HIV-1 and opiates dysregulate human neural progenitor function

HIV-1 和阿片类药物会导致人类神经祖细胞功能失调

• 批准号: 9064561
• 负责人: Joyce Balinang
• 金额: $ 2.68万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-10 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9064561
• 关键词: Adenylate Cyclase; Affect; Affinity; Alternative Splicing; Astrocytes; Behavior; Biochemical; Biological Assay; CMV promoter; Cell Cycle; Cell Death; Cell Density; Cells; Co-Immunoprecipitations; Complication; Consequences of HIV; Coupling; Cyclic AMP; Data; Development; Disease; Disease Progression; Drug abuse; Event; Exposure to; Family; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Genetic Transcription; HIV; HIV-1; HIV-associated neurocognitive disorder; Heroin; Heroin Abuse; Human; Immunoprecipitation; Lead; Light; MAPK Signaling Pathway Pathway; Mediating; Microglia; Microscopic; Mitogen-Activated Protein Kinases; Molecular; Morphine; Morphine Abuse; Mus; Neurocognitive Deficit; Neuroglia; Neurons; Nuclear Pore Complex; Opiates; Opioid; Opioid Receptor; Outcome; Oxycodone; Pathway interactions; Patients; Phenotype; Pilot Projects; Play; Population; Prevalence; Proteins; RNA Splicing; Reporting; Research; Role; S Phase; Severities; Signal Pathway; Signal Transduction; Small Interfering RNA; Testing; Time; Undifferentiated; Variant; antiretroviral therapy; brain tissue; cell motility; cell type; drug of abuse; expression vector; functional outcomes; genetic manipulation; gliogenesis; knock-down; member; nerve stem cell; neurogenesis; neurotoxicity; novel; opioid abuse; overexpression; precursor cell; progenitor; protein structure; public health relevance; receptor; response; self renewing cell; sensor

 DESCRIPTION (provided by applicant): Although the advent of combination antiretroviral therapy (cART) has significantly reduced the severity of the CNS deficits in most patients, the prevalence of milder deficits, collectively referred to as HIV-associated neurocognitive disorders (HAND), remains at pre-CART levels. Increasing evidence suggests that opiate drug abuse, including heroin and morphine abuse, enhances the development and progression of HAND. This is thought to occur via the interaction of opiates with opioid receptor-expressing microglia, astrocytes, and neurons, ultimately leading to a cascade of cellular events that directly/indirectl impacts surrounding neurons. Opiate drugs of abuse chiefly activate the µ-opioid receptor (MOR) subtype, expressed by various CNS cells. MOR is encoded by the OPRM1 gene, which has been shown to undergo extensive alternative splicing to generate at least 21 splice variants. Neural progenitor cells, which are the undifferentiated precursor cells of CNS neurons and glial cells, are known to be affected by HIV and opiates independently, but we recently showed an interactive effect between HIV and morphine on the proliferation of mouse NPCs and immortalized human NPC (hNPCs), as well as on the populations of cells that they produce. It remains entirely unknown whether similar effects of HIV and morphine occur in primary hNPCs, and if other essential functions of hNPCs are affected. The proposed studies test the hypothesis that HIV and opiates have interactive effects on the survival, proliferation, motility, and differentiation of primary hNPCs that differentially involve MOR splice variants. To support the hypotheses, our preliminary data showed that: 1) HIV-1 and morphine co-exposure significantly decreased the number of hNPCs entering S phase, reduced hNPCs cell density, and significantly increased hNPCs doubling time without affecting hNPC survival; 2) hNPCs express MOR splice variants MOR(1-2) and MOR-1K and their expression was differently modulated by HIV-1. The hypotheses are tested in three specific aims. Aim 1 examines the effects of HIV-1 and morphine co-exposure on essential hNPC functions such as proliferation, differentiation, and motility using multiple biochemical and histochemical assays. Aim 2 tests the functional significance of MOR splice variant signaling in HIV-1 ± morphine-mediated outcomes by genetic manipulation of expression using siRNAs and over-expression vectors. Resulting phenotypes/behaviors will be examined as described in Aim 1. Aim 3 examines the signaling pathways that are activated by MOR(1-2) versus MOR-1K when NPCs respond to HIV ± morphine. A combination of functional (e.g. cAMP activation assay) and biochemical (e.g. immunoprecipitation) approaches examine specific signaling through MAP-kinases and PI3K/Akt. Findings from the proposed studies will provide novel information concerning how hNPCs in the CNS are affected by HIV-opiate co-exposure and the involvement of particular MOR splice variants. Our studies may be fundamental to understanding HIV-MOR interactions in other cell types as well, since the separate functional roles of MOR splice variants have not been carefully tested.

 描述（由适用提供）：尽管组合抗逆转录病毒疗法（CART）的进步显着降低了大多数患者中枢神经系统缺陷的严重程度，但米勒缺乏症的患病率统称为HIV相关的神经认知疾病（手），但仍处于前盘前的水平。越来越多的证据表明，包括海洛因和吗啡滥用在内的优化药物滥用，可以增强手的发展和进展。人们认为这是通过表达精选受体的小胶质细胞，星形胶质细胞和神经元的相互作用来发生的，最终导致一系列直接/indirectl影响周围神经元的细胞事件。阿片类药物的滥用药物主要激活由各种CNS细胞表达的µ-阿片受体（MOR）亚型。 MOR由OPRM1基因编码，该基因已被证明经历了广泛的替代剪接，以产生至少21个剪接变体。已知神经祖细胞是中枢神经系统神经元和神经胶质细胞的未分化的前体细胞，受HIV的影响并独立进行优化，但是我们最近在HIV和吗啡之间表现出对小鼠NPC的增殖和永生的人NPC（HNPCS）的相互作用，以及在其上产生的细胞，它们会产生它们的含量。艾滋病毒和吗啡在原发性HNPC中的发生是否相似，以及HNPC的其他基本功能是否受到影响。提出的研究检验了艾滋病毒和优化对原代HNPC的生存，增殖，运动和分化的互动影响的假设，这些互动效果涉及MOR剪接变体。为了支持假设，我们的初步数据表明：1）HIV-1和吗啡共暴露显着减少了进入S相的HNPC的数量，HNPCS细胞密度降低，并显着增加HNPC，而无需影响HNPC存活而使HNPC倍增了一倍。 2）HNPC表达MOR剪接变体MOR（1-2）和MOR-1K及其表达受HIV-1的不同调节。假设以三个特定目的进行了检验。 AIM 1检查了HIV-1和吗啡共暴露对使用多种生化和组织化学测定的基本HNPC功能（例如增殖，分化和运动）的影响。 AIM 2通过使用siRNA和过表达载体对表达的遗传操纵来测试MOR剪接变体信号传导在HIV-1±吗啡介导的结果中的功能意义。如AIM 1所述，将检查产生的表型/行为。AIM 3检查NPC对HIV±吗啡的反应时，MOR（1-2）与MOR-1K激活的信号传导途径。功能（例如CAMP激活测定法）和生化（例如免疫沉淀）方法的组合通过MAP-激酶和PI3K/AKT检查特定的信号传导。拟议研究的发现将提供有关中枢神经系统中HNPC如何受到HIV侵蚀性侵害和特定MOR剪接变体的参与的新信息。我们的研究也可能是理解其他细胞类型中HIV-MOR相互作用的基础，因为尚未仔细测试MOR剪接变体的单独功能作用。