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受到感染在脆弱的亚人群中的传播驱动药物滥用障碍（SUD）的高患病率，包括使用诸如甲基苯丙胺之类的兴奋剂（甲基）和可卡因（COC）。兴奋剂使用会加速系统性疾病，并可以推动变化神经病发生，增加风险神经精神病的合并症并加速认知能力下降，多叠岩有效的艺术。尽管PLWH中刺激性使用率很高，但刺激剂的机制影响疾病进展的定义很差。在中枢神经系统中尤其如此，因为有实质性建模小胶质细胞感染和与神经元的相互作用涉及的技术挑战以及随后的改变这对神经元功能的变化。所有兴奋剂都会增加CNS多巴胺释放，暴露髓样群体达到高度升高的多巴胺水平。数据表明它是释放的多巴胺而不是兴奋剂本身，它可以驱动小胶质细胞感染和功能的变化。我们的数据支持这一点，显示刺激性诱导的多巴胺水平增加了HIV的进入并增强了髓样炎症，细胞因子释放的增加以及NF-κB和NRLP3炎症体活性在体外和NHP中 CNS。受感染的CNS髓样群驱动的神经炎症是HIV神经病发生的核心艺术擦除，神经元功能障碍和神经免疫性交流中的干扰认知障碍和行为变化。我们假设刺激性使用加剧了HIV- 通过多巴胺受体激活相关的小胶质细胞注射，导致神经元神经联体功能障碍。为了解决这个问题，我们建议开发人类的合成共同培养 IPSC衍生的小胶质细胞（IMG）和IPSC衍生的多巴胺神经元（IDAN）。至关重要的是，这些Idan将释放多巴胺在体外响应刺激性暴露。共培养将基于我们现有的IMG协议和IDAN分化，并将在R61阶段开发和优化用于高通量分析。在R61期间，我们将感染HIV并用刺激剂+/- ART进行治疗，然后使用高内容成像，单个细胞RNA-seq / atac-seq和alphalisas评估病毒动力学的变化（AIM 1B），基因表达和染色质可及性（AIM 1C）和炎症途径（NF-κB，AP-1，STAT和NLRP3活性，AIM 1D）。将R61结果作为读数，R33阶段将使用药物抑制和CRISPR来识别每个读数涉及的特定多巴胺受体（AIM 2），并检查神经元功能和通过评估神经免疫相互作用（目标3）；用斑块静止的膜电位和神经元发射速率使用夹具电生理学，树突状脊柱密度和形态和小胶质细胞神经元接触共聚焦成像和Neurolucida360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以及可以针对改善的小胶质细胞或神经元功能通过操纵多巴胺能活性，SUD对PLWH炎症的影响。