S-glutathionylation profile of microglia stimulated by amyloid oligomers.

Introduction. Microglia are the resident macrophage-like cells of the central nervous system with a broad role in the brain's innate immunity and in inflammatory neuropathologies. A central role for these cells in the onset and progression of neurodegenerative pathologies, such as Alzheimer’s disease, has been assessed. Since oxidative stress is a condition associated with this disease, we investigated if treatment of microglia by amyloid beta (Aβ) oligomers could affect cellular redox balance and alter the profile of redox-dependent post-translational modification. Methods. Murine microglial cell line BV2 were treated with 50 µM of Aβ25-35 peptide. Glutathione was measured by HPLC equipped with an electrochemical detector. Carbonyls content was assessed by colorimetric assay with 2,4-dinitrophenylhydrazine. S-glutathionylated proteins were identified isolating glutathionylated peptides by an affinity column. Proteins were then identified by a “bottom up” approach using an LTQ-Orbitrap mass spectrometer. Results. BV2 stimulated by Aβ oligomers showed an increase in reactive carbonyls of proteins and in oxidized glutathione while the total content of glutathione was significantly decreased indicating a steady redox unbalance. Thus we further investigated whether this scenario might change the redox-dependent S-glutathionylation profile in these cells. For this purpose, we use a combined approach of affinity labelling and mass spectrometry. We were able to indentify more than 20 modified proteins specific for the proteome of activated BV2 with respect to control cells. These differentially modified proteins belong to different functional classes, such as cytoskeletal and chaperone known to be involved in microglia activation. Conclusion. These findings indicate that the redox unbalance induced by Aβ oligomers in microglia may cause a novel pattern of S-glutathionylation of cellular proteins, that can modulate their function and possibly switch towards new signalling pathways