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: 9754573
• 负责人: Aaron Joseph Barbour
• 金额: $ 2.3万
• 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/9754573
• 关键词: 3-Dimensional; AIDS dementia; 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; Structure; 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.

项目摘要 采用联合抗逆转录病毒疗法降低了艾滋病毒相关痴呆症的发病率， 但较轻的艾滋病相关神经认知障碍（HAND）的患病率有所增加。CNS高度 通过释放神经毒性HIV蛋白、炎症因子和兴奋性毒素，容易受到HIV-1的伤害， 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感染共病，有证据表明， 使用者中增强的手神经学，可能是由于会聚细胞信号传导途径导致增加的 神经元功能障碍尽管进行了大量的研究，但HIV-1和阿片类药物之间的相互作用导致神经元 损伤仍然是难以捉摸的，并且大多数这项工作是在动物模型中进行的。由于HIV-1是人类特有的 疾病，最重要的是验证和扩大在非人类模型中的研究结果，使用人类 模型为此，我们分化了人神经祖细胞，以开发两种原代人CNS培养物 模型：i）含有谷氨酸能和GABA能神经元的分离系统;和ii）三维脑 聚集体（BrAgg）模型，其包含所有主要CNS细胞类型。使用非侵入性电生理学 包括遗传编码电压和Ca 2+指标（GEVI/GECI）和光遗传学技术，我们将 检测感染性HIV-1、达特和gp 120 ±吗啡对原代人类神经元功能的影响。通过 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（KCC 2）维持低的神经元[Cl-]i，这是α-氨基丁酸A型受体所必需的 （GABAAR）介导的抑制。KCC 2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集以降低神经元KCC 2活性，从而导致 电生理和突触兴奋-抑制不平衡的原代人类神经元。我们的数据 证明了KCC 2的显著损失和随后响应GABA从超极化到去极化的转变 在暴露于HIV-1、达特和gp 120 ±吗啡的人类神经元中。药物干预提高KCC 2 水平逆转了这些影响。目标1将利用GEVI和GECI来确定功能反应， 感染性HIV-1、达特和gp 120（R5-、X4-和双嗜性）暴露于分离的人神经元， 检查KCC 2和相关上游调节因子（如NMDAR、CCR 5、CXCR 4、阿片受体）的调节 以挽救明显的赤字。目的2将利用稳定的BrAgg，其含有所有主要的CNS细胞类型，并且可以被主动地 感染HIV-1。目标1中描述的工具和测量将用于检查慢性， HIV-1感染的环境和阐明慢性，急性和戒断吗啡之间的差异 暴露范例。这些研究使用创新的模型和技术来确定活HIV-1的影响， 达特和gp 120 ±吗啡对原代人神经元功能的影响及HIV-1和吗啡的新作用点 信号收敛和干预的目标，以减轻手±共病阿片类药物使用的症状。