Free Radicals and Methamphetamine Abuse

描述(申请人提供)：甲基苯丙胺是滥用最多的毒品之一，会导致神经损伤，并与有利于接触艾滋病毒的行为有关，艾滋病毒是一种穿透中枢神经系统导致神经艾滋病的病毒。由于对神经元的影响，感染艾滋病毒的冰毒使用者通常会出现一种更严重的神经艾滋病。已知自由基参与了脑组织病理的增强，但细胞类型以及甲硫氨酸影响的途径导致了自由基介导的应激。这一提议背后的假设是，将探索Meth与神经元以外的其他细胞类型的相互作用，特别是关于其诱导和/或调节活性氧和氮中间体的潜力。我们将研究甲基苯丙胺(Meth)对氧化代谢的作用，直接作用于巨噬细胞，以及在体外和体内，在共病因素相互作用的条件下对其他类型细胞的作用。为此，我们将通过调查药物如何影响氧化爆发的强度和质量，并在ROI和RNI途径中寻找在Meth暴露时表现出转录模式变化的基因，来研究Meth对细胞系的作用。这种转录变化将通过寻找从感染SIV的Meth处理动物获得的细胞中的类似变化来验证，这些细胞以前已经被表征为CNS疾病参数。该项目的结果将为Meth通过作用于ROI和RNI诱导通路，不仅在中枢神经系统，而且在外周器官中参与氧化应激介导的病理过程提供重要线索。 公共卫生相关性：我们将研究甲基苯丙胺(Meth)对氧化代谢的作用，直接作用于巨噬细胞，以及在体外对其他类型的细胞，以及在共病因素相互作用的情况下对体内的作用。这一建议背后的假设是，除了对神经元的作用外，蛋氨酸还通过直接作用于巨噬细胞和/或神经胶质细胞，影响氧化爆发的强度和质量，不仅在中枢神经系统，而且在外周器官中，促进氧化应激介导的病理的发展。首先，我们将重点研究冰毒对各种人和小鼠巨噬细胞(THP1，RAW264.7)，小胶质细胞(MG5，EoC2)，星形胶质细胞(C8-D1A，CCF-STTG1)和神经元(SH-SY5Y，SK-N-MC)细胞产生O2_2，H_2O_2和其他活性氧中间体(ROI)，以及活性氮中间体(RNI)的影响。与冰毒相互作用时的线粒体活动也将在冰毒刺激的细胞上得到解决。在描述了不同的细胞系在对Meth的反应中产生自由基和线粒体变化的特征之后，我们将选择良好的响应者来研究由Meth的作用引起的ROI和RNI途径中分子的转录变化。为此，我们将使用多重技术，以及酶检查点抑制策略。这些方法将有助于剖析Meth诱导呼吸爆发的分子基础，并将区分Meth是否干扰线粒体、过氧化物酶体或两者兼而有之。在使用细胞系的体外方法之后，我们打算在病理学的背景下验证这些发现。我们将使用冷冻保存的细胞和组织，这些细胞和组织来自感染了SIV的猴子(作为HIV感染的模型，这是一种常见的共病情况)，遵循Meth治疗计划。我们将通过qRT-PCR鉴定来自SIV感染Meth处理的猴子的冷冻保存的脑和外周组织中转录变化的氧化途径。这带来了与人类病理学的相关性。目前的建议将确定甲基苯丙氨酸使用者大脑和其他器官中氧化应激的基础。冰毒是滥用人数最多的街头毒品之一，使吸毒者暴露在艾滋病毒感染之下，并有可能促进病毒穿过血脑屏障(BBB)进入大脑。在这项提案中，我们将剖析Meth诱导和调节自由基产生的机制，导致与脑组织中病毒存在相关的损害的严重程度加重，从而加剧艾滋病相关的中枢神经系统功能障碍。在这项建议中获得的结果将允许产生关于药物对细胞的直接影响、不同细胞类型对直接作用的相对敏感性以及在途径上的数据，旨在未来R01在体内Meth滥用中对氧化应激的调节应用。这些将导致开发用于个人康复的重要治疗工具，特别是出现艾滋病毒感染等共病因素的患者。

DESCRIPTION (provided by applicant): Methamphetamine is one of the most growing drugs of abuse, causing neurological impairments, and associated with behaviors that favor exposure to HIV, a virus that penetrates the CNS leading to neuroAIDS. Meth users infected with HIV usually present a more severe form of neuroAIDS, due to effects on neurons. IT is known that free radicals participate in the enhancement of brain pathology, but the cell types as well as pathways affected by Meth that lead to free-radical mediated stress. The hypothesis behind this proposal is that Meth interacts with cell types other than neurons will be explored, particularly regarding its potential to induce and/or modulate reactive oxygen and nitrogen intermediates. We will investigate the action of methamphetamine (Meth) on oxidative metabolism, directly on macrophages, and on other cell types in vitro, as well as in vivo, in conditions of co-morbid factor interaction. For that we will examine the action of Meth on cell lines, by investigating how the drug affects the intensity and quality of the oxidative burst, and searching for genes within ROI and RNI pathways that exhibit transcriptional pattern changes upon Meth exposure. Such transcriptional changes will be validated by looking for similar changes in cells obtained from Meth-treated animals infected with SIV, which have been previously characterized for CNS disease parameters. The results from this project will offer important clues on the participation of Meth in pathology mediated by oxidative stress by acting on ROI and RNI induction pathways, not only in the Central Nervous System, but also in peripheral organs. PUBLIC HEALTH RELEVANCE: We will investigate the action of methamphetamine (Meth) on oxidative metabolism, directly on macrophages, and on other cell types in vitro, as well as in vivo in conditions of co-morbid factor interaction. The hypothesis behind this proposal is that Meth affects the intensity and quality of the oxidative burst by acting directly on macrophages and/or glial cells, in addition to its action on neurons, contributing to the development of pathology mediated by oxidative stress, not only in the Central Nervous System, but also in peripheral organs. Initially, we will focus on the effect of meth on various human and mouse macrophage (THP1, RAW264.7), microglia (MG5, EOC 2), astrocyte (C8-D1A, CCF-STTG1), and neuronal (SH-SY5Y, SK- N-MC) cell lines, regarding the production of O2_, H2O2, and other reactive oxygen intermediates (ROI), as well as Reactive Nitrogen Intermediates (RNI) using various chromogenic methods. Mitochondria activity upon interaction with meth will be also addressed on cells stimulated with Meth. Following a characterization of the different cell lines regarding production of free radicals and mitochondrial changes in reaction to Meth, we will choose good responders to investigate transcriptional changes on molecules within ROI and RNI pathways, caused by action of Meth. For that we will use multiplex technology, as well as enzymatic checkpoint inhibition strategies. These approaches will help dissect the molecular basis of respiratory burst induction by Meth, and will distinguish whether Meth interferes over mitochodria, peroxisome, or both. Following the in vitro approach using cell lines, we intend to validate findings in the context of pathology. We will use cryopreserved cells and tissues from monkeys infected with SIV (as a model for HIV infection, a common co-morbid condition) subjected to a Meth treatment schedule. We will identify transcriptionally altered oxidative pathways on cryopreserved brain and peripheral tissues derived from SIV-infected Meth-treated monkeys by qRT-PCR. This brings relevance to pathology in humans. The present proposal will identify the basis of oxidative stress in the brain and other organs in Meth users. Meth is one of the most growing street drugs of abuse, exposing users to HIV infection, and potentially facilitating the penetration of virus across the blood brain barrier (BBB) into the brain. In this proposal we will dissect mechanisms by which Meth can induce and modulate free radical production, causing an aggravation on the severity of damage associated with virus presence in the brain tissue, and consequently aggravate AIDS-associated CNS dysfunctions. The results obtained in this proposal will allow the generation of data on direct effects of the drug on cells, relative susceptibility of different cell types to direct actions, and on pathways, aiming a future R01 application on modulation of oxidative stress in Meth abuse in vivo. These will lead to the development of important therapeutic tools for rehabilitating individuals, especially patients presenting co-morbid factors, such as HIV infection.