Quantitative clinical proteomic study of autopsied human infarcted brain specimens to elucidate the deregulated pathways in ischemic stroke pathology
Graphical abstract
Introduction
Ischemic stroke, still lacking an effective neuroprotective therapy, continues to be a major socioeconomic burden throughout the world. The successive clinical failures in new drug development along with a bleak epidemiological landscape have led to the emergence of several novel concepts. Increased emphasis has been given on the neurovascular unit and the interaction between its different components instead of the neuron alone. The participation of peripheral organs through bidirectional communications with the brain following an ischemic stroke has been highlighted [1]. Consequently, a combination therapy instead of a mono-therapy or the incorporation of a multi-target drug has been suggested [2]. Therefore, a global comprehensive study of the altered system(s) is the prerequisite to understand and tackle this multifactorial disorder.
Quantitative neuroproteomics has emerged as an advanced technique in the post-genomic era for unbiased probing of perturbed pathways in complex biological systems which is not possible by traditional reductionist approaches. Recently an iTRAQ-based neuroproteomic strategy has been applied to improve our mechanistic understanding of axon injury and Alzheimer's disease (AD) [3], [4]. In the area of ischemic stroke, we pioneered the employment of the iTRAQ-2D-LC–MS/MS-based quantitative proteomic strategy to study an in vitro neuronal model of ischemic penumbra [5] and an in vivo rodent model of transient focal stroke [6] to delineate the molecular complexities and to propose potential therapeutic targets for further studies. However, the direct translation of a hypothesis generated through pre-clinical models into clinical application is limited by the complexity and fundamental difference of the biological systems as exemplified by successive clinical failures in translating the targets. The reasons may be related to the extreme heterogeneity of human stroke, the absence of long-term environmental influence or co-morbidity or risk factors in the pre-clinical models or flaws in the clinical trial design [7]. Moreover, the majority of the animal studies used rodents that are phylogenetically separated from humans millions of years ago [8]. Hence, a wealth of bedside-back-to-bench data using post-mortem brain specimens is needed to exclude model-specific artifacts and to identify stroke-specific ‘target space’ encompassing different pathological events and brain cell types. However, studying the molecular pathophysiology of ischemic stroke using clinical samples is confronted with various difficulties and challenges that may account for pre-analytical or ex vivo stresses [9]. Additionally, for a pathological condition like stroke, considerable neuropathological and diagnostic expertise is needed as only specific locations have to be targeted for isolation. Unsurprisingly, although several methodological studies using different sub-structures of human brain have been reported for profiling either whole proteome [10], [11], [12] or sub-proteome [13], only a few studies addressed pathological questions related to ischemic stroke [14], [15]. The first one used a targeted approach by combining laser microdissection with protein array to focus on different matrix metalloproteinases [14]. The other one is the only reported profiling study to examine different areas of ischemic brain where 2DE with an off-line MALDI MS/MS was applied [15]. Using a complementary online shotgun proteomic approach, such as the multi-dimensional protein identification technology [16] with isobaric labeling of peptides (i.e. iTRAQ) will allow simultaneous quantification of proteins in up to 8 samples by avoiding intra-experimental variation.
Given that the iTRAQ-2D-LC–MS/MS strategy has already been successfully applied on the pre-clinical models of ischemic stroke in our laboratory [5], [6] here we extend to quantitatively study three well-documented and pathologically-characterized infarcted tissues of human brain by an 8-plex iTRAQ experiment. Ethnicity-, age-, sex-, location- and post-mortem interval (PMI)-matched areas of the non-infarcted brains were used as control for an appropriate comparison. The location-matched perturbed proteome obtained from the three pairs of infarct–control specimens were validated by comparing the expression of each protein with an independent control and infarcted sample included in the same iTRAQ experiment during the data analysis. Mining the data set revealed a mitochondrial dysfunction that may have been caused by the uncoupling of cytosolic and mitochondrial metabolism along with an iron-mediated oxidative imbalance.
Section snippets
Reagents
Unless indicated, all reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Autopsy and sampling of brain tissues
Post-mortem brain samples of 7 subjects (infarct, n = 3; control, n = 4) were obtained from the brain bank of the Choju Medical Institute of Fukushimura Hospital (Toyohashi, Aichi, Japan). The protocols utilized were approved by the local ethics committee of the Fukushimura Hospital. The scientific use of human material was conducted in accordance with the Declaration of Helsinki, and informed consent was obtained from
Quality control of the iTRAQ data set
To minimize the false positive identification of proteins, an unused ProtScore ≥ 2 (confidence > 99%) was used as the qualification criteria. With this criterion, 1520 proteins were identified with a FDR of 0.1% (Supplemental Table 2). The average number of unique peptides (having a confidence level of > 95%) detected per protein was 6.96 and more than 30% of the proteins had ≥ 5 unique peptides that was similar to our previous findings [5], [6]. The quality of the sample preparation was determined
Discussion
Proteomic characterization of post-mortem samples has been used widely in the recent past to generate valuable bedside data from subjects of different neurodegenerative or psychiatric disorders (e.g. Alzheimer's, Parkinson's disease, and schizophrenia) [28], [32]. In contrast, the area of clinical proteomics of ischemic stroke is lagging behind as few proteomic studies used human brain tissue [14], [15]. Here, we functionally analyzed the deregulated consensus proteome of human brain infarcts
Limitations
These patients are a surviving population following a non-fatal attack of ischemic stroke, thus representing cases of chronic ischemia. Hence, the duration between the onset of stroke symptoms and death is variable and not controlled. The patency of the arteries in the infarcted region was also not known at the time of death [15]. In addition, the small sample size and advanced age group (average age > 80 y) of the subjects could limit the general applicability of the data generated from this
Concluding remarks
This study reports the deregulated infarct proteome perturbed irrespective of location using an iTRAQ-2D-LC–MS/MS based quantitative proteomic profiling approach. The failure of mitochondrial energy metabolism is apparent through the down-regulation of the MAS/PDC-complex, enzymes of glycolysis and OxPhos that is most likely caused by the death of brain cells and loss of electrophysiological activities of the neurons in the infarct. The significant elevation of ferritin may be related to the
Conflict of interest statement
The authors have declared no conflict of interest.
Acknowledgment
We thank Mr. T. Kanesaka, Mr. N. Ogawa, Mr. Y. Taniand and Ms. C. Taniguchi (all at the Choju Medical Institute, Fukushimura Hospital) for the technical assistance, patient care, sampling and tissue data acquisition. This research is supported by the Singapore National Research Foundation under its NMRC-CBRG and administered by the Singapore Ministry of Health's National Medical Research Council (NMRC/CBRG/0004/2012).
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