Defining molecular mechanisms by which stimulant evoked dopamine drives inflammation and neuronal dysfunction in neuroHIV

定义兴奋剂诱发多巴胺驱动神经艾滋病毒炎症和神经元功能障碍的分子机制

• 批准号: 10685160
• 负责人: Peter Jesse Gaskill
• 金额: $ 57.03万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685160
• 关键词: 3-Dimensional; ATAC-seq; Acceleration; Address; Atlases; Automobile Driving; Behavioral; Biological Assay; Brain; Cell Culture Techniques; Cell Survival; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Cocaine; Coculture Techniques; Cognitive; Communication; Data; Data Correlations; Dendritic Spines; Disease; Disease Progression; Dopamine; Dopamine Receptor; Electrophysiology (science); Evaluation; Exposure to; Gene Expression; Gene Expression Profile; Genes; Genetic; HIV; HIV Infections; Health; High Prevalence; Human; Image; Impaired cognition; Impairment; In Vitro; Infection; Inflammasome; Inflammation; Inflammatory; Intervention; Knowledge; Macrophage; Mediating; Membrane Potentials; Methamphetamine; Microglia; Modeling; Molecular; Morphology; Myelogenous; Myeloid Cells; Neurobiology; Neurocognitive Deficit; Neuroimmune; Neuronal Differentiation; Neuronal Dysfunction; Neurons; Neuropathogenesis; Pathway interactions; Persons; Phase; Physiological; Population; Process; Production; Protocols documentation; Publishing; Receptor Activation; Rest; Risk; Stimulant; Substance abuse problem; System; Systemic disease; Testing; Transcription Factor AP-1; Viral; antiretroviral therapy; cell type; comorbidity; confocal imaging; cytokine; density; dopaminergic neuron; high throughput analysis; high throughput screening; in vitro activity; induced pluripotent stem cell; knock-down; multi-electrode arrays; neuroAIDS; neuroinflammation; neuropathology; neuropsychiatry; nonhuman primate; pandemic disease; patch clamp; pharmacologic; prevent; receptor; response; single-cell RNA sequencing; stimulant exposure; stimulant use; transcription factor

The HIV pandemic is increasingly driven by the spread of infection in vulnerable sub-populations with a relatively high prevalence of substance abuse disorders (SUD), including the use of stimulants such as Methamphetamine (Meth) and Cocaine (Coc). Stimulant use accelerates systemic disease, and can drive changes in neuropathogenesis, increase risk neuropsychiatric comorbidities and accelerate cognitive decline, despite effective ART. Despite the high prevalence of stimulant use among PLWH, the mechanisms by which stimulants impact disease progression are poorly defined. This is particularly true in the CNS, as there are substantial technical challenges involved in modeling microglial infection and interaction with neurons, as well as the subsequent changes that this has on neuronal function. All stimulants increase CNS dopamine release, exposing myeloid populations to highly elevated dopamine levels. Data indicate that it is the exposure to released dopamine, rather than the stimulants themselves, which drives changes in microglial infection and function. Our data support this, showing stimulant induced dopamine levels increase HIV entry and enhance myeloid inflammation, increasing cytokine release, and NF-κB and NRLP3 inflammasome activity in vitro and in the NHP CNS. Neuroinflammation driven by infected CNS myeloid populations is central to HIV neuropathogenesis in the ART era, underlying the neuronal dysfunction and disruptions in neuroimmune communication that lead to cognitive impairment and behavioral changes. We hypothesize that stimulant use exacerbates HIV- associated microglial inflammation through dopamine receptor activation, leading to neuronal dysfunction in neuroHIV. To address this, we propose to develop tractable, syngeneic co-cultures of human iPSC-derived microglia (iMG) and iPSC-derived dopamine neurons (iDAN). Critically, these iDAN will release dopamine in vitro in response to stimulant exposure. Co-cultures will be based on our existing protocols for iMG and iDAN differentiation and will be developed and optimized for high-throughput analysis during the R61 phase. During the R61, we will infect with HIV and treat with stimulants +/- ART, then use high content imaging, single cell RNA-seq / ATAC-seq and Alphalisas to evaluate changes in viral dynamics (Aim 1b), gene expression and chromatin accessibility (Aim 1c) and inflammation pathways (NF-κB, AP-1, STAT and NLRP3 activity, Aim 1d). Using the R61 results as readouts, the R33 phase will use pharmacologic inhibition and CRISPR to identify the specific dopamine receptors involved in each readout (Aim 2), and to examine neuronal function and neuroimmune interaction (Aim 3) by evaluating; resting membrane potential and neuron firing rate with patch clamp electrophysiology, dendritic spine density and morphology and microglial-neuronal contacts using confocal imaging and Neurolucida360 analysis, and neuronal network activity using multielectrode arrays. This will define specific dopamine receptors and microglial or neuronal functions that can be targeted to ameliorate the impact of SUD on inflammation in PLWH by manipulating dopaminergic activity.

艾滋病毒流行病越来越多地受到感染在脆弱亚群体中传播的驱动， 药物滥用病症（SUD）的高流行率，包括使用甲基苯丙胺等兴奋剂 （Meth）和钴（Coc）。兴奋剂的使用加速了全身性疾病，并可以推动 神经发病机制，增加神经精神共病的风险，并加速认知能力下降，尽管 尽管在PLWH中使用兴奋剂的流行率很高，但兴奋剂 影响疾病进展定义不明确。在CNS中尤其如此，因为存在大量的 技术挑战涉及建模小胶质细胞感染和与神经元的相互作用， 这对神经元功能的影响。所有的兴奋剂都会增加中枢神经系统多巴胺的释放， 骨髓细胞群多巴胺水平升高。数据表明，这是暴露于释放 多巴胺，而不是兴奋剂本身，这驱动了小胶质细胞感染和功能的变化。我们 数据支持这一点，表明兴奋剂诱导的多巴胺水平增加了HIV的进入，并增强了骨髓 炎症，增加细胞因子释放，以及体外和NHP中NF-κB和NRLP 3炎性体活性 CNS。由感染的CNS髓样群体驱动的神经炎症是HIV神经发病机制的核心， ART时代，潜在的神经元功能障碍和神经免疫通讯中断，导致 认知障碍和行为改变。我们假设使用兴奋剂会加剧艾滋病- 相关的小胶质细胞炎症通过多巴胺受体激活，导致神经元 neuroHIV的功能障碍为了解决这一问题，我们建议开发易于处理的人类同源共培养物， iPSC衍生的小胶质细胞（iMG）和iPSC衍生的多巴胺神经元（iDAN）。关键是，这些iDAN将释放 多巴胺在体外对兴奋剂暴露的反应。共培养将基于我们现有的iMG方案 和iDAN分化，并将在R61阶段开发和优化高通量分析。 在R61期间，我们将感染HIV并使用兴奋剂+/- ART治疗，然后使用高内容成像，单次 细胞RNA-seq / ATAC-seq和Alphalisas，以评估病毒动力学（Aim 1b）、基因表达和 染色质可及性（Aim 1c）和炎症通路（NF-κB、AP-1、STAT和NLRP 3活性，Aim 1d）。 使用R61结果作为读数，R33阶段将使用药理学抑制和CRISPR来识别 每个读数中涉及的特异性多巴胺受体（目的2），并检查神经元功能， 通过评估神经免疫相互作用（目标3）;使用贴片的静息膜电位和神经元放电率 钳夹电生理学、树突棘密度和形态以及小胶质细胞-神经元接触， 共聚焦成像和Neurolucida 360分析，以及使用多电极阵列的神经元网络活性。这 将定义特定的多巴胺受体和小胶质细胞或神经元功能，可以有针对性地改善 SUD通过操纵多巴胺能活性对PLWH炎症的影响。