Proteomic and metabolomic profiling of a trait anxiety mouse model implicate affected pathways

Abstract

Depression and anxiety disorders affect a great number of people worldwide. Whereas singular factors have been associated with the pathogenesis of psychiatric disorders, growing evidence emphasizes the significance of dysfunctional neural circuits and signaling pathways. Hence, a systems biology approach is required to get a better understanding of psychiatric phenotypes such as depression and anxiety. Furthermore, the availability of biomarkers for these disorders is critical for improved diagnosis and monitoring treatment response. In the present study, a mouse model presenting with robust high versus low anxiety phenotypes was subjected to thorough molecular biomarker and pathway discovery analyses. Reference animals were metabolically labeled with the stable (15)N isotope allowing an accurate comparison of protein expression levels between the high anxiety-related behavior versus low anxiety-related behavior mouse lines using quantitative mass spectrometry. Plasma metabolomic analyses identified a number of small molecule biomarkers characteristic for the anxiety phenotype with particular focus on myo-inositol and glutamate as well as the intermediates involved in the tricarboxylic acid cycle. In silico analyses suggested pathways and subnetworks as relevant for the anxiety phenotype. Our data demonstrate that the high anxiety-related behavior and low anxiety-related behavior mouse model is a valuable tool for anxiety disorder drug discovery efforts.

Fig. 1.

Quantification of glyoxalase-I in HAB and LAB hippocampus. Mass spectrometry total ion current data for the hippocampal Glx1-derived tryptic peptide GLAFIQDPDGYWIEILNPNK. ¹⁵N-labeled NAB hippocampal specimens were used as a reference and either mixed with unlabeled HAB or LAB material. An indirect comparison of the HAB/NAB (left) and LAB/NAB (right) datasets revealed that Glx1 expression in LAB mice is ∼fivefold higher than in HAB mice.

Fig. 2.

Relative protein quantification in HAB/LAB mice. A, D, G, Eluted chromatographic profiles for carbonic anhydrase 2 (CA2, IPI00121534) tryptic peptide AVQQPDGLAVLGIFLK, transthyretin (TTR, IPI00127560) tryptic peptide TAESGELHGLTTDEK, serum amyloid P-component (SAP, IPI00309214) tryptic peptide GRDNELLIYKEK, respectively. The peak areas are used for the ¹⁴N/¹⁵N signal quantification. In all cases ¹⁵N-labeled NAB proteins were used as a reference and either mixed with unlabeled HAB or LAB material; B, E, H, Western blot analyses of CA2, TTR and SAP protein levels; C, F, I, Western blot protein band density quantification for CA2 (p = 0.0025), TTR (p < 0.0001) and SAP (p = 0.0008), respectively. In all cases the Western blot analyses confirm the relative protein expression level data obtained by mass spectrometry.

Fig. 3.

Plasma allo-inostitol, myo-inositol, and glutamate levels in HAB and LAB mice. The intensity mean and standard error of mean for the metabolites are indicated.

Fig. 4.

Correlation between hippocampal protein regulation versus KEGG pathways (left), and protein regulation versus GO cellular component (right). The blue boxes at the top indicate relative HAB/LAB protein expression levels as log2 ratios. Proteins were divided into five bins and analyzed with respect to KEGG pathways and GO cellular component. p values were transformed to z-scores, indicating bin-specific enrichments. (because of the limited image resolution the pathway names are not legible; a higher resolution figure is available in the electronic file.)

Fig. 5.

TCA cycle pathways and biological subnetworks enriched with proteins and metabolites. A, TCA cycle; enzymes and metabolites labeled in red are expressed at higher levels in HAB compared with LAB mice. B, Subnetwork related to dexamethasone; C, subnetworks related to glycinergic synaptic transmission and myelin maintenance. (because of the limited image resolution the protein and metabolite names are not legible; a higher resolution figure is available in the electronic file.)