Opiate drug abuse & HIV-induced excitotoxicity in striatal neurons

描述（由申请人提供）：注射阿片类药物的HIV-1感染者表现出更多的认知缺陷，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的发病机制。我们发现，在神经元死亡之前，有一段较长时间的突触剔除、功能丧失和树突病理，这些被认为是可逆的。重要的是，阿片类药物滥用通过协同增加树突病理（静脉曲张形成、串珠、断裂、修剪），同时促进附加树突脊柱损失（可塑性），从而增强了HIV-1的神经发病机制。这在纹状体的中棘神经元（MSN）中得到了证实，并在电子显微镜下证实了突触修剪。此外，在没有明显神经元死亡的情况下，运动活动中的行为缺陷伴随着突触丧失和树突病理，这表明亚致死性神经元损伤和突触连通性降低是阿片类药物加重hiv -1相关神经系统疾病（HAND）的能力的基础。虽然死亡本身是重要的，但在治疗上，中断神经元死亡之前的事件可能更具战略性。这笔拨款将通过研究阿片类药物+/- hiv诱导的兴奋性毒性的潜在生理机制，重点研究MSN的功能水平。据推测，阿片类药物通过一系列涉及or介导通路的复杂事件加剧了细胞内稳态和MSN兴奋性的变化。通过评估阿片类药物和HIV-1 tat诱导的离解皮质纹状体细胞培养神经毒性的影响，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通过评估动作电位以及钠、钾、AMPA、NMDA和钙（Ca2+）电流进行。OR的作用将通过药理学（OR拮抗剂）、遗传学（OR敲除小鼠）和沉默（沉默NMDAR）策略来阐明，以确定阿片类药物与HIV蛋白相互作用的机制。为了弄清阿片类药物是否通过OR对MSN加剧纹状体中Tat的兴奋毒性作用，我们将使用两种Cre-lox小鼠进行体内实验。有条件地删除关键部位的OR将确定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（运动活动）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.