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

基本信息

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
项目类别：
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

项目摘要

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.

艾滋病毒大流行越来越多地受到感染在相对脆弱的亚人群中传播的推动。药物滥用障碍 (SUD) 患病率很高，包括使用甲基苯丙胺等兴奋剂 (Meth) 和可卡因 (Coc) 的使用会加速全身性疾病，并可能导致身体状况发生变化。神经发病机制，增加神经精神合并症的风险并加速认知能力下降，尽管尽管艾滋病病毒感染者中兴奋剂的使用率很高，但兴奋剂的作用机制仍然存在。疾病进展的影响尚不清楚，这在中枢神经系统中尤其如此，因为存在大量的影响。模拟小胶质细胞感染和与神经元相互作用所涉及的技术挑战，以及这对神经功能产生的后续变化所有兴奋剂都会增加中枢神经系统多巴胺的释放，从而暴露。数据表明，骨髓细胞中的多巴胺水平显着升高。多巴胺，而不是兴奋剂本身，驱动了小胶质细胞感染和功能的变化。数据支持这一点，显示兴奋剂诱导的多巴胺水平增加了艾滋病毒的进入并增强了骨髓细胞炎症、增加细胞因子释放以及体外和 NHP 中的 NF-κB 和 NRLP3 炎性体活性中枢神经系统 (CNS) 受感染的中枢神经系统髓系细胞群驱动的神经炎症是 HIV 神经发病机制的核心。 ART 时代，神经元功能障碍和神经免疫通讯中断导致我们勇敢地面对兴奋剂的使用会加剧艾滋病毒的传播。通过多巴胺受体激活相关的小胶质细胞炎症，导致神经元为了解决这个问题，我们建议开发易于处理的、同基因的人类共培养物。 iPSC 衍生的小胶质细胞 (iMG) 和 iPSC 衍生的多巴胺神经元 (iDAN) 至关重要，这些 iDAN 将释放。多巴胺体外对刺激物暴露的反应将基于我们现有的 iMG 方案。和 iDAN 差异化，并将在 R61 阶段针对高通量分析进行开发和优化。在R61期间，我们将感染HIV并用兴奋剂+/- ART进行治疗，然后使用高内涵成像，单次细胞 RNA-seq / ATAC-seq 和 Alphalisas 评估病毒动力学（目标 1b）、基因表达和染色质可及性（目标 1c）和炎症通路（NF-κB、AP-1、STAT 和 NLRP3 活性，目标 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定多巴胺受体（目标 2），并检查神经功能和通过评估静息膜电位和神经元放电率来实现神经免疫相互作用（目标 3）；使用钳位电生理学、树突棘密度和形态以及小胶质细胞-神经元接触共焦成像和 Neurolucida360 分析，以及使用多电极阵列的神经网络活动。将定义可以针对性改善的特定多巴胺受体和小胶质细胞或神经元功能 SUD 通过控制多巴胺能活性对 PLWH 炎症的影响。