The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione -transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

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We have found recently that dendritic spine extension is inhibited through acrolein conjugation with αand β-tubulin proteins during brain infarction. In this current study, we looked for other acrolein-conjugated proteins in the 100,000g precipitate fraction, to clarify how cytoskeleton structure is modified by acrolein. Acroleinconjugated proteins were sought from acrolein-treated mouse FM3A and Neuro2a cells and from tissues isolated from mouse brain infarction. It was found that vimentin was conjugated with acrolein, and the conjugated amino acid residue was Cys328, which is the only Cys residue in vimentin. It was also found that Cys207, 257, 285, and Lys118 in actin, another cytoskeleton protein, were conjugated with acrolein. The structure and localization of vimentin and actin filaments were changed greatly in infarct brain in photochemically induced thrombosis model mice and in acrolein-treated Neuro2a cells. In addition, degradation of cytoskeleton proteins was accelerated in the order vimentin > tubulin > actin in mouse brain infarction. These findings indicate that a dysfunction of the cytoskeleton by acrolein is strongly involved in the tissue damage during brain infarction, together with the apoptosis caused by glyceraldehyde-3-phosphate dehydrogenase and protein degradation by matrix metalloproteinase-9.

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx — a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells — functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans (GAGs), a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis (PIT) mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine (NAC)+GAG oligosaccharides as compared to NAC administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR modified amino acid residues of proHPSE using nano LC-MS/MS, suggesting that ACR modification of Lys139 (6- kDa linker), and Lys107 and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part, is attributed to the activation of proHPSE. Since proHPSE, but not HPSE, localizes outside cells by binding with HS proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

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