Mechanism of action of hydroxyl-docosahexaenoic acid molecule in the treatment of Alzheimer’s disease: identification of potential receptors in brain

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder very alive in today’s society. One of the drugs tested in animals which has shown neuroprotective properties is LP226A1 (a DHA α-hydroxylated derivative). The aim of this work was focused on the investigation of the mechanism of action of this drug. Specifically, our work was focused on searching for and isolating of LP226A1 binding proteins (LP226BPs) by separation of membrane proteins from mouse cortex by ion exchange (IEX) and hydrophobicity (HIC) chromatography using different sodium chloride (NaCl) concentrations as eluent. The resulting fractions from those techniques were tested in radio-binding assays to LP226A1 in order to test the presence of LP226BPs. Interestingly, our results showed two different protein fractions from IEX with evident binding to LP226A1. One of them was further separated by HIC in order to get LP226BP-enriched fractions. This additional analysis revealed other two fractions which showed binding to LP226A1 and that might be considered as semi-purified LP226BPs. On the other hand, affinity chromatography was addressed to isolate LP226BPs from soluble protein fractions. For this purpose, the drug was anchored to nitrocellulose. Such anchorage was confirmed by gas chromatography. This LP226A1-bound nitrocellulose was used as stationary phase for affinity chromatography, and indeed, this was incubated with soluble protein from mouse cortex and treated under different conditions in order to promote interaction between anchored LP226A1 with soluble proteins from the medium. Captured proteins were eluted and the resulting samples were analysed by electrophoresis and coomassie blue staining. The results showed that binding of soluble proteins was not via LP226A1, and in fact, they were non-specifically bound to the nitrocellulose membrane. Finally, western blot analysis was performed to analyse FA2H expression (enzyme in charge of fatty acid α-hydroxylation) in human brain samples from AD patients and healthy controls. These results showed a percentage of AD patients with up-regulated levels of FA2H as compared with healthy controls which might indicate a neuronal response to counteract the early onset of the neuropathology