Therapeutic efficacy of Ibudilast to attenuate inflammation at the synapse

摘要 尽管早期的研究已经显示了HIV诱导的炎症和兴奋性毒性的总体影响， 甲基苯丙胺（冰毒）在艾滋病毒相关的神经认知障碍的进展，一个显着的差距， 目前的研究重点是识别突触蛋白靶点，包括通过治疗药物对其进行调节，以改善这种 突触处的炎症反应和谷氨酸兴奋毒性。这份R21提案的重点是这样一个 突触靶向磷脂酰乙醇胺结合蛋白1（PEBP 1），我们已经确定了从以前的 研究发现，HIV（达特，gp 120）和metha导致的其表达减少可通过抗- 炎症药物异丁司特，磷酸二酯酶抑制剂。基于PEBP 1的突触定位，我们 测试了使用siRNA敲除PEBP 1对混合的皮层培养物的因果作用， 包括分化的神经元和星形胶质细胞。我们在体外（DIV 14）处理了14天的混合皮质 用针对PEBP 1的siRNA培养物，然后单独和组合达特/甲基处理24小时。同时， 对一组24小时达特/甲基处理的培养物给予异丁司特处理24小时。达特和冰毒混合 降低谷氨酸转运体EAAT 2的表达，同时增加囊泡的表达。 谷氨酸转运蛋白1（vGLUT 1）的蛋白质印迹，从而表明兴奋性毒性。的这种增加 谷氨酸水平与促炎细胞因子IL 6和TNF-α水平的增加相证实， 通过ELISA测定的细胞上清液。有趣的是，这些作用在用 异丁司特24小时。基于HIV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PEBP 1增加炎症和谷氨酸毒性， 加重了突触-树突损伤，这可以通过异丁司特治疗逆转。我们将检查我们的 一种由易处理的临床前动物补充的混合皮质培养物体外模型中的假设 模型系统HIV-Tg大鼠使用生化，分子，成像和电生理方法。到 总之，我们的研究已经确定了一个潜在的突触蛋白靶点和一种抗炎药物， 减轻与HIV期间突触下调相关的畸变的治疗功效， 冰毒导致的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.