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.

艾滋病毒的流行越来越多地是由于感染在弱势亚群体中的传播造成的