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

总结：抗逆转录病毒药物诱导的人iPSC衍生的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 联合抗逆转录病毒治疗（cART）显著提高了生存率和生活质量 HIV-1感染者。越来越多的人担心，虽然cART通过降低HIV-1的神经毒性， 减少病毒载量，抗逆转录病毒药物本身可能产生神经毒性。在一些患者中，抗逆转录病毒药物 药物会引起抑郁和焦虑的症状。这些症状，像许多神经精神疾病， 与突触接触数量的减少有关。我们开发了一种高内涵的成像技术 方法来量化培养的神经元之间的功能性突触。这种高度自动化的 高含量分析（HCA）测定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。在本提案中，我们描述了该测定的修改，以评估 人诱导多能干细胞（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存在突触抑制剂的抗逆转录病毒药物的突触毒性。 与精神疾病风险相关的修饰语。提出了三个具体目标。在目标1中， 优化用于检测hiPSC衍生的神经元之间的突触的HCA测定。一种自动化检测方法， 量化活体人类神经元之间的突触连接将对研究神经元的功能有广泛的应用。 神经突触变化的机制，是精神疾病的基础。在目标2中，我们将确定 抗逆转录病毒药物对hiPSC衍生的皮质神经元之间的突触是有毒的。这些结果将确定 抗逆转录病毒药物具有损害突触功能的特殊倾向。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。一个有限的屏幕上的存在 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 单独和与抗逆转录病毒药物组合进行，以确定潜在的协同作用。 这些研究将为使用HCA测定法确定可能导致肝硬化的风险因素建立概念验证。 抗逆转录病毒药物诱导的人类皮层神经元之间突触的丧失。该项目的成果将 确定使用HCA测定来鉴定突触变化的可行性， 神经精神副作用，并将为未来的研究提供基础，以探讨机制， 突触丢失的潜在逆转。确定抗逆转录病毒药物组合和风险因素， 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，进一步验证 该测定可通过鉴定潜在的副作用来帮助开发下一代HIV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.