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 nenronal 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 level 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.

我们的目的是研究AIT - 082对培养的嗜铬细胞瘤（PC12）细胞中突触素水平的影响。药物AIT - 082是一种独特的嘌呤次黄嘌呤衍生物，正处于开发用于治疗阿尔茨海默病（AD）的过程中。我们将突触素蛋白作为突触数量和密度以及间接神经元传递的指标进行分析。PC12细胞用神经生长因子（NGF）（50纳克/毫升）和/或从加利福尼亚州新疗法公司获得的不同剂量的AIT - 082（5 - 50纳克/毫升）进行处理。在条件培养基和细胞裂解物的蛋白质免疫印迹中，我们检测到突触素为36 - 40千道尔顿的蛋白条带。当PC12细胞用NGF处理且在处理后24或48小时对样本进行分析时，条件培养基中突触素的分泌急剧减少。在NGF处理的样本中，突触素的细胞内水平也显著降低。相比之下，当PC12细胞用AIT - 082处理时，与对照组相比，条件培养基中突触素的分泌增加。在AIT - 082处理的培养物中，突触素的细胞内水平也显著增加。NGF处理使PC12细胞呈现交感神经元表型。由于已知突触素蛋白的免疫反应性与突触末端的密度相关，我们的结果表明AIT - 082处理可增强突触前末端的神经递质释放，这可能在阿尔茨海默病患者认知改善中起作用。