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.

Variability assessment of (15)N metabolic labeling-based proteomics workflow in mouse plasma and brain.

(15)N metabolic labeling-based quantitative proteomics is used for the identification of disease- and phenotype-related alterations in live organisms. The variability of (15)N metabolic labeling proteomics workflows has been assessed in plants and bacteria. However, no study has addressed this topic in mice. We have investigated the repeatability of a quantitative in vivo(15)N metabolic labeling proteomics workflow in mice by assessing LC variability, peptide and protein profiling characteristics and overall (15)N/(14)N protein quantification accuracy in technical replicates of plasma and brain specimens. We furthermore examined how sample preparation affects these parameters in plasma and brain. We found that specimen type (i.e. plasma or brain) influences the variability of the (15)N metabolic labeling workflow in an LC-independent manner.

Psychiatric patient stratification using biosignatures based on cerebrospinal fluid protein expression clusters.

Psychiatric disorders are caused by perturbed molecular pathways that affect brain circuitries. The identification of specific biosignatures that are the result of altered pathway activities in major depression, bipolar disorder and schizophrenia can contribute to a better understanding of disease etiology and aid in the implementation of diagnostic assays. In the present study we identified disease-specific protein biosignatures in cerebrospinal fluid of depressed (n: 36), bipolar (n: 27) and schizophrenic (n: 35) patients using the Reverse Phase Protein Microarray technology. These biosignatures were able to stratify patient groups in an objective manner according to cerebrospinal fluid protein expression patterns. Correct classification rates were over 90%. At the same time several protein sets that play a role in neuronal growth, proliferation and differentiation (NEGR1, NPDC1), neurotransmission (SEZ6) and protection from oxidative damage (GPX3) were able to distinguish diseased from healthy individuals (n: 35) indicating a molecular signature overlap for the different psychiatric phenotypes. Our study is a first step toward implementing a psychiatric patient stratification system based on molecular biosignatures. Protein signatures may eventually be of use as specific and sensitive biomarkers in clinical trials not only for patient diagnostic and subgroup stratification but also to follow treatment response.

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.

Characterization of the B-Raf interactome in mouse hippocampal neuronal cells.

B-Raf links a variety of extracellular stimuli downstream of cell surface receptors, constituting a determining factor in the ability of neurons to activate ERK. A detailed study of the B-Raf interactome is necessary to clarify the intricacy of B-Raf-dependent signal transduction. We used a mouse hippocampal cell line (HT22) that expresses B-Raf at high levels, to identify B-Raf associated proteins under endogenous expression conditions, avoiding artificial interactions from overexpression studies. We used stringent procedures to co-immunoprecipitate proteins that specifically associate with endogenous B-Raf with the help of gel electrophoresis separation and off-line LC-MALDI-MS/MS proteomic analysis. Our stringent protein identification criteria allowed confident identification of B-Raf interacting proteins under non-stimulating conditions. The presence of previously reported B-Raf interactors among the list of proteins identified confirms the quality of proteomic data. We identified tubulin and actin as B-Raf interactors for the first time, among structural and accessory proteins of cell cytoskeleton, molecular chaperones (Hsc70, GRP78), and cellular components involved in aspects of mRNA metabolism and translation. Interactions were validated in HT22 cells and in the neuronal cell line Neuro-2a providing further evidence that the identified proteins are B-Raf interactors, which constitute a basis for understanding MAPK pathway regulation in neurons.

Prefrontal cortex shotgun proteome analysis reveals altered calcium homeostasis and immune system imbalance in schizophrenia.

Schizophrenia is a complex disease, likely to be caused by a combination of serial alterations in a number of genes and environmental factors. The dorsolateral prefrontal cortex (Brodmann's Area 46) is involved in schizophrenia and executes high-level functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts and attitudes, correct social behavior and personality expression. Global proteomic analysis of post-mortem dorsolateral prefrontal cortex samples from schizophrenia patients and non-schizophrenic individuals was performed using stable isotope labeling and shotgun proteomics. The analysis resulted in the identification of 1,261 proteins, 84 of which showed statistically significant differential expression, reinforcing previous data supporting the involvement of the immune system, calcium homeostasis, cytoskeleton assembly, and energy metabolism in schizophrenia. In addition a number of new potential markers were found that may contribute to the understanding of the pathogenesis of this complex disease.

Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis.

Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease.

Proteome analysis of human dorsolateral prefrontal cortex using shotgun mass spectrometry.

The prefrontal cortex executes important functions such as differentiation of conflicting thoughts, correct social behavior and personality expression, and is directly implicated in different neurodegenerative diseases. We performed a shotgun proteome analysis that included IEF fractionation, RP-LC, and MALDI-TOF/TOF mass spectrometric analysis of tryptic digests from a pool of seven human dorsolateral prefrontal cortex protein extracts. In this report, we present a catalog of 387 proteins expressed in these samples, identified by two or more peptides and high confidence search scores. These proteins are involved in different biological processes such as cell growth and/or maintenance, metabolism/energy pathways, cell communication/signal transduction, protein metabolism, transport, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism, and immune response. This analysis contributes to the knowledge of the human brain proteome by adding sample diversity and protein expression data from an alternative technical approach. It will also aid comparative studies of different brain areas and medical conditions, with future applications in basic and clinical research.