An integrated proteomic and metabolomic approach to investigate cerebral ischemic preconditioning

Scornavacca, Giacomo Maria (2013). An integrated proteomic and metabolomic approach to investigate cerebral ischemic preconditioning. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ef06

Abstract

The molecular mechanism that leads to ischemic preconditioning and hence to ischemic tolerance, are not completely understood although it is clear that multiple effectors and pathways contribute to the instauration of this neuroprotective profile. To study the mechanism/pathway involved in the ischemic tolerance, brain proteins, plasma proteins and plasma metabolites were analyzed in preconditioning stimulus (7'Middle Cerebral Artery occlusion or 7'MCAo), in severe stroke and (permanent Middle Cerebral Artery occlusion or pMCAo) and in preconditioned (7'MCAo/pMCAo) mouse model.

A conventional 2-DE approach was used to study technical replicates of pooled brain proteins revealing an involvement of energy metabolism, mitochondrial electron transport, synaptic vesicle transport and antioxidant processes; moreover network analysis suggested an involvement of the androgen receptor that was validated on technical replicates of pooled brain proteins by western blot analysis revealing an increased expression in preconditioned stimulus animals (7'MCAo).

Plasma proteins were analyzed using a i-DE LC-MS/MS approach on technical replicates of pooled plasma proteins revealing decreased levels of epidermal growth factor receptor (EGFR) and increased levels of insuline like growth factor acid labile subunit (IGFALS), which expression was paralleled by increased insulin like growth factor 1 (IGFi) plasma concentration, as validated by ELISA on biological replicates, in preconditioning stimulus animals (7'MCAo).

Finally an untarget metabolomics analysis was applied to technical replicates of pooled plasma proteins revealing fatty acid oxidation and branched-chain aminoacid metabolism as the main biological processes modulated in ischemic tolerance and highlighted an involvement of the aminoacid leucine, carnitine esters and adenosine.

The results described in this thesis represents the first application of both proteomic and metabolomic approaches in cerebral ischemic sets, highlighting the androgen receptor as an important mediator between proteins and metabolites and adding new evidence to the current knowledge on ischemic preconditioning that may represent a starting point for future experiments on investigating candidate pathways that relate to the Androgen receptor.

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