Antiretroviral Drug-Induced Changes in Synapses between Human iPSC-Derived Cortical Neurons to Assess Risk and Mechanisms of Neuropsychiatric Adverse Effects

综述：抗逆转录病毒药物诱导的人IPSC来源皮质神经元之间突触的变化 评估神经精神科不良反应的风险和机制 联合抗逆转录病毒疗法(CART)显著提高了患者的存活率和生活质量。 针对HIV-1感染患者。人们越来越担心，虽然CART通过以下方式减少HIV-1的神经毒性 降低病毒载量，抗逆转录病毒药物本身可能会产生神经毒性。在一些患者中，抗逆转录病毒 药物会引起抑郁和焦虑的症状。这些症状，像许多神经精神障碍一样，可能 与突触接触次数的减少有关。我们开发了一种高含量的成像 量化培养中神经元间功能性谷氨酸能突触的方法。这是高度自动化的 高含量分析(Hca)对降低树突棘密度的试剂非常敏感。 在原代培养的大鼠神经元之间。在这项建议中，我们描述了对该检测方法的修改，以评估 人类诱导多能干细胞(HiPSC)来源的皮质神经元之间的突触接触。对.的使用 人类的神经元是有理由的，因为人类的突触对一些神经毒性的侮辱是独特的敏感。我们 将使用这种测试来评估单独的抗逆转录病毒药物和在突触存在的情况下的突触毒性 与精神疾病风险相关的修饰物。提出了三个具体目标。在目标1中，我们将 优化HCA法检测HIPSC来源神经元之间的突触。一种自动化的化验方法 量化活体人类神经元之间的突触联系将在研究 导致精神障碍的突触变化的机制。在目标2中，我们将确定是否 抗逆转录病毒药物对HiPSC衍生的皮质神经元之间的突触具有毒性。这些结果将确定 具有损害突触功能的特殊倾向的抗逆转录病毒药物。在目标3中，我们将评估突触 抗逆转录病毒诱导的突触毒性风险改变的修饰剂。在存在的情况下的有限屏幕 促炎细胞因子增加、应激激素升高或神经营养因子水平降低 将单独和与抗逆转录病毒药物联合使用，以确定潜在的协同效应。 这些研究将为使用HCA分析识别增强的危险因素建立概念证明。 抗逆转录病毒药物引起的人皮质神经元之间突触的丢失。这个项目的结果将是 确定使用HCA分析来确定突触变化与潜在的 神经精神病学的副作用，并将为未来的研究提供基础，以探讨其机制和 可能逆转突触丢失。确认抗逆转录病毒药物的组合和危险因素 产生突触丢失可以为艾滋病毒携带者的临床护理提供信息。最后，通过进一步的验证， 这项试验可能通过确定潜在的不良反应来帮助开发下一代艾滋病毒疗法。

Summary: Antiretroviral Drug-Induced Changes in Synapses between Human iPSC-Derived Cortical Neurons to Assess Risk and Mechanisms of Neuropsychiatric Adverse Effects Combined antiretroviral therapy (cART) has dramatically increased the survival and improved the quality of life for HIV-1 infected patients. There is growing concern that while cART decreases HIV-1 neurotoxicity by reducing viral load, antiretroviral drugs themselves may produce neurotoxicity. In some patients, antiretroviral drugs elicit symptoms of depression and anxiety. These symptoms, like many neuropsychiatric disorders may be associated with a reduction in the number of synaptic contacts. We developed a high content imaging approach to quantify functional glutamatergic synapses between neurons in culture. This highly automated high content analysis (HCA) assay is exquisitely sensitive to agents that reduce dendritic spine density between rat neurons in primary culture. In this proposal, we describe the modification of this assay to assess synaptic contacts between human induced pluripotent stem cell (hiPSC)–derived cortical neurons. The use of human neurons is warranted because human synapses are uniquely sensitive to some neurotoxic insults. We will use this assay to evaluate the synaptic toxicity of antiretroviral drugs alone and in the presence of synaptic modifiers associated with risk of psychiatric disease. Three specific aims are proposed. In aim 1, we will optimize the HCA assay for detecting synapses between hiPSC-derived neurons. An automated assay to quantify synaptic connections between live human neurons will have broad application for studying the mechanism of synaptic changes that underlie psychiatric disorders. In aim 2, we will determine whether antiretroviral drugs are toxic to synapses between hiPSC-derived cortical neurons. These results will identify antiretroviral drugs with a particular propensity to impair synaptic function. In aim 3, we will evaluate synaptic modifiers for altered risk of antiretroviral-induced synaptic toxicity. A limited screen in the presence of increased pro-inflammatory cytokines, elevated stress hormones, or reduced levels of neurotrophic factors alone and in combination with antiretroviral drugs will be performed to determine potential synergistic effects. These studies will establish a proof of concept for using the HCA assay to identify risk factors that potentiate antiretroviral drug-induced loss of synapses between human cortical neurons. The results from this project will determine the feasibility of using an HCA assay to identify synaptic changes with the potential to produce neuropsychiatric side effects and will provide a foundation for future studies to probe the mechanism and potential reversal of synapse loss. Identification of combinations of antiretroviral drugs and risk factors that produce synapse loss could inform the clinical care of persons living with HIV. Finally, with further validation this assay may assist in developing next-generation HIV therapies by identifying potential adverse effects.