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

艾滋病毒大流行越来越多地受到感染在脆弱亚群中传播的推动，相对 药物滥用障碍的高流行率，包括使用甲基苯丙胺等兴奋剂 (冰毒)和可卡因(可卡因)。兴奋剂的使用会加速全身性疾病，并可能推动 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 有效的艺术。尽管兴奋剂在PLWH中的使用率很高，但兴奋剂的作用机制 对疾病进展的影响没有明确的定义。这在中南地区尤其如此，因为有大量的 建模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 这对神经元功能产生了后续的变化。所有兴奋剂都会增加中枢多巴胺的释放，暴露在 骨髓系人群的多巴胺水平显著升高。数据表明，它是暴露在释放的 多巴胺，而不是兴奋剂本身，这推动了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髓系 炎症，增加细胞因子的释放，以及体外和正常大鼠核因子-κB和NRLP3的炎症体活性 中枢神经系统。中枢神经系统髓系感染所致的神经炎症是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沟通中断导致 认知障碍和行为改变。我们假设兴奋剂的使用会加剧艾滋病毒- 通过激活多巴胺受体导致小胶质细胞炎症，导致神经元 神经艾滋病毒的功能障碍。为了解决这一问题，我们建议发展易驯化的、同基因的人类共培养。 IPSC来源的小胶质细胞(IMG)和IPSC来源的多巴胺神经元(IDAN)。关键是，这些Idan将发布 多巴胺在体外对兴奋剂暴露的反应。混合文化将基于我们现有的IMG协议 和IDAN差异化，并将为R61阶段的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治疗，然后使用高内容成像，单一 细胞RNA-seq/atac-seq和Alphalisas以评估病毒动力学(目标1b)、基因表达和 染色质可及性(Aim 1c)和炎症途径(核因子-κB、AP-1、STAT和NLRP3活性，Aim 1d)。 使用R61结果作为读数，R33阶段将使用药物抑制和CRISPR来识别 每个读数中涉及的特定多巴胺受体(目标2)，并检查神经元功能和 用贴片评估静息膜电位和神经元放电速率的神经免疫相互作用(目标3) 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NeuroLucida360分析，以及使用多电极阵列的神经元网络活动。这 将定义特定的多巴胺受体和小胶质细胞或神经元功能，这些功能可以被靶向改善 SUD通过调节多巴胺能活动对PLWH炎症反应的影响。

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.