Opiate drug abuse & HIV-induced excitotoxicity in striatal neurons

描述(申请人提供)：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的发病。我们已经发现，神经元死亡之前会有一段长时间的突触剔除、功能丧失和树突病理，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(静脉曲张形成、珠状、碎裂、修剪)，同时促进相加的树突棘丢失(可塑性)，加强了艾滋病毒-1的神经发病。这在纹状体的中棘神经元(MSN)中得到了证实，突触修剪在电子显微镜下也得到了证实。此外，在没有明显神经元死亡的情况下，运动活动的行为缺陷伴随着突触丢失和树突病理，这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相关神经疾病(HAND)的能力的基础。虽然死亡本身意义重大，但中断神经元死亡之前的事件可能在治疗上更具战略性。这笔赠款将通过研究阿片类药物+/-HIV诱导的兴奋毒性的潜在生理机制，将重点放在MSN的功能水平上。假设TAT引起MSN细胞内稳态和兴奋性的改变，而阿片类药物通过涉及OR介导的通路的一系列复杂事件加剧了这种改变。通过评估阿片类药物和HIV-1TAT在分离的皮质-纹状体细胞培养中诱导的神经毒性的影响，提出了体外方法。全细胞膜片钳记录将在电压和电流钳模式下进行，通过评估动作电位和钠、钾、AMPA、NMDA和钙(Ca2+)电流。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)策略来确定阿片类药物和HIV蛋白相互作用的潜在机制来阐明。为了弄清阿片类药物是否通过或通过MSN加重纹状体内TAT的兴奋毒性效应，我们将使用两种类型的Cre-lox小鼠进行体内实验。有条件地删除关键部位的OR将定义阿片类药物加剧纹状体神经元兴奋性(动作电位、离子通道活性、离子成像、线粒体膜电位)、损伤(包括树突病理和脊柱密度)和行为缺陷(运动活性)的靶点和相关机制。

DESCRIPTION (provided by applicant): 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 wil 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.