Leveraging Directly Reprogrammed Human Neurons to Investigate the Molecular Impact of Age and Distinct Antiretroviral Therapies in Individuals Living with HIV-1

项目总结/摘要 尽管临床护理取得了进展，但接受长期抗逆转录病毒治疗（ART）的HIV-1感染者（PLWH）仍然无法获得有效的治疗。 存在认知和行为缺陷以及HIV-1相关神经认知障碍的患病率 （手）是30%-50%。HIV诱导的中枢神经系统（CNS）功能障碍的机制， 这些患者是复杂的、多因素的，并且难以使用当前的方法进行研究。在本提案中，我们将 研究HIV-1中神经元老化的可改变的机制途径， 参与者衍生的细胞模型。近年来，从神经元产生直接诱导神经元（iN）的能力受到了广泛的关注。 患者来源的成纤维细胞。不像从大脑中产生的未成熟神经元群体， 诱导多能干细胞（iPSC），iNs保留神经元特异性，衰老相关基因表达 捐赠者的特点。因此，这些iN代表了一项重大的技术进步。我们的初步 数据表明我们有能力从PLWH的皮肤活组织检查中产生iN。据我们所知，这使我们 第一个将这项技术应用于研究HIV-1的神经健康的小组。我们现在建议使用这些工具 以确定基因表达的差异是否反映了加速的年龄-和/或其他艾滋病毒疾病- 相关的转录签名是显而易见的，通过转录表型的iN来源于 年龄匹配的年轻和老年艾滋病毒阴性和艾滋病毒阳性队列。iN的功能分析将测试 神经元老化的病理机制。越来越多的科学证据也表明， 抗逆转录病毒药物（ARV）广泛用于HIV-1治疗，已知可穿过血脑屏障， 程度可能对神经元健康具有类别和/或药物特异性有害影响。因此，我们在 探索性次要分析，假设存在潜在的可改变的机制途径， HIV-1中的神经元损伤也是ARV类依赖性的。我们将在两个特定的领域研究我们的假设。 目标。在AIM 1中，我们将从临床特征良好的参与者中生成患者来源的iN。我们将 根据Mertens等人（Cell Stem Cell，2015），从6个临床明确定义的队列的皮肤活检中生成iN 按年龄、HIV-1状态和使用蛋白酶抑制剂（PI）与整合酶链分层 在AIM 2中，我们将确定神经元相关基因的表达， 通过对来自健康人的iN进行转录谱分析， 确定的临床队列。将在队列中进行差异基因表达分析，以尝试 在次要探索性分析中，确定年龄（老年vs.年轻），HIV-1（阳性vs.阴性）和ARV (PI vs. p53 I）对基因转录的影响。然后我们将确定年龄依赖性核质 区室化（NCC）损伤表征来自较年长的HIV-1阳性群组的iN。我们的新型 将这种iNs技术应用于PLWH的转化研究，使我们有机会填补我们的空白， 了解HIV-1中的神经元老化，并确定改善认知的治疗靶点。

PROJECT SUMMARY/ABSTRACT Despite advances in clinical care, people living with HIV-1 (PLWH) on long-term antiretroviral therapy (ART) still present with cognitive and behavioral deficits and the prevalence of HIV-1 associated neurocognitive disorders (HANDs) is 30%-50%. The mechanisms underlying HIV-induced central nervous system (CNS) dysfunction in these patients are complex, multifactorial and difficult to study using current methods. In this proposal, we will investigate modifiable mechanistic pathways of neuronal aging in HIV-1 using physiologically relevant, participant-derived cell models. In recent years, the ability to generate directly induced neurons (iNs) from patient-derived fibroblasts has been demonstrated. Unlike the immature neuronal populations generated from induced pluripotent stem cells (iPSCs), iNs retain neuron-specific, aging-associated gene-expression characteristics of the donor. As a result, these iNs represent a major technological advance. Our preliminary data demonstrates our ability to generate iNs from the skin biopsies of PLWH. To our knowledge, this makes us the first group to apply this technology to study of neuronal health in HIV-1. We now propose to use these tools to determine if differences in gene expression reflective of accelerated age- and/or additional HIV disease- associated transcriptional signatures are evident through the transcriptional phenotyping of iNs derived from age-matched young and older HIV-negative and HIV-positive cohorts. Functional analyses of the iNs will test hypothesized pathomechanisms of neuronal aging. A growing body of scientific evidence also suggests that antiretroviral drugs (ARVs) widely used in HIV-1 therapy and known to cross the blood-brain barrier to varying degrees may have class- and/or drug-specific deleterious effects on neuronal health. We therefore, in exploratory secondary analyses, hypothesize that there are potentially modifiable, mechanistic pathways of neuronal injury in HIV-1 that are also ARV class dependent. We will investigate our hypotheses in two specific aims. In AIM 1, we will generate patient-derived iNs from clinically well-characterized participants. We will generate iNs as per Mertens et al. (Cell Stem Cell, 2015) from the skin biopsies of 6 clinically well-defined cohorts stratified by age, HIV-1 status and use of protease-inhibitor (PI)- versus Integrase strand transfer inhibitor (INSTI)-based ART. In AIM 2, we will determine neuronal-associated gene-expression pathways modulated by age, HIV-1 status and cART usage through transcriptional profiling of iNs from well- defined clinical cohorts. Differential gene expression analysis will be performed across cohorts in attempts to determine, age (old vs. young), and in secondary exploratory analyses, HIV-1 (positive vs. negative) and ARV (PI vs. INSTI) effects on gene transcription. We will then determine if age-dependent nucleocytoplasmic compartmentalization (NCC) impairments characterize iNs from older, HIV-1 positive cohorts. Our novel application of this iNs technology to the translational study of PLWH allows us the opportunity to fill a gap in our understanding of neuronal aging in HIV-1 and identify therapeutic targets for improved cognition.