Long-lived Drosophila larvae for studies of synaptic growth, decay, and repair

用于研究突触生长、衰退和修复的长寿果蝇幼虫

• 批准号: 8282203
• 负责人: BARRY S GANETZKY
• 金额: $ 22.17万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8282203
• 关键词: Acute; Address; Age; Aging; Area; Axon; Axonal Transport; Biological Metamorphosis; Biological Models; Development; Disease; Dissection; Distal; Drosophila genus; Experimental Models; Frequencies; Goals; Growth; Human; Impairment; Individual; Injury; Investigation; Larva; Life; Longevity; Maintenance; Measurement; Measures; Membrane; Modeling; Molecular Genetics; Morphology; Motor; Motor Neurons; Motor Pathways; Muscle; Mutation; Natural regeneration; Nerve; Nerve Crush; Nerve Degeneration; Neurobiology; Neurodegenerative Disorders; Neuroglia; Neuromuscular Junction; Neurons; Peripheral Nerves; Peripheral Nervous System; Peripheral nerve injury; Process; Regulation; Research Design; Signal Pathway; Signal Transduction; Site; Staining method; Stains; Structure; Swelling; Synapses; Synaptic Potentials; System; Time; Tissues; Vesicle; Wit; axon regeneration; base; cell type; density; genetic variant; injured; nerve supply; nervous system disorder; novel; prevent; repaired; research study; response; response to injury; synaptic function; time interval; tool; trafficking

DESCRIPTION (provided by applicant): For decades, the Drosophila larval neuromuscular junction (NMJ) has been a powerful model system for genetic and molecular dissection of synaptic growth, structure, and function. More recently the peripheral nervous system of third instar larvae has been employed to study acute neuronal responses to axon damage and disease. However, due to the short time interval between the third larval instar and pupariation, the system is not well suited to study processes that extend over a longer time period. Recent studies demonstrate that third instar larvae mount a rapid initial response to axon damage and display tantalizing beginnings of axonal regrowth. However, the onset of metamorphosis with replacement of most larval tissues precludes more complete analysis of the response to injury - including involvement of glia and possible axonal repair - over time. Similarly, the window of observation in experiments probing mechanisms that maintain NMJ structure and function over time, or how these are compromised with age or by disease, is significantly limited by the onset of pupariation. The goal of this application is to characterize and demonstrate the utility of an experimental system we are developing that overcomes these time constraints while preserving the features of the larval NMJ that makes it such a powerful model. We exploit genetic variants in which larvae develop normally but subsequently remain in the third instar for up to 10 days (4 times longer than normal), during which time they continue to grow before finally undergoing metamorphosis and eclosion. On the basis of our preliminary results, we are confident that the expanded third instar lifespan provides a novel and powerful opportunity for experiments that probe time-dependent neurobiological processes. To establish the validity and utility of this Extended Larval Life-span (ELL) model, we propose experiments that aim to answer the following questions: (1) Is NMJ growth normal in ELL larvae during development? Does the NMJ continue to grow along with the increase in larval size during ELL? Do the key signaling pathways known to regulate NMJ growth during normal development continue to function during ELL? (2) Does the NMJ remain structurally and functionally intact throughout ELL? (3) Can we prove the utility of the ELL system as an experimental tool by employing it to expand our understanding of the injury response in larval motor axons and peripheral nerve glia over an extended time frame? We believe that this novel experimental system has enormous potential to greatly expand the power of the larval NMJ as a model system and enable us to make unique inroads in studies of axonal regeneration and synaptic maintenance, both of which are highly relevant for understanding and treatment of a number of human neurological disorders. PUBLIC HEALTH RELEVANCE: Many human neurological disorders are associated with impairment of motor pathways either as a result of physical damage to nerves caused by injury or as a result of various diseases that perturb the structure or function of synapses where nerves relay electrical signals to muscles. To fully understand these and related disorders and to develop rational therapies, we require a model system in which the necessary genetic, molecular, and cellular analyses can be carried out. We are developing a novel experimental system, long-lived Drosophila larvae, that offers numerous advantages for studies of this type and we are applying it to study the process of nerve regrowth after injury.

描述(申请人提供)：几十年来，果蝇幼虫神经肌肉接头(NMJ)一直是突触生长、结构和功能的遗传和分子解剖的强大模型系统。最近，三龄幼虫的外周神经系统被用来研究轴突损伤和疾病的急性神经元反应。然而，由于三龄幼虫和蛹之间的时间间隔很短，该系统不太适合研究持续较长时间的过程。最近的研究表明，三龄幼虫对轴突损伤有快速的初始反应，并显示出诱人的轴突再生开始。然而，随着大多数幼虫组织的替换而开始变态，排除了对损伤反应的更全面的分析，包括随着时间的推移，神经胶质细胞的参与和可能的轴突修复。同样，在实验中，探索随时间保持NMJ结构和功能的机制，或者这些机制是如何随着年龄或疾病而受损的，观察的窗口明显受到羽化开始的限制。这个应用程序的目标是描述和展示我们正在开发的实验系统的实用性，该系统克服了这些时间限制，同时保留了使其成为如此强大的模型的幼虫NMJ的特征。我们利用幼虫正常发育的遗传变异，但随后在三龄幼虫中停留长达10天(比正常长4倍)，在此期间它们继续生长，最后经历变态和羽化。根据我们的初步结果，我们相信扩大的三龄寿命为探索依赖时间的神经生物学过程的实验提供了一个新的和强大的机会。为了建立这种延长幼虫寿命(ELL)模型的有效性和实用性，我们提出了旨在回答以下问题的实验：(1)ELL幼虫在发育过程中NMJ的生长正常吗？在ELL期间，NMJ是否随着幼虫大小的增加而继续生长？已知在正常发育过程中调节NMJ生长的关键信号通路在ELL过程中继续发挥作用吗？(2)整个ELL过程中NMJ在结构和功能上保持完整吗？(3)我们能否通过使用ELL系统来扩展我们对幼虫运动轴突和周围神经胶质细胞损伤反应的理解，来证明ELL系统作为实验工具的有效性？我们相信，这一新颖的实验系统具有巨大的潜力，可以极大地扩大NMJ幼虫作为模型系统的能力，并使我们能够在轴突再生和突触维持的研究中取得独特的进展，这两项研究都与理解和治疗许多人类神经疾病高度相关。 与公共卫生相关：许多人类神经疾病与运动通路受损有关，要么是由于损伤对神经造成的物理损害，要么是由于各种疾病扰乱了突触的结构或功能，在突触中，神经将电信号传递给肌肉。为了充分了解这些和相关的疾病并开发合理的治疗方法，我们需要一个模型系统，在这个模型系统中可以进行必要的遗传、分子和细胞分析。我们正在开发一种新的实验系统--长寿命果蝇幼虫，它为这类研究提供了许多优势，我们正在将其应用于研究损伤后神经再生的过程。