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

描述（申请人提供）：几十年来，果蝇幼虫神经肌肉连接（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，三龄幼虫的周围神经系统被用于研究轴突损伤和疾病的急性神经元反应。然而，由于第三幼虫和蛹化之间的时间间隔很短，该系统不太适合研究更长时间的过程。最近的研究表明，三龄幼虫对轴突损伤有快速的初始反应，并表现出诱人的轴突再生开始。然而，随着时间的推移，大多数幼体组织发生蜕变，这阻碍了对损伤反应的更完整分析，包括神经胶质的参与和可能的轴突修复。同样，在实验中探索维持NMJ结构和功能的机制，或这些机制如何随年龄或疾病而受损的观察窗口，也受到化蛹的发生的显著限制。本申请的目的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NMJ幼虫的特征，使其成为如此强大的模型。我们利用遗传变异，使幼虫正常发育，但随后在第三龄停留长达10天（比正常时间长4倍），在此期间它们继续生长，最后经历变态和羽化。基于我们的初步结果，我们相信，延长的第三岁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有力的机会。为了验证延长幼虫寿命（ELL）模型的有效性和实用性，我们提出了以下问题的实验：(1)ELL幼虫在发育过程中NMJ生长是否正常？在ELL期间，NMJ是否会随着幼虫大小的增加而继续增长？已知在正常发育期间调节NMJ生长的关键信号通路在ELL期间继续发挥作用吗？(2)在整个ELL过程中，NMJ在结构和功能上是否保持完整？(3)我们能否证明ELL系统作为一种实验工具的实用性，利用它来扩大我们对幼虫运动轴突和周围神经胶质损伤反应的理解？我们相信，这个新的实验系统具有巨大的潜力，可以极大地扩展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，这两者都与理解和治疗许多人类神经系统疾病高度相关。

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.