Optical electrophysiology of human primary neurons: role of KCC2 in hyperexcitability induced by HIV-1, Tat, and gp120 and morphine exposure

人类原代神经元的光学电生理学：KCC2 在 HIV-1、Tat、gp120 和吗啡暴露诱导的过度兴奋中的作用

• 批准号: 9623656
• 负责人: Aaron Joseph Barbour
• 金额: $ 3.71万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-10 至 2020-01-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9623656
• 关键词: 3-Dimensional; AIDS Dementia Complex; Acute; Address; Affect; Aminobutyric Acids; Animal Model; Astrocytes; Brain; Brain-Derived Neurotrophic Factor; CCR5 gene; CXCR4 Receptors; Calcium; Cells; Chloride Ion; Chronic; Comorbidity; Data; Disease; Drug usage; Electrophysiology (science); Enhancers; Environment; Equilibrium; Excitatory Neurotoxins; Exposure to; Functional disorder; Glutamates; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; HIV-associated neurocognitive disorder; Human; Image; In Vitro; Individual; Infection; Inflammation Mediators; Inflammatory; Intervention; Lead; Light; Maintenance; Measures; Mediating; Mediator of activation protein; Microglia; Modeling; Morphine; N-Methyl-D-Aspartate Receptors; Neuronal Dysfunction; Neurons; Opioid; Opioid Receptor; Optics; Outcome; Pathology; Pharmacology; Physiological; Play; Prevalence; Property; Proteins; Protocols documentation; Receptor Activation; Receptor Protein-Tyrosine Kinases; Research; Role; Signal Pathway; Signal Transduction; Symptoms; Synapses; System; T-Lymphocyte; Techniques; Testing; Withdrawal; Work; antiretroviral therapy; astrocyte progenitor; calcium indicator; cell type; chemokine receptor; chloride-cotransporter potassium; exposed human population; fetal; gamma-Aminobutyric Acid; gamma-Chemokines; human model; inhibitor/antagonist; innovation; macrophage; mu opioid receptors; nerve stem cell; nervous system disorder; neuronal excitability; neurotoxic; new therapeutic target; novel; opioid use; opioid use disorder; optogenetics; receptor; recruit; reduce symptoms; relating to nervous system; response; tool; voltage

PROJECT SUMMARY The introduction of combined antiretroviral therapy has decreased the prevalence of HIV-associated dementia, but prevalence of milder HIV-associated neurocognitive disorders (HAND) has increased. The CNS is highly vulnerable to insult from HIV-1 via release of neurotoxic HIV proteins, inflammatory factors, and excitotoxins by infected astrocytes and microglia. Opiate use is often comorbid with HIV-1 infection and there is evidence for enhanced HAND symptomology in users, likely due to convergent cell signaling pathways resulting in increased neuronal dysfunction. Despite intense research the interactions between HIV-1 and opiates that lead to neuronal damage remain elusive, and most of this work is conducted in animal models. Since HIV-1 is a human-specific disease, it is paramount to validate and expand upon research findings in nonhuman models using human models. To this end, we have differentiated human neural progenitors to develop two primary human CNS culture models: i) a dissociated system containing glutamatergic and GABAergic neurons; and ii) a 3-dimensional brain aggregate (BrAgg) model that contains all major CNS cell types. Using non-invasive electrophysiological techniques including genetically encoded voltage and Ca2+ indicators (GEVI/GECI), and optogenetics we will examine the effects of infectious HIV-1, Tat, and gp120 ± morphine on primary human neuron function. By studying HIV-1 ± morphine effects on neuronal excitability we have uncovered a novel therapeutic target. K-Cl cotransporter 2 (KCC2) maintains low neuronal [Cl-]i necessary for -aminobutyric acid type A receptor (GABAAR)-mediated inhibition. Loss of KCC2 activity resulting in loss of GABAAR hyperpolarization, and even GABA-induced depolarization, has been implicated in a variety of neurological disorders. This project tests the hypothesis that HIV-1 and morphine-induced signaling converge to reduce neuronal KCC2 activity leading to electrophysiological and synaptic excitatory-inhibitory imbalance in primary human neurons. Our data have demonstrated significant loss of KCC2 and subsequent shift from hyper- to depolarization in response to GABA in HIV-1, Tat, and gp120 ± morphine-exposed human neurons. Pharmacological intervention to raise KCC2 levels has reversed these effects. Aim 1 will utilize GEVI and GECI to determine functional responses to infectious HIV-1, Tat, and gp120 (R5-, X4-, and dual-tropic) exposure on dissociated human neurons and examine modulation of KCC2 and relevant upstream regulators (e.g. NMDAR, CCR5, CXCR4, opioid receptors) to rescue noted deficits. Aim 2 will utilize stable BrAgg which contain all major CNS cell types and can be actively infected by HIV-1. Tools and measures described in Aim 1 will be used to examine neuronal function in a chronic, HIV-1-infected environment and elucidate differences between chronic, acute, and withdrawal morphine exposure paradigms. These studies use innovative models and techniques to determine effects of live HIV-1, Tat, and gp120 ± morphine on primary human neuron function and identify novel points of HIV-1 and morphine signaling convergence and targets for intervention to alleviate the symptoms of HAND ± comorbid opiate use.

项目概要 联合抗逆转录病毒疗法的引入降低了艾滋病毒相关痴呆症的患病率， 但与艾滋病毒相关的轻度神经认知障碍 (HAND) 患病率有所增加。中枢神经系统高度 通过释放神经毒性 HIV 蛋白、炎症因子和兴奋毒素，容易受到 HIV-1 的损害 感染的星形胶质细胞和小胶质细胞。阿片类药物的使用通常与 HIV-1 感染并存，并且有证据表明 用户的 HAND 症状增强，可能是由于细胞信号通路趋同导致增加 神经元功能障碍。尽管进行了大量研究，但 HIV-1 和阿片类药物之间的相互作用会导致神经元 损害仍然难以捉摸，而且大部分工作都是在动物模型中进行的。由于 HIV-1 是人类特异性的 疾病，至关重要的是使用人类在非人类模型中验证和扩展研究结果 模型。为此，我们分化了人类神经祖细胞，开发了两种原代人类中枢神经系统培养物 模型：i) 包含谷氨酸能和 GABA 能神经元的分离系统； ii) 3维大脑 包含所有主要 CNS 细胞类型的聚合 (BrAgg) 模型。使用非侵入性电生理学 技术，包括基因编码电压和 Ca2+ 指示剂 (GEVI/GECI) 以及光遗传学，我们将 检查传染性 HIV-1、Tat 和 gp120 ± 吗啡对人类初级神经元功能的影响。经过 通过研究 HIV-1±吗啡对神经元兴奋性的影响，我们发现了一个新的治疗靶点。氯化钾 协同转运蛋白 2 (KCC2) 维持 γ-氨基丁酸 A 型受体所需的低神经元 [Cl-]i (GABAAR) 介导的抑制。 KCC2 活性丧失导致 GABAAR 超极化丧失，甚至 GABA 诱导的去极化与多种神经系统疾病有关。该项目测试了 假设 HIV-1 和吗啡诱导的信号传导会聚合以减少神经元 KCC2 活性，从而导致 原代人类神经元的电生理学和突触兴奋抑制失衡。我们的数据有 表现出 KCC2 的显着缺失，并随后响应 GABA 从超极化转变为去极化 HIV-1、Tat 和 gp120 ± 吗啡暴露的人类神经元中。提高 KCC2 的药物干预 水平扭转了这些影响。目标 1 将利用 GEVI 和 GECI 来确定对 传染性 HIV-1、Tat 和 gp120（R5-、X4- 和双向）暴露于分离的人类神经元和 检查 KCC2 和相关上游调节因子（例如 NMDAR、CCR5、CXCR4、阿片受体）的调节 以挽救明显的赤字。目标 2 将利用稳定的 BrAgg，其中包含所有主要 CNS 细胞类型，并且可以主动 感染 HIV-1。目标 1 中描述的工具和措施将用于检查慢性、 HIV-1 感染环境并阐明慢性、急性和戒断吗啡之间的差异 曝光范例。这些研究使用创新模型和技术来确定活 HIV-1 的影响， Tat 和 gp120 ± 吗啡对人类初级神经元功能的影响并确定 HIV-1 和吗啡的新点 信号趋同和干预目标，以减轻 HAND ± 合并阿片类药物使用的症状。