攀登缺陷的神经退行性疾病的果蝇模型定量分析
Summary
We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.
Abstract
从神经变性疾病造成机车的缺陷可以是疾病的晚期发作症状，随后的几年亚临床变性的，因此目前的治疗策略是不能治愈。通过使用全基因组测序的，越来越多的基因已被确定，以在人类运动的作用。尽管确定这些基因中，它是不知道这些基因如何是至关重要的正常机车运作。因此，可靠的测定法，其利用模式生物以阐明，以便确定治疗性利益的新靶标，这些基因的作用，需要比以往更。我们设计了负趋地性测定在先允许对较温和的缺陷的检测，并具有随着时间的推移，以评估这些缺陷的能力的致敏版本。在玻璃进行测定量筒中，被密封与蜡阻挡膜。通过提高门槛的距离要攀升至17.5Ç米和增加实验的持续时间为2分钟，我们观察到在检测温和流动性功能障碍更大的灵敏度。该测定是成本有效的，不需要大量的训练，以获得高度可重复的结果。这使得它一个很好的技术来筛选候选药物在果蝇突变体运动缺陷。
Introduction
破坏性神经变性疾病如帕金森氏病，肌萎缩性侧索硬化，和遗传性痉挛性截瘫日益认识到。不幸的是，大多数这些神经变性疾病的仍然没有治疗方法。的广泛临床应用的全基因组，无偏基因测试，如全基因组测序已导致了越来越多的基因受到牵连在人类运动障碍。尽管这样的进步，从早期到晚期的病理进展，仍然难以在这些疾病。 果蝇提供一个具有遗传工具来研究基因在需求控制的空间和时间的方式。此外， 果蝇已经证明在筛选药物对神经系统疾病，如帕金森氏1，阿尔茨海默2，智力障碍3,4-和癫痫5,6-等等是有用的。我们的目标是建立一个符合成本效益可靠的检测，将允许高通量分析，将仍然非常敏感，可以检测电机性能的微小变化。
有用于量化对果蝇攀登行为的基因突变和/或环境条件的影响几个实验。大多数的测定的利用苍蝇爬，称为负趋地性，或攀登测定的自然趋势。本泽7在1967年提出，用于趋光性的研究逆流装置也可以用来研究gravitaxis。自那时以来，Ganetsky 8和许多其他9 -12已经建立在初始测定。其原理是放置在小瓶中的苍蝇的已知数量和强烈挖掘小瓶在硬表面上，造成苍蝇下降到小瓶的底部。因为它是一个先天行为，苍蝇将试图攀爬到小瓶的顶部，相对的重力。该测定是定量的测量仪表水库有多少苍蝇在一个规定的时间段过去攀上小瓶标记。速度，而不是对苍蝇攀登总数的测量已成为一个可靠的参数和的情况下的苍蝇准则的数目并不显著13中所示的缺陷。
攀登测定在许多神经变性疾病，包括帕金森氏病14的研究证明是有益的。然而，我们注意到，机车的缺陷未必在时间被检测到，其中神经变性是已经看到在病理研究14。因此，使用传统的测定法的可能限制，研究疾病发病的早期阶段的能力。机车缺陷在以后的病理阶段的出现可能反映了其疾病进展是太先进完整的救援。
这就提出了与传统的攀登测定的灵敏度的潜在问题。的俗的电位不能tional爬坡法检测温和机车缺陷可以归因于向其中的苍蝇都需要攀登高度。传统的试验15,16措施蝇的数目，成功地翻越2至5厘米的高度在10至20秒。
Protocol
Representative Results
Discussion
果蝇已经被证明是在帕金森氏病14和其他神经变性病症1,2-极好模型。除了 在果蝇中可用的遗传工具，其基因组中是高度保守的对参与神经障碍19基因。全基因组基因筛查方法（包括全外显子组测序）的出现很可能继续提供与人类运动障碍相关的候选基因较大名单。的治疗这些疾病的发展将需要的动物模型，以增加我们涉及神经变性的早期阶段的病理学的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.
Materials
| Drosophila stocks | The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet. | ||
| Video camera | Any digital camcorder will do. Make sure they can focus on close object. | ||
| Graduated cylinder | Kimble | 20028W | Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height. |
| computer | Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point |
References
- Auluck, P. K., Bonini, N. M. Pharmacological prevention of Parkinson disease in Drosophila. Nature medicine. 8, 1185-1186 (2002).
- Bonini, N. M., Fortini, M. E. Human neurodegenerative disease modeling using Drosophila. Annual review of neuroscience. 26, 627-656 (2003).
- Bolduc, F. V., Bell, K., Cox, H., Broadie, K. S., Tully, T. Excess protein synthesis in Drosophila fragile X mutants impairs long-term memory. Nature neuroscience. 11, 1143-1145 (2008).
- McBride, S. M., et al. Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome. Neuron. 45, 753-764 (2005).
- Parker, L., Padilla, M., Du, Y., Dong, K., Tanouye, M. A. Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures. Genetics. 187, 523-534 (2011).
- Marley, R., Baines, R. A. Increased persistent Na+ current contributes to seizure in the slamdance bang-sensitive Drosophila mutant. Journal of neurophysiology. 106, 18-29 (2011).
- Benzer, S. Behavioral Mutants Of Drosophila Isolated By Countercurrent Distribution. Proceedings of the National Academy of Sciences of the United States of America. 58, 1112-1119 (1967).
- Ganetzky, B., Flanagan, J. R. On the relationship between senescence and age-related changes in two wild-type strains of Drosophila melanogaster. Experimental gerontology. 13, 189-196 (1978).
- Nichols, C. D., Becnel, J., Pandey, U. B. Methods to assay Drosophila behavior. Journal of visualized experiments : JoVE. , (2012).
- Toma, D. P., White, K. P., Hirsch, J., Greenspan, R. J. Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait. Nature genetics. 31, 349-353 (2002).
- Inagaki, H. K., Kamikouchi, A., Ito, K. Methods for quantifying simple gravity sensing in Drosophila melanogaster. Nature protocols. 5, 20-25 (2010).
- Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila. Experimental gerontology. 40, 386-395 (2005).
- Botella, J. A., et al. The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration. Current biology : CB. 14, 782-786 (2004).
- Feany, M. B., Bender, W. W. A Drosophila model of Parkinson’s disease. Nature. 404, 394-398 (2000).
- Chakraborty, R., et al. Characterization of a Drosophila Alzheimer’s disease model: pharmacological rescue of cognitive defects. PLoS One. 6, e20799 (2011).
- Orso, G., et al. Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine. J Clin Invest. 115, 3026-3034 (2005).
- Lehmann, E. L., D’Abrera, H. J. M. . Nonparametrics : statistical methods based on ranks. , (2006).
- Sherwood, N. T., Sun, Q., Xue, M., Zhang, B., Zinn, K. Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS biology. 2, e429 (2004).
- Inlow, J. K., Restifo, L. L. Molecular and comparative genetics of mental retardation. Genetics. 166, 835-881 (2004).
- Palladino, M. J., Hadley, T. J., Ganetzky, B. Temperature-sensitive paralytic mutants are enriched for those causing neurodegeneration in Drosophila. Genetics. 161, 1197-1208 (2002).
