20 individually matched controls were evaluated under similar methodological conditions. Ten schizophrenia subjects were antipsychotic-free at death. Saturation curve analyses were performed by non-linear regression to obtain a maximal density of binding sites (Bmax) and the affinity of the respective radiotracers (Kd). In schizophrenia subjects, 5-HT2AR density was decreased when quantified by [18F]altanserin binding, whereas increased when evaluated by [3H]LSD binding. However, [3H]MDL100907 binding was unaltered. A slight loss of affinity (higher Kd) was observed exclusively in [3H]LSD binding. The findings were more evident in antipsychotic-free subjects than in antipsychotic-treated subjects. In conclusion, a higher proportion of the 5-HT2AR-active functional conformation, which is rather identified by agonist radiotracers, was observed in schizophrenia patients. A consequent reduction of the inactive 5-HT2AR conformation, which is preferentially identified by inverse agonist radiotracers, was also obtained. Antagonist radiotracers do not distinguish between molecular conformations of the receptor, and accordingly, the absence of changes was shown. These results are compatible with the proposed increased functional activity of brain cortical 5-HT2ARs in schizophrenia.

GRK3 deficiency elicits brain immune activation and psychosis

The G protein-coupled receptor kinase (GRK) family member protein GRK3 has been linked to the pathophysiology of schizophrenia and bipolar disorder. Expression, as well as protein levels, of GRK3 are reduced in post-mortem prefrontal cortex of schizophrenia subjects. Here, we investigate functional behavior and neurotransmission related to immune activation and psychosis using mice lacking functional Grk3 and utilizing a variety of methods, including behavioral, biochemical, electrophysiological, molecular, and imaging methods. Compared to wildtype controls, the Grk3−/− mice show a number of aberrations linked to psychosis, including elevated brain levels of IL-1β, increased turnover of kynurenic acid (KYNA), hyper-responsiveness to D-amphetamine, elevated spontaneous firing of midbrain dopamine neurons, and disruption in prepulse inhibition. Analyzing human genetic data, we observe a link between psychotic features in bipolar disorder, decreased GRK expression, and increased concentration of CSF KYNA. Taken together, our data suggest that Grk3−/− mice show face and construct validity relating to the psychosis phenotype with glial activation and would be suitable for translational studies of novel immunomodulatory agents in psychotic disorders.

Sex-specific DNA methylation differences in Alzheimer\u2019s disease pathology

Sex is an important factor that contributes to the clinical and biological heterogeneities in Alzheimer’s disease (AD), but the regulatory mechanisms underlying sex disparity in AD are still not well understood. DNA methylation is an important epigenetic modification that regulates gene transcription and is known to be involved in AD. We performed the first large-scale sex-specific meta-analysis of DNA methylation differences in AD neuropathology, by re-analyzing four recent epigenome-wide association studies totaling more than 1000 postmortem prefrontal cortex brain samples using a uniform analytical pipeline. For each cohort, we employed two complementary analytical strategies, a sex-stratified analysis that examined methylation-Braak stage associations in male and female samples separately, and a sex-by-Braak stage interaction analysis that compared the magnitude of these associations between different sexes. Our analysis uncovered 14 novel CpGs, mapped to genes such as TMEM39A and TNXB that are associated with the AD Braak stage in a sex-specific manner. TMEM39A is known to be involved in inflammation, dysregulated type I interferon responses, and other immune processes. TNXB encodes tenascin proteins, which are extracellular matrix glycoproteins demonstrated to modulate synaptic plasticity in the brain. Moreover, for many previously implicated genes in AD neuropathology, such as MBP and AZU1, our analysis provided the new insights that they were predominately driven by effects in only one sex. These sex-specific DNA methylation differences were enriched in divergent biological processes such as integrin activation in females and complement activation in males. Our study implicated multiple new loci and biological processes that affected AD neuropathology in a sex-specific manner.

Cortical copper transporter expression in schizophrenia: interactions of risk gene dysbindin-1.

Schizophrenia susceptibility factor dysbindin-1 is associated with cognitive processes. Downregulated dysbindin-1 expression is associated with lower expression of copper transporters ATP7A and CTR1, required for copper transport to the central nervous system. We measured dysbindin-1 isoforms-1A and -1BC, CTR1, and ATP7A via Western blots of the postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenia subjects (n = 28) and matched controls (n = 14). In addition, we subdivided the schizophrenia group by treatment status and comorbidity of alcohol use disorder (AUD) and assessed the relationships between proteins. Schizophrenia subjects exhibited similar protein levels to that of controls, with no effect of antipsychotic treatment. We observed a shift towards more dysbindin-1A expression in schizophrenia, as revealed by the ratio of dysbindin-1 isoforms. Dysbindin-1A expression was negatively correlated with ATP7A in schizophrenia, with no correlation present in controls. AUD subjects exhibited less dysbindin-1BC and CTR1 than those without AUD. Our results, taken together with previous data, suggest that alterations in dysbindin-1 and copper transporters are brain-region specific. For example, protein levels of ATP7A, dysbindin 1BC, and CTR1 are lower in the substantia nigra in schizophrenia subjects. AUD in the DLPFC was associated with lower protein levels of dysbindin-1 and CTR1. Changes in dysbindin-1 isoform ratio and relationships appear to be prevalent in the disease, potentially impacting symptomology.

5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated activation of Gαq/11 in postmortem dorsolateral prefrontal cortex of opiate addicts.

Chronic exposure to opiates causes the development of tolerance and physical dependence as well as persistent brain neuroplasticity. Despite a wealth of postmortem human studies for opiate addicts, little direct information regarding the functional status of serotonergic and cholinergic receptor-mediated signaling pathways in the human brain of opiate addicts is yet available. Functional activation of Gαq/11 proteins coupled to 5-HT2A and M1 type muscarinic acetylcholine receptor (mAChR) was assessed by using the method named [35S]GTPγS binding/immunoprecipitation in frontal cortical membrane preparations from postmortem human brains obtained from opiate addicts and matched controls. Concentration-response curves for 5-HT and carbachol in individual subjects were analyzed according to a nonlinear regression model, which generated the values of maximum percent increase (%Emax), negative logarithm of the half-maximal effect (pEC50) and slope factor. As for 5-HT2A receptor-mediated Gαq/11 activation, the %Emax values were reduced significantly and the pEC50 values were decreased significantly in opiate addicts as compared to the control group. Regarding carbachol-induced Gαq/11 activation, no significant difference in %Emax or pEC50 values was detected between the both groups, whereas the slope factor was increased significantly in opiate addicts as compared to the control group. Our data demonstrate that the signaling pathways mediated by Gαq/11 proteins coupled with 5-HT2A receptors and M1 mAChRs in prefrontal cortex are functionally altered in opiate addicts in comparison with control subjects. These alterations may underpin some aspects of addictive behavior to opiate as well as neuropsychological consequences or comorbid mental disorders associated with opioid use.

SGK1 knockdown in the medial prefrontal cortex reduces resistance to stress-induced memory impairment

Down-regulation of serum and glucocorticoid-regulated kinase1 (SGK1) expression has been reported in the postmortem prefrontal cortex (PFC) of subjects with post-traumatic stress disorder. Furthermore, experimental treatments that reduce SGK1 function in the medial prefrontal cortex (mPFC) cause depressive-like behaviors and synaptic dysfunction. Therefore, we examined the effect of SGK1 down-regulation in the mPFC on resistance to stress-induced cognitive impairment. Rats with viral-mediated knockdown of SGK1 in the mPFC were subjected to either a brief 20-min restraint plus 20 intermittent tail shocks or a prolonged 60-min restraint plus 60 intermittent tail shocks, after which their performance in an object recognition task was assessed. Recognition memory remained intact in control rats following the brief stress, but was impaired in rats with SGK1 knockdown in the mPFC. Prolonged stress impaired recognition memory in both control rats and rats with SGK1 knockdown. Our findings indicate that altered mPFC SGK1 signaling is a potential mechanism for resistance to stress-induced cognitive impairment.

Sex Differences in Molecular Rhythms in the Human Cortex

BackgroundDiurnal rhythms in gene expression have been detected in the human brain. Previous studies found that males and females exhibit 24-hour rhythms in known circadian genes, with earlier peak expression in females. Whether there are sex differences in large-scale transcriptional rhythms in the cortex that align with observed sex differences in physiological and behavioral rhythms is currently unknown. MethodsDiurnal rhythmicity of gene expression was determined for males and females using RNA sequencing data from human postmortem dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC). Sex differences among rhythmic genes were determined using significance cutoffs, threshold-free analyses, and R2 difference. Phase concordance was assessed across the DLPFC and ACC for males and females. Pathway and transcription factor analyses were also conducted on significantly rhythmic genes. ResultsCanonical circadian genes had diurnal rhythms in both sexes with similar amplitude and phase. When analyses were expanded to the entire transcriptome, significant sex differences in transcriptional rhythms emerged. There were nearly twice as many rhythmic transcripts in the DLPFC in males and nearly 4 times as many rhythmic transcripts in the ACC in females. Results suggest a diurnal rhythm in synaptic transmission specific to the ACC in females (e.g., GABAergic [gamma-aminobutyric acidergic] and cholinergic neurotransmission). For males, there was phase concordance between the DLPFC and ACC, while phase asynchrony was found in females. ConclusionsThere are robust sex differences in molecular rhythms of genes in the DLPFC and ACC, providing potential mechanistic insights into how neurotransmission and synaptic function are modulated in a circadian-dependent and sex-specific manner.

45698 Molecular Signatures of Cocaine Neurotoxicity in Human Brain Models

ABSTRACT IMPACT: This project will use human neuron models and bioinformatics techniques to elucidate mechanisms of cocaine neurotoxicity, which will allow treatments to be developed for minimizing or preventing neurological damage caused by cocaine abuse and overdose. OBJECTIVES/GOALS: The goals of this project are to identify genes and gene networks altered by cocaine exposure in neurons (short term), and to use these pathways to understand mechanisms of cocaine neurotoxicity for the establishment of therapeutic targets (long term). METHODS/STUDY POPULATION: To study the molecular effects of cocaine, we generated preliminary proteomics and next-generation RNA sequencing (RNAseq) data from human postmortem prefrontal cortex (Broadmann area 9 or BA9) of 12 cocaine overdose subjects and 17 controls. Future directions for this project include RNAseq analysis of neuronal nuclei sorted from human postmortem BA9 and a human induced pluripotent stem cell-derived neuron (hiPSN) model of cocaine exposure from the same postmortem subjects from whom we have brain samples. RESULTS/ANTICIPATED RESULTS: We found alterations in neuronal synaptic protein levels and gene expression, including the serotonin transporter SLC6A4, and synaptic proteins SNAP25, SYN2, SYNGR3. Pathway analysis of our results revealed alterations in specific pathways involved with neuronal function including voltage-gated calcium channels, and GABA receptor signaling. In the future, we expect to see an enhancement in neuron-specific gene expression signatures in our sorted neuronal nuclei and our hiPSN model of cocaine exposure. The hiPSN model will help elucidate which effects are due to acute versus chronic exposure of cocaine. DISCUSSION/SIGNIFICANCE OF FINDINGS: Transcriptomic signatures found with this analysis can help us understand mechanisms of cocaine neurotoxicity in human neurons. With this work and future proposed studies, we can discover targetable molecular pathways to develop drugs that can reduce or reverse cocaine-related impairment.

Identification of major depressive disorder disease-related genes and functional pathways based on system dynamic changes of network connectivity

BackgroundMajor depressive disorder (MDD) is a leading psychiatric disorder that involves complex abnormal biological functions and neural networks. This study aimed to compare the changes in the network connectivity of different brain tissues under different pathological conditions, analyzed the biological pathways and genes that are significantly related to disease progression, and further predicted the potential therapeutic drug targets.MethodsExpression of differentially expressed genes (DEGs) were analyzed with postmortem cingulate cortex (ACC) and prefrontal cortex (PFC) mRNA expression profile datasets downloaded from the Gene Expression Omnibus (GEO) database, including 76 MDD patients and 76 healthy subjects in ACC and 63 MDD patients and 63 healthy subjects in PFC. The co-expression network construction was based on system network analysis. The function of the genes was annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Human Protein Reference Database (HPRD, http://www.hprd.org/) was used for gene interaction relationship mapping.ResultsWe filtered 586 DEGs in ACC and 616 DEGs in PFC for further analysis. By constructing the co-expression network, we found that the gene connectivity was significantly reduced under disease conditions (P = 0.04 in PFC and P = 1.227e−09 in ACC). Crosstalk analysis showed that CD19, PTDSS2 and NDST2 were significantly differentially expressed in ACC and PFC of MDD patients. Among them, CD19 and PTDSS2 have been targeted by several drugs in the Drugbank database. KEGG pathway analysis demonstrated that the function of CD19 and PTDSS2 were enriched with the pathway of Glycerophospholipid metabolism and T cell receptor signaling pathway.ConclusionCo-expression network and tissue comparing analysis can identify signaling pathways and cross talk genes related to MDD, which may provide novel insight for understanding the molecular mechanisms of MDD.

Standardised ginseng extract G115® potentiates the antidepressant-like properties of fluoxetine in the forced swim test.

Ginsenosides, biologically active components of the root of Panax ginseng, have been reported to have therapeutic benefits in a number of disease states including psychiatric conditions such as major depressive disorder. Our objective was to determine if a standardised commercial ginseng extract, G115®, could reduce the signs of behavioural despair commonly observed in animal models of depression either alone or in combination with the selective serotonin reuptake inhibitor (SSRI) fluoxetine. Male Sprague-Dawley (SD) rats (N = 51) were divided into four groups: vehicle control, G115® ginseng root extract, fluoxetine and fluoxetine plus G115®. Rats were trained to voluntarily consume treatments twice daily for 14 days and were then tested in an open field (OF), elevated plus maze (EPM) and forced swim test (FST). Post-mortem hippocampal and prefrontal cortex tissue was analysed for expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) by western blot. One-way Analysis of Variance revealed no significant group differences in the OF or plus-maze performance on any variable examined. In the FST, fluoxetine significantly reduced immobility time and increased latency to immobility. The effects of fluoxetine were further significantly potentiated by co-administration of G115®. Post-mortem tissue analysis revealed significant group differences in BDNF expression in the left hippocampus and left prefrontal cortex without any accompanying changes in TrkB expression. We conclude that oral G115® significantly potentiates the antidepressant-like effect of fluoxetine in the FST in the absence of potentially confounding effects on locomotion and anxiety.

Characteristics of Epigenetic Clocks Across Blood and Brain Tissue in Older Women and Men.

Epigenetic clocks are among the most promising biomarkers of aging. It is particularly important to establish biomarkers of brain aging to better understand neurodegenerative diseases. To advance application of epigenetic clocks—which were largely created with DNA methylation levels in blood samples—for use in brain, we need clearer evaluation of epigenetic clock behavior in brain, including direct comparisons of brain specimens with blood, a more accessible tissue for research. We leveraged data from the Religious Orders Study and Rush Memory and Aging Project to examine three established epigenetic clocks (Horvath, Hannum, PhenoAge clocks) and a newer clock, trained in cortical tissue. We calculated each clock in three different specimens: (1) antemortem CD4+ cells derived from blood (n = 41); (2) postmortem dorsolateral prefrontal cortex (DLPFC, n = 730); and (3) postmortem posterior cingulate cortex (PCC, n = 186), among older women and men, age 66–108 years at death. Across all clocks, epigenetic age calculated from blood and brain specimens was generally lower than chronologic age, although differences were smallest for the Cortical clock when calculated in the brain specimens. Nonetheless, we found that Pearson correlations of epigenetic to chronologic ages in brain specimens were generally reasonable for all clocks; correlations for the Horvath, Hannum, and PhenoAge clocks largely ranged from 0.5 to 0.7 (all p < 0.0001). The Cortical clock outperformed the other clocks, reaching a correlation of 0.83 in the DLFPC (p < 0.0001) for epigenetic vs. chronologic age. Nonetheless, epigenetic age was quite modestly correlated across blood and DLPFC in 41 participants with paired samples [Pearson r from 0.21 (p = 0.2) to 0.32 (p = 0.05)], indicating that broader research in neurodegeneration may benefit from clocks using CpG sites better conserved across blood and brain. Finally, in analyses stratified by sex, by pathologic diagnosis of Alzheimer disease, and by clinical diagnosis of Alzheimer dementia, correlations of epigenetic to chronologic age remained consistently high across all groups. Future research in brain aging will benefit from epigenetic clocks constructed in brain specimens, including exploration of any advantages of focusing on CpG sites conserved across brain and other tissue types.

Trajectory of change in brain complement factors from neonatal to young adult humans.

Immune system components also regulate synapse formation and refinement in neurodevelopment. The complement pathway, associated with cell lysis and phagocytosis, is implicated in synaptic elimination. Aberrant adolescent synaptic pruning may underpin schizophrenia onset; thus, changes in cortical complement activity during human development are of major interest. Complement is genetically linked to schizophrenia via increased C4 copy number variants, but the developmental trajectory of complement expression in the human brain is undetermined. As complement increases during periods of active synaptic engulfment in rodents, we hypothesized that complement expression would increase during postnatal development in humans, particularly during adolescence. Using human postmortem prefrontal cortex, we observed that complement activator (C1QB and C3) transcripts peaked in early neurodevelopment, and were highest in toddlers, declining in teenagers (all ANCOVAs between F=2.41 -3.325, p=.01-0.05). We found that C4 protein was higher at 1-5years (H=16.378, p=.012), whereas C3 protein levels were unchanged with age. The microglial complement receptor subunit CD11b increased in mRNA early in life and peaked in the toddler brain (ANCOVA: pH, F=4.186, p=.003). Complement inhibitors (CD46 and CD55) increased at school age, but failed to decrease like complement activators (both ANCOVAs, F>4.4, p<.01). These data suggest the activation of complement in the human prefrontal cortex occurs between 1 and 5years. We did not find evidence of induction of complement factors during adolescence and instead found increased or sustained levels of complement inhibitor mRNA at maturation. Dysregulation of these typical patterns of complement may predispose the brain to neurodevelopmental disorders such as autism or schizophrenia.

SVA insertion in X-linked Dystonia Parkinsonism alters histone H3 acetylation associated with TAF1 gene.

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.

Spinophilin expression in postmortem prefrontal cortex of schizophrenic subjects: Effects of antipsychotic treatment

Schizophrenia has been associated with alterations in neurotransmission and synaptic dysfunction. Spinophilin is a multifunctional scaffold protein that modulates excitatory synaptic transmission and dendritic spine morphology. Spinophilin can also directly interact with and regulate several receptors for neurotransmitters, such as dopamine D2 receptors, which play a role in the pathophysiology of schizophrenia and are targets of antipsychotics. Several studies have thus suggested an implication of spinophilin in schizophrenia. In the present study spinophilin protein expression was determined by western blot in the postmortem dorsolateral prefrontal cortex of 24 subjects with schizophrenia (12 antipsychotic-free and 12 antipsychotic-treated subjects) and 24 matched controls. Experiments were performed in synaptosomal membranes (SPM) and in postsynaptic density fractions (PSD). As previously reported, two specific bands for this protein were observed: an upper 120–130 kDa band and a lower 80–95 kDa band. The spinophilin lower band showed a significant decrease in schizophrenia subjects compared to matched controls, both in SPM and PSD fractions (-15%, p = 0.007 and -15%, p = 0.039, respectively). When schizophrenia subjects were divided by the presence or absence of antipsychotics in blood at death, the lower band showed a significant decrease in antipsychotic-treated schizophrenia subjects (-24%, p = 0.003 for SPM and -26%, p = 0.014 for PSD), but not in antipsychotic-free subjects, compared to their matched controls. These results suggest that antipsychotics could produce alterations in spinophilin expression that do not seem to be related to schizophrenia per se. These changes may underlie some of the side effects of antipsychotics.

5-HT2A receptor-mediated Gαq/11 activation in psychiatric disorders: A postmortem study

Objectives Serotonin-2A (5-HT2A) receptors play an important role in the regulation of many brain functions that are disturbed in patients with such psychiatric diseases as mood disorders and schizophrenia. The objective of this study was to evaluate 5-HT2A receptor-mediated signalling pathway through Gαq/11 activation in psychiatric patients by using post-mortem brain samples. Methods Functional activation of Gαq/11 proteins coupled to 5-HT2A receptors was determined by means of [35S]GTPγS binding/immunoprecipitation assay in post-mortem prefrontal cortex of psychiatric patients diagnosed as bipolar disorder (BP), major depressive disorder (MDD), and schizophrenia, and individually matched controls. The effects of antipsychotic treatment as well as suicide were also analysed. Results There was no significant difference in maximum percent increase (%Emax) or slope factor among the four groups. The negative logarithm of concentration eliciting the half-maximal effect (pEC50) was significantly reduced in BP and schizophrenia patients as compared to controls. These alterations were attributable to antipsychotic medication. The pEC50 values in ‘non-suicide’ group of schizophrenia, but not in ‘suicide’ group, were significantly reduced as compared with controls. Conclusions Altered 5-HT2A receptor-mediated signalling pathway through Gαq/11 proteins in prefrontal cortex might be apparently involved in pathophysiology and pharmacotherapy of BP and schizophrenia. In schizophrenic patients, these alterations as a result of successful treatment with antipsychotic agents may help in prevention of suicidal behaviour.

P.589 Alteration of G-alpha protein subtypes expression in postmortem prefrontal cortex of subjects with schizophrenia

Infections often precede the onset of autoimmune disease and molecular (or epitope) mimicry is a plausible link. Cross-reacting epitopes are common between an infecting microorganism and the host because negative selection of self-reactive T-cells and B-cells is frequently incomplete. Complete eradication could lead to major voids in the immunologic repertoire. The association of an autoimmune disease with a microbial epitope may signify a causal relationship with the organism, an indirect connection through bystander effects, persistent infection or coincidence. There are well-established examples of a microbial mimic inducing a defined model of autoimmune disease in experimental animals but such instances are still relatively rare in humans. Establishing epitope mimicry as a direct cause opens opportunities for preventing the disease. Current approaches to cancer immunotherapy provides new examples of epitope mimicry between cancer antigens and normal tissue antigens.

Identifying Plasma Biomarkers with high specificity for major depressive disorder: A multi-level proteomics study

BackgroundThere are currently no objective diagnostic biomarkers for major depressive disorder (MDD) due to the biological complexity of the disorder. The existence of blood-based biomarkers with high specificity would be convenient for the clinical diagnosis of MDD. MethodsA comprehensive plasma proteomic analysis was conducted in a highly homogeneous cohort [7 drug-naïve MDD patients and 7 healthy controls (HCs)], with bioinformatics analysis combined with machine learning used to screen candidate proteins. Verification of reproducibility and specificity was conducted in independent cohorts [60 HCs and 74 MDD, 42 schizophrenia (SZ) and 39 bipolar I disorder (BD-I) drug-naïve patients]. Furthermore, verification of consistency was accomplished by proteomic analysis of postmortem brain tissue from 16 MDD patients and 16 HCs. Results: Levels of C-reactive protein (CRP), antithrombin III (ATIII), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and vitamin D-binding protein (VDB) were significantly higher in MDD patients, both in the discovery cohort and independent replication cohort. In comparison with SZ or BD-I patients, two proteins (VDB and ITIH4) were significantly elevated only in MDD patients. In addition, increased VDB and ITIH4 were observed consistently in both plasma and postmortem dorsolateral prefrontal cortex tissues of MDD patients. Furthermore, a panel consisting of all four plasma proteins was able to distinguish MDD patients from HCs or SZ or BD-I patients with the highest accuracy. ConclusionPlasma ITIH4 and VDB may be potential plasma biomarkers of MDD with high specificity. The four-protein panel is more suitable as a potential clinical diagnostic marker for MDD.

Key role of the 5-HT1A receptor addressing protein Yif1B in serotonin neurotransmission and SSRI treatment

Altered function of serotonin receptor 1A (5-HT1AR) has been consistently implicated in anxiety, major depressive disorder and resistance to antidepressants. Mechanisms by which the function of 5-HT1AR (expressed as an autoreceptor in serotonergic raphe neurons and as a heteroreceptor in serotonin [5-HT] projection areas) is altered include regulation of its expression, but 5-HT1AR trafficking may also be involved. We investigated the consequences of the lack of Yif1B (the 5-HT1AR trafficking protein) on 5-HT neurotransmission in mice, and whether Yif1B expression might be affected under conditions known to alter 5-HT neurotransmission, such as anxious or depressive states or following treatment with fluoxetine (a selective serotonin reuptake inhibitor) in humans, monkeys and mice. Compared with wild-type mice, Yif1B-knockout mice showed a significant decrease in the forebrain density of 5-HT projection fibres and a hypofunctionality of 5-HT1A autoreceptors expressed on raphe 5-HT neurons. In addition, social interaction was less in Yif1B-knockout mice, which did not respond to the antidepressant-like effect of acute fluoxetine injection. In wild-type mice, social defeat was associated with downregulated Yif1B mRNA in the prefrontal cortex, and chronic fluoxetine treatment increased Yif1B expression. The expression of Yif1B was also downregulated in the postmortem prefrontal cortex of people with major depressive disorder and upregulated after chronic treatment with a selective serotonin reuptake inhibitor in monkeys. We found sex differences in Yif1B expression in humans and monkeys, but not in mice under the tested conditions. These data support the concept that Yif1B plays a critical role in 5-HT1AR functioning and brain 5-HT homeostasis. The opposite changes in its expression observed in anxious or depressive states and after therapeutic fluoxetine treatment suggest that Yif1B might be involved in vulnerability to anxiety and depression, and fluoxetine efficacy.

Neuroinflammation and histone H3 citrullination are increased in X-linked Dystonia Parkinsonism post-mortem prefrontal cortex

Neuroinflammation plays a pathogenic role in neurodegenerative diseases and recent findings suggest that it may also be involved in X-linked Dystonia-Parkinsonism (XDP) pathogenesis. Previously, fibroblasts and neuronal stem cells derived from XDP patients demonstrated hypersensitivity to TNF-α, dysregulation in NFκB signaling, and an increase in several pro-inflammatory markers. However, the role of inflammatory processes in XDP patient brain remains unknown. Here we demonstrate that there is a significant increase in astrogliosis and microgliosis in human post-mortem XDP prefrontal cortex (PFC) compared to control. Furthermore, there is a significant increase in histone H3 citrullination (H3R2R8R17cit3) with a concomitant increase in peptidylarginine deaminase 2 (PAD2) and 4 (PAD4), the enzymes catalyzing citrullination, in XDP post-mortem PFC. While there is a significant increase in myeloperoxidase (MPO) levels in XDP PFC, neutrophil elastase (NE) levels are not altered, suggesting that MPO may be released by activated microglia or reactive astrocytes in the brain. Similarly, there was an increase in H3R2R8R17cit3, PAD2 and PAD4 levels in XDP-derived fibroblasts. Importantly, treatment of fibroblasts with Cl-amidine, a pan inhibitor of PAD enzymes, reduced histone H3 citrullination and pro-inflammatory chemokine expression, without affecting cell survival. Taken together, our results demonstrate that inflammation is increased in XDP post-mortem brain and fibroblasts and unveil a new epigenetic potential therapeutic target.

FOXP2 expression and gray matter density in the male brains of patients with schizophrenia

Common genetic variants of FOXP2 may contribute to schizophrenia vulnerability, but controversial results have been reported for this proposal. Here we evaluated the potential impact of the common FOXP2 rs2396753 polymorphism in schizophrenia. It was previously reported to be part of a risk haplotype for this disease and to have significant effects on gray matter concentration in the patients. We undertook the first examination into whether rs2396753 affects the brain expression of FOXP2 and a replication study of earlier neuroimaging findings of the influence of this genetic variant on brain structure. FOXP2 expression levels were measured in postmortem prefrontal cortex samples of 84 male subjects (48 patients and 36 controls) from the CIBERSAM Brain and the Stanley Foundation Array Collections. High-resolution anatomical magnetic resonance imaging was performed on 79 male subjects (61 patients, 18 controls) using optimized voxel-based morphometry. We found differences in FOXP2 expression and brain morphometry depending on the rs2396753, relating low FOXP2 mRNA levels with reduction of gray matter density. We detected an interaction between rs2396753 and the clinical groups, showing that heterozygous patients for this polymorphism have gray matter density decrease and low FOXP2 expression comparing with the heterozygous controls. This study shows the importance of independent replication of neuroimaging genetic studies of FOXP2 as a candidate gene in schizophrenia. Furthermore, our results suggest that the FOXP2 rs2396753 affects mRNA levels, thus providing new knowledge about its significance as a potential susceptibility polymorphism in schizophrenia.