Opiate abuse and sex steroids influence neuroAIDS pathology

 描述(由申请人提供)：人类免疫缺陷病毒(HIV)携带者经历与一般人群相比更严重的运动/情绪/认知障碍(统称为“神经艾滋病”)的脑部病理，这些影响在阿片剂滥用者中加剧。此外，存在性别差异，与滥用阿片类药物的男性相比，滥用阿片类药物的女性感染艾滋病毒的风险更大；然而，一旦感染发生，一些女性可能比男性经历较轻的神经艾滋病症状。我们在小鼠身上总结了这些效应，并已开始阐明与阿片类药物相互作用的性类固醇激素，以保护神经艾滋病。目前的建议将开始揭示性激素与鸦片类药物和艾滋病毒相互作用的影响和机制，这一翻译方法利用培养的 小鼠和人类神经细胞，以及转基因全动物小鼠模型。这一独立之路奖(K99/R00)的目标是为Jason Paris博士提供培训，以识别性类固醇环境可能影响神经艾滋病病理中阿片类药物/HIV相互作用的细胞/分子机制。帕里斯博士将接受小鼠和人类神经细胞培养和成像技术方面的高级培训，以检测亚致命神经元的致病性、细胞内离子和线粒体膜的完整性。在K99培训阶段，帕里斯博士将在库尔特·豪泽博士(首席导师)的指导下工作，检验性类固醇可能对阿片剂与小鼠神经共培养中的HIV-1病毒毒素TAT(目标1a)或gp120(目标1b)之间的协同作用产生的潜在保护作用。将通过免疫细胞化学检测存活/退化的神经元、星形胶质细胞和小胶质细胞，将评估充满生物细胞素的纹状体中棘神经元(MSN)的形态亚致死变化，并将评估培养上清中细胞因子/趋化因子、活性氧和亚硝酸盐的产生。在小鼠(目标2a和2b)和人类(目标2c)神经培养中，将评估类固醇对阿片类药物与Tat(2a)、gp120(2b)或X4/R5嗜性艾滋病毒(2c)相互作用的保护作用对MSN形态、MSN胞体和树突内[Ca]i和[Na]I的离子成像的影响，2以及线粒体膜失稳(通过罗丹明123荧光成像)。在R00独立阶段，Paris博士将使用有条件地表达中枢HIV-1 Tat(目标3a)或结构性表达中枢gp120(目标3b)的全动物模型，研究中枢类固醇形成对体内阿片剂/艾滋病毒蛋白相互作用的保护作用。雄性和雌性小鼠将接受类似神经艾滋病的运动、情感和认知行为的评估。纹状体组织将被评估目标1中描述的终点。最后，将在从TAT或gp120转基因小鼠获得的小鼠神经共培养中检查类固醇、阿片剂和艾滋病毒蛋白之间重叠的药效靶点的影响(目标3c)。将用药理拮抗剂对培养物进行预处理，并如目标1和2中所述进行检查。这项提议是及时的，提供了新的培训，并将使创新和独立研究计划的发展成为可能。

 DESCRIPTION (provided by applicant): Individuals with human immunodeficiency virus (HIV) experience brain pathology associated with greater motor/mood/cognitive disorders (collectively termed "neuroAIDS") than the general population and these effects are exacerbated among opiate abusers. Moreover, gender differences exist with opiate-abusing women at greater risk to contract HIV compared to opiate-abusing men; yet, some women may experience lesser neuroAIDS symptomology than men once infection occurs. We recapitulated these effects in mice and have begun to elucidate the sex steroid hormones that interact with opiates to confer protection to neuroAIDS. The present proposal will begin to reveal the effects and mechanisms of sex steroid interactions with opiates and HIV in a translational approach that utilizes cultured murine and human neural cells, as well as transgenic whole-animal murine models. The goal of this Pathway to Independence Award (K99/R00) is to provide training to Dr. Jason Paris to discern the cellular/molecular mechanisms by which sex steroid milieu may influence opiate/HIV interactions for neuroAIDS pathology. Dr. Paris will receive advanced training in murine and human neural cell culture and imaging techniques for detection of sub lethal neuronal pathogenicity, intracellular ions, and mitochondrial membrane integrity. In the K99 training phase, Dr. Paris will work under the tutelage of Dr. Kurt Hauser (Primary Mentor) to examine the potentially-protective effects that sex steroids may exert over synergistic actions between opiates and the HIV-1 virotoxins, Tat (Aim 1a) or gp120 (Aim 1b) in murine neural co-cultures. Viable/degenerating neurons, astrocytes, and microglia will be detected via immunocytochemistry, biocytin-filled striatal medium spiny neurons (MSNs) will be assessed for sub lethal changes in morphology, and supernatants will be assessed for production of cytokines/ chemokines, reactive oxygen species, and nitrites. In murine (Aims 2a and 2b) and human (Aim 2c) neural cultures, steroid protection against opiate interactions with Tat (2a), gp120 (2b), or X4-/R5-tropic HIV (2c) will be assessed for effects on MSN morphology, ion imaging for [Ca] i and [Na] i within MSN soma and dendrites, 2+ + and mitochondrial membrane destabilization (via Rhodamine 123 fluorescence imaging). In the R00 independent phase, Dr. Paris will examine the protective effects of central steroid formation on opiate/HIV protein interactions in vivo using whole-animal models that conditionally-express central HIV-1 Tat (Aim 3a) or constitutively-express central gp120 (Aim 3b). Male and female mice will be assessed for neuroAIDS-like motor, affective, and cognitive behavior. Striatal tissue will be assessed for endpoints described in Aim 1. Lastly, the influence of pharmacodynamic targets that overlap between steroids, opiates, and HIV proteins will be examined in murine neural co-cultures obtained from Tat- or gp120-transgenic mice (Aim 3c). Cultures will be pretreated with pharmacological antagonists and examined as in Aims 1 and 2. This proposal is timely, provides novel training, and will enable the development of an innovative and independent research program.