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

摘要：抗逆转录病毒药物引起的人 iPSC 衍生皮质神经元之间突触的变化 评估神经精神不良反应的风险和机制 联合抗逆转录病毒疗法 (cART) 显着提高了生存率并改善了生活质量 对于 HIV-1 感染患者。人们越来越担心，虽然 cART 通过降低 HIV-1 神经毒性 减少病毒载量，抗逆转录病毒药物本身可能会产生神经毒性。在某些患者中，抗逆转录病毒药物 药物会引起抑郁和焦虑症状。这些症状，就像许多神经精神疾病一样，可能会导致 与突触接触数量的减少有关。我们开发了高内涵成像 量化培养物中神经元之间功能性谷氨酸突触的方法。这种高度自动化的 高内涵分析 (HCA) 检测对降低树突棘密度的药物非常敏感 原代培养的大鼠神经元之间。在本提案中，我们描述了对该测定法的修改以评估 人类诱导多能干细胞（hiPSC）衍生的皮质神经元之间的突触接触。使用 人类神经元是有必要的，因为人类突触对某些神经毒性损伤特别敏感。我们 将使用该测定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因素。提出了三个具体目标。在目标 1 中，我们将 优化用于检测 hiPSC 衍生神经元之间突触的 HCA 测定。自动化测定 量化活体人类神经元之间的突触连接将在研究 精神疾病的突触变化机制。在目标 2 中，我们将确定是否 抗逆转录病毒药物对 hiPSC 衍生的皮层神经元之间的突触有毒。这些结果将确定 具有特定损害突触功能倾向的抗逆转录病毒药物。在目标 3 中，我们将评估突触 改变抗逆转录病毒引起的突触毒性风险的修饰剂。有限的屏幕 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 将单独进行或与抗逆转录病毒药物联合进行以确定潜在的协同效应。 这些研究将建立一个概念证明，使用 HCA 测定来识别增强的风险因素 抗逆转录病毒药物引起的人类皮质神经元之间突触的丧失。该项目的结果将 确定使用 HCA 测定来识别可能产生突触变化的可行性 神经精神副作用，并将为未来的研究奠定基础，探讨其机制和 突触损失的潜在逆转。确定抗逆转录病毒药物组合和危险因素 产生突触损失可以为艾滋病毒感染者的临床护理提供信息。最后，经过进一步验证 该检测可能通过识别潜在的副作用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.