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).

注射吸毒增加感染艾滋病毒的可能性，阿片类药物使用障碍(OUD)加速艾滋病毒- 1通过免疫抑制和直接中枢神经系统作用感染。谷氨酸能兴奋毒性是一个主要因素 在HIV依赖的中枢神经系统损伤中，但新的证据也表明，在 神经艾滋病毒。氯−动态平衡和GABA能张力的平行丧失将恶化兴奋性结果，因为 “去抑制”的结果是净兴奋。如果这是真的，那么保护抑制系统的干预措施 预计至少会部分抵消艾滋病毒的“兴奋毒性”效应。KCC2是主要的运输者，负责 在成人中枢神经系统中维持[Cl−]i动态平衡和GABA能功能是目前研究的重点。 尽管去抑制作为驱动力的想法被接受用于其他神经疾病(例如，自闭症， 某些癫痫、阿片类药物依赖、创伤性脑/脊髓损伤)，它代表着关于 神经性HIV患者突触树突起功能障碍的机制。神经元内氯−浓度([Cl−]i) Cl-−翻转电位接近静止膜电位。因此，中的细微更改 [CL−]i可以极大地影响抑制性(例如，GaBAA)传递的强度和极性。NKCC1(氯−摄取) 和KCC2(Cl−外排)共转运体是[Cl−]i的关键调节因子，KCC2的表达/功能是 成体神经元存活。它们的平衡可以由来自胶质细胞的细胞因子/营养因子(例如，BDNF)来调节， 治疗学(CLP290)、遗传学和阿片类药物。重要的是，我们发现增加KCC2水平/功能 强烈保护人类和小鼠神经元免受TAT、gp120、传染性艾滋病毒和阿片类药物的影响。在……里面 体内，维持KCC2磷酸化可以使KCC2在表达Drd2的纹状体介质中的定位正常化 刺状神经元(MSN)细胞膜和逆转由HIV-1TAT引起的运动缺陷。AIM 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动态平衡，失调d1/d2msn兴奋性，如何触发突触树突损伤， 和保护性策略。尽管KCC2很重要，但它在兴奋/抑制(E-I)中的作用 在艾滋病毒/艾滋病的背景下，从未探讨过失衡和变化的ECL和EGABA。Aim 2扩展了 体内/体外研究。2个转基因模型(TAT+/-，HIVTg26)，两者都模拟临床病理，具有相当大的 将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种群。检查急性(2wk)和慢性(8wk)艾滋病毒/TAT暴露时间；分开 一组小鼠(不分性别)同时、逐渐暴露于吗啡(S.C.)。综合研究 [Cl−]i在MSN中的调节、突触树突起损伤和行为 与纹状体功能相关的是以KCC2增强子前药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。中的GCaMP8f表达式 在清醒、行为正常的小鼠中，d1/d2 msns将纹状体输出活动与行为变化联系起来(内窥镜微型显微镜)。