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

线虫秀丽线虫已成为研究分子和细胞功能的宝贵模式生物。我们正在利用线虫来研究人类的基因功能，因为它与成瘾有关。我们感兴趣的是，参与神经递质多巴胺的生物合成、运输、释放、重摄取、信号传递和分解代谢的基因的等位基因变异如何在滥用药物(如甲基苯丙胺)的易感性中发挥作用。在人类和其他哺乳动物中，多巴胺是“奖赏通路”的主要神经递质，“奖赏通路”强化有利于有机体生存的行为。这是最受滥用药物影响的奖励途径，因为它们加强了他们的消费和相关行为。线虫有八个多巴胺神经元，控制特定的运动和生殖行为。在过去的一年里，我们采用了游泳诱导麻痹试验(SWIP)，并开发了一种运动跟踪试验来检测对甲基苯丙胺的行为反应。我们还利用这第一年为我们的研究购买了设备和线虫品系。我们聘请了一位有7年线虫及相关分子生物学和转基因技术工作经验的研究助理。在我们的暑期学生联谊会期间，她能够获得剂量依赖的甲基苯丙胺诱导的行为变化的数据。我们正在完成这项研究，以便出版。 存在与多巴胺生物学有关的保守基因，再加上与这8个多巴胺神经元相关的明确行为，可以测试人类基因功能对药物滥用的反应。线虫基因操作的简便性使我们可以用人类多巴胺转运蛋白的变体来取代线虫的基因，例如多巴胺转运蛋白。然后可以分析“人性化”的蠕虫对药物反应的变化。最终，通过制造人类基因的基因标记版本，我们可以筛选药物暴露后行为的变化，并识别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调节对滥用药物的改变反应，并将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，我们希望利用基于遗传的脆弱性来开发更个性化的治疗成瘾的方法。为了实现这些目标，我们已经获得了几种人类多巴胺转运蛋白的变体，它们在亲和力和转运方面存在已知的差异。我们正在以蠕虫多巴胺转运蛋白基因敲除为背景来产生转基因蠕虫。 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退化模型来研究对多巴胺能神经元发育、存活和纤维再生至关重要的潜在分子。例如，我们一直在研究一种保守的基因--中脑星形胶质细胞衍生神经营养因子(MANF)，它最近被鉴定为哺乳动物中的一种多巴胺能神经营养因子。从我们对线虫的研究中，我们已经能够确定这种蛋白质可能的神经保护机制。在过去的一年里，我们已经产生了几个证明MANF的细胞位置的转基因动物，2)鉴定了MANF基因敲除的蠕虫，3)产生并鉴定了用于研究蠕虫中MANF的抗体，以及4)鉴定了对诱导细胞应激的药物的新反应。我们的一些工作在2009年芝加哥神经科学学会会议上发表在NIDA小型研讨会上。我们目前正在准备总结我们对MANF发现的手稿。从我们对秀丽线虫和MANF的研究中，我们将回到帕金森氏病和甲基苯丙胺中毒的哺乳动物模型，以研究治疗多巴胺能神经元退化的新治疗方法。 综上所述，我们能够利用线虫作为人类基因功能的模型，在成瘾和帕金森病中受到影响的多巴胺能神经元。

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.