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.

项目概要 足有一半的艾滋病毒感染者继续表现出某种（通常较温和）的艾滋病毒相关症状 尽管采用了抗逆转录病毒疗法（ART），但仍出现神经认知障碍（HAND）。超过十分之一的人 这些人在接受 ART 治疗后会表现出进行性神经功能恶化。更严重的手部疾病形式，包括 与艾滋病毒相关的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疗法的个人中。 手部疾病很可能是由大脑慢性炎症导致神经元功能障碍引起的。难题是 尽管 ART 有效抑制了病毒复制，但这种炎症反应如何持续存在。我们 相信小胶质细胞的潜在艾滋病毒感染可能起着核心作用。小胶质细胞占所有细胞的 10-15% 中枢神经系统，并作为大脑的“恒定园丁”，通过突触修剪和塑造神经元可塑性 剥离；小胶质细胞还参与与紧密缠绕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（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 以公正的方式研究这些 CM 来定义基因表达 配置文件和 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单元中测量这些参数。我们假设小胶质细胞是 潜伏感染并且持续的神经元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）。 重新激活的病毒颗粒的释放可能直接引发慢性炎症反应。另外，当这些 病毒在细胞间传播，由于 RT 的作用，可能会出现 HIV 感染的流产形式 ART 中存在抑制剂。 IFI16 DNA 传感器可以检测这些导致炎症小体的 RT 产物 组装、caspase-1 激活、IL-1β 和 IL-18 的产生以及细胞焦亡（高度炎症）导致的死亡 程序性细胞死亡的形式。因为焦亡会产生更多的焦亡，所以这种前馈形式 炎症可能会产生对 ART 具有抵抗力的慢性炎症反应。最后，我们迫不及待地想探索一下 两种针对中枢神经系统定制的方法来攻击小胶质细胞中潜在的艾滋病毒储存库。首先，病毒将被清除 具有中枢神经系统渗透性的 LRA 和产生病毒 RNA 的细胞将通过诱导 RIG-I 依赖性而被选择性杀死 细胞凋亡。第二步，将测试 HIV 原病毒的持久、序列特异性转录沉默 使用 CRISPR 干扰促进 H3K9me3 和 DNA 甲基化——这两种表观遗传修饰都是 需要长期沉默。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。