Juliana S. Cassoli

Zika virus disrupts molecular fingerprinting of human neurospheres

Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

The protein interactome of collapsin response mediator protein-2 (CRMP2/DPYSL2) reveals novel partner proteins in brain tissue

Collapsin response mediator protein‐2 (CRMP2) is a CNS protein involved in neuronal development, axonal and neuronal growth, cell migration, and protein trafficking. Recent studies have linked perturbations in CRMP2 function to neurodegenerative disorders such as Alzheimers disease, neuropathic pain, and Batten disease, and to psychiatric disorders such as schizophrenia. Like most proteins, CRMP2 functions though interactions with a molecular network of proteins and other molecules.

Effect of MK-801 and Clozapine on the Proteome of Cultured Human Oligodendrocytes

Separate lines of evidence have demonstrated the involvement of N-methyl-D-aspartate (NMDA) receptor and oligodendrocyte dysfunctions in schizophrenia. Here, we have carried out shotgun mass spectrometry proteome analysis of oligodendrocytes treated with the NMDA receptor antagonist MK-801 to gain potential insights into these effects at the molecular level. The MK-801 treatment led to alterations in the levels of 68 proteins, which are associated with seven distinct biological processes. Most of these proteins are involved in energy metabolism and many have been found to be dysregulated in previous proteomic studies of post-mortem brain tissues from schizophrenia patients. Finally, addition of the antipsychotic clozapine to MK-801-treated oligodendrocyte cultures resulted in changes in the levels of 45 proteins and treatment with clozapine alone altered 122 proteins and many of these showed opposite changes to the MK-801 effects. Therefore, these proteins and the associated energy metabolism pathways should be explored as potential biomarkers of antipsychotic efficacy. In conclusion, MK-801 treatment of oligodendrocytes may provide a useful model for testing the efficacy of novel treatment approaches.

Differential proteome and phosphoproteome may impact cell signaling in the corpus callosum of schizophrenia patients

Schizophrenia is a multifactorial disease in both clinical and molecular terms. Thus, depicting the molecular aspects of the disease will contribute to the understanding of its biochemical mechanisms and consequently may lead to the development of new treatment strategies. The protein phosphorylation/dephosphorylation switch acts as the main mechanism for regulating cellular signaling. Moreover, approximately onethird of human proteins are phosphorylable. Thus, identifying proteins differentially phosphorylated in schizophrenia postmortem brains may improve our understanding of the molecular basis of brain function in this disease. Hence, we quantified the phosphoproteome of corpus callosum samples collected post mortem from schizophrenia patients and healthy controls. We used state-of-the-art, bottom-up shotgun mass spectrometry in a two-dimensional liquid chromatography-tandem mass spectrometry setup in the MSE mode with label-free quantification. We identified 60,634 peptides, belonging to 3283 proteins. Of these, 68 proteins were differentially phosphorylated, and 56 were differentially expressed. These proteins are mostly involved in signaling pathways, such as ephrin B and ciliary neurotrophic factor signaling. The data presented here are novel because this was the very first phosphoproteome analysis of schizophrenia brains. They support the important role of glial cells, especially astrocytes, in schizophrenia and help to further the understanding of the molecular aspects of this disease. Our findings indicate a need for further studies on cell signaling, which might shape the development of treatment strategies.

Consensus paper of the WFSBP Task Force on Biological Markers: Criteria for biomarkers and endophenotypes of schizophrenia, part III: Molecular mechanisms

Abstract Objectives: Despite progress in identifying molecular pathophysiological processes in schizophrenia, valid biomarkers are lacking for both the disease and treatment response. Methods: This comprehensive review summarises recent efforts to identify molecular mechanisms on the level of protein and gene expression and epigenetics, including DNA methylation, histone modifications and micro RNA expression. Furthermore, it summarises recent findings of alterations in lipid mediators and highlights inflammatory processes. The potential that this research will identify biomarkers of schizophrenia is discussed. Results: Recent studies have not identified clear biomarkers for schizophrenia. Although several molecular pathways have emerged as potential candidates for future research, a complete understanding of these metabolic pathways is required to reveal better treatment modalities for this disabling condition. Conclusions: Large longitudinal cohort studies are essential that pair a thorough phenotypic and clinical evaluation for example with gene expression and proteome analysis in blood at multiple time points. This approach might identify biomarkers that allow patients to be stratified according to treatment response and ideally also allow treatment response to be predicted. Improved knowledge of molecular pathways and epigenetic mechanisms, including their potential association with environmental influences, will facilitate the discovery of biomarkers that could ultimately be effective tools in clinical practice.

Employing proteomics to unravel the molecular effects of antipsychotics and their role in schizophrenia.

Schizophrenia is an incurable neuropsychiatric disorder managed mostly by treatment of the patients with antipsychotics. However, the efficacy of these drugs has remained only low to moderate despite intensive research efforts since the early 1950s when chlorpromazine, the first antipsychotic, was synthesized. In addition, antipsychotic treatment can produce often undesired severe side effects in the patients and addressing these remains a large unmet clinical need. One of the reasons for the low effectiveness of these drugs is the limited knowledge about the molecular mechanisms of schizophrenia, which impairs the development of new and more effective treatments. Recently, proteomic studies of clinical and preclinical samples have identified changes in the levels of specific proteins in response to antipsychotic treatment, which have converged on molecular pathways such as cell communication and signaling, inflammation and cellular growth, and maintenance. The findings of these studies are summarized and discussed in this review and we suggest that this provides validation of proteomics as a useful tool for mining drug mechanisms of action and potentially for pinpointing novel molecular targets that may enable development of more effective medications.

Proteomics and molecular tools for unveiling missing links in the biochemical understanding of schizophrenia

Psychiatric disorders are one of the biggest burdens to society, with significant personal and economical costs. Schizophrenia (SCZ), among them, is still poorly understood, and its molecular characterization is crucial to improve patients’ diagnosis and treatment. The combination of genetic, biochemical, and environmental factors leads to systemic alterations, which are yet to be fully comprehended. Thus, understanding those missing links by connecting some molecular reports of SCZ is essential. From postmortem brain to animal models and cell culture, new tools are emerging, including recent advances in proteomics, and there is a need to apply them to solve these problems. Here, we review some of those features, mainly related to where proteomics could help, and discuss whether those new technologies could and should be applied to psychiatric disorder studies.

MK-801-Treated Oligodendrocytes as a Cellular Model to Study Schizophrenia

Glutamate is the most important excitatory neurotransmitter in the brain. The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is found both in neurons and glial cells such as oligodendrocytes, which have been shown to be dysfunctional in schizophrenia. For this reasons, the oligodendrocyte MO3.13 cell line has been used to study glutamatergic dysfunction as a model of schizophrenia using the NMDA receptor antagonists such as MK-801 to block receptor function. Here, we describe a comprehensive protocol for culturing and carrying out proteomic analyses of MK-801-treated MO3.13 cells as a means of identifying potential new biomarkers and targets for drug discovery in schizophrenia research.

Co-immunoprecipitation for Deciphering Protein Interactomes

A single protein is often capable of binding with many partners, enabling potential effects on either protein, such as modifying its expression or activity. However, due to the complex nature of in vivo systems, it is often difficult to perform nontargeted assays with a protein of interest. Methods in discovery proteomics must be used to find potential interactors to pave the way for additional, more focused studies. This protocol describes the biological steps needed to create an interactome focused on a single protein target through co-immunoprecipitation.

Unveiling alterative splice diversity from human oligodendrocyte proteome data

Oligodendrocytes produce and maintain the myelin sheath of axons in the central nervous system. Because misassembled myelin sheaths have been associated with brain disorders such as multiple sclerosis and schizophrenia, recent advances have been made towards the description of the oligodendrocyte proteome. The identification of splice variants represented in the proteome is as important as determining the level of oligodendrocyte-associated proteins. Here, we used an oligodendrocyte proteome dataset deposited in ProteomeXchange to search against a customized protein sequence file containing computationally predicted splice variants. Our approach resulted in the identification of 39 splice variants, including one variant from the GTPase KRAS gene and another from the human glutaminase gene family. We also detected the mRNA expression of five selected splice variants and demonstrated that a fraction of these have their canonical proteins participating in direct protein-protein interactions. In conclusion, we believe our findings contribute to the molecular characterization of oligodendrocytes and may encourage other research groups working with central nervous system disorders to investigate the biological significance of these splice variants. The splice variants identified in this study may encode proteins that could be targeted in novel treatment strategies and diagnostic methods. SIGNIFICANCE Several disorders of the central nervous system (CNS) are associated with misassembled myelin sheaths, which are produced and maintained by oligodendrocytes (OL). Recently, the OL proteome has been explored to identify key proteins and molecular functions associated with CNS disorders. We developed an innovative approach to select, with a higher level of confidence, a relevant list of splice variants from a proteome dataset and detected the mRNA expression of five selected variants: EEF1D, KRAS, MFF, SDR39U1, and SUGT1. We also described splice variants extracted from OL proteome data. Among the splice variants identified, some are from genes previously linked to CNS and related disorders. Our findings may contribute to oligodendrocyte characterization and encourage other research groups to investigate the biological role of splice variants and to improve current treatments and diagnostic methods for CNS disorders.