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).

注射药物的使用增加了感染HIV的可能性，而阿片类药物使用障碍（OUD）会加速HIV- 1通过免疫抑制和直接CNS作用感染。谷氨酸能兴奋性是主要因素 在HIV依赖性的CNS损伤中，但新出现的证据也表明抑制性GABA能功能丧失 Neurohiv。 Cl-稳态和GABA能量的平行丧失将引起兴奋的结果，因为 “抑制作用”导致净兴奋。如果这是真的，那么保护抑制系统的干预措施是 预测至少会部分否定艾滋病毒的“兴奋性”作用。 KCC2是负责 在成年中枢神经系统中维持[Cl-] I稳态和GABA能功能，并且是拟议研究的重点。 尽管对其他神经系统疾病（例如自闭症，， 某些癫痫，阿片类药物依赖性，大脑创伤/脊髓损伤），它代表了关于 神经hiv中突触性功能障碍的机制。神经内的Cl-浓度（[Cl-] i） 很小，Cl-反转电位（ECL）接近静膜电位。那是微小的变化 [Cl-]我可以极大地影响抑制性（例如GABAA）传播的强度和极性。 NKCC1（CL-吸收） 和KCC2（Cl-feflux）共转运剂是[Cl-] I的关键调节剂，KCC2表达/功能对于 成人神经元生存。他们的平衡可以由细胞因子/营养因子（例如BDNF）调节， 在治疗上（CLP290），通常是阿片类药物。重要的是，我们发现增加KCC2水平/功能 强烈保护人和小鼠神经元中的TAT，GP120，感染性HIV和阿片类药物的暴露。在 体内，维持KCC2磷酸化可以使表达DRD2的纹状体培养基中的KCC2定位 刺神经元（MSN）细胞膜和反向运动因HIV-1 TAT而定义。 AIM 1在体外模型中使用 包括IPSC MSN，感染性HIV和光学生理学，以识别HIV和/或的机制 OPIOIDS ALTER [CL-] I稳态，失调D1/D2 MSN兴奋性，该如何触发突触损伤， 和受保护的策略。尽管具有根本重要性，但KCC2在兴奋性/抑制性（E-I）中的作用 在HIV/OUD的背景下，从未探索过失衡和ECL和Egaba的变化。目标2扩展了 体内/ex Vivo的研究。两种模仿临床病理学的转基因模型（TAT +/-，HIVTG26） 用表达DRD1A-TDOMATO（D1）和DRD2-EGFP（D2）的转基因小鼠越过忠诚度 纹状体MSN种群。检查了急性（2周）和慢性（8周）HIV/TAT暴露时间；分离 小鼠队列（两性）同时接触吗啡（S.C.）。综合研究 MSN中的[Cl-] I调节（Gramicidin perforfated Patch pysology），Synaptodondritic损伤和行为 与纹状体功能相关的是±KCC2增强子前药CLP290作为干预策略。 还测试了通过NKCC1，TMEM16A和CLC-1的替代[Cl-] I调节。 GCAMP8F表达 D1/D2 MSN在清醒中，行为小鼠将纹状体输出活动与行为变化（Inscopix Miniscopes）联系起来。