Insulin signaling effects on 2-arachidonoylglycerol hydrolysis in synaptic terminals exposed to amyloid beta oligomers

Abstract

2-arachidonoylglycerol (2-AG) behaves as a neuroprotective agent in Alzheimer’s disease (AD). Aβ oligomers (OAβ) are responsible for the synaptic dysfunction observed in AD and were shown to disrupt the synaptosomal membrane and to diminish 2-AG availability. Insu- lin (Ins) is involved in synaptic plasticity and its signaling was shown to be downregulated in AD. OAβ can bind to insulin receptor (IR) and can, therefore, be internalized into neurons, while Ins prevents this binding and thus its neurotoxicity. Here, we aimed to study Ins preincubation effects on 2-AG hydrolysis in cerebral cortex synapto- somes (Syn) exposed to OAβ. To this end, Syn were isolated by dif- ferential centrifugation, purified in ficoll gradients, and preincubated with 10 μM LY294002 (phosphatidylinositol-3-kinase -PI3K- inhibi- tor) or 100 μM genistein (tyrosine kinase -TK- Inhibitor) for 10 min, and subsequently incubated with 0.2 mM vanadate (protein-tyrosine phosphatase inhibitor), 100 nM Ins, or 0.2 mM vanadate plus 100 nM Ins, for 30 min. Syn were then incubated for 10 min with or with- out 0.1 μM OAβ, and for 20 min with [3 H]monoacylglycerol, to as- say 2-AG hydrolysis. It was observed that Ins and vanadate -either separately or coincubated- decreased 2-AG hydrolysis (p<0.01), and that their effect was not seen if Syn were preincubated with LY (p>0.05). On the other hand, in the presence of OAβ, while Ins and vanadate failed to alter 2-AG hydrolysis (p>0.05), LY increased this activity (p<0.001). However, the presence of Ins plus vanadate after incubation with LY could restore the activity (p<0.001) to basal lev- els in Syn treated with OAβ. Additionally, the presence of genistein previous to OAβ did not change the activity (p>0.05). Our results show a regulation of 2-AG hydrolysis by Ins, possibly decreasing its availability via IR and involving PI3K pathway, which is abolished by OAβ. The effect of OAβ appears to be independent of TK receptors and to involve PI3K activity.