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: 10678898
• 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/10678898
• 关键词: 3-Dimensional; AIDS dementia; Antiretroviral resistance; Apoptosis; Bar Codes; Brain; Breeding; 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; DNMT3a; 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; IL18 gene; Individual; Infection; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1 beta; Joints; 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; Productivity; Proto-Oncogene Proteins c-akt; Proviruses; Reporter; Role; Severities; Shapes; Signal Transduction; Symptoms; System; T-Cell Depletion; Testing; Tretinoin; Viral; Viral reservoir; Virion; Virus; Virus Activation; Virus Latency; Virus Replication; analog; antiretroviral therapy; cell type; chemokine; clinically relevant; cofactor; combinatorial; cytokine; effectiveness evaluation; endonuclease; excitatory neuron; fetal; improved; indexing; induced pluripotent stem cell; inhibitor; insight; latent HIV reservoir; latent virus activation; neuroinflammation; neuron development; neuron loss; neurotransmission; neurotropic; novel; novel therapeutics; opioid epidemic; opioid use; permissiveness; personalized approach; purge; response; sensor; single-cell RNA sequencing; synaptic pruning; therapy resistant; tool; transcription factor; viral RNA; viral transmission

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 在所有艾滋病毒感染者中，有整整一半继续表现出某种（通常是较温和的）艾滋病毒相关性疾病。 神经认知障碍（HAND），尽管引入了抗逆转录病毒疗法（ART）。超过十分之一的 这些人在ART治疗中会表现出进行性神经功能恶化。 艾滋病毒相关痴呆症在发展中国家仍然很常见，特别是在未接受抗逆转录病毒治疗的人群中。 手可能是由大脑慢性炎症导致神经元功能障碍引起的。难题在于 尽管ART有效抑制了病毒复制，但这种炎症反应是如何持续的。 认为潜伏的艾滋病毒感染的小胶质细胞可能发挥了核心作用。小神经胶质细胞占脑内所有细胞的10-15%。 中枢神经系统，并作为大脑的“不断园丁”，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导。如何最好地学习 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，其被工程化以表达强力霉素诱导的转录因子，所述转录因子足以驱动 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 细胞形成脑微器官（CM），概括了许多细胞结构特征和功能 胎儿的大脑。我们将使用scRNA-seq以无偏倚的方式研究这些CM以定义基因表达 图和scATAC-sec来询问染色质可及性。使用组合索引系统 条形码将允许在同一细胞中测量这些参数。我们假设小胶质细胞 潜伏感染，持续的神经递质信号可能足以重新激活病毒 表达加上暴露于阿片样物质将进一步增强再活化（病毒产生不受ART损害）。 重新激活的病毒体的释放可直接触发慢性炎症反应。此外，当这些 当病毒通过细胞间传播时，由于RT的作用， IFI 16 DNA传感器可以检测这些导致炎性小体的RT产物。 组装，半胱天冬酶-1激活，IL-1β和IL-18的产生以及由焦亡引起的死亡， 程序性细胞死亡。因为焦亡会产生更多的焦亡，这种前馈形式的 炎症可能会产生对ART耐药的慢性炎症反应。最后，我们渴望探索 两种CNS定制的攻击小胶质细胞中潜伏的HIV库的方法。首先，病毒将被清除 使用CNS渗透剂LRA，产生病毒RNA的细胞将通过诱导RIG-I依赖性的 凋亡在第二个实验中，将测试HIV前病毒的持久的、序列特异性的转录沉默 使用CRISPR干扰来促进H3 K9 me 3和DNA甲基化-这两种表观遗传修饰都是 需要长期保持沉默。总之，这些研究有望为HAND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。