Direct and indirect striatal pathway selective vulnerability to Tat and morphine

 描述(申请人提供)：联合抗逆转录病毒疗法(CART)的出现大大延长了艾滋病毒-1患者的寿命；然而，艾滋病毒-1相关神经认知障碍(HAND)的流行率总体上没有改变，这种疾病可能会因阿片类药物滥用而加剧。许多手部症状被认为是由于在感染HIV-1期间特定大脑区域的选择性退化引起的，例如纹状体。纹状体由约90%-95%的中等棘神经元(MSN)组成。MSN群体是异质性的，可根据其表面标志物和蛋白质的不同组合表达而分为亚型，如多巴胺1和2(D1，D2)、腺苷A2a和M受体，以及P物质、强啡肽和脑啡肽，其中表达D1和D2的MSN为主要亚型。评估MSN亚型对艾滋病毒-1/阿片类药物使用的脆弱性可以增强我们对艾滋病毒-1和共病阿片类药物使用如何导致手部症状的理解，并建议改进预防性和功能性治疗。建议的研究将分别利用多巴胺DRD1(D1)或drd2(D2)受体启动子驱动的tdTomato或EFGP表达的小鼠系来鉴定表达d1和d2受体的MSN。这些小鼠品系，以及野生型对照，将被用于研究HIV-1蛋白转录反式激活因子(TAT)和阿片类药物对MSN亚型的致死和亚致死效应，使用体外和体内模型。原代混合皮质-纹状体、神经元-神经胶质细胞培养将被用来确定TAT和吗啡对这些亚型的选择性直接和神经胶质诱导的间接神经毒性和突触树突变性效应。然后，我们将使用一个表达番茄和D2-EGFP的小鼠品系与一个多西环素可诱导的表达TAT的小鼠品系杂交，来探索之前提到的TAT和/或吗啡诱导的运动障碍是否选择性地通过突触树突变性和/或特定MSN子集的电生理变化来介导。这将使用一组小鼠运动分析、全细胞膜片钳和表达d1和d2的MSN的三维重建来完成。综上所述，这些实验结果将表明，MSN的一个亚群是否推动了前面提到的由TAT和吗啡诱导的致命性和突触树突变性效应，以及它们与运动障碍的相关性。体外方法允许精确控制实验条件，体内测试将显示功能/形态相关性，这两种方法的结合将首次提供有关MSN亚型对艾滋病毒-1和/或阿片类药物选择性易感性的见解。

 DESCRIPTION (provided by applicant): The advent of combined antiretroviral therapy (cART) has substantially extended the lifespan of HIV-1 patients; yet, the prevalence of HIV-1 associated neurocognitive disorders (HAND), which can be exacerbated by co-morbid opioid drug abuse, overall has not changed. Many HAND symptoms are thought to be caused by the selective degeneration of particular brain regions, such as the striatum, during HIV-1 infection. The striatum is comprised of approximately 90-95% medium spiny neurons (MSNs). The MSN population is heterogeneous, and can be divided in subtypes based on their expression of different combinations of surface markers and proteins, such as dopamine 1 and 2 (D1, D2), adenosine A2A, and muscarinic M4 receptors, as well as, substance P, dynorphin, and enkephalin, with D1- and D2-expressing MSN as the main subtypes. Assessment of MSN subtype vulnerability to HIV-1/opioid-use could enhance our understanding of how HIV-1 and co-morbid opioid use lead to HAND symptoms, and suggest refined preventative and functional treatments. The proposed studies will take advantage of dopamine Drd1 (D1) or Drd2 (D2) -receptor promoter-driven tdTomato- or EFGP-expressing mouse lines, respectively, to identify D1- and D2-receptor- expressing MSNs. These mouse lines, along with wild-type controls, will be used to investigate lethal and sub-lethal effects of the HIV-1 protein transactivator of transcription (Tat) and opioids on the subtypes of MSNs using both in vitro and in vivo models. Primary mixed cortical-striatal, neuron-glia cultures will be employed to determine the selective direct and glial-induced indirect neurotoxic and synaptodendritic degenerative effects of Tat and morphine on these subtypes. We will then use a D1-tdTomato- and D2-EGFP-expressing mouse line crossed with a doxycycline-inducible Tat-expressing mouse line to explore if previously noted Tat- and/or morphine-induced motor disturbances are selectively mediated through synaptodendritic degeneration, and/or electrophysiological changes in a particular subset of MSNs. This will be accomplished using a battery of mouse-motor assays, whole-cell patch clamp, and three-dimensional reconstruction of D1- and D2-expressing MSNs. Taken together, these experimental results will show if a subgroup of MSNs is driving the previously noted Tat- and morphine-induced lethal and synaptodendritic degenerative effects, and their correlation with motor impairments. The combination of in vitro approaches, which allow for precise control of experimental conditions, and in vivo tests, which will show a functional/morphological correlation, will provide the first described insights into the selective vulnerability of MSN subtypes to HIV-1 and/or opioids.