Cholinesterase Inhibitors, Axonal transport, and Memory

描述(申请人提供)：接触有毒有机磷(OP)杀虫剂和化学战剂继续危及世界上许多人口。这种暴露的一个潜在可怕后果是认知功能的长期损害。到目前为止，对OP的机制研究主要集中在公开毒性剂量的影响上，然而，对于反复接触这些没有明显急性毒性迹象的药物的细胞和行为后果(即阈值以下剂量)，人们知之甚少。这一问题非常重要，因为可检测到的有机磷农药水平可能会在环境中保留较长时间。因此，我们的长期目标是进一步阐明OP的机制，以便为遭受暴露的患者开发更有效的治疗策略。这项应用的目的是在实验动物模型中确定反复阈值下暴露于有机磷农药的细胞和生化表现与认知功能之间的特定关系。我们有来自大鼠研究的令人信服的初步证据表明，与重复阈值下OP暴露相关的记忆障碍的一个机制是与运动蛋白Kinesin相互作用导致的快速轴突运输受损。由于轴突运输在神经元功能中起着如此重要的作用，而且大脑中的胆碱能系统对认知过程如此重要，我们提出了一个假设，即随着时间的推移，OPs对快速轴突运输的损害会导致大脑敏感区域胆碱能大分子的表达不足，从而导致记忆功能的损害。这项研究的理论基础是，清楚地了解亚阈值暴露于有机磷农药的认知影响的机制将有助于我们设计逆转这些药物影响的治疗方法。为了验证这一假设，我们提出了两个具体的目标：1)在一个实验动物模型中，评估重复阈值下暴露于神经毒性和非神经毒性有机磷农药的残留行为表现(特别是认知效应)。2)：确定OP引起的认知改变与快速轴突运输损伤之间的具体关系。我们将使用水迷宫任务来测量空间学习，使用8臂径向臂迷宫任务来评估工作记忆，并分别使用微管运动分析和视频增强差示干涉显微镜来研究OP对动蛋白和轴突运输的影响。免疫印迹法和受体放射自显影将被用来测量OP对脑内关键胆碱能标志物表达的影响，器官培养方法将被用来测量OP对海马区的毒性。在这项研究完成后，我们希望确定重复阈值以下暴露于两种有代表性的OP(DFP和毒死蜱)所导致的时间细胞变化及其与认知变化的相关性。这些研究具有重要意义，因为它们将有助于更好地了解与一类继续对全球数百万人构成重大环境风险的制剂相关的毒性。

DESCRIPTION (provided by applicant): The exposure to toxic organophosphate (OP) insecticides and chemical warfare agents continues to endanger many of the world's population. One potentially dire consequence of such exposure is the prolonged impairment of cognitive function. Mechanistic studies of OPs to date have focused primarily the effects of overtly toxic doses, however, little is known about the cellular and behavioral consequences of repeated exposure to doses of these agents that produce no overt signs of acute toxicity (i.e., subthreshold doses). This issue is very important since detectible levels of OPs can remain in the environment for extended periods. Accordingly, our long-term goal is further elucidate OP mechanisms such that more effective therapeutic strategies can be developed for patients suffering from exposure. The objective of this application is to identify specific relationships between cellular and biochemical manifestations of repeated, subthreshold exposures to OPs and cognitive function in an experimental animal model. We have compelling preliminary evidence from rat studies suggesting that one mechanism underlying memory dysfunction associated with repeated, subthreshold OP exposure is the impairment of fast axonal transport resulting from interactions with the motor protein, kinesin. Since axonal transport plays such a fundamental role in neuronal function, and since the cholinergic system in the brain is so important for cognitive processes, we have developed the hypothesis that the compromise of fast axonal transport by OPs, over time, leads to deficiencies in the expression of cholinergic macromolecules in sensitive regions of the brain that result in the impairment of memory function. The rationale for the proposed research is that a clear understanding of the mechanisms underlying the cognitive effects of subthreshold exposures to OPs will help us in designing treatments to reverse the effects of these agents. To test the hypothesis we propose two specific aims: 1): To evaluate the residual behavioral manifestations (especially cognitive effects) of repeated, subthreshold, exposures to both neurotoxic and non-neurotoxic OPs in an experimental animal model. 2): To identify specific relationships between OP-induced cognitive changes and impairments in fast axonal transport. We will use a water maze task to measure spatial learning, an 8-arm radial arm maze task to assess working memory, and a microtubule motility assay and video enhanced-differential interference contrast microscopy to study OP effects on kinesin and axonal transport, respectively. Immunoblotting methods and receptor autoradiography will be used to measure OP effects on the expression of key cholinergic markers in the brain, and organotypic culture methods will be employed to measure toxicity of OPs to the hippocampus. At the completion of this research we expect to identify temporal cellular changes resulting from repeated, subthreshold exposures to two representative OPs (DFP and chlorpyrifos) and their correlation with cognitive changes. These studies are significant because they will contribute to a better understanding of the toxicity associated with a class of agents that continues to pose a significant environmental risk to millions of people worldwide.