Human genetics and drugs of abuse using the nematode, C. elegans.

使用线虫，秀丽隐杆线虫的人类遗传学和药物滥用。

• 批准号: 8148582
• 负责人: Brandon Harvey
• 金额: $ 22.31万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148582
• 关键词: Caenorhabditis elegans; Human Genetics; Nematoda; drug of abuse

The nematode, Caenorhabditis elegans has become an invaluable model organism for studying molecular and cellular functions. We are employing C. elegans to study human gene function as it relates to addiction. We are interested in how allelic variations in genes involved in the biosynthesis, transport, release, reuptake, signaling and catabolism of the neurotransmitter dopamine play a role in the susceptibilities to drugs of abuse such as methamphetamine. In humans and other mammals, dopamine is the primary neurotransmitter of the "reward pathway" which reinforces behaviors that are beneficial to the survival of an organism. It is the reward pathway that is most affected by drugs of abuse as they reinforce their consumption and associated behaviors. C. elegans has eight dopamine neurons that control specific movement and reproductive behaviors. This past year, we have adopted a swimming induced paralysis assay (SWIP) assay and developed a movement tracking assay to examine behavioral response to methamphetamine. We have also used this first year to acquire equipment and strains of C. elegans for our studies. We have hired a research associate with 7 years of experience working with C. elegans and associated molecular biology and transgenic techniques. During our summer students fellowship, she was able to acquire data on dose-dependent methamphetamine-induced changes in behavior. We are in the process of completing the study for publication. The presence of conserved genes involved in dopamine biology coupled with defined behaviors associated with these 8 dopamine neurons allows for testing human gene function in response to drugs of abuse. The ease of genetic manipulation in C. elegans allows us to replace the C. elegans gene such as the dopamine transporter with variants of human dopamine transporter. The "humanized" worms can then be analyzed for changes in drug response. Ultimately, by making genetically tagged versions of the human genes, we can screen for altered behavior after drug exposure and identify the combination of human alleles. We hypothesize that specific combinations of human gene variants or alleles can mediate altered responses to drugs of abuse and will indicate "vulnerabilities" to drugs such as methamphetamine or cocaine. From these studies, we hope use genetic based vulnerabilities to develop more individualized approaches to treating addiction. Towards these goals, we have acquired several variants of the human dopamine transporter with known differences in affinity and transport. We are in the process of generating transgenic worms on a worm dopamine transporter knockout background. In addition to studying gene function and drugs of abuse in the dopaminergic system, we can use models of dopaminergic neurodegeneration to study potential molecules important for dopaminergic neuron development, survival and regeneration of fibers. For example, we have been working on a conserved gene, mesencephalic astrocyte-derived neurotrophic factor (MANF) which was recently identified as a dopaminergic neurotrophic factor in mammals. From our studies in C elegans, we have been able to identify possible mechanisms of neuroprotection for this protein. This past year, we have 1) generated several transgenic animals that demonstrate the cellular location of MANF, 2) characterized MANF knockout worms, 3) generated and characterized antibodies for studying MANF in worms, and 4) identified a novel response to drugs that induce cellular stress. Some of our work was presented at the NIDA MiniSymposium at the 2009 Society for Neurosciene Meeting in Chicago. We are currently preparing the manuscript summarizing our findings of MANF. From our work with C.elegans and MANF, we will return to our mammalian models of Parkinsons disease and methamphetamine toxicity to examine new therapeutic approaches to treating the degeneration of dopaminergic neurons. In summary, we are able to employ C. elegans as a model for human gene function in dopaminergic neurons which are affected in addiction and Parkinsons disease.

线虫秀丽隐杆线虫已成为研究分子和细胞功能的宝贵模型生物。 我们正在使用秀丽隐杆线虫来研究与成瘾有关的人类基因功能。 我们对与甲基苯丙胺等滥用药物的敏感性（如甲基苯胺）的敏感性有关，我们对参与基因的等位基因变异如何在神经递质多巴胺的生物合成，释放，再摄取，信号传导和分解代谢中发挥作用。 在人类和其他哺乳动物中，多巴胺是“奖励途径”的主要神经递质，它增强了对生物生存的有益行为。 这是奖励途径，最受滥用药物的影响，因为它们加强了他们的消费和相关的行为。 秀丽隐杆线虫具有八个多巴胺神经元，可控制特定运动和生殖行为。 在过去的一年中，我们采用了游泳诱导的瘫痪测定法（SWIP）测定法，并开发了一种运动跟踪测定法，以检查对甲基苯丙胺的行为反应。 我们还利用第一年来获取秀丽隐杆线虫的设备和菌株进行研究。 我们聘请了一名研究助理，具有7年的经验，与秀丽隐杆线虫以及相关的分子生物学和转基因技术合作。 在我们的夏季学生奖学金中，她能够获取有关依赖剂量的甲基苯丙胺引起的行为变化的数据。 我们正在完成该研究以进行发表。 与这8种多巴胺神经元有关的多巴胺生物学涉及的保守基因的存在，可以测试人类基因的作用，以响应滥用药物。 秀丽隐杆线虫中的遗传操作的易度性使我们能够用人多巴胺转运蛋白的变体代替秀丽隐杆线虫基因，例如多巴胺转运蛋白。 然后可以分析“人性化”蠕虫的药物反应变化。 最终，通过制作人类基因的遗传标记版本，我们可以筛选出药物暴露后的行为改变，并确定人类等位基因的组合。 我们假设人类基因变异或等位基因的特定组合可以介导对滥用药物的反应改变，并表明对甲基苯丙胺或可卡因等药物的“脆弱性”。 从这些研究中，我们希望使用基于遗传的脆弱性来开发更个性化的方法来治疗成瘾。 朝向这些目标，我们获得了人类多巴胺转运蛋白的几种变体，其亲和力和运输方面有已知差异。 我们正在在蠕虫多巴胺转运蛋白敲除背景上产生转基因蠕虫。 除了研究多巴胺能系统中的基因功能和滥用药物外，我们还可以使用多巴胺能神经变性的模型研究对多巴胺能神经元发展，纤维生存和再生至关重要的潜在分子。 例如，我们一直在研究保守的基因中脑星形细胞衍生的神经营养因子（MANF），该因子最近被确定为哺乳动物中多巴胺能神经营养因子。 从我们在C秀丽隐杆线虫的研究中，我们能够确定该蛋白质神经保护的可能机制。 在过去的一年中，我们有1）产生了几种转基因动物，这些动物证明了MANF的细胞位置，2）表征MANF敲除蠕虫，3）产生并表征了用于在蠕虫中研究MANF的抗体，而4）确定了对诱导细胞应激的药物的新反应。 我们的一些工作是在芝加哥举行的2009年神经西科生会议学会的NIDA Minisymposium上提出的。 我们目前正在准备汇总MANF发现的手稿。 从与C.Elegans和Manf的工作中，我们将回到帕金森氏病和甲基苯丙胺毒性的哺乳动物模型，以检查治疗多巴胺能神经元退化的新治疗方法。 总而言之，我们能够利用秀丽隐杆线虫作为在成瘾和帕金森氏病影响的多巴胺能神经元中人类基因功能的模型。