Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning.

Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundance of WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.

Unraveling the Serum Metabolomic Profile of Post-partum Depression.

Post-partum depression (PPD) is a severe psychiatric disorder affecting ∼15% of young mothers. Early life stressful conditions in periconceptual, fetal and early infant periods or exposure to maternal psychiatric disorders, have been linked to adverse childhood outcomes interfering with physiological, cognitive and emotional development. The molecular mechanisms of PPD are not yet fully understood. Unraveling the molecular underpinnings of PPD will allow timely detection and establishment of effective therapeutic approaches. To investigate the underlying molecular correlates of PPD in peripheral material, we compared the serum metabolomes of an in detail characterized group of mothers suffering from PPD and a control group of mothers, all from Heraklion, Crete in Greece. Serum samples were analyzed by a mass spectrometry platform for targeted metabolomics, based on selected reaction monitoring (SRM), which measures the levels of up to 300 metabolites. In the PPD group, we observed increased levels of glutathione-disulfide, adenylosuccinate, and ATP, which associate with oxidative stress, nucleotide biosynthesis and energy production pathways. We also followed up the metabolomic findings in a validation cohort of PPD mothers and controls. To the very best of our knowledge, this is the first metabolomic serum analysis in PPD. Our data show that molecular changes related to PPD are detectable in peripheral material, thus paving the way for additional studies in order to shed light on the molecular correlates of PPD.

Exposure-induced changes of plasma metabolome and gene expression in patients with panic disorder.

BACKGROUND: Anxiety disorders including panic disorder (PD) are the most prevalent psychiatric diseases leading to high disability and burden in the general population. Acute panic attacks are distinctive for PD but also frequent in other anxiety disorders. The neurobiology or specific molecular changes leading to and present during panic attacks are insufficiently known so far. METHODS: In the present pilot study, we investigated dynamic metabolomic and gene expression changes in peripheral blood of patients with PD (n = 25) during two exposure-induced acute panic attacks. RESULTS: The results show that the metabolite glyoxylate was dynamically regulated in peripheral blood. Additionally, glyoxylate levels were associated with basal anxiety levels and showed gender-related differences at baseline. As glyoxylate is part of the degradation circuit of cholecystokinin, this suggests that this neuropeptide might be directly involved in exposure-induced panic attacks. Only gene expression changes of very small magnitude were observed in this experimental setting. CONCLUSIONS: From this first metabolome and gene expression study in exposure-induced acute panic attacks in PD we conclude that metabolites can potentially serve as dynamic markers for different anxiety states. However, these findings have to be replicated in cohorts with greater sample sizes.

Detection of Posttranslational Modifications by Fluorescent Staining of Two-Dimensional Gels.

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as multiple sclerosis (MS), using an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level resulting in valuable insights into the pathophysiological mechanism of EAE and MS.

The ABRF Metabolomics Research Group 2013 Study: Investigation of Spiked Compound Differences in a Human Plasma Matrix.

Metabolomics is an emerging field that involves qualitative and quantitative measurements of small molecule metabolites in a biological system. These measurements can be useful for developing biomarkers for diagnosis, prognosis, or predicting response to therapy. Currently, a wide variety of metabolomics approaches, including nontargeted and targeted profiling, are used across laboratories on a routine basis. A diverse set of analytical platforms, such as NMR, gas chromatography-mass spectrometry, Orbitrap mass spectrometry, and time-of-flight-mass spectrometry, which use various chromatographic and ionization techniques, are used for resolution, detection, identification, and quantitation of metabolites from various biological matrices. However, few attempts have been made to standardize experimental methodologies or comparative analyses across different laboratories. The Metabolomics Research Group of the Association of Biomolecular Resource Facilities organized a "round-robin" experiment type of interlaboratory study, wherein human plasma samples were spiked with different amounts of metabolite standards in 2 groups of biologic samples (A and B). The goal was a study that resembles a typical metabolomics analysis. Here, we report our efforts and discuss challenges that create bottlenecks for the field. Finally, we discuss benchmarks that could be used by laboratories to compare their methodologies.

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.

Detection of post-translational modifications by fluorescent staining of two-dimensional gels.

Post-translational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial in the discovery of biomarkers of disease. Recent commercial availability of fluorescent dyes specifically staining PTMs of proteins such as phosphorylation and glycosylation enables the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular and signalling interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as Multiple Sclerosis (MS), using its animal model, Experimental autoimmune encephalomyelitis (EAE), we have applied the phopshorylation specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent Colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level that has given valuable insights into the patho-physiological mechanism of EAE and possibly also MS.

SHIP family inositol phosphatases interact with and negatively regulate the Tec tyrosine kinase.

The Tec family of protein-tyrosine kinases (PTKs), that includes Tec, Itk, Btk, Bmx, and Txk, plays an essential role in phospholipase Cgamma (PLCgamma) activation following antigen receptor stimulation. This function requires activation of phosphatidylinositol 3-kinase (PI 3-kinase), which promotes Tec membrane localization through phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P(3)) generation. The mechanism of negative regulation of Tec family PTKs is poorly understood. In this study, we show that the inositol 5' phosphatases SHIP1 and SHIP2 interact preferentially with Tec, compared with other Tec family members. Four lines of evidence suggest that SHIP phosphatases are negative regulators of Tec. First, SHIP1 and SHIP2 are potent inhibitors of Tec activity. Second, inactivation of the Tec SH3 domain, which is necessary and sufficient for SHIP binding, generates a hyperactive form of Tec. Third, SHIP1 inhibits Tec membrane localization. Finally, constitutively targeting Tec to the membrane relieves SHIP1-mediated inhibition. These data suggest that SHIP phosphatases can interact with and functionally inactivate Tec by de-phosphorylation of local PtdIns 3,4,5-P(3) and inhibition of Tec membrane localization.

Decrease in hnRNP A/B expression during erythropoiesis mediates a pre-mRNA splicing switch.

A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE(16)) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model pre-mRNAs showed that CE(16) can repress splicing of upstream introns, and that mutagenesis or replacement of CE(16) can relieve this inhibition. An affinity selection assay with biotinylated CE(16) RNA demonstrated specific binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract significantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-specific activation of the E16 splicing switch in concert with a dramatic and specific down-regulation of hnRNP A/B protein expression. These findings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in pre-mRNA splicing.