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: 10006808
• 负责人: Warner C. Greene
• 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/10006808
• 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; Effectiveness; Engineering; Epigenetic Process; Event; Exhibits; Exposure to; Expression Profiling; 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; endonuclease; excitatory neuron; fetal; indexing; induced pluripotent stem cell; inhibitor/antagonist; 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 在所有感染HIV的人中，完全有一半的人继续显示某些（通常是米勒）形式的HIV相关形式 尽管引入了抗逆转录病毒疗法（ART），但神经认知障碍（手）。十分之一的超过十分之一 这些人将揭露艺术的渐进神经系统定义。更严重的手，包括 与艾滋病毒相关的痴呆症在发展中国家中仍然很普遍，尤其是在不接受艺术的人中。 手很可能是由于大脑的慢性炎症引起的，导致神经元功能障碍。难题是 尽管有效地抑制了ART的病毒复制，但如何持续这种炎症反应。我们 认为小胶质细胞的潜在艾滋病毒感染可能起着核心作用。小胶质细胞在所有细胞中完成10-15％ 中枢神经系统并充当大脑的“恒定园丁”，通过突触修剪和 剥离；小胶质细胞还参与具有紧密相互交织神经元的双向信号传导。如何最好地学习 这些小胶质细胞，与神经元的相互作用以及HIV感染的影响？我们建议共同培养两个IPSC- 派生的子线设计，旨在表达多西环素诱导的转录因子，足以驱动 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 细胞形成脑微有机体（CMS），该细胞概括了许多细胞结构特征和功能 我们将使用SCRNA-SEQ以公正的方式研究这些CMS来定义基因表达 配置文件和SCATAC-SEC询问染色质的可及性。使用组合索引系统 条形码将允许在同一单元格中测量这些参数。我们假设小胶质细胞是 受到潜在感染和持续的神经递质信号传导可能足以重新激活病毒 表达加上阿片类药物的暴露将进一步增强重新激活（ART不会损害病毒的产生）。 重新激活病毒的释放可能直接引发慢性炎症反应。另外，当这些 病毒是传播细胞到细胞的，可能导致艾滋病毒感染的流产形式 艺术中存在的抑制剂。 IFI16 DNA传感器可以检测到这些RT产品导致炎症体 组装，caspase-1激活，IL-1β和IL-18的产生以及高度炎性的死亡（一种高度炎症） 编程细胞死亡的形式。由于凋亡会繁殖更多的凋亡，因此这种馈送形式的形式 炎症可能是一种对艺术的耐药性抗药性反应。最后，我们渴望探索 两种CNS级方法，用于攻击小胶质细胞中潜在的HIV储藏剂。首先，将清除病毒 使用CNS-PENETRANT LRA和产生病毒RNA的细胞将通过诱导RIG-I依赖性选择性杀死 凋亡。在第二个，将测试HIV病毒的耐用，序列特异性的转录沉默 使用CRISPR干扰促进H3K9me3和DNA甲基化 - 表观遗传修饰是 长期沉默所必需的。这些研究共同承诺将提供新的和令人兴奋的见解 发病机理，大脑中的艾滋病毒潜伏期，阿片类药物的作用以及这些过程之间的潜在联系。