Multi-Omics Analysis Reveals Myelin, Presynaptic and Nicotinate Alterations in the Hippocampus of G72/G30 Transgenic Mice.

The primate-specific G72/G30 gene locus has been associated with major psychiatric disorders, such as schizophrenia and bipolar disorder. We have previously generated transgenic mice which carry the G72/G30 locus and express the longest G72 splice variant (LG72) protein encoded by this locus with schizophrenia-related symptoms. Here, we used a multi-omics approach, including quantitative proteomics and metabolomics to investigate molecular alterations in the hippocampus of G72/G30 transgenic (G72Tg) mice. Our proteomics analysis revealed decreased expression of myelin-related proteins and NAD-dependent protein deacetylase sirtuin-2 (Sirt2) as well as increased expression of the scaffolding presynaptic proteins bassoon (Bsn) and piccolo (Pclo) and the cytoskeletal protein plectin (Plec1) in G72Tg compared to wild-type (WT) mice. Metabolomics analysis indicated decreased levels of nicotinate in G72Tg compared to WT hippocampi. Decreased hippocampal protein expression for selected proteins, namely myelin oligodentrocyte glycoprotein (Mog), Cldn11 and myelin proteolipid protein (Plp), was confirmed with Western blot in a larger population of G72Tg and WT mice. The identified molecular pathway alterations shed light on the hippocampal function of LG72 protein in the context of neuropsychiatric phenotypes.

Behavioral and Metabolome Differences between C57BL/6 and DBA/2 Mouse Strains: Implications for Their Use as Models for Depression- and Anxiety-Like Phenotypes.

Mouse models are widely used to study behavioral phenotypes related to neuropsychiatric disorders. However, different mouse strains vary in their inherent behavioral and molecular characteristics, which needs to be taken into account depending on the nature of the study. Here, we performed a detailed behavioral and molecular comparison of C57BL/6 (B6) and DBA/2 (DBA) mice, two inbred strains commonly used in neuropsychiatric research. We analyzed anxiety-related and depression-like traits, quantified hippocampal and plasma metabolite profiles, and assessed total antioxidant capacity (ΤAC). B6 mice exhibit increased depression-like and decreased anxiety-related behavior compared to DBA mice. Metabolite level differences indicate alterations in amino acid, nucleotide and mitochondrial metabolism that are accompanied by a decreased TAC in B6 compared to DBA mice. Our data reveal multiple behavioral and molecular differences between B6 and DBA mouse strains, which should be considered in the experimental design for phenotype, pharmacological and mechanistic studies relevant for neuropsychiatric disorders.

Ketamine's antidepressant effect is mediated by energy metabolism and antioxidant defense system.

Fewer than 50% of all patients with major depressive disorder (MDD) treated with currently available antidepressants (ADs) show full remission. Moreover, about one third of the patients suffering from MDD does not respond to conventional ADs and develop treatment-resistant depression (TRD). Ketamine, a non-competitive, voltage-dependent N-Methyl-D-aspartate receptor (NMDAR) antagonist, has been shown to have a rapid antidepressant effect, especially in patients suffering from TRD. Hippocampi of ketamine-treated mice were analysed by metabolome and proteome profiling to delineate ketamine treatment-affected molecular pathways and biosignatures. Our data implicate mitochondrial energy metabolism and the antioxidant defense system as downstream effectors of the ketamine response. Specifically, ketamine tended to downregulate the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) metabolite ratio which strongly correlated with forced swim test (FST) floating time. Furthermore, we found increased levels of enzymes that are part of the 'oxidative phosphorylation' (OXPHOS) pathway. Our study also suggests that ketamine causes less protein damage by rapidly decreasing reactive oxygen species (ROS) production and lend further support to the hypothesis that mitochondria have a critical role for mediating antidepressant action including the rapid ketamine response.

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.

Stable isotope metabolic labeling suggests differential turnover of the DPYSL protein family.

PURPOSE: In this work, we discuss how in vivo 15 N metabolic labeling in combination with MS simultaneously provides information on protein expression and protein turnover. EXPERIMENTAL DESIGN: We metabolically labeled mice with the stable nitrogen isotope 15 N using a 15 N-enriched diet and analyzed unlabeled (14 N) versus 15 N-labeled brain tissue with LC-MS/MS. We then compared the 14 N versus 15 N peptide isotopologue clusters of 14 N and 15 N-labeled dihydropyrimidinase-related (DPYSL) proteins. RESULTS: We present a workflow assessing protein expression and turnover at different time points of mouse brain development. Our data demonstrate distinct protein turnover patterns of DPYSL3 and DPYSL5 compared to other quantified proteins. We report the presence of two DPYSL3 and DPYSL5 populations with different 15 N incorporation rates, indicating altered protein turnover during development. CONCLUSIONS AND CLINICAL RELEVANCE: In vivo 15 N metabolic labeling allows the simultaneous investigation of protein expression and turnover, enabling detailed protein dynamics studies. We report for the first time protein turnover data for the DPYSL2, DPYSL3, and DPYSL5 protein family members. As DPYSL proteins have important functions for nervous system maturation, our data provide useful information on their molecular fate during brain development.

Selective Mitochondrial Targeting Exerts Anxiolytic Effects In Vivo.

Current treatment strategies for anxiety disorders are predominantly symptom-based. However, a third of anxiety patients remain unresponsive to anxiolytics highlighting the need for more effective, mechanism-based therapeutic approaches. We have previously compared high vs low anxiety mice and identified changes in mitochondrial pathways, including oxidative phosphorylation and oxidative stress. In this work, we show that selective pharmacological targeting of these mitochondrial pathways exerts anxiolytic effects in vivo. We treated high anxiety-related behavior (HAB) mice with MitoQ, an antioxidant that selectively targets mitochondria. MitoQ administration resulted in decreased anxiety-related behavior in HAB mice. This anxiolytic effect was specific for high anxiety as MitoQ treatment did not affect the anxiety phenotype of C57BL/6N and DBA/2J mouse strains. We furthermore investigated the molecular underpinnings of the MitoQ-driven anxiolytic effect and found that MitoQ treatment alters the brain metabolome and that the response to MitoQ treatment is characterized by distinct molecular signatures. These results indicate that a mechanism-driven approach based on selective mitochondrial targeting has the potential to attenuate the high anxiety phenotype in vivo, thus paving the way for translational implementation as long-term MitoQ administration is well-tolerated with no reported side effects in mice and humans.

Behavioral extremes of trait anxiety in mice are characterized by distinct metabolic profiles.

No comprehensive metabolic profile of trait anxiety is to date available. To identify metabolic biosignatures for different anxiety states, we compared mice selectively inbred for ∼ 40 generations for high (HAB), normal (NAB) or low (LAB) anxiety-related behavior. Using a mass spectrometry-based targeted metabolomics approach, we quantified the levels of 257 unique metabolites in the cingulate cortex and plasma of HAB, NAB and LAB mice. We then pinpointed affected molecular systems in anxiety-related behavior by an in silico pathway and network prediction analysis followed by validation of in silico predicted alterations with molecular assays. We found distinct metabolic profiles for different trait anxiety states and detected metabolites with altered levels both in cingulate cortex and plasma. Metabolomics data revealed common candidate biomarkers in cingulate cortex and plasma for anxiety traits and in silico pathway analysis implicated amino acid metabolism, pyruvate metabolism, oxidative stress and apoptosis in the regulation of anxiety-related behavior. We report characteristic biosignatures for trait anxiety states and provide a network map of pathways involved in anxiety-related behavior. Pharmacological targeting of these pathways will enable a mechanism-based approach for identifying novel therapeutic targets for anxiety disorders.

Myelination and oxidative stress alterations in the cerebellum of the G72/G30 transgenic schizophrenia mouse model.

G72/G30 is a primate-specific locus that has been repeatedly implicated as a risk factor in genetic studies of schizophrenia. The function of the longest G72 splice variant (LG72 protein) encoded by this locus is not fully understood. To investigate the role of the LG72 protein in vivo, we have generated transgenic (G72Tg) mice carrying the G72/G30 locus that exhibit schizophrenia-like symptoms. We investigated protein expression alterations in the cerebella of G72Tg compared to wild type (WT) mice using a proteomics approach based on in vivo(15)N metabolic labeling and quantitative mass spectrometry (MS). Our data revealed expression level differences of proteins involved in myelin-related processes, oxidative stress and mitochondrial function. Furthermore, in silico pathway analyses suggested common regulators and targets for the observed protein alterations. Our work sheds light on the functional role of the LG72 protein and pinpoints molecular correlates of schizophrenia-like behavior.

The 15N isotope effect in Escherichia coli: a neutron can make the difference.

Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the (15) N isotope effect on Escherichia coli cultures that were grown in either unlabeled ((14) N) or (15) N-labeled media by LC-ESI-MS/MS-based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between (14) N and (15) N-labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between (14) N and (15) N-labeled bacteria. Our data demonstrate for the first time that the introduction of the (15) N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.

The (15)N isotope effect as a means for correlating phenotypic alterations and affected pathways in a trait anxiety mouse model.

Stable isotope labeling techniques hold great potential for accurate quantitative proteomics comparisons by MS. To investigate the effect of stable isotopes in vivo, we metabolically labeled high anxiety-related behavior (HAB) mice with the heavy nitrogen isotope (15)N. (15)N-labeled HAB mice exhibited behavioral alterations compared to unlabeled ((14)N) HAB mice in their depression-like phenotype. To correlate behavioral alterations with changes on the molecular level, we explored the (15)N isotope effect on the brain proteome by comparing protein expression levels between (15)N-labeled and (14)N HAB mouse brains using quantitative MS. By implementing two complementary in silico pathway analysis approaches, we were able to identify altered networks in (15)N-labeled HAB mice, including major metabolic pathways such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Here, we discuss the affected pathways with regard to their relevance for the behavioral phenotype and critically assess the utility of exploiting the (15)N isotope effect for correlating phenotypic and molecular alterations.

Cerebrospinal fluid biomarkers for major depression confirm relevance of associated pathophysiology.

Individual characteristics of pathophysiology and course of depressive episodes are at present not considered in diagnostics. There are no biological markers available that can assist in categorizing subtypes of depression and detecting molecular variances related to disease-causing mechanisms between depressed patients. Identification of such differences is important to create patient subgroups, which will benefit from medications that specifically target the pathophysiology underlying their clinical condition. To detect characteristic biological markers for major depression, we analyzed the cerebrospinal fluid (CSF) proteome of depressed vs control persons, using two-dimensional polyacrylamide gel electrophoresis and time-of-flight (TOF) mass spectrometry peptide profiling. Proteins of interest were identified by matrix-assisted laser desorption ionization TOF mass spectrometry (MALDI-TOF-MS). Validation of protein markers was performed by immunoblotting. We found 11 proteins and 144 peptide features that differed significantly between CSF from depressed patients and controls. In addition, we detected differences in the phosphorylation pattern of several CSF proteins. A subset of the differentially expressed proteins implicated in brain metabolism or central nervous system disease was validated by immunoblotting. The identified proteins are involved in neuroprotection and neuronal development, sleep regulation, and amyloid plaque deposition in the aging brain. This is one of the first hypothesis-free studies that identify characteristic protein expression differences in CSF of depressed patients. Proteomic approaches represent a powerful tool for the identification of disease markers for subgroups of patients with major depression.

Proteomics and metabolomics analysis of a trait anxiety mouse model reveals divergent mitochondrial pathways.

BACKGROUND: Although anxiety disorders are the most prevalent psychiatric disorders, no molecular biomarkers exist for their premorbid diagnosis, accurate patient subcategorization, or treatment efficacy prediction. To unravel the neurobiological underpinnings and identify candidate biomarkers and affected pathways for anxiety disorders, we interrogated the mouse model of high anxiety-related behavior (HAB), normal anxiety-related behavior (NAB), and low anxiety-related behavior (LAB) employing a quantitative proteomics and metabolomics discovery approach. METHODS: We compared the cingulate cortex synaptosome proteomes of HAB and LAB mice by in vivo (15)N metabolic labeling and mass spectrometry and quantified the cingulate cortex metabolomes of HAB/NAB/LAB mice. The combined data sets were used to identify divergent protein and metabolite networks by in silico pathway analysis. Selected differentially expressed proteins and affected pathways were validated with immunochemical and enzymatic assays. RESULTS: Altered levels of up to 300 proteins and metabolites were found between HAB and LAB mice. Our data reveal alterations in energy metabolism, mitochondrial import and transport, oxidative stress, and neurotransmission, implicating a previously nonhighlighted role of mitochondria in modulating anxiety-related behavior. CONCLUSIONS: Our results offer insights toward a molecular network of anxiety pathophysiology with a focus on mitochondrial contribution and provide the basis for pinpointing affected pathways in anxiety-related behavior.

Profiling of mouse synaptosome proteome and phosphoproteome by IEF.

Synapses play important roles in neurotransmission and neuroplasticity. For an in-depth analysis of the synaptic proteome and phosphoproteome, synaptosomal proteins from whole mouse brain were analyzed by IEF and MS resulting in the largest synaptosome proteome described to date, with 2980 unique proteins identified with two or more peptides. At the same time, 118 synaptosomal phosphoproteins were identified, eight of which are reported for the first time as phosphorylated. Expression of selected proteins in synaptosomes was investigated by Western blot. We demonstrate that IEF is a powerful method to interrogate complex samples such as brain tissue both at the proteome and the phosphoproteome level without the need of additional enrichment for phosphoproteins. The detailed synaptoproteome data set reported here will help to elucidate the molecular complexity of the synapse and contribute to our understanding of synaptic systems biology in health and disease.

Phosphopeptide enrichment by IEF.

In our efforts to improve the identification of phosphopeptides by MS we have used peptide IEF on IPG strips. Phosphopeptides derived from trypsin digests of single proteins as well as complex cellular protein mixtures can be enriched by IEF and recovered in excellent yields at the acidic end of an IPG strip. IPG peptide fractionation in combination with MS/MS analysis has allowed us to identify phosphopeptides from tryptic digests of a cellular protein extract.

Protein biomarkers in a mouse model of extremes in trait anxiety.

Brain proteome analysis of mice selectively bred for either high or low anxiety-related behavior revealed quantitative and qualitative protein expression differences. The enzyme glyoxalase-I was consistently expressed to a higher extent in low anxiety as compared with high anxiety mice in several brain areas. The same phenotype-dependent difference was also found in red blood cells with normal and cross-mated animals showing intermediate expression profiles of glyoxalase-I. Another protein that showed a different mobility during two-dimensional gel electrophoresis was identified as enolase phosphatase. The presence of both protein markers in red or white blood cells, respectively, creates the opportunity to screen for their expression in clinical blood specimens from patients suffering from anxiety.

The quest for brain disorder biomarkers.

The identification of disease markers in tissues and body fluids requires an extensive and thorough analysis of its protein constituents. In our efforts to identify biomarkers for affective and neurological disorders we are pursuing several different strategies. On one hand we are using animal models that represent defined phenotypes characteristic for the respective disorder in humans. In addition, we are analyzing human specimens from carefully phenotyped patient groups. Several fractions representing different protein classes from human cerebrospinal fluid obtained by lumbar puncture are used for this purpose. Our biomarker identification efforts range from classical proteomics approaches such as two dimensional gel electrophoresis and mass spectrometry to phage display screens with cerebrospinal fluid antibodies.