Innovative therapeutic approaches to address excitotoxic CNS/neuronal damage in opioid-neuroHIV comorbidity

解决阿片类药物-神经艾滋病毒合并症中的兴奋性中枢神经系统/神经元损伤的创新治疗方法

• 批准号: 10573827
• 负责人: Kurt F Hauser
• 金额: $ 67.87万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10573827
• 关键词: AMPA Receptors; Acute; Address; Adult; Affect; Agonist; Animals; Architecture; Behavior; Behavioral; Biochemical; Brain; Brain-Derived Neurotrophic Factor; CLC Gene; Cell membrane; Cells; Chronic; Clinical Pathology; Contracts; Corpus striatum structure; Data; Disinhibition; Dopamine Receptor; Duct (organ) structure; Electrophysiology (science); Enhancers; Epilepsy; Equilibrium; Event; Exposure to; Functional disorder; Glutamates; Gramicidin; HIV; HIV Envelope Protein gp120; HIV-1; Heroin; Homeostasis; Human; Immunosuppression; Impaired cognition; In Vitro; Incidence; Infection; Inflammation; Injury; Intervention; Intractable Epilepsy; Link; Membrane Potentials; Minor; Morphine; Mus; N-Methyl-D-Aspartate Receptors; Neuroglia; Neurologic; Neuronal Injury; Neurons; Opiate Addiction; Opioid; Optics; Outcome; Output; Pattern; Phosphorylation; Physiological; Physiology; Population; Probability; Prodrugs; Property; Proteins; Ramp; Regulation; Reporter; Rest; Role; Severities; Signal Transduction; Spinal cord injury; System; Temporal Lobe; Testing; Therapeutic; Therapeutic Intervention; Time; Transgenic Mice; Transgenic Model; autism spectrum disorder; awake; behavior measurement; central nervous system injury; cohort; comorbidity; cytokine; deafness; driving force; excitotoxicity; experimental study; genetic regulatory protein; in vitro Model; in vivo; induced pluripotent stem cell; inhibitor; injection drug use; innovation; motor behavior; motor deficit; mu opioid receptors; nervous system disorder; neuroAIDS; neuronal survival; neurotoxic; opioid abuse; opioid exposure; opioid use disorder; optical imaging; painful neuropathy; patch clamp; sex; sodium-potassium-chloride cotransporter 1 protein; spinal cord and brain injury; symporter; theories; therapy design; trafficking; transmission process; uptake; voltage

Injection drug use increases the probability of contracting HIV, and opioid use disorder (OUD) accelerates HIV- 1 infection through immune suppression and direct CNS actions. Glutamatergic excitotoxicity is a major factor in HIV-dependent CNS injury, but emerging evidence also suggests a loss of inhibitory GABAergic function in neuroHIV. The parallel loss of Cl− homeostasis and GABAergic tone will worsen excitatory outcomes since ‘disinhibition’ results in net excitation. If this is true, then interventions that protect inhibitory systems are predicted to at least partially negate ‘excitotoxic’ effects of HIV. KCC2 is the main transporter responsible for maintaining [Cl−]i homeostasis and GABAergic function in the adult CNS and is the focus of proposed studies. Although the idea of disinhibition as a driving force is embraced for other neurological disorders (e.g., autism, certain epilepsies, opioid dependence, traumatic brain/spinal cord injury), it represents a conceptual shift about mechanisms underlying synaptodendritic dysfunction in neuroHIV. The Cl− concentration inside neurons ([Cl−]i) is small and the Cl− reversal potential (ECl) is close to the resting membrane potential. Thus, minor changes in [Cl−]i can greatly affect the strength and polarity of inhibitory (e.g., GABAA) transmission. NKCC1 (Cl− uptake) and KCC2 (Cl− efflux) co-transporters are key regulators of [Cl−]i, and KCC2 expression/function is essential for adult neuron survival. Their balance can be regulated by cytokines/trophic factors (e.g., BDNF) from glia, therapeutically (CLP290), genetically, and by opioids. Importantly, we find that increasing KCC2 levels/function strongly protects against exposure to Tat, gp120, infectious HIV, and opioids in human and mouse neurons. In vivo, maintaining KCC2 phosphorylation can normalize KCC2 localization in Drd2-expressing striatal medium spiny neuron (MSN) cell membranes and reverse motor deficits due to HIV-1 Tat. Aim 1 uses in vitro models including iPSC MSNs, infective HIV, and optical electrophysiology to identify mechanisms by which HIV and/or opioids alter [Cl−]i homeostasis, dysregulate D1/D2 MSN excitability, how this triggers synaptodendritic injury, and protective strategies. Despite its fundamental importance, the role of KCC2 in excitatory/inhibitory (E-I) imbalances and altered ECl and EGABA have never been explored in the context of HIV/OUD. Aim 2 extends the studies in vivo/ex vivo. 2 transgenic models (Tat+/-, HIVTg26) that both mimic clinical pathology with considerable fidelity are crossed with transgenic mice expressing Drd1a-tdTomato (D1) and Drd2-eGFP (D2) to identify both striatal MSN populations. Acute (2 wk) and chronic (8 wk) HIV/Tat exposure times are examined; separate cohorts of mice (both sexes) receive concurrent, ramping exposure to morphine (s.c.). Comprehensive studies of [Cl−]i regulation in MSNs (gramicidin-perforated patch physiology), synaptodendritic injury, and behavior related to striatal function are performed ± the KCC2 enhancer prodrug CLP290 as an intervention strategy. Alternative [Cl−]i regulation through NKCC1, TMEM16A, and CLC-1 are also tested. GCaMP8f expression in D1/D2 MSNs in awake, behaving mice links striatal output activity to behavioral change (Inscopix miniscopes).

注射吸毒会增加感染 HIV 的可能性，而阿片类药物使用障碍 (OUD) 会加速 HIV 感染 1 通过免疫抑制和直接中枢神经系统作用而感染。谷氨酸兴奋性毒性是主要因素 HIV 依赖性中枢神经系统损伤，但新出现的证据也表明，抑制性 GABA 能功能丧失 神经艾滋病毒。 Cl− 稳态和 GABA 能张力的同时丧失将使兴奋性结果恶化，因为 “去抑制”导致净兴奋。如果这是真的，那么保护抑制系统的干预措施就是 预计至少可以部分消除艾滋病毒的“兴奋毒性”作用。 KCC2 是负责的主要运输者 维持成人 CNS 中的 [Cl−]i 稳态和 GABA 能功能，是拟议研究的重点。 尽管去抑制作为驱动力的想法也被其他神经系统疾病（例如自闭症、 某些癫痫症、阿片类药物依赖、创伤性脑/脊髓损伤），它代表了关于 神经艾滋病毒突触树突功能障碍的机制。神经元内的 Cl− 浓度 ([Cl−]i) 很小，Cl− 反转电位 (ECl) 接近静息膜电位。因此，微小的变化 [Cl−]i 可以极大地影响抑制性（例如 GABAA）传输的强度和极性。 NKCC1（Cl− 摄取） 和 KCC2（Cl− 流出）协同转运蛋白是 [Cl−]i 的关键调节因子，KCC2 表达/功能对于 成年神经元存活。它们的平衡可以通过神经胶质细胞的细胞因子/营养因子（例如 BDNF）来调节， 治疗（CLP290）、遗传和阿片类药物。重要的是，我们发现增加 KCC2 水平/功能 强烈防止人类和小鼠神经元接触 Tat、gp120、传染性 HIV 和阿片类药物。在 体内，维持 KCC2 磷酸化可以使 KCC2 在表达 Drd2 的纹状体培养基中的定位正常化 棘神经元 (MSN) 细胞膜并逆转 HIV-1 Tat 引起的运动缺陷。目标 1 使用体外模型 包括 iPSC MSN、感染性 HIV 和光学电生理学，以确定 HIV 和/或 阿片类药物改变 [Cl−]i 稳态，调节 D1/D2 MSN 兴奋性，这如何触发突触树突损伤， 和保护策略。尽管 KCC2 具有根本重要性，但其在兴奋/抑制 (E-I) 中的作用 从未在 HIV/OUD 的背景下探讨过不平衡以及 ECI 和 EGABA 的改变。目标 2 扩展了 体内/离体研究。 2 种转基因模型（Tat+/-、HIVTg26）均模仿临床病理学，具有相当大的 fidelity 与表达 Drd1a-tdTomato (D1) 和 Drd2-eGFP (D2) 的转基因小鼠杂交，以鉴定两者 纹状体 MSN 群体。检查急性（2 周）和慢性（8 周）HIV/Tat 暴露时间；分离 小鼠组（男女）同时接受吗啡（SC）逐渐增加的暴露。综合研究 [Cl−]i 在 MSN（短杆菌肽穿孔斑生理学）、突触树突损伤和行为中的调节 进行与纹状体功能相关的±KCC2增强剂前药CLP290作为干预策略。 还测试了通过 NKCC1、TMEM16A 和 CLC-1 进行的替代 [Cl−]i 调节。 GCaMP8f 表达于 清醒、有行为的小鼠中的 D1/D2 MSN 将纹状体输出活动与行为变化联系起来（Inscopix 微型显微镜）。