The underappreciated diversity of bile acid modifications.

The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.

Prefrontal Cortex Cytosolic Proteome and Machine Learning-Based Predictors of Resilience toward Chronic Social Isolation in Rats.

Chronic social isolation (CSIS) generates two stress-related phenotypes: resilience and susceptibility. However, the molecular mechanisms underlying CSIS resilience remain unclear. We identified altered proteome components and biochemical pathways and processes in the prefrontal cortex cytosolic fraction in CSIS-resilient rats compared to CSIS-susceptible and control rats using liquid chromatography coupled with tandem mass spectrometry followed by label-free quantification and STRING bioinformatics. A sucrose preference test was performed to distinguish rat phenotypes. Potential predictive proteins discriminating between the CSIS-resilient and CSIS-susceptible groups were identified using machine learning (ML) algorithms: support vector machine-based sequential feature selection and random forest-based feature importance scores. Predominantly, decreased levels of some glycolytic enzymes, G protein-coupled receptor proteins, the Ras subfamily of GTPases proteins, and antioxidant proteins were found in the CSIS-resilient vs. CSIS-susceptible groups. Altered levels of Gapdh, microtubular, cytoskeletal, and calcium-binding proteins were identified between the two phenotypes. Increased levels of proteins involved in GABA synthesis, the proteasome system, nitrogen metabolism, and chaperone-mediated protein folding were identified. Predictive proteins make CSIS-resilient vs. CSIS-susceptible groups linearly separable, whereby a 100% validation accuracy was achieved by ML models. The overall ratio of significantly up- and downregulated cytosolic proteins suggests adaptive cellular alterations as part of the stress-coping process specific for the CSIS-resilient phenotype.

Antidepressant treatment effects on hippocampal protein turnover: Molecular and spatial insights from mass spectrometry.

A major challenge in managing depression is that antidepressant drugs take a long time to exert their therapeutic effects. For the development of faster-acting treatments, it is crucial to get an improved understanding of the molecular mechanisms underlying antidepressant mode of action. Here, we used a novel mass spectrometry-based workflow to investigate how antidepressant treatment affects brain protein turnover at single-cell and subcellular resolution. We combined stable isotope metabolic labeling, quantitative Tandem Mass Spectrometry (qTMS) and Multi-isotope Imaging Mass Spectrometry (MIMS) to simultaneously quantify and image protein synthesis and turnover in hippocampi of mice treated with the antidepressant paroxetine. We identified changes in turnover of individual hippocampal proteins that reveal altered metabolism-mitochondrial processes and report on subregion-specific turnover patterns upon paroxetine treatment. This workflow can be used to investigate brain protein turnover changes in vivo upon pharmacological interventions at a resolution and precision that has not been possible with other methods to date. Our results reveal acute paroxetine effects on brain protein turnover and shed light on antidepressant mode of action.

Multi-omics analysis identifies mitochondrial pathways associated with anxiety-related behavior.

Stressful life events are major environmental risk factors for anxiety disorders, although not all individuals exposed to stress develop clinical anxiety. The molecular mechanisms underlying the influence of environmental effects on anxiety are largely unknown. To identify biological pathways mediating stress-related anxiety and resilience to it, we used the chronic social defeat stress (CSDS) paradigm in male mice of two inbred strains, C57BL/6NCrl (B6) and DBA/2NCrl (D2), that differ in their susceptibility to stress. Using a multi-omics approach, we identified differential mRNA, miRNA and protein expression changes in the bed nucleus of the stria terminalis (BNST) and blood cells after chronic stress. Integrative gene set enrichment analysis revealed enrichment of mitochondrial-related genes in the BNST and blood of stressed mice. To translate these results to human anxiety, we investigated blood gene expression changes associated with exposure-induced panic attacks. Remarkably, we found reduced expression of mitochondrial-related genes in D2 stress-susceptible mice and in exposure-induced panic attacks in humans, but increased expression of these genes in B6 stress-susceptible mice. Moreover, stress-susceptible vs. stress-resilient B6 mice displayed more mitochondrial cross-sections in the post-synaptic compartment after CSDS. Our findings demonstrate mitochondrial-related alterations in gene expression as an evolutionarily conserved response in stress-related behaviors and validate the use of cross-species approaches in investigating the biological mechanisms underlying anxiety disorders.

Long-Term Fluoxetine Administration Causes Substantial Lipidome Alteration of the Juvenile Macaque Brain.

Fluoxetine is an antidepressant commonly prescribed not only to adults but also to children for the treatment of depression, obsessive-compulsive disorder, and neurodevelopmental disorders. The adverse effects of the long-term treatment reported in some patients, especially in younger individuals, call for a detailed investigation of molecular alterations induced by fluoxetine treatment. Two-year fluoxetine administration to juvenile macaques revealed effects on impulsivity, sleep, social interaction, and peripheral metabolites. Here, we built upon this work by assessing residual effects of fluoxetine administration on the expression of genes and abundance of lipids and polar metabolites in the prelimbic cortex of 10 treated and 11 control macaques representing two monoamine oxidase A (MAOA) genotypes. Analysis of 8871 mRNA transcripts, 3608 lipids, and 1829 polar metabolites revealed substantial alterations of the brain lipid content, including significant abundance changes of 106 lipid features, accompanied by subtle changes in gene expression. Lipid alterations in the drug-treated animals were most evident for polyunsaturated fatty acids (PUFAs). A decrease in PUFAs levels was observed in all quantified lipid classes excluding sphingolipids, which do not usually contain PUFAs, suggesting systemic changes in fatty acid metabolism. Furthermore, the residual effect of the drug on lipid abundances was more pronounced in macaques carrying the MAOA-L genotype, mirroring reported behavioral effects of the treatment. We speculate that a decrease in PUFAs may be associated with adverse effects in depressive patients and could potentially account for the variation in individual response to fluoxetine in young people.

Interactome Studies of Psychiatric Disorders.

High comorbidity and complexity have precluded reliable diagnostic assessment and treatment of psychiatric disorders. Impaired molecular interactions may be relevant for underlying mechanisms of psychiatric disorders but by and large remain unknown. With the help of a number of publicly available databases and various technological tools, recent research has filled the paucity of information by generating a novel dataset of psychiatric interactomes. Different technological platforms including yeast two-hybrid screen, co-immunoprecipitation-coupled with mass spectrometry-based proteomics, and transcriptomics have been widely used in combination with cellular and molecular techniques to interrogate the psychiatric interactome. Novel molecular interactions have been identified in association with different psychiatric disorders including autism spectrum disorders, schizophrenia, bipolar disorder, and major depressive disorder. However, more extensive and sophisticated interactome research needs to be conducted to overcome the current limitations such as incomplete interactome databases and a lack of functional information among components. Ultimately, integrated psychiatric interactome databases will contribute to the implementation of biomarkers and therapeutic intervention.

FKBP5 Gene Expression Predicts Antidepressant Treatment Outcome in Depression.

Adverse experiences and chronic stress are well-known risk factors for the development of major depression, and an impaired stress response regulation is frequently observed in acute depression. Impaired glucocorticoid receptor (GR) signalling plays an important role in these alterations, and a restoration of GR signalling appears to be a prerequisite of successful antidepressant treatment. Variants in genes of the stress response regulation contribute to the vulnerability to depression in traumatized subjects. Consistent findings point to an important role of FKBP5, the gene expressing FK506-binding protein 51 (FKBP51), which is a strong inhibitor of the GR, and thus, an important regulator of the stress response. We investigated the role of FKBP5 and FKB51 expression with respect to stress response regulation and antidepressant treatment outcome in depressed patients. This study included 297 inpatients, who participated in the Munich Antidepressant Response Signature (MARS) project and were treated for acute depression. In this open-label study, patients received antidepressant treatment according to the attending doctor's choice. In addition to the FKBP5 genotype, changes in blood FKBP51 expression during antidepressant treatment were analyzed using RT-PCR and ZeptoMARKTM reverse phase protein microarray (RPPM). Stress response regulation was evaluated in a subgroup of patients using the combined dexamethasone (dex)/corticotropin releasing hormone (CRH) test. As expected, increased FKBP51 expression was associated with an impaired stress response regulation at baseline and after six weeks was accompanied by an elevated cortisol response to the combined dex/CRH test. Further, we demonstrated an active involvement of FKBP51 in antidepressant treatment outcome. While patients responding to antidepressant treatment had a pronounced reduction of FKBP5 gene and FKBP51 protein expression, increasing expression levels were observed in nonresponders. This effect was moderated by the genotype of the FKBP5 single nucleotide polymorphism (SNP) rs1360780, with carriers of the minor allele showing the most pronounced association. Our findings demonstrate that FKBP5 and, specifically, its expression product FKBP51 are important modulators of antidepressant treatment outcome, pointing to a new, promising target for future antidepressant drug development.

Partially 13C-labeled mouse tissue as reference for LC-MS based untargeted metabolomics.

The inclusion of stable isotope-labeled reference standards in the sample is an established method for the detection and relative quantification of metabolic features in untargeted metabolomics. In order to quantify as many metabolites as possible, the reference should ideally include the same metabolites in their stable isotope-labeled form as the sample under investigation. We present here an attempt to use partially 13C-labeled mouse material as internal standard for relative metabolite quantification of mouse and human samples in untargeted metabolomics. We fed mice for 14 days with a13C-labeled Ralstonia eutropha based diet. Tissue and blood amino acids from these mice showed 13C enrichment levels that ranged from 6% to 75%. We used MetExtract II software to automatically detect native and labeled peak pairs in an untargeted manner. In a dilution series and with the implementation of a correction factor, partially 13C-labeled mouse plasma resulted in accurate relative quantification of human plasma amino acids using liquid chromatography coupled to mass spectrometry, The coefficient of variation for the relative quantification is reduced from 27% without internal standard to 10% with inclusion of partially 13C-labeled internal standard. We anticipate the method to be of general use for the relative metabolite quantification of human specimens.

Tau Deletion Prevents Stress-Induced Dendritic Atrophy in Prefrontal Cortex: Role of Synaptic Mitochondria.

Tau protein in dendrites and synapses has been recently implicated in synaptic degeneration and neuronal malfunction. Chronic stress, a well-known inducer of neuronal/synaptic atrophy, triggers hyperphosphorylation of Tau protein and cognitive deficits. However, the cause-effect relationship between these events remains to be established. To test the involvement of Tau in stress-induced impairments of cognition, we investigated the impact of stress on cognitive behavior, neuronal structure, and the synaptic proteome in the prefrontal cortex (PFC) of Tau knock-out (Tau-KO) and wild-type (WT) mice. Whereas exposure to chronic stress resulted in atrophy of apical dendrites and spine loss in PFC neurons as well as significant impairments in working memory in WT mice, such changes were absent in Tau-KO animals. Quantitative proteomic analysis of PFC synaptosomal fractions, combined with transmission electron microscopy analysis, suggested a prominent role for mitochondria in the regulation of the effects of stress. Specifically, chronically stressed animals exhibit Tau-dependent alterations in the levels of proteins involved in mitochondrial transport and oxidative phosphorylation as well as in the synaptic localization of mitochondria in PFC. These findings provide evidence for a causal role of Tau in mediating stress-elicited neuronal atrophy and cognitive impairment and indicate that Tau may exert its effects through synaptic mitochondria.

Biomarkers for Psychiatry: The Journey from Fantasy to Fact, a Report of the 2013 CINP Think Tank.

BACKGROUND: A think tank sponsored by the Collegium Internationale Neuropsychopharmacologium (CINP) debated the status and prospects of biological markers for psychiatric disorders, focusing on schizophrenia and major depressive disorder. METHODS: Discussions covered markers defining and predicting specific disorders or domains of dysfunction, as well as predicting and monitoring medication efficacy. Deliberations included clinically useful and viable biomarkers, why suitable markers are not available, and the need for tightly-controlled sample collection. RESULTS: Different types of biomarkers, appropriate sensitivity, specificity, and broad-based exploitability were discussed. Whilst a number of candidates are in the discovery phases, all will require replication in larger, real-life cohorts. Clinical cost-effectiveness also needs to be established. CONCLUSIONS: Since a single measure is unlikely to suffice, multi-modal strategies look more promising, although they bring greater technical and implementation complexities. Identifying reproducible, robust biomarkers will probably require pre-competitive consortia to provide the resources needed to identify, validate, and develop the relevant clinical tests.

Deciphering the human brain proteome: characterization of the anterior temporal lobe and corpus callosum as part of the Chromosome 15-centric Human Proteome Project.

Defining the proteomes encoded by each chromosome and characterizing proteins related to human illnesses are among the goals of the Chromosome-centric Human Proteome Project (C-HPP) and the Biology and Disease-driven HPP. Following these objectives, we investigated the proteomes of the human anterior temporal lobe (ATL) and corpus callosum (CC) collected post-mortem from eight subjects. Using a label-free GeLC-MS/MS approach, we identified 2454 proteins in the ATL and 1887 in the CC through roughly 7500 and 5500 peptides, respectively. Considering that the ATL is a gray-matter region while the CC is a white-matter region, they presented proteomes specific to their functions. Besides, 38 proteins were found to be differentially expressed between the two regions. Furthermore, the proteome data sets were classified according to their chromosomal origin, and five proteins were evidenced at the MS level for the first time. We identified 70 proteins of the chromosome 15 - one of them for the first time by MS - which were submitted to an in silico pathway analysis. These revealed branch point proteins associated with Prader-Willi and Angelman syndromes and dyskeratosis congenita, which are chromosome-15-associated diseases. Data presented here can be a useful for brain disorder studies as well as for contributing to the C-HPP initiative. Our data are publicly available as resource data to C-HPP participant groups at http://yoda.iq.ufrj.br/Daniel/chpp2013. Additionally, the mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD000547 for the corpus callosum and PXD000548 for the anterior temporal lobe.

Analysis of quantitative trait loci in mice suggests a role of Enoph1 in stress reactivity.

Significant progress in elucidating the genetic etiology of anxiety and depression has been made during the last decade through a combination of human and animal studies. In this study, we aimed to discover genetic loci linked with anxiety as well as depression in order to reveal new candidate genes. Therefore, we initially tested the behavioral sensitivity of 543 F2 animals derived from an intercross of C57BL/6J and C3H/HeJ mice in paradigms for anxiety and depression. Next, all animals were genotyped with 269 microsatellite markers with a mean distance of 5.56 cM. Finally, a Quantitative Trait Loci (QTL) analysis was carried out, followed by selection of candidate genes. The QTL analysis revealed several new QTL on chromosome 5 with a common core interval of 19 Mb. We further narrowed this interval by comparative genomics to a region of 15 Mb. A database search and gene prioritization revealed Enoph1 as the most significant candidate gene on the prioritization list for anxiety and also for depression fulfilling our selection criteria. The Enoph1 gene, which is involved in polyamine biosynthesis, is differently expressed in parental strains, which have different brain spermidine levels and show distinct anxiety and depression-related phenotype. Our result suggests a significant role in polyamines in anxiety and depression-related behaviors.

S100B is downregulated in the nuclear proteome of schizophrenia corpus callosum.

Here we report the downregulation of S100B in the nuclear proteome of the corpus callosum from nine schizophrenia patients compared to seven mentally healthy controls. Our data have been obtained primarily by mass spectrometry and later confirmed by Western blot. This is an intriguing finding coming from a brain region which is essentially composed by white matter, considering the potential role of S100B in the control of oligodendrocyte maturation. This data reinforce the importance of oligodendrocytes in schizophrenia, shedding more light to its pathobiology.

Prefrontal cortical synaptoproteome profile combined with machine learning predicts resilience towards chronic social isolation in rats.

Chronic social isolation (CSIS) of rats serves as an animal model of depression and generates CSIS-resilient and CSIS-susceptible phenotypes. We aimed to investigate the prefrontal cortical synaptoproteome profile of CSIS-resilient, CSIS-susceptible, and control rats to delineate biochemical pathways and predictive biomarker proteins characteristic for the resilient phenotype. A sucrose preference test was performed to distinguish rat phenotypes. Class separation and machine learning (ML) algorithms support vector machine with greedy forward search and random forest were then used for discriminating CSIS-resilient from CSIS-susceptible and control rats. CSIS-resilient compared to CSIS-susceptible rat proteome analysis revealed, among other proteins, downregulated glycolysis intermediate fructose-bisphosphate aldolase C (Aldoc), and upregulated clathrin heavy chain 1 (Cltc), calcium/calmodulin-dependent protein kinase type II (Cam2a), synaptophysin (Syp) and fatty acid synthase (Fasn) that are involved in neuronal transmission, synaptic vesicular trafficking, and fatty acid synthesis. Comparison of CSIS-resilient and control rats identified downregulated mitochondrial proteins ATP synthase subunit beta (Atp5f1b) and citrate synthase (Cs), and upregulated protein kinase C gamma type (Prkcg), vesicular glutamate transporter 1 (Slc17a7), and synaptic vesicle glycoprotein 2 A (Sv2a) involved in signal transduction and synaptic trafficking. The combined protein differences make the rat groups linearly separable, and 100% validation accuracy is achieved by standard ML models. ML algorithms resulted in four panels of discriminative proteins. Proteomics-data-driven class separation and ML algorithms can provide a platform for accessing predictive features and insight into the molecular mechanisms underlying synaptic neurotransmission involved in stress resilience.

Long-lived proteins and DNA as candidate predictive biomarkers for tissue associated diseases.

Protein turnover is an important mechanism to maintain proteostasis. Long-lived proteins (LLPs) are vulnerable to lose their function due to time-accumulated damages. In this study we employed in vivo stable isotope labeling in mice from birth to postnatal day 89. Quantitative proteomics analysis of ten tissues and plasma identified 2113 LLPs, including widespread and tissue-specific ones. Interestingly, a significant percentage of LLPs was detected in plasma, implying a potential link to age-related cardiovascular diseases. LLPs identified in brains were related to neurodegenerative diseases. In addition, the relative quantification of DNA-derived deoxynucleosides from the same tissues provided information about cellular DNA renewal and showed good correlation with LLPs in the brain. The combined data reveal tissue-specific maps of mouse LLPs that may be involved in pathology due to a low renewal rate and an increased risk of damage. Tissue-derived peripheral LLPs hold promise as biomarkers for aging and age-related diseases.

Lumbar puncture increases Alzheimer's disease biomarker levels in cerebrospinal fluid of rhesus monkeys.

Cerebrospinal fluid (CSF) samples are commonly collected via lumbar puncture (LP) in both clinical and research settings for measurement of biomarkers of Alzheimer's disease (AD). To determine the effects of LP on CSF AD biomarkers, we collected CSF samples at seven different time points after an LP in rhesus monkeys. We find that amyloid-beta (Aß) and Tau levels increased significantly on day 1, peaked on day 3, and returned to baseline on day 10 after LP. The NFL levels increased significantly on day 5, peaked on day 10, and returned to baseline after day 30. The increased AD biomarker levels were mainly due to CSF outflow and deep intrathecal invasion during LP. Therefore, if LPs are repeated within a short period of time, prior LP can affect Aß and Tau levels within 10 days and NFL levels within 30 days, which may lead to clinical misdiagnosis or incorrect scientific conclusions.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

Proteome profiling of peripheral mononuclear cells from human blood.

Peripheral blood mononuclear cells (MNCs) are accessible through blood collection and represent a useful source for investigations on disease mechanisms and treatment response. Aiming to build a reference proteome database, we generated three proteome data sets from MNCs using a combination of SDS-PAGE and nanoflow LC-MS. Experiments were performed in triplicates and 514 unique proteins were identified by at least two non-redundant peptides with 95% confidence for all replicates. Identified proteins are associated with a range of dermatologic, inflammatory and neurological conditions as well as molecular processes, such as free radical scavenging and cellular growth and proliferation. Mapping the MNC proteome provides a valuable resource for studies on disease pathogenesis and the identification of therapeutic targets.

The human oligodendrocyte proteome.

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS-PAGE and after in-gel digestion subjected to nanoflow LC-MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nine distinct biological processes and various molecular classes and functions. OL-specific proteins, such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), as well as other proteins involved in multiple sclerosis and schizophrenia were also identified and are discussed. Proteins of this dataset have also been classified according to their chromosomal origin for providing useful data to the Chromosome-centric Human Proteome Project (C-HPP). Given the importance of OLs in the etiology of demyelinating and oligodendrogial disorders, the MO3.13 proteome database is a valuable data resource. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000263 (http://proteomecentral.proteomexchange.org/dataset/PXD000263).