Effect of a memory-enhancing drug, AIT-082, on the level of synaptophysin

Our objective is to study the effect of AIT-082 on the level of synaptophysin in cultured pheochromocytoma (PC12) cells. The drug AIT-082, a unique purine hypoxanthine derivative, is under development for the treatment of Alzheimer's disease (AD). We analyzed synaptophysin protein as an index of synaptic numbers and density and indirectly neuronal transmission. PC12 cells were treated with nerve growth factor (NGF) (50 ng/ml) and/or different doses of AIT-082 (5-50 ng/ml) obtained from NeoTherapeutics, CA. In the western immunoblots of conditioned media and cell lysates, we detected synaptophysin as 36-40 kDa protein bands. When PC12 cells were treated with NGF and samples were analyzed at 24 or 48 hours after treatment, the secretion of synaptophysin was drastically reduced in the conditioned medium. A significant reduction in the intracellular levels of synaptophysin in NGF-treated samples was also noted. By contrast, when PC12 cells were treated with AIT-082, the secretion of synaptophysin was increased in the conditioned medium as compared to the control. There was also a significant increase in the intracellular levels of synaptophysin in AIT-082-treated cultures. NGF treatment resulted in sympathetic neuronal phenotypes in PC12 cells. As it is known that the immunoreactivity of the synaptophysin protein correlates with the density of the synaptic terminal, our results suggest that treatment by AIT-082 could enhance neurotransmitter release at the presynaptic terminal, which may play a role in the improvement of cognition seen in AD subjects.