Therapeutic efficacy of Ibudilast to attenuate inflammation at the synapse

摘要 尽管早期的研究表明，艾滋病毒和艾滋病引起的炎症和兴奋毒性的总体影响 甲基苯丙胺(METH)在HIV相关神经认知障碍进展中的作用 RESTESS正在识别突触蛋白靶点，包括治疗药物对它们的调节，以改善这种情况 突触的炎症反应和谷氨酸兴奋性毒性。这份R21提案关注的是这样一个 突触靶向磷脂酰乙醇胺结合蛋白1(PEBP1)，我们已经从以前的 研究发现，HIV(TAT，gp120)和冰毒对其表达的抑制可被抗病毒药物逆转。 炎症性药物异丁司特，一种磷酸二酯酶抑制剂。基于PEBP1的突触定位，我们 使用siRNA在混合的大脑皮层培养物上测试了PEBP1基因敲除的更具因果关系的影响 由分化的神经元和星形胶质细胞组成。我们在体外治疗14天(DIV14)混合大脑皮层 用针对PEBP1的siRNA培养后，TAT/METH单独和联合处理24小时。同时， 24小时TAT/METH处理的一部分培养物给予异丁司特处理24小时。纹身和冰毒的组合 降低谷氨酸转运体EAAT2的表达，同时增加囊泡的表达 蛋白质印迹显示裂解物中有谷氨酸转运体1(VGLUT1)，提示有兴奋性毒性。这一增长 谷氨酸水平与促炎细胞因子IL-6和肿瘤坏死因子-水平的升高相一致 用酶联免疫吸附试验测定细胞上清液。有趣的是，这些影响在培养过程中被减弱。 异丁司特24小时。基于艾滋病毒和冰毒引起的炎症和兴奋性毒性的公认教条，我们 假设HIV和冰毒下调PEBP1的表达会增加炎症和谷氨酸毒性 加重突触树突状细胞损伤，而异丁司特治疗可以逆转这种损伤。我们将检查我们的 由易驯化的临床前动物补充的混合大脑皮层培养的体外模型中的假设 利用生化、分子、成像和电生理方法建立HIV-TG大鼠模型系统。至 综上所述，我们的研究已经确定了一个潜在的突触蛋白靶点和一种具有 减轻HIV和HIV期间突触下调相关的异常的治疗效果 冰毒致中枢神经系统功能障碍。

Abstract Despite earlier studies that have shown the overall impact of inflammation and excitotoxicity induced by HIV and methamphetamine (meth) in the progression of HIV-Associated Neurocognitive Disorders, a significant gap that remains is identifying synaptic protein targets including their modulation by therapeutic drugs to ameliorate such inflammatory responses and glutamate excitotoxicity at the synapse. This R21 proposal focuses on one such synaptic target phosphatidylethanolamine-binding protein 1 (PEBP1) which we have identified from a previous study and whose reduced expression by HIV (Tat, gp120) and meth was reversed by treatment with the anti- inflammatory drug Ibudilast, a phosphodiesterase inhibitor. Based on the synaptic localization of PEBP1, we tested a more causal effect of knock down of PEBP1 using siRNA on mixed cerebrocortical cultures that comprise of differentiating neurons and astrocytes. We treated 14 days in vitro (DIV14) mixed cerebrocortical cultures with siRNA against PEBP1 followed by Tat/meth treatments alone and in combination for 24h. In parallel, a subset of 24h Tat/meth treated cultures were given ibudilast treatment for 24h. Tat and meth in combination decreased the expression of the glutamate transporter EAAT2 and a concurrent increase in the vesicular glutamate transporter 1 (vGLUT1) in the lysates by western blot thus suggesting excitotoxicity. This increase in glutamate levels corroborated with increase in the levels of the proinflammatory cytokines IL6 and TNF-in the cell supernatants as determined by ELISA. Interestingly, these effects were attenuated in cultures treated with Ibudilast for 24h. Based on the well-established dogma of inflammation and excitotoxicity by HIV and meth, we hypothesize that PEBP1 down regulation by HIV and meth increases inflammation and glutamate toxicity thus exacerbating synaptodendritic damage which can be reversed by ibudilast treatment. We will examine our hypothesis in an in vitro model of mixed cerebrocortical cultures complemented by a tractable preclinical animal model system HIV-Tg rat using biochemical, molecular, imaging and electrophysiological approaches. To summarize, our studies have identified a potential synaptic protein target and an anti-inflammatory drug with a therapeutic efficacy to mitigate aberrations associated with its downregulation at the synapse during HIV and meth induced CNS dysfunction.