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（由申请人提供）：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，第三龄幼虫的周围神经系统已被用来研究对轴突损伤和疾病的急性神经元反应。然而，由于幼虫第三龄和化蛹之间的时间间隔很短，该系统不太适合研究较长时间段的过程。最近的研究表明，三龄幼虫对轴突损伤产生快速的初始反应，并显示出轴突再生的诱人开始。然而，随着时间的推移，大多数幼虫组织发生变态，无法对损伤反应进行更完整的分析，包括神经胶质细胞的参与和可能的轴突修复。同样，在实验中探索随着时间的推移维持 NMJ 结构和功能的机制，或者这些机制如何随着年龄或疾病而受到损害的实验观察窗口，受到蛹化的开始的显着限制。本应用的目标是表征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，同时保留了幼虫 NMJ 的特征，使其成为如此强大的模型。我们利用遗传变异，幼虫正常发育，但随后在第三龄停留长达 10 天（比正常长四倍），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，我们相信延长的第三龄寿命为探索时间依赖性神经生物学过程的实验提供了一个新颖而强大的机会。为了确定这种延长幼虫寿命（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生长是否正常？在 ELL 期间，NMJ 是否会随着幼虫体型的增大而继续生长？已知在正常发育过程中调节 NMJ 生长的关键信号通路在 ELL 过程中是否继续发挥作用？ (2) NMJ 在整个 ELL 中是否保持结构和功能完整？ (3) 我们能否通过使用 ELL 系统来扩大我们对幼虫运动轴突和周围神经胶质细胞在较长时间内的损伤反应的理解来证明 ELL 系统作为实验工具的实用性？我们相信，这种新颖的实验系统具有巨大的潜力，可以极大地扩展幼虫 NMJ 作为模型系统的能力，并使我们能够在轴突再生和突触维护的研究中取得独特的进展，这两者都与理解和治疗神经胶质瘤高度相关。一些人类神经系统疾病。 公共健康相关性：许多人类神经系统疾病与运动通路损伤有关，这可能是由于损伤引起的神经物理损伤，也可能是由于各种疾病扰乱了神经将电信号传递到肌肉的突触的结构或功能。为了充分了解这些和相关疾病并开发合理的治疗方法，我们需要一个可以进行必要的遗传、分子和细胞分析的模型系统。我们正在开发一种新颖的实验系统，即长寿果蝇幼虫，它为此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。