Exploring HIV-associated Neurocognitive Disorder (HAND) and HIV Latency at the Single Cell Level in Cerebral Organoids

在脑类器官的单细胞水平上探索 HIV 相关神经认知障碍 (HAND) 和 HIV 潜伏期

• 批准号: 10466829
• 负责人: Melanie Maria Ott
• 金额: $ 70.8万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10466829
• 关键词: 3-Dimensional; AIDS dementia; Antiretroviral resistance; Apoptosis; Bar Codes; Brain; CASP1 gene; CD4 Positive T Lymphocytes; CRISPR interference; Cell Differentiation process; Cell Line; Cells; Cerebrum; Cessation of life; Characteristics; Chromatin; Chronic; Coculture Techniques; DNA; DNA Methylation; DNA Modification Methylases; DNA cassette; Data; Deterioration; Development; Doxycycline; Engineering; Epigenetic Process; Event; Exhibits; Exposure to; FRAP1 gene; Fluorescence; Frequencies; Functional disorder; Gene Expression; Gene Silencing; Goals; HIV; HIV Infections; HIV-associated neurocognitive disorder; Individual; Infection; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1 beta; Interleukin-18; Joints; Lead; Link; Lymphoid Tissue; Measurement; Medical; Microglia; Modification; Monitor; Neurocognitive Deficit; Neurologic; Neuronal Dysfunction; Neuronal Plasticity; Neurons; Neurotransmitters; Opioid; Organoids; Pathogenesis; Pathway interactions; Patients; Peripheral; Permeability; Physiological; Play; Process; Production; Proto-Oncogene Proteins c-akt; Proviruses; Reporter; Resistance; Role; Severities; Shapes; Signal Transduction; Symptoms; System; Testing; Tretinoin; Viral; Viral reservoir; Virion; Virus; Virus Latency; Virus Replication; analog; antiretroviral therapy; cell type; chemokine; clinically relevant; combinatorial; cytokine; effectiveness evaluation; endonuclease; excitatory neuron; fetal; indexing; induced pluripotent stem cell; inhibitor; insight; latent HIV reservoir; neuroinflammation; neuron development; neuron loss; neurotransmission; neurotropic; novel; novel therapeutics; opioid epidemic; opioid use; personalized approach; purge; response; sensor; single-cell RNA sequencing; synaptic pruning; tool; transcription factor; viral RNA

Project Summary Fully half of all HIV-infected individuals continue to display some (often milder) form of HIV-associated neurocognitive disorder (HAND) despite the introduction of antiretroviral therapy (ART). More than one in 10 of these individuals will exhibit progressive neurologic deterioration on ART. More severe forms of HAND, including HIV-associated dementia, remain common in the developing world, especially in individuals not receiving ART. HAND is likely caused by chronic inflammation in the brain leading to neuronal dysfunction. The conundrum is how this inflammatory response is sustained despite effective suppression of viral replication with ART. We believe latent HIV infection of microglia likely plays a central role. Microglia comprise 10-15% of all cells in the CNS and serve as the brain's "constant gardeners" shaping neuronal plasticity through synaptic pruning and stripping; microglia also participate in bidirectional signaling with closely intertwined neurons. How best to study these microglia, their interplay with neurons, and the effects of HIV infection? We propose to coculture two iPSC- derived sub-lines engineered to express doxycycline-inducible transcription factors that are sufficient to drive differentiation into either microglia or excitatory neurons. When induced and cocultured in 3D conditions, these cells form cerebral microorganoids (CMs) that recapitulate many of the cytoarchitectural features and functions of the fetal brain. We will study these CMs in an unbiased manner using scRNA-seq to define gene expression profiles and scATAC-sec to interrogate chromatin accessibility. Use of a combinatorial indexing system of barcodes will allow measurement of these parameters in the same cell. We hypothesize that microglia are latently infected and that sustained neuronal neurotransmitter signaling is likely sufficient to reactivate virus expression plus exposure to opioids will further enhance reactivation (virus production is not impaired by ART). Release of reactivated virions may directly trigger a chronic inflammatory response. Additionally, when these viruses are transmitted cell-to-cell, an abortive form of HIV infection may ensue due to the action of the RT inhibitors present in ART. The IFI16 DNA sensor may detect these RT products leading to inflammasome assembly, caspase-1 activation, production of IL-1β and IL-18 and death by pyroptosis, a highly inflammatory form of programmed cell death. Because pyroptosis breeds more pyroptosis, this feed-forward form of inflammation could a create chronic inflammatory response resistant to ART. Finally, we are eager to explore two CNS-tailored approaches for attacking the latent HIV reservoir in microglia. In the first, virus will be purged with a CNS-penetrant LRA and cells producing viral RNA will be selectively killed by induction of RIG-I-dependent apoptosis. In the second, durable, sequence-specific transcriptional silencing of HIV proviruses will be tested using CRISPR interference to promote H3K9me3 and DNA methylation––both epigenetic modifications are needed for long term silencing. Together, these studies promise to provide new and exciting insights into HAND pathogenesis, HIV latency in the brain, effects of opioids, and the potential link between these processes.

项目摘要 整整一半的艾滋病毒感染者继续表现出某种形式的(通常是较轻微的)艾滋病毒相关 神经认知障碍(手)，尽管引入了抗逆转录病毒治疗(ART)。超过十分之一的人 这些人会在ART中表现出进行性的神经退化。更严重的手部形态，包括 艾滋病毒相关性痴呆症在发展中国家仍然很常见，特别是在没有接受抗逆转录病毒治疗的人中。 手很可能是由大脑中的慢性炎症引起的，导致神经元功能障碍。难题是 在ART有效抑制病毒复制的情况下，这种炎症反应是如何持续的。我们 相信潜伏的HIV感染小胶质细胞可能起着核心作用。小胶质细胞占所有细胞的10%-15%。 中枢神经系统作为大脑的“恒定园丁”，通过突触修剪和 剥离；小胶质细胞也参与与紧密交织的神经元的双向信号传递。如何学习才是最好的 这些小胶质细胞，它们与神经元的相互作用，以及艾滋病毒感染的影响？我们建议共培养两个IPSC- 衍生的亚系被设计为表达足以驱动多西环素诱导的转录因子 分化为小胶质细胞或兴奋性神经元。当在3D条件下诱导和共培养时，这些 细胞形成脑微器官(CMS)，它概括了许多细胞结构的特征和功能 胎儿的大脑。我们将以公正的方式研究这些CMS，使用scRNA-seq来定义基因表达。 Profile和scatac-sec用于询问染色质的可及性。一个组合标引系统的使用 条形码将允许在同一单元中测量这些参数。我们假设小胶质细胞是 潜伏感染，持续的神经递质信号可能足以重新激活病毒 表达加上接触阿片类药物将进一步增强重新激活(病毒产生不受抗逆转录病毒治疗的影响)。 重新激活的病毒粒子的释放可能直接引发慢性炎症反应。此外，当这些 病毒在细胞间传播，由于RT的作用，可能会导致艾滋病毒感染的流产形式 艺术品中存在的抑制物。IFI16DNA传感器可能会检测到这些导致炎症的RT产物 组装、caspase-1激活、IL-1β和IL-18的产生以及由高度炎症性下垂引起的死亡 一种程序性细胞死亡的形式。由于下垂会滋生更多的下垂，这种前馈形式的 炎症可能会产生对ART具有抵抗力的慢性炎症反应。最后，我们渴望探索 两种针对中枢神经系统量身定做的方法，用于攻击小胶质细胞中潜在的艾滋病毒储备库。在第一阶段，病毒将被清除 对于CNS穿透性LRA和产生病毒RNA的细胞将通过诱导依赖RIG-I而选择性地被杀死 细胞凋亡。在第二个实验中，将测试HIV前病毒的持久、序列特异性转录沉默 利用CRISPR干扰促进H3K9me3和DNA甲基化--这两种表观遗传修饰 需要长时间的沉默。总之，这些研究有望为我们提供新的、令人兴奋的见解。 发病机制、艾滋病毒在大脑中的潜伏期、阿片类药物的影响以及这些过程之间的潜在联系。