Daniel Martins-de-Souza

Prefrontal cortex shotgun proteome analysis reveals altered calcium homeostasis and immune system imbalance in schizophrenia

Schizophrenia is a complex disease, likely to be caused by a combination of serial alterations in a number of genes and environmental factors. The dorsolateral prefrontal cortex (Brodmanns Area 46) is involved in schizophrenia and executes high-level functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts and attitudes, correct social behavior and personality expression. Global proteomic analysis of post-mortem dorsolateral prefrontal cortex samples from schizophrenia patients and non-schizophrenic individuals was performed using stable isotope labeling and shotgun proteomics. The analysis resulted in the identification of 1,261 proteins, 84 of which showed statistically significant differential expression, reinforcing previous data supporting the involvement of the immune system, calcium homeostasis, cytoskeleton assembly, and energy metabolism in schizophrenia. In addition a number of new potential markers were found that may contribute to the understanding of the pathogenesis of this complex disease.

Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis

Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease.

Proteome analysis of the thalamus and cerebrospinal fluid reveals glycolysis dysfunction and potential biomarkers candidates for schizophrenia

Schizophrenia (SCZ) is the result of DNA alterations and environmental factors, which together lead to differential protein expression and ultimately to the development of the illness. The diagnosis is based on clinical symptoms, and the molecular background of SCZ is not completely understood. The thalamus, whose dysfunction has been associated with SCZ based in diverse lines of evidences, plays for instance a pivotal role in the central nervous system as a relay center by re-distributing auditory and visual stimuli from diverse brain regions to the cerebral cortex. We analyzed the proteome of postmortem mediodorsal thalamus (MDT) samples from 11 SCZ patients and 8 non-SCZ individuals by using quantitative shotgun-mass spectrometry and two-dimensional gel electrophoresis. Our analyses identified 551 proteins, 50 of which showed significant differential expression. The main pathways affected by the differentially expressed proteins include energy metabolism, oligodendrocyte metabolism, and cytoskeleton assembly. The potential protein biomarkers candidates myelin basic protein and myelin oligodendrocyte protein were validated by Western blot in the MDT samples and also in cerebrospinal fluid from a separate set of samples of 17 first-episode SCZ patients and 10 healthy controls. The differential expression of μ-crystallin, protein kinase C-gamma, and glial fibrillary acidic protein were confirmed in MDT. Because we found several glycolysis enzymes to be differentially expressed, we measured the levels of pyruvate and NADPH and found them to be altered in MDT. The protein changes described here corroborate the importance of myelin/oligodendrocyte and energy metabolism in SCZ and highlight new potential biomarkers candidates that may contribute to the understanding of the pathogenesis of this complex disease.

Identification of proteomic signatures associated with depression and psychotic depression in post-mortem brains from major depression patients

Major depressive disorder (MDD) is a leading cause of disability worldwide and results tragically in the loss of almost one million lives in Western societies every year. This is due to poor understanding of the disease pathophysiology and lack of empirical medical tests for accurate diagnosis or for guiding antidepressant treatment strategies. Here, we have used shotgun proteomics in the analysis of post-mortem dorsolateral prefrontal cortex brain tissue from 24 MDD patients and 12 matched controls. Brain proteomes were pre-fractionated by gel electrophoresis and further analyzed by shotgun data-independent label-free liquid chromatography-mass spectrometry. This led to identification of distinct proteome fingerprints between MDD and control subjects. Some of these differences were validated by Western blot or selected reaction monitoring mass spectrometry. This included proteins associated with energy metabolism and synaptic function and we also found changes in the histidine triad nucleotide-binding protein 1 (HINT1), which has been implicated recently in regulation of mood and behavior. We also found differential proteome profiles in MDD with (n=11) and without (n=12) psychosis. Interestingly, the psychosis fingerprint showed a marked overlap to changes seen in the brain proteome of schizophrenia patients. These findings suggest that it may be possible to contribute to the disease understanding by distinguishing different subtypes of MDD based on distinct brain proteomic profiles.

The Role of Energy Metabolism Dysfunction and Oxidative Stress in Schizophrenia Revealed by Proteomics

Schizophrenia is a psychiatric illness that affects approximately 30 million people worldwide. Converging lines of evidence suggest that mitochondrial function may be compromised in this disorder, and this can lead to perturbations in calcium buffering, oxidative phosphorylation, increased production of reactive oxygen species, and apoptotic factors, which can, in turn, affect neuronal processes such as neurotransmitter synthesis and synaptic plasticity. Proteomics studies in brain and peripheral tissues of schizophrenia patients have provided considerable evidence and identified biomarker fingerprints corresponding to such pathways. Here we review the results of these studies with a focus on the biomarker pattern depicting alterations in energy metabolism and oxidative stress in this debilitating illness.

Proteome analysis of schizophrenia brain tissue

Abstract Objectives. Proteome analysis has emerged as a promising strategy to the identification of potential biomarkers and to further confirm the importance of certain pathways in the schizophrenia (SCZ) pathophysiology. Reviewing the results of 13 proteome studies in SCZ brain tissue, we aimed to provide information regarding potential proteins biomarkers as well as information about the pathophysiology of the disease. Methods and results. Using two-dimensional gel electrophoresis and shotgun mass spectrometry, 31 proteins were consistently found differentially expressed in the brains of SCZ patients. The most frequent protein alterations reported in SCZ were related to brain energy metabolism, brain plasticity, and synaptic function, processes that are thought to belong to the core of the biology of this disease. The recurrent identification and validation of inter-related protein clusters, determined in different samples and approaches, strongly reinforces the putative involvement of certain pathways in SCZ. Conclusions. The availability of reliable markers not only paves the way to the development of new therapeutic strategies but also points out the possibility of their use as peripheral blood markers that may potentially contribute to the early SCZ detection and early therapeutic intervention, both of which can reduce the social and cognitive consequences of the disease.

2DE : the phoenix of proteomics

UNLABELLED Given the rapid developments in mass spectrometry (MS) in terms of sensitivity, mass accuracy, and throughput, some have suggested that two-dimensional gel electrophoresis (2DE) may no longer be a method of choice for proteomic analyses. However, as recognition of issues with these newer shotgun-MS approaches grows, there is a fresh and growing regard for the maturity of 2DE-MS as a genuine top-down analytical approach, particularly as it resolves thousands of intact protein species in a single run, enabling the simultaneous analysis of total protein complement, including isoforms and post-translational modifications. Given the strengths of both, it is most appropriate to view these as complementary or at least parallel approaches: as proteins encompass a myriad of physico-chemical properties, and the real aim is to explore proteomes as deeply as possible, all available resolving strategies must be considered in terms of the complexity encountered. It is time to critically and constructively focus on the optimization and integration of existing techniques rather than simplistically suggesting that one should replace the other. Our intention here is thus to present an overview of protein resolving techniques, focusing on milestones associated with 2DE, including pros, cons, advances and variations, in particular relative to shotgun proteomic approaches. BIOLOGICAL SIGNIFICANCE Proteomic researchers recognize the importance of 2DE in the history of proteomics. But the latest developments in mass spectrometry-based techniques have led some researchers to retire 2DE in their labs. However, we argue here that 2DE-MS is a genuine top-down analytical approach. The significance of this discussion is to make proteomic researchers aware of the importance of this technique in a proteomic pipeline. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Sex-specific proteome differences in the anterior cingulate cortex of schizophrenia

Molecular knowledge about schizophrenia--a psychotic, multifactorial mental disorder that affects about 1% of the population worldwide--is limited and no diagnostic biomarkers are available. The comparative proteome analysis of human brain tissue from patients with schizophrenia and healthy controls may supply useful information on both the disorder and potential biomarkers candidates. Here, we present the results of our investigation of anterior cingulate cortex samples from 11 patients and 8 controls. We used two-dimensional gel electrophoresis combined with mass spectrometry, the most traditional approach to studying the proteome, to reveal the differentially expressed proteins in schizophrenia, and western blot to validate some interesting potential biomarker candidates such as dihydropyrimidinase-like 2 and alpha-crystallin, involved in a number of processes such as cytoskeleton arrangement. Most interesting is that our additional sex-specific proteome comparison showed that male and female schizophrenia patients present different patterns of proteome regulation, for instance for the proteins aldolase C, an enzyme of glycolysis, and glutamine synthetase that synthesizes glutamine, responsible for maintain glutamate levels. Our findings not only support previous findings but also indicate areas that warrant further study in schizophrenia.

Quantitative proteomics for investigating psychiatric disorders

The underlying pathophysiology of psychiatric disorders remains elusive. The use of quantitative proteomics to investigate disease‐specific protein signatures holds great promise to improve the understanding of psychiatric disorders and identify relevant biomarkers. In this review, we discuss quantitative proteomic approaches for elucidating molecular mechanisms of psychiatric disorders, i.e. anxiety, schizophrenia, bipolar disorder and depression, by studying specimens from animal models and patients. We present gel‐based, label‐free and stable isotope‐labeling methodologies and evaluate their strengths and limitations in the context of psychiatric research, with a focus on 15N metabolic labeling of live animals due to its increased accuracy and potential for future applications. We also review biomarker candidate validation methods and present quantitative proteomic studies from the literature that aim to disentangle the molecular pathobiology of psychiatric disorders and identify candidate biomarkers. Finally, we explore the applicability of implementing proteomic methods as a routine diagnostic tool in the clinical laboratory.

The role of proteomics in depression research

Depression is a severe neuropsychiatric disorder affecting approximately 10% of the world population. Despite this, the molecular mechanisms underlying the disorder are still not understood. Novel technologies such as proteomic-based platforms are beginning to offer new insights into this devastating illness, beyond those provided by the standard targeted methodologies. Here, we will show the potential of proteome analyses as a tool to elucidate the pathophysiological mechanisms of depression as well as the discovery of potential diagnostic, therapeutic and disease course biomarkers.