Cholinesterase Inhibitors, Axonal transport, and Memory

描述（由申请人提供）：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的可怕后果是认知功能的长期损害。迄今为止，OP的机制研究主要集中在明显毒性剂量的影响上，然而，对于重复暴露于不产生明显急性毒性迹象的这些药剂的剂量的细胞和行为后果知之甚少（即，阈下剂量）。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长期存在。因此，我们的长期目标是进一步阐明OP机制，以便为遭受暴露的患者开发更有效的治疗策略。本申请的目的是在实验动物模型中确定重复、阈下暴露于OP的细胞和生化表现与认知功能之间的特定关系。我们有令人信服的初步证据，从大鼠的研究表明，一个机制的记忆功能障碍与重复，阈下OP暴露是损害的快速轴突运输与运动蛋白，驱动蛋白的相互作用。由于轴突运输在神经元功能中起着如此重要的作用，并且由于大脑中的胆碱能系统对于认知过程如此重要，我们已经发展了这样的假设，即随着时间的推移，OP对快速轴突运输的妥协导致大脑敏感区域中胆碱能大分子表达的不足，从而导致记忆功能的损害。这项拟议研究的基本原理是，清楚地了解阈下暴露于OP的认知影响的机制将有助于我们设计治疗方法来逆转这些药物的影响。为了验证这一假设，我们提出了两个具体的目标：1）：在实验动物模型中评估重复的、阈下的、暴露于神经毒性和非神经毒性OP的残余行为表现（特别是认知效应）。2）：确定OP诱导的认知变化与快速轴突运输障碍之间的特定关系。我们将使用水迷宫任务来测量空间学习，8臂径向臂迷宫任务来评估工作记忆，微管运动分析和视频增强微分干涉对比显微镜来研究OP对驱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用于测量OP对脑中关键胆碱能标志物表达的影响，器官型培养方法将用于测量OP对海马的毒性。在这项研究完成后，我们希望确定时间细胞的变化所造成的重复，阈下暴露于两个代表性的OP（DFP和毒死蜱）及其与认知变化的相关性。这些研究意义重大，因为它们将有助于更好地了解与一类继续对全世界数百万人构成重大环境风险的制剂有关的毒性。

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.