Opiate drug abuse & HIV-induced excitotoxicity in striatal neurons

项目摘要 注射阿片类药物的HIV-1感染者表现出认知缺陷增加，并经历 加速发展为艾滋病的速度。越来越多的证据表明，阿片类药物滥用本质上 加剧了HIV-1的致病作用。我们发现神经元死亡之前会有一段较长的时间。 突触剔除、功能丧失和树突状病变，这些都被认为是可逆的。重要的是，阿片类药物 滥用通过协同增加树突状病变(静脉曲张)加强HIV-1的神经发病 形成、珠状、碎裂、修剪)，同时促进附加树突棘损失(可塑性)。这 已证实纹状体中棘神经元(MSN)和突触修剪已被证实 电子显微镜下。此外，运动活动中的行为缺陷伴随着突触 在没有可证明的神经元死亡的情况下，丢失和树突病理，表明亚致死性 神经元损伤和突触连接减少是阿片类药物加重HIV-1相关的能力的基础 神经性疾病(手)。虽然死亡本身是重要的，但神经元之前的事件的中断 从治疗角度来说，死亡可能更具战略意义。这笔赠款将重点放在MSN的功能层面上，通过 探讨阿片类药物±HIV兴奋毒性的潜在生理机制。这是假设的 TAT引起MSN细胞内稳态和兴奋性的变化，而阿片类药物则加剧了这种变化 药物通过一系列复杂的事件涉及OR介导的途径。体外实验方法正在进行中 通过评估阿片类药物和HIV-1 TAT在分离的皮质-TAT诱导的神经毒性的影响而提出的 纹状体细胞培养。全细胞膜片钳记录将在电压和电流钳制模式下进行 通过评估动作电位以及钠、钾、AMPA、NMDA和钙(Ca~(2+))电流。这个 OR的作用将通过应用药理学(OR拮抗剂)、遗传学(OR基因敲除小鼠)和 沉默(沉默NMDAR)战略，以确定阿片类药物与艾滋病毒蛋白相互作用的潜在机制。至 梳理阿片类药物是否通过或对MSN加重纹状体中TAT的兴奋毒性效应 使用两种类型的Cre-lox小鼠进行体内实验。有条件地删除关键站点的OR将定义 阿片类药物加重神经元兴奋性(动作电位， 离子通道活性、离子成像、线粒体膜电位)、损伤(包括树突病理和 脊椎密度)和纹状体的行为缺陷(运动活动)。

Project Summary HIV-1 infected individuals who are injecting opioid drugs show increased cognitive defects and undergo an accelerated rate of progression to AIDS. Accumulating evidence suggests that opioid drug abuse intrinsically exacerbates the pathogenesis of HIV-1. We have found that neuronal death is preceded by a prolonged period of synaptic culling, functional losses, and dendritic pathology that are presumed reversible. Importantly, opioid abuse potentiates the neuropathogenesis of HIV-1 by synergistically increasing dendritic pathology (varicosity formation, beading, fragmentation, pruning), while promoting additive dendritic spine losses (plasticity). This has been verified in medium spiny neurons (MSN) of the striatum and synaptic pruning has been confirmed electron microscopically. Moreover, behavioral defects in locomotor activity are accompanied by synaptic losses and dendritic pathology in the absence of demonstrable neuron death, suggesting that sublethal neuronal injury and reduced synaptic connectivity underlie the ability of opioids to aggravate HIV-1-associated neurological disorders (HAND). While death per se is significant, the interruption of events preceding neuron death may be more strategic therapeutically. This grant will focus on the functional level of MSN by investigating the underlying physiological mechanisms of opioid ± HIV-induced excitotoxicity. It is hypothesized that Tat induces changes in the cellular homeostasis and excitability of MSN, that are exacerbated by opioid drugs through a complex sequence of events involving OR-mediated pathways. In vitro approaches are being proposed by assessing the effects of opioid drug and HIV-1 Tat-induced neurotoxicity in dissociated cortical- striatal cell cultures. Whole-cell patch-clamp recordings will be conducted in voltage- and current-clamp mode by assessing action potentials as well as sodium, potassium, AMPA, NMDA, and calcium (Ca2+) currents. The role of OR will be elucidated by applying pharmacological (OR antagonists), genetic (OR knockout mice) and silencing (silencing NMDAR) strategies to identify mechanisms underlying opioid + HIV protein interaction. To sort out whether opioids exacerbate the excitotoxic effects of Tat in the striatum via OR on MSN we will conduct experiments in vivo using two types of Cre-lox mice. Conditionally deleting OR at key sites will define the targets and associated mechanisms by which opioids exacerbate neuronal excitability (action potentials, ion channel activity, ion imaging, mitochondrial membrane potential), injury (including dendritic pathology and spine density), and behavioral defects (locomotor activity) in the striatum.