Dataset Information

Single Cell Immuno-laser Microdissection Coupled to Label-free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia

ABSTRACT: Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological disorders. The use of biomarkers in stroke might have a major impact in patients’ management, both in the diagnosis and the prognosis. Thus, the analysis of the brain proteome is a straightforward approach for the identification of specific biomarkers and for the detection of potential new therapeutic targets for neuroprotection or recovery strategies. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed in a label-free manner on a LTQ Orbitrap Velos Pro MS following a gas phase fractionation approach for precursor ion selection. Ninety one proteins were identified only in neurons, 261 proteins only in BBB and 260 proteins in both cell types (peptides≥2). Gene ontology analyses revealed the alteration of metabolic processes after brain ischemia and also changes regarding catalytic activity and binding. Antioxidant activity and enzyme regulator activity appeared as cell-specific altered molecular functions. A total of 25 proteins showing p<0.05 and fold-change>|2| between IC and CL areas were considered significant in this study: 11 in neurons, 11 in BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistochemistry in independent brains. The MS findings were verified for neuronal SAHH2 and SRSF1 and for EAA2 in both cell types. In addition, SAHH2 showed its potential as prognostic biomarker when analyzed early in plasma of ischemic stroke patients. Therefore, our results will contribute to increase the knowledge about the molecular mechanisms of ischemic stroke pathology and launch new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Neuron, Endothelial Cell

DISEASE(S): Ischemic Stroke

SUBMITTER: Víctor Llombart

LAB HEAD: Joan Montaner

PROVIDER: PXD003519 | Pride | 2017-12-04

REPOSITORIES: pride

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			Action	DRS
	2012_03_06_CAP_OT_06_TEG_16_02_1.raw	Raw
	2012_03_06_CAP_OT_07_TEG_16_02_2.raw	Raw
	2012_03_06_CAP_OT_08_TEG_16_02_3.raw	Raw
	2012_03_06_CAP_OT_09_TEG_16_02_4.raw	Raw
	2012_03_06_CAP_OT_11_TEG_16_11_1.raw	Raw

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Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia.

García-Berrocoso Teresa T Llombart Víctor V Colàs-Campàs Laura L Hainard Alexandre A Licker Virginie V Penalba Anna A Ramiro Laura L Simats Alba A Bustamante Alejandro A Martínez-Saez Elena E Canals Francesc F Sanchez Jean-Charles JC Montaner Joan J

Molecular & cellular proteomics : MCP 20171113 1

Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contral ...[more]

PMID: 29133510

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Project description:1. Rationale: Stroke is still a major cause of death and disability worldwide. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches, such as proteomics and transcriptomics, might aid to deepen the knowledge of stroke at the molecular level. Moreover, the integration of omics data is needed to depict the existing interaction between different molecular units and interpret the results in a global biological context. 2. Objective: Our aim was to identify and verify protein and gene expression changes that occur in the human brain after ischemia through an integrative approach to join and combine the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. 3. Methods and results: Proteomics and transcriptomics discovery studies were performed in human brain samples from deceased stroke patients, unveiling 128 proteins and 2716 genes significantly dysregulated after cerebral ischemia. Afterwards, integrative bioinformatics analyses joining both datasets exposed several canonical pathways altered in the ischemic area, such as neurotransmitters release or matrisome regulation, highlighting the most influential molecules in these pathways. To conduct the verification phase, 28 genes and 9 proteins were selected to be validated in independent human brain samples by means of orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2 and IL8. Finally, the blood circulating levels of the validated protein candidates were explored in ischemic stroke patients over the acute phase of the disease. Protein fluctuations along the first week after onset were detected for A1AG1 and A1AT. 4. Conclusion: The results presented here expand the knowledge of stroke pathology, revealing new key molecules that can be further explored as biomarkers and/or therapeutic targets of ischemic stroke.

Project description:Stroke remains a major leading cause of death and disability worldwide. Despite continuous advances, the identification of key molecular signatures of ischemic stroke within the hyper-acute phase of the disease is still of primary interest for a real translational research on stroke diagnosis, prognosis and treatment. High-throughput - omics technologies are enabling large-scale studies on stroke pathology at different molecular levels. Data integration resulting from these -omics approaches is becoming crucial to unravel the interactions among all different molecular elements and highly contribute to interpret all findings in a complex biological context. Here, we have used advanced data integration methods for multi-level joint analysis of transcriptomics and proteomics datasets depicted from the mouse brain 2h after cerebral ischemia. By modeling network-like correlation structures, we identified a set of differentially expressed genes and proteins by ischemia with a relevant association in stroke pathology. The ischemia-induced deregulation of 10 of these inter-correlated elements was successfully verified in a new cohort of ischemic mice, and changes in their expression pattern were also evaluated at a later time-point after cerebral ischemia. Of those, CLDN20, GADD45G, RGS2, BAG5 and CTNND2 were highlighted and evaluated as potential blood biomarkers of cerebral ischemia in blood samples from ischemic and sham-control mice and from ischemic strokes and other patients presenting stroke-mimicking conditions. Our findings indicated that CTNND2 and GADD45G levels in blood within the first hours after ischemic stroke might be potentially useful to discriminate ischemic strokes from mimics and to predict patients’ poor outcome after stroke, respectively. In summary, we have here used for the first time an integrative approach to elucidate by means of biostatistical tools key elements of the initial stages of the stroke pathophysiology, highlighting new outstanding proteins that might be further considered as blood biomarkers of ischemic stroke.

Project description:Cerebral ischemia/reperfusion injury (CI/RI), including neurological behavior deficits, cerebral infarction, blood-brain barrier (BBB) dysfunction, and neuroinflammation, et al. is a severe challenge in treatment of ischemic stroke. Traditional Chinese medicine (TCM) with the characteristics of multi-components and multi-functions for multiple targets/pathways has been historically used in the treatment of stroke and post stroke recovery in China. QiShenYiQi (QSYQ), a representative component-based Chinese medicine, is capable of reducing organ injury caused by ischemia/reperfusion via multiple mechanisms. Recently, we have previously reported that the positive action of QSYQ against the acute phase of CI/RI is partly via modulation of neuroinflammatory response. However, the effects and underlying mechanisms of QSYQ on subacute phase of CI/RI remain unknown, which are aimed to investigate in present study. The pharmacological action of QSYQ treatment on brain damage was estimated in the experimental stroke rats underwent 90 min ischemia and 8 days reperfusion by assessing the neurological and locomotor deficits, cerebral infarction, brain edema, and BBB integrity. Furthermore, we used TMT-based quantitative proteomics technology to identify significantly differentially expressed proteins following QSYQ treatment, which could give important clues for revealing the possible mechanism underlying the positive role of QSYQ in CI/RI. Besides, immunohistochemistry, western blot analysis, RT-qPCR, and rat ELISA kits were executed to further verify the accuracy of proteomics analysis results. As a consequence, we found that treatment with QSYQ (600mg/kg) for 7 days obviously improved neurological and behavioral impairment, attenuated infarct volume and brain edema, and alleviated BBB breakdown in stroke rats. Bioinformatics analysis suggested that differentially expressed protein galectin-3 mediated inflammatory response was closely related to the beneficial effect of QSYQ. Results of immunohistochemistry, western blot analysis and RT-qPCR showed that the model rats treated with QSYQ (600mg/kg) markedly downregulated the mRNA and protein expression level of galectin-3 in brain tissues compared to the untreated stroke rats. In addition, the decrease of mRNA level in brain tissues and contents in serum of TNF-α and IL-6 following QSYQ treatment were also observed. These interesting finding manifested that the effective action of QSYQ against subacute phase of CI/RI was partly via regulating galectin-3 mediated inflammatory reaction.