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.

项目摘要 联合抗逆转录病毒疗法的引入降低了与HIV相关痴呆的患病率， 但是米勒HIV相关的神经认知疾病（手）的患病率增加了。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损害的影响。 感染的星形胶质细胞和小胶质细胞。使用使用通常与HIV-1感染合并，并且有证据表明 用户的手动症状增强，可能是由于收敛性细胞信号通路导致增加 神经元功能障碍。尽管进行了激烈的研究，但HIV-1之间的相互作用并优化导致神经元的相互作用 损坏仍然难以捉摸，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，使用人类在非人类模型中验证和扩展研究结果至关重要 型号。为此，我们有区分人类神经祖细胞来发展两种主要的人类中枢神经系统培养物 模型：i）一个含有谷氨酸能和GABA能神经元的解离系统； ii）三维大脑 包含所有主要CNS细胞类型的骨料（Bragg）模型。使用非侵入性电生理学 包括遗传编码的电压和Ca2+指标（GEVI/GECI）和光遗传学在内的技术，我们将 检查感染性HIV-1，TAT和GP120±吗啡对原代人神经元功能的影响。经过 研究HIV-1±吗啡对神经元刺激的影响，我们发现了一个新型的治疗靶标。 k-cl cotransporter 2（KCC2）维持低神经元[Cl-] i对于A型A型接收器所必需 （Gabaar）介导的抑制作用。 KCC2活性的丧失导致Gabaar超极化的丧失，甚至 GABA诱导的去极化已隐含在多种神经系统疾病中。该项目测试 假设HIV-1和吗啡诱导的信号传导会融合以减少神经元KCC2活性导致 原发性人神经元中电生理和突触兴奋性抑制性失衡。我们的数据有 表现出KCC2的显着损失，随后从高度转向GABA的转移 在HIV-1，TAT和GP120±吗啡暴露的人类神经元中。提高KCC2的药理干预措施 水平扭转了这些影响。 AIM 1将利用Gevi和Geci来确定对 感染性HIV-1，TAT和GP120（R5-，X4-和双疗法）暴露于解离的人类神经元和 检查KCC2和相关上游调节剂的调节（例如NMDAR，CCR5，CXCR4，阿片受体） AIM 2将利用稳定的Bragg，其中包含所有主要CNS单元类型，并且可以主动 由HIV-1感染。 AIM 1中描述的工具和度量将用于检查慢性， HIV-1感染的环境以及慢性，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实时HIV-1的影响， TAT和GP120±吗啡在原代人神经元功能上，并鉴定HIV-1和吗啡的新点 信号收敛和干预目标，以减轻手部症状±合并症优化使用。