Quantitative clinical proteomic study of autopsied human infarcted brain specimens to elucidate the deregulated pathways in ischemic stroke pathology.

Ischemic stroke, still lacking an effective neuroprotective therapy is the third leading cause of global mortality and morbidity. Here, we have applied an 8-plex iTRAQ-based 2D-LC-MS/MS strategy to study the commonly regulated infarct proteome from three different brain regions (putamen, thalamus and the parietal lobe) of female Japanese patients. Infarcts were compared with age-, post-mortem interval- and location-matched control specimens. The iTRAQ experiment confidently identified 1520 proteins with 0.1% false discovery rate. Bioinformatics data mining and immunochemical validation of pivotal perturbed proteins revealed a global failure of the cellular energy metabolism in the infarcted tissues as seen by the parallel down-regulation of proteins related to glycolysis, pyruvate dehydrogenase complex, TCA cycle and oxidative phosphorylation. The concomitant down-regulation of all participating proteins (SLC25A11, SLC25A12, GOT2 and MDH2) of malate-aspartate shuttle might be responsible for the metabolic in-coordination between the cytosol and mitochondria resulting in the failure of energy metabolism. The levels of proteins related to reactive gliosis (VIM, GFAP) and anti-inflammatory response (ANXA1, ANXA2) showed an increasing trend. The elevation of ferritin (FTL, FTH1) may indicate an iron-mediated oxidative imbalance aggravating the mitochondrial failure and neurotoxicity. The deregulated proteins could be useful as potential therapeutic targets or biomarkers for ischemic stroke. BIOLOGICAL SIGNIFICANCE: Clinical proteomics of stroke has been lagging behind other areas of clinical proteomics like Alzheimer's disease or schizophrenia. Our study is the first quantitative clinical proteomics study where iTRAQ-2D-LC-MS/MS has been utilized in the area of ischemic stroke to obtain a comparative profile of human ischemic infarcts and age-, sex-, location- and post-mortem interval-matched control brain specimens. Different pathological attributes of ischemic stroke well-known through basic and pre-clinical research such as failure of cellular energy metabolism, reactive gliosis, activation of anti-inflammatory response and aberrant iron metabolism have been observed at the bedside. Our dataset could act as a reference for similar studies done in the future using ischemic brain samples from various brain banks across the world. A meta-analysis of these studies could help to map the pathological proteome specific to ischemic stroke that will guide the scientific community to better evaluate the pros and cons of the pre-clinical models for efficacy and mechanistic studies. Infarct being the core of injury should have the most intense regulation for several key proteins involved in the pathophysiology of ischemic stroke. Hence, a part of the up-regulated proteome could leak into the general circulation that may offer candidates of interest as potential biomarkers. In support of our proposed hypothesis, we report ferritin in the current study as one of the most elevated proteins in the infarct, which has been documented as a biomarker in the context of ischemic stroke by an independent study. Overall, our approach has the potential to identify probable therapeutic targets and biomarkers in the area of ischemic stroke.