Numerous investigations support decreased glutamatergic signaling as a pathogenic mechanism of schizophrenia: yet molecular underpinnings for such dysregulation are largely unknown. In the postmortem dorsolateral prefrontal cortex, we found striking decreases in tyrosine phosphorylation of N-methyl-D aspartate (NMDA) receptor subunit 2 (GluN2), which is critical for neuroplasticity. The decreased GluN2 activity in schizophrenia may not be due to downregulation of NMDA receptors since MK-801 binding and NMDA receptor complexes in the PSD were in fact increased in schizophrenia cases. At the post-receptor level, however, we found striking reductions in the protein kinase C, Pyk 2 and Src kinase activity, which in tandem can decrease GluN2 activation. Given that Src serves as a hub of various signaling mechanisms impacting GluN2 phosphorylation, we postulated that Src hypoactivity may result from convergent alterations of various schizophrenia susceptibility pathways and thus mediate their impacts on NMDA receptor signaling. Indeed, the DLPFC of schizophrenia cases exhibit increased PSD-95 and erbB4 and decreased RPTPa and dysbindin-1, each of which reduces Src activity via protein interaction with Src. To test genomic underpinnings for Src hypoactivity, we examined genome wide association study results, incorporating 13,394 cases and 34,676 controls, which yielded no significant association of individual variants of Src and its direct regulators with schizophrenia. However, a wider protein-protein interaction based network centered on Src, showed significant enrichment of gene-level associations with schizophrenia compared to other psychiatric illnesses. Our results together demonstrate striking decreases in NMDA receptor signaling at the post-receptor level and propose Src as a nodal point of convergent dysregulations impacting NMDA receptor pathway via protein-protein associations.

Abbreviations: ammonium bicarbonate: ABC; electron-transfer/higher-energy collisional dissociation: EThcD; higher-energy collisional dissociation: HCD; hydrophilic interaction chromatography: HILIC; multi-lectin weak affinity chromatography: M-LWAC; posttranslational modification: PTM; reverse phase: RP Abstract Protein glycosylation represents one of the most common and heterogeneous posttranslational modifications (PTMs) in human biology. Herein, an approach for the enrichment of glycopeptides using multi-lectin weak affinity chromatography (M-LWAC), followed by fractionation of the enriched material, and multi-mode fragmentation LC/MS is described. Two fragmentation methods, high-energy collision induced dissociation (HCD) and electron transfer dissociation (EThcD), were independently analyzed. While each fragmentation method provided similar glycopeptide coverage, there was some dependence on the glycoform identity. From these data a total of 7,503 unique glycopeptides belonging to 666 glycoproteins from the combined tissue types, human serum and brain, were identified. Of these, 617 glycopeptides (192 proteins) were found in both tissues; 2,006 glycopeptides (48 proteins) were unique to serum, and 4,880 glycopeptides (426 proteins) were unique to brain tissue. From 379 unique glycoforms, 1,420 unique sites of glycosylation were identified, with an average of four glycans 2 per site. Glycan occurrences were significantly different between tissue types: serum showed greater glycan diversity whereas brain tissue showed a greater abundance of the high mannose family. Glycosylation co-occurrence rates were determined, which enabled us to infer differences in underlying biosynthetic pathways. Experimental procedures Brain Lysate Sample PreparationBrain specimens from all subjects were obtained during autopsies conducted at the Allegheny County Office of the Medical Examiner after receiving consent from the next-of-kin.Procedures were approved by the University of Pittsburgh Institutional Review Board and Committee for Oversight of Research Involving the Dead. Grey matter was harvested from the auditory cortex as previously described (40,41): Tissue slabs containing the superior temporal gyrus with Heschl's Gyrus located medial to the planum temporale were identified, and the superior temporal gyrus removed as single block. Grey matter (100 mg) was collected from HG by taking 100 μm frozen sections (40). Grey matter was homogenized in 1 mL of 8M urea with a FastPrep-24 benchtop homogenizer. Samples were stored at -20 ˚C. Protein PreparationSera (Sigma-Aldrich, St. Louis, MO) was denatured using 8 M urea (Sigma-Aldrich, St.Louis MO) in 100 mM ammonium bicarbonate (pH 7.4, Sigma-Aldrich, St. Louis MO).Disulfide bonds were reduced using 1 mM tris(2-carboxyethyl)phosphine (Sigma-Aldrich, St. Louis, MO) for 1 hour at 65 ˚C. Reduced cysteine residues were modified using 4.4 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature. Following this, the sample was digested overnight at 37 ˚C with tryps...

Importance: Findings from unbiased genetic studies have consistently implicated synaptic protein networks in Schizophrenia (Sz), but the molecular pathology at these networks and their potential contribution to the synaptic and circuit deficits thought to underlie disease symptoms remain unknown. Objective:To determine if protein levels are altered within synapses from primary auditory cortex (A1) of subjects with Sz; and if so, are these differences restricted to the synapse or present throughout the grey matter?Design: A paired case-control design was utilized for this study. Biochemical fractionaltargeted Mass Spectrometry (MS) was used to measure the levels of >350 proteins in A1 grey matter homogenate and synaptosome preparations, respectively. All experimenters were blinded to diagnosis at every stage of sample preparation, MS analysis, and raw data processing. The effects of postmortem interval (PMI) and antipsychotic drug treatment on protein levels were assessed in mouse and monkey models, respectively.Setting: All cases were recruited from a single site, The Allegheny County Office of the Medical Examiner, and all tissues were processed at the University of Pittsburgh.Participants: Brain specimens from all subjects were obtained during autopsies conducted at the Allegheny County Office of the Medical Examiner after receiving consent from the next-of-kin. An independent panel of experienced clinicians made consensus Diagnostic and Statistical Manual of Mental Disorders Fourth Edition diagnoses. Unaffected comparison subjects underwent identical assessments and were determined to be free of lifetime psychiatric illness. Each Sz subject was matched by sex, and as closely as possible for age and PMI, with one unaffected comparison subject. Main Outcomes and Measures:Primary measures were homogenate and synaptosome protein levels and their co-regulation network features. Prior to data collection we hypothesized: 1. That levels of canonical postsynaptic proteins in A1 synaptosome preparations would differ between Sz and control subjects; and 2. That these differences would not be explained by changes in total A1 homogenate protein levels.Results: Mean subject age was 48 years for both groups with a range of 17-83; each group included 35 males and 13 females; mean PMI was 17.7 hours in controls and 17.9 in Sz. We observed robust alterations (q < 0.05) in synaptosome levels of canonical mitochondrial and postsynaptic proteins that were highly co-regulated and not readily explained by postmortem interval, antipsychotic drug treatment, synaptosome yield, or underlying alterations in homogenate protein levels. Conclusions and Relevance:Our findings indicate a robust and highly coordinated rearrangement of the synaptic proteome likely driven by aberrant synaptic, not cell-wide, proteostasis. In line with unbiased genetic findings, our results identified alterations in synaptic levels of postsynaptic proteins, providing a road map to identify the specific cells and circuits that are impaired in Sz A1.