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

项目摘要/摘要 尽管临床护理取得了进步，但接受长期抗逆转录病毒治疗(ART)的HIV-1感染者(PLWH)仍然 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)功能障碍机制 这些患者是复杂的，多因素的，很难用现有的方法进行研究。在这项提案中，我们将 利用生理学上的相关性研究HIV-1中神经元老化的可改变的机制途径， 参与者派生的细胞模型。近年来，直接诱导神经元(INS)的产生能力 患者来源的成纤维细胞已经被证实。与未成熟的神经元群体不同的是 诱导的多能干细胞(IPSCs)，INS保持神经元特异性的衰老相关基因表达 捐赠者的特征。因此，这些INS代表着一项重大的技术进步。我们的预赛 数据表明，我们有能力从PLWH的皮肤活检中产生INS。据我们所知，这使我们 第一个将这项技术应用于HIV-1神经元健康研究的小组。我们现在建议使用这些工具 为了确定基因表达的差异是否反映了年龄和/或其他艾滋病毒疾病的加速- 相关的转录特征明显通过转录表型的INS来源 年龄匹配的年轻人和老年人艾滋病毒阴性和艾滋病毒阳性队列。惯导系统的功能分析将测试 假想的神经元衰老的病理机制。越来越多的科学证据也表明 抗逆转录病毒药物(ARV)广泛应用于HIV-1的治疗，并已知能跨越血脑屏障而变化 学位可能会对神经元健康产生特定类别和/或药物的有害影响。因此，我们在 探索性的二次分析，假设存在潜在的可修改的、机械的路径 HIV-1中的神经元损伤也是ARV类依赖的。我们将从两个具体的方面来研究我们的假设 目标。在目标1中，我们将从临床特征良好的参与者中生成患者衍生的INS。我们会 按照Merten等人的要求生成INS。(细胞干细胞，2015)来自6个临床定义明确的队列的皮肤活检 按年龄、HIV-1状态和使用蛋白酶抑制物(PI)与整合酶链进行分层 转移抑制物(INSTI)为基础的抗逆转录病毒治疗。在AIM 2中，我们将确定神经元相关基因的表达 受年龄、HIV-1状态和购物车使用调控的途径--通过Well-Ins的转录图谱 确定的临床队列。将在队列中进行差异基因表达分析，试图 确定年龄(老年人与年轻人)，在二次探索性分析中，HIV-1(阳性与阴性)和ARV (PI与INSTI)对基因转录的影响。然后我们将确定依赖于年龄的核质 来自较老的HIV-1阳性队列的INS的特征是分区(NCC)损害。我们的小说 将这项INS技术应用于PLWH的翻译研究使我们有机会填补我们在 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.