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: 10684110
• 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/10684110
• 关键词: AMPA Receptors; Acceleration; Acute; Address; Adult; Affect; Agonist; Animals; Architecture; Behavior; Behavioral; Biochemical; Brain; Brain-Derived Neurotrophic Factor; Breeding; CLC Gene; Cell membrane; Cells; Chronic; Clinical Pathology; Contracts; Corpus striatum structure; Data; Disinhibition; Dopamine Receptor; 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; Opioid Receptor; Optics; Outcome; Output; Pattern; Perforation; 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; 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; nervous system disorder; neuroAIDS; neuronal excitability; 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).

注射吸毒增加了感染艾滋病毒的可能性，阿片类药物使用障碍（OUD）加速了艾滋病毒的传播。 1例通过免疫抑制和直接CNS作用感染。谷氨酸能兴奋毒性是一个主要因素 但新的证据也表明，在HIV依赖性CNS损伤中， 神经HIV。Cl−稳态和GABA能张力的平行丧失将使兴奋性结局恶化， “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 预计至少可以部分抵消HIV的“兴奋毒性”作用。KCC 2是主要的运输机， 维持成人CNS中[Cl−]i的稳态和GABA能功能，是拟议研究的重点。 尽管解除抑制作为驱动力的想法被接受用于其他神经系统疾病（例如，孤独症， 某些癫痫，阿片类药物依赖，创伤性脑/脊髓损伤），它代表了一个概念的转变， 神经HIV中突触树突功能障碍的潜在机制。神经元内的Cl−浓度（[Cl−]i） Cl-逆转电位（ECl）接近静息膜电位。因此， [Cl- ]i可以极大地影响抑制的强度和极性（例如，GABAA）传输。NKCC 1（Cl−摄取） 和KCC 2（Cl−流出）协同转运蛋白是[Cl−]i的关键调节因子，KCC 2的表达/功能对于 成体神经元存活它们的平衡可以通过细胞因子/营养因子（例如，BDNF）从神经胶质细胞， 治疗（CLP 290），遗传学和阿片类药物。重要的是，我们发现增加KCC 2水平/功能 在人类和小鼠神经元中强烈保护免于暴露于达特、gp 120、传染性HIV和阿片类药物。在 在体内，维持KCC 2磷酸化可以使KCC 2在表达Drd 2的纹状体培养基中的定位正常化 多刺神经元（MSN）细胞膜和逆转HIV-1达特引起的运动缺陷。Aim 1使用体外模型 包括iPSC MSN，感染性HIV和光电生理学，以确定HIV和/或 阿片类药物改变[Cl−]i稳态，D1/D2 MSN兴奋性失调，这如何触发突触树突损伤， 和保护策略。尽管KCC 2具有根本的重要性，但其在兴奋/抑制（E-I） 在HIV/OUD的背景下，从未探索过不平衡和改变的EC 1和EGABA。目标2扩展了 体内/离体研究。2种转基因模型（达特+/-，HIV Tg 26），均模拟临床病理学， Fidelity与表达Drd 1a-tdTomato（D1）和Drd 2-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/达特暴露时间;分别 一组小鼠（两种性别）同时接受吗啡（s.c.）的斜升暴露。综合研究 [Cl−]i在MSN（短杆菌肽穿孔贴片生理学）、突触树突损伤和行为中的调节 与纹状体功能相关的一系列干预策略± KCC 2增强剂前药CLP 290作为干预策略进行。 还测试了通过NKCC 1、TMEM 16 A和CLC-1的替代[Cl−]i调节。GCaMP 8 f表达 清醒的行为小鼠中的D1/D2 MSN将纹状体输出活动与行为变化联系起来（Insopix微型镜）。