Exploring cellular markers of metabolic syndrome in peripheral blood mononuclear cells across the neuropsychiatric spectrum.

Recent evidence suggests that comorbidities between neuropsychiatric conditions and metabolic syndrome may precede and even exacerbate long-term side-effects of psychiatric medication, such as a higher risk of type 2 diabetes and cardiovascular disease, which result in increased mortality. In the present study we compare the expression of key metabolic proteins, including the insulin receptor (CD220), glucose transporter 1 (GLUT1) and fatty acid translocase (CD36), on peripheral blood mononuclear cell subtypes from patients across the neuropsychiatric spectrum, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions (n = 25/condition), relative to typical controls (n = 100). This revealed alterations in the expression of these proteins that were specific to schizophrenia. Further characterization of metabolic alterations in an extended cohort of first-onset antipsychotic drug-naïve schizophrenia patients (n = 58) and controls (n = 63) revealed that the relationship between insulin receptor expression in monocytes and physiological insulin sensitivity was disrupted in schizophrenia and that altered expression of the insulin receptor was associated with whole genome polygenic risk scores for schizophrenia. Finally, longitudinal follow-up of the schizophrenia patients over the course of antipsychotic drug treatment revealed that peripheral metabolic markers predicted changes in psychopathology and the principal side effect of weight gain at clinically relevant time points. These findings suggest that peripheral blood cells can provide an accessible surrogate model for metabolic alterations in schizophrenia and have the potential to stratify subgroups of patients with different clinical outcomes or a greater risk of developing metabolic complications following antipsychotic therapy.

Exploring the neuropsychiatric spectrum using high-content functional analysis of single-cell signaling networks.

Neuropsychiatric disorders overlap in symptoms and share genetic risk factors, challenging their current classification into distinct diagnostic categories. Novel cross-disorder approaches are needed to improve our understanding of the heterogeneous nature of neuropsychiatric diseases and overcome existing bottlenecks in their diagnosis and treatment. Here we employ high-content multi-parameter phospho-specific flow cytometry, fluorescent cell barcoding and automated sample preparation to characterize ex vivo signaling network responses (n = 1764) measured at the single-cell level in B and T lymphocytes across patients diagnosed with four major neuropsychiatric disorders: autism spectrum condition (ASC), bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ; n = 25 each), alongside matched healthy controls (n = 100). We identified 25 nodes (individual cell subtype-epitope-ligand combinations) significantly altered relative to the control group, with variable overlap between different neuropsychiatric diseases and heterogeneously expressed at the level of each individual patient. Reconstruction of the diagnostic categories from the altered nodes revealed an overlapping neuropsychiatric spectrum extending from MDD on one end, through BD and SCZ, to ASC on the other end. Network analysis showed that although the pathway structure of the epitopes was broadly preserved across the clinical groups, there were multiple discrete alterations in network connectivity, such as disconnections within the antigen/integrin receptor pathway and increased negative regulation within the Akt1 pathway in CD4+ T cells from ASC and SCZ patients, in addition to increased correlation of Stat1 (pY701) and Stat5 (pY694) responses in B cells from BD and MDD patients. Our results support the "dimensional" approach to neuropsychiatric disease classification and suggest potential novel drug targets along the neuropsychiatric spectrum.

Drug discovery for psychiatric disorders using high-content single-cell screening of signaling network responses ex vivo.

There is a paucity of efficacious new compounds to treat neuropsychiatric disorders. We present a novel approach to neuropsychiatric drug discovery based on high-content characterization of druggable signaling network responses at the single-cell level in patient-derived lymphocytes ex vivo. Primary T lymphocytes showed functional responses encompassing neuropsychiatric medications and central nervous system ligands at established (e.g., GSK-3ß) and emerging (e.g., CrkL) drug targets. Clinical application of the platform to schizophrenia patients over the course of antipsychotic treatment revealed therapeutic targets within the phospholipase Cγ1-calcium signaling pathway. Compound library screening against the target phenotype identified subsets of L-type calcium channel blockers and corticosteroids as novel therapeutically relevant drug classes with corresponding activity in neuronal cells. The screening results were validated by predicting in vivo efficacy in an independent schizophrenia cohort. The approach has the potential to discern new drug targets and accelerate drug discovery and personalized medicine for neuropsychiatric conditions.

Temporal proteomic profiling of postnatal human cortical development.

Healthy cortical development depends on precise regulation of transcription and translation. However, the dynamics of how proteins are expressed, function and interact across postnatal human cortical development remain poorly understood. We surveyed the proteomic landscape of 69 dorsolateral prefrontal cortex samples across seven stages of postnatal life and integrated these data with paired transcriptome data. We detected 911 proteins by liquid chromatography-mass spectrometry, and 83 were significantly associated with postnatal age (FDR < 5%). Network analysis identified three modules of co-regulated proteins correlated with age, including two modules with increasing expression involved in gliogenesis and NADH metabolism and one neurogenesis-related module with decreasing expression throughout development. Integration with paired transcriptome data revealed that these age-related protein modules overlapped with RNA modules and displayed collinear developmental trajectories. Importantly, RNA expression profiles that are dynamically regulated throughout cortical development display tighter correlations with their respective translated protein expression compared to those RNA profiles that are not. Moreover, the correspondence between RNA and protein expression significantly decreases as a function of cortical aging, especially for genes involved in myelination and cytoskeleton organization. Finally, we used this data resource to elucidate the functional impact of genetic risk loci for intellectual disability, converging on gliogenesis, myelination and ATP-metabolism modules in the proteome and transcriptome. We share all data in an interactive, searchable companion website. Collectively, our findings reveal dynamic aspects of protein regulation and provide new insights into brain development, maturation, and disease.

Variation in serum biomarkers with sex and female hormonal status: implications for clinical tests.

Few serum biomarker tests are implemented in clinical practice and recent reports raise concerns about poor reproducibility of biomarker studies. Here, we investigated the potential role of sex and female hormonal status in this widespread irreproducibility. We examined 171 serum proteins and small molecules measured in 1,676 participants from the Netherlands Study of Depression and Anxiety. Concentrations of 96 molecules varied with sex and 66 molecules varied between oral contraceptive pill users, postmenopausal females, and females in the follicular and luteal phases of the menstrual cycle (FDR-adjusted p-value <0.05). Simulations of biomarker studies yielded up to 40% false discoveries when patient and control groups were not matched for sex and up to 41% false discoveries when premenopausal females were not matched for oral contraceptive pill use. High accuracy (over 90%) classification tools were developed to label samples with sex and female hormonal status where this information was not collected.

Sex Differences in Serum Markers of Major Depressive Disorder in the Netherlands Study of Depression and Anxiety (NESDA).

Women have a consistently higher prevalence of major depressive disorder (MDD) than men. Hypotheses implicating hypothalamic-pituitary -adrenal, -gonadal, and -thyroid axes, immune response, genetic factors, and neurotransmitters have emerged to explain this difference. However, more evidence for these hypotheses is needed and new explanations must be explored. Here, we investigated sex differences in MDD markers using multiplex immunoassay measurements of 171 serum molecules in individuals enrolled in the Netherlands Study of Depression and Anxiety (NMDD = 231; Ncontrol = 365). We found 28 sex-dependent markers of MDD, as quantified by a significant interaction between sex and log2-transformed analyte concentration in a logistic regression with diagnosis (MDD/control) as the outcome variable (p<0.05; q<0.30). Among these were a number of male-specific associations between MDD and elevated levels of proteins involved in immune response, including C-reactive protein, trefoil factor 3, cystatin-C, fetuin-A, ß2-microglobulin, CD5L, FASLG receptor, and tumor necrosis factor receptor 2. Furthermore, only male MDD could be classified with an accuracy greater than chance using the measured serum analytes (area under the ROC curve = 0.63). These findings may have consequences for the generalization of inflammatory hypotheses of depression to males and females and have important implications for the development of diagnostic biomarker tests for MDD. More studies are needed to validate these results, investigate a broader range of biological pathways, and integrate this data with brain imaging, genetic, and other relevant data.

Serum proteomic analysis identifies sex-specific differences in lipid metabolism and inflammation profiles in adults diagnosed with Asperger syndrome.

BACKGROUND: The higher prevalence of Asperger Syndrome (AS) and other autism spectrum conditions in males has been known for many years. However, recent multiplex immunoassay profiling studies have shown that males and females with AS have distinct proteomic changes in serum. METHODS: Here, we analysed sera from adults diagnosed with AS (males = 14, females = 16) and controls (males = 13, females = 16) not on medication at the time of sample collection, using a combination of multiplex immunoassay and shotgun label-free liquid chromatography mass spectrometry (LC-MSE). The main objective was to identify sex-specific serum protein changes associated with AS. RESULTS: Multiplex immunoassay profiling led to identification of 16 proteins that were significantly altered in AS individuals in a sex-specific manner. Three of these proteins were altered in females (ADIPO, IgA, APOA1), seven were changed in males (BMP6, CTGF, ICAM1, IL-12p70, IL-16, TF, TNF-alpha) and six were changed in both sexes but in opposite directions (CHGA, EPO, IL-3, TENA, PAP, SHBG). Shotgun LC-MSE profiling led to identification of 13 serum proteins which had significant sex-specific changes in the AS group and, of these, 12 were altered in females (APOC2, APOE, ARMC3, CLC4K, FETUB, GLCE, MRRP1, PTPA, RN149, TLE1, TRIPB, ZC3HE) and one protein was altered in males (RGPD4). The free androgen index in females with AS showed an increased ratio of 1.63 compared to controls. CONCLUSION: Taken together, the serum multiplex immunoassay and shotgun LC-MSE profiling results indicate that adult females with AS had alterations in proteins involved mostly in lipid transport and metabolism pathways, while adult males with AS showed changes predominantly in inflammation signalling. These results provide further evidence that the search for biomarkers or novel drug targets in AS may require stratification into male and female subgroups, and could lead to the development of novel targeted treatment approaches.

Identification of an age-dependent biomarker signature in children and adolescents with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASDs) are neurodevelopmental conditions with symptoms manifesting before the age of 3, generally persisting throughout life and affecting social development and communication. Here, we have investigated changes in protein biomarkers in blood during childhood and adolescent development. METHODS: We carried out a multiplex immunoassay profiling analysis of serum samples from 37 individuals with a diagnosis of ASD and their matched, non-affected siblings, aged between 4 and 18 years, to identify molecular pathways affected over the course of ASDs. RESULTS: This analysis revealed age-dependent differences in the levels of 12 proteins involved in inflammation, growth and hormonal signaling. CONCLUSIONS: These deviations in age-related molecular trajectories provide further insight into the progression and pathophysiology of the disorder and, if replicated, may contribute to better classification of ASD individuals, as well as to improved treatment and prognosis. The results also underline the importance of stratifying and analyzing samples by age, especially in ASD and potentially other developmental disorders.

Distinct molecular phenotypes in male and female schizophrenia patients.

BACKGROUND: In schizophrenia, sex specific dimorphisms related to age of onset, course of illness and response to antipsychotic treatment may be mirrored by sex-related differences in the underlying molecular pathways. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have carried out multiplex immunoassay profiling of sera from 4 independent cohorts of first episode antipsychotic naive schizophrenia patients (n = 133) and controls (n = 133) to identify such sex-specific illness processes in the periphery. The concentrations of 16 molecules associated with hormonal, inflammation and growth factor pathways showed significant sex differences in schizophrenia patients compared with controls. In female patients, the inflammation-related analytes alpha-1-antitrypsin, B lymphocyte chemoattractant BLC and interleukin-15 showed negative associations with positive and negative syndrome scale (PANSS) scores. In male patients, the hormones prolactin and testosterone were negatively associated with PANSS ratings. In addition, we investigated molecular changes in a subset of 33 patients before and after 6 weeks of treatment with antipsychotics and found that treatment induced sex-specific changes in the levels of testosterone, serum glutamic oxaloacetic transaminase, follicle stimulating hormone, interleukin-13 and macrophage-derived chemokine. Finally, we evaluated overlapping and distinct biomarkers in the sex-specific molecular signatures in schizophrenia, major depressive disorder and bipolar disorder. CONCLUSIONS/SIGNIFICANCE: We propose that future studies should investigate the common and sex-specific aetiologies of schizophrenia, as the current findings suggest that different therapeutic strategies may be required for male and female patients.

Molecular sex differences in human serum.

BACKGROUND: Sex is an important factor in the prevalence, incidence, progression, and response to treatment of many medical conditions, including autoimmune and cardiovascular diseases and psychiatric conditions. Identification of molecular differences between typical males and females can provide a valuable basis for exploring conditions differentially affected by sex. METHODOLOGY/PRINCIPAL FINDINGS: Using multiplexed immunoassays, we analyzed 174 serum molecules in 9 independent cohorts of typical individuals, comprising 196 males and 196 females. Sex differences in analyte levels were quantified using a meta-analysis approach and put into biological context using k-means to generate clusters of analytes with distinct biological functions. Natural sex differences were established in these analyte groups and these were applied to illustrate sexually dimorphic analyte expression in a cohort of 22 males and 22 females with Asperger syndrome. Reproducible sex differences were found in the levels of 77 analytes in serum of typical controls, and these comprised clusters of molecules enriched with distinct biological functions. Analytes involved in fatty acid oxidation/hormone regulation, immune cell growth and activation, and cell death were found at higher levels in females, and analytes involved in immune cell chemotaxis and other indistinct functions were higher in males. Comparison of these naturally occurring sex differences against a cohort of people with Asperger syndrome indicated that a cluster of analytes that had functions related to fatty acid oxidation/hormone regulation was associated with sex and the occurrence of this condition. CONCLUSIONS/SIGNIFICANCE: Sex-specific molecular differences were detected in serum of typical controls and these were reproducible across independent cohorts. This study extends current knowledge of sex differences in biological functions involved in metabolism and immune function. Deviations from typical sex differences were found in a cluster of molecules in Asperger syndrome. These findings illustrate the importance of investigating the influence of sex on medical conditions.