System-based proteomic and metabonomic analysis of the Df(16)A+/- mouse identifies potential miR-185 targets and molecular pathway alterations

Abstract

Deletions on chromosome 22q11.2 are a strong genetic risk factor for development of schizophrenia and cognitive dysfunction. We employed shotgun liquid chromatography-mass spectrometry (LC-MS) proteomic and metabonomic profiling approaches on prefrontal cortex (PFC) and hippocampal (HPC) tissue from Df(16)A^+/- mice, a model of the 22q11.2 deletion syndrome. Proteomic results were compared with previous transcriptomic profiling studies of the same brain regions. The aim was to investigate how the combined effect of the 22q11.2 deletion and the corresponding miRNA dysregulation affects the cell biology at the systems level. The proteomic brain profiling analysis revealed PFC and HPC changes in various molecular pathways associated with chromatin remodelling and RNA transcription, indicative of an epigenetic component of the 22q11.2DS. Further, alterations in glycolysis/gluconeogenesis, mitochondrial function and lipid biosynthesis were identified. Metabonomic profiling substantiated the proteomic findings by identifying changes in 22q11.2 deletion syndrome (22q11.2DS)-related pathways, such as changes in ceramide phosphoethanolamines, sphingomyelin, carnitines, tyrosine derivates and panthothenic acid. The proteomic findings were confirmed using selected reaction monitoring mass spectrometry, validating decreased levels of several proteins encoded on 22q11.2, increased levels of the computationally predicted putative miR-185 targets UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT1) and kinesin heavy chain isoform 5A and alterations in the non-miR-185 targets serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform, neurofilament light chain and vesicular glutamate transporter 1. Furthermore, alterations in the proteins associated with mammalian target of rapamycin signalling were detected in the PFC and with glutamatergic signalling in the hippocampus. Based on the proteomic and metabonomic findings, we were able to develop a schematic model summarizing the most prominent molecular network findings in the Df(16)A^+/- mouse. Interestingly, the implicated pathways can be linked to one of the most consistent and strongest proteomic candidates, (OGT1), which is a predicted miR-185 target. Our results provide novel insights into system-biological mechanisms associated with the 22q11DS, which may be linked to cognitive dysfunction and an increased risk to develop schizophrenia. Further investigation of these pathways could help to identify novel drug targets for the treatment of schizophrenia.

Figure 1

(a) Venn diagrams of the overlap of all identified proteins (left) and of all significantly altered proteins (right) identified in label-free LC-MS^E (liquid chromatography–mass spectrometry) proteomic profiling studies of Df(16)A^+/− mice compared with wild-type mice (Supplementary Table S4 for full list). (b) Percentages of significantly increased (red) and decreased (green) proteins in Df(16)A^+/− mice across all proteomic studies. Mean percentages show significantly increased protein levels in the prefrontal cortex (PFC; p<0.05, t-test). PFC combined are samples from both cohorts combined and in-gel digested (see Material and methods). As the hippocampal (HPC) was only investigated from one cohort, significant levels could not be established. (c) Top ingenuity pathway analysis: altered pathways in the PFC and HPC. (d) Detailed information of the overlapping proteins identified as significantly changing across all proteomic screens. Proteins were sorted by the number of proteomic studies in which they have been detected as significantly altered. Only proteins with a mean ratio of >1.1 or <0.9 across all identified studies are displayed. P-values were determined using MSstats and corrected to control for multiple hypothesis testing after Benjamini–Hochberg. (Supplementary Table S3 for full information). Proteins identified by one peptide (italic) were included if they were overlapping between the proteomic screens. Results were compared with the transcriptomic results using Affymetrix chips published by Stark et al. and Feneleon et al. Stark reported 716 transcripts altered in the FC and 85 transcripts in the HPC (false-discovery rate=0.01). Significantly changed transcripts are marked with an ▴ and ▾ according to their reported fold change direction in comparison to wild-type mice. Four different bioinformatic miRNA target prediction tools were used to assess if the upregulated proteins are potential Mir-185 targets. Xof4, predicted in X of the four predictions; D, downregulation; PC, positive control.

Figure 2

Significantly enriched pathways identified by gene set enrichment analysis using GOstats (proteins per pathway >3). The top 15 significant Gene Ontotlogy (GO) terms are displayed. GO terms falling into the category of chromosomal regulation are highlighted in grey. A full colour version of this figure is available at the Molecular Psychiatry journal online.

Figure 3

Schematic model summarizing the findings of the proteomic and metabonomic profiling analyses. Green boxes: altered levels of molecules determined by proteomic or metabonomic profiling. Orange boxes: altered pathways determined by in silico pathway analysis using IPKB or gene set enrichment analysis. Increased levels of O-GlcNAc transferase subunit p110 (OGT1) lead to abnormal O-GlcNAcylation, which affects chromatin remodelling and transcriptional regulation. Levels of the OGT1 substrate UDP-GlcNac are generated by glycolysis and gluconeogenesis (both found to be enriched). Both glutamatergic and Ca²⁺ signalling are mainly affected in the hippocampus (HPC). (R)=regulates. IPKB, Ingenuity Protein Knowledge Base; PFC, prefrontal cortex.