Proteomic Analysis of Site Specific Changes in Brain of Schizophrenics

Abstract

Schizophrenia is a complex multifactorial neuropsychiatric disorder which affects 1% of the population worldwide. It is typically characterized by hallucinations, delusions, social withdrawal and cognitive decline with disrupted neuronal excitability and impaired structural and functional connectivity. Despite extensive research for decades, the underlying molecular mechanisms associated with pathology of schizophrenia still remain ambiguous and needs further investigation. Recent efforts through proteomics based studies, suggested a breakthrough in improving our understanding of the dysregulated proteins attenuating the physiological conditions. The present study was undertaken to compare regional brain proteome in schizophrenic patients and age-matched controls, specifically the substantia nigra region which has never been reported earlier despite its role in dopamine transmission. In addition, the aim was to examine whether changes in protein expression of brain regions can be linked with their interacting partners, through Ingenuity Pathway Analysis (IPA) and to the extent of pathology. Proteomic analysis aims at simultaneous separation, quantification and identification of proteins among three distinct brain regions i.e. substantia nigra, hippocampus, and prefrontal cortex. The regional brain proteome expression profiling can potentially uncover perturbations associated with this complex psychiatric disorder, using two dimensional electrophoresis, in combination with Nano LC-MS/MS. The data provides a conceptual insight into the molecular mechanisms associated with the differential protein expression and have established a preliminary proteome map of site specific changes in schizophrenia. The proteomic findings were correlated with the pathway enrichment and cross talk of co and contra regulated proteins of the three brain regions studied. A total of 1443 protein spots were detected on 2DE gels in both Schizophrenic and control brain, out of which fifty eight were found to be significantly differentially expressed (p<0.05), representing twenty six proteins of substantia nigra, fourteen of hippocampus and eighteen of prefrontal cortex. A comparison of regional proteome maps revealed thirteen co/contra regulated proteins of substantia nigra, hippocampus, and prefrontal cortex. The seven differentially expressed proteins namely ETFB, DCXR, HAGH, BLVRB, MYDGF, DEST and VPS29 are reported for the very first time with regionally variable expression in schizophrenic patients. xxi An extensive set of functional annotation pathways using IPA and PANTHER software revealed the enrichment of identified proteins in energy metabolism, cytoskeleton, transportation, cell cycle, redox dysregulation, apoptosis, ubiquitination and signaling cascades. Interestingly, the merge network obtained from IPA indicates several molecules appearing as prime molecules including APP, NFkb complex, ERK1/2, TNF, and Akt. It may suggest that the unconventional activities of NF kb complex, APP, ERK1/2 and glutathione such as metabolic regulation and redox imbalance may also be a critical feature in the pathophysiology of schizophrenia. Moreover, the overlapping networks with top interactions of identified focused molecules and their interacting partners, exhibit a complex molecular cross talk at functional platform, which may clearly demonstrate the complex nature of this disorder. Furthermore, this may suggest that not a single pathway is responsible; rather multiple pathways are interconnected and disrupted to initiate the deleterious effects associated with schizophrenia. The study further encompasses the role of glycosylation, an important post translational modification (PTM) of proteins, in schizophrenia pathology. Interesting observations were also made by analyzing the regional profile of glycosylated proteins utilizing Digoxigenin glycan kit method. Evidence of significant alterations in the glycosylated patterns of co and contra regulated proteins of the three regions specifically support the notion that there is a substantial global contribution of glycosylation in the pathology of schizophrenia. These functionally aberrant glycosylated proteins are enriched in cytoskeleton, mitochondrial integrity processes, cell adhesion, protein ubiquitination and calcium metabolism pathways. To conclude, the results emphasize the importance of studying substantia nigra proteome and its alteration in comparison with differentially expressed proteins of hippocampus and prefrontal cortex. By better understanding the potential role of proteins and involvement of related aberrant pathways in schizophrenia, new aspects of the pathology and therapeutic targets may be discovered.