Regulation of axonal arbor and synaptic dynamics by the apoptotic pathway: Interactions with the cytoskeleton, downstream substrates and upstream regulators

神经元之间适当连接的形成和维持对神经系统的正常功能至关重要。这些过程中的缺陷会导致行为紊乱和改变。在中枢神经系统（CNS）中，突触的形成依赖于突触前和突触后轴突和树突状分支的精细调控和相互作用。因此，树突化和突触发生对于确保突触连接的正确模式至关重要，同时对于定义神经元的复杂和分区几何结构至关重要。这种区隔化使细胞信号通路的划分成为可能，这是至关重要的，特别是当信号级联反应具有多效性或甚至可能损害整个细胞时。我最近的研究表明，半胱天冬酶是凋亡的关键介质，在年轻的、动态的轴突乔木的分支点被局部激活，是维持乔木生长过程中轴突和突触动力学的关键。为此，我利用了斑马鱼的幼虫，由于它们的体积小，透明和遗传延展性，为研究脊椎动物中枢神经系统的神经细胞生物学提供了前所未有的机会。长期以来，对连接视网膜和中脑顶盖的视网膜顶盖投影的特异性关注一直被认为是突触特异性的重要发育模型。在这个应用中，我建议解决我们关于局部信号通路在乔木动力学中的作用的知识中的一个重要空白：我们不了解半胱天冬酶是如何介导分支动力学的，这必须涉及细胞骨架的调节，细胞生物学如何将这种细胞骨架变化转化为形态发生事件，或者它是如何被上游信号调节以避免细胞死亡的。也不知道神经活动独立和活动依赖的调节乔木生长的机制是否有相似的机制。通过结合树杈和突触形成的体内实时成像、基因功能操纵和视觉刺激模式，我建议在发育和经验依赖的树杈和突触动力学过程中研究半胱天冬酶的细胞和分子生物学。我的具体计划是：1)定义树突和突触发生的潜在细胞骨架动力学，并确定caspase激活如何以及在何处促进树突和突触前重塑的潜在细胞骨架变化。2)。鉴定下游半胱天冬酶靶点及其如何修饰细胞骨架。3)。确定在乔木中，caspase活性是如何通过外在或内在的细胞凋亡途径和视觉体验进行动态和局部调节的。我的研究将为神经元树突和突触形成提供关键的机制见解，我的研究将确定神经元中的一组细胞和分子成分和原理，这些成分和原理可能会转移到其他细胞环境中的非凋亡过程中。

The formation and maintenance of appropriate connections between neurones are critical to the normal functioning of the nervous system. Defects in these processes can lead to disorders and alterations in behaviour. In the central nervous system (CNS), synapse formation depends on the regulated elaboration and interaction of pre- and post-synaptic axonal and dendritic branches. Arborisation and synaptogenesis are hence critical in ensuring the proper pattern of synaptic connections and at the same time in defining the complex and compartmentalised geometry of neurones. This compartmentalisation enables the partitioning of cellular signaling pathways, which is critical especially if signaling cascades involved have pleiotropic effects or that could even harm the cell as a whole. I have recently shown that caspases, key mediators of apoptosis, are activated locally at branch points of young, dynamic axonal arbors and are key in maintaining axonal and synaptic dynamics during arbor growth. For this, I took advantage of zebrafish larvae, which owing to their small size, transparency and genetic malleability, offer an unprecedented opportunity to study neuronal cell biology in vivo in the vertebrate CNS. The specific focus on the retinotectal projection, which connects the retina to the midbrain tectum, has long served as an important developmental model of synaptic specificity. In this application, I propose to address an important gap in our knowledge about the role of local signaling pathways in arbor dynamics: We do not understand how caspases mediate branch dynamics, which must involve modulation of the cytoskeleton, what cell biology translates such cytoskeletal changes into morphogenic events or how it is regulated by upstream signals to avoid cell death. It is also not known whether neural activity-independent and activity-dependent mechanisms regulating arbor growth share similar mechanisms. Via a combination of in vivo live imaging of arborisation and synapse formation, manipulation of gene function and visual stimulation patterns, I propose to study the cell and molecular biology of caspases during developmental and experience-dependent arbor and synaptic dynamics. Specifically I plan: 1.) To define the underlying cytoskeletal dynamics of arborisation and synaptogenesis and to identify how and where caspase activation promotes the cytoskeletal changes underlying arbor and presynaptic remodeling. 2.) To identify downstream caspase targets and how they modify the cytoskeleton. 3.) To determine how caspase activity is dynamically and locally regulated in arbors via the extrinsic or intrinsic apoptosis pathways and visual experience. My studies will provide key mechanistic insights into neuronal arborisation and synapse formation and my research will identify a set of cellular and molecular components and principles in neurones, which may be transferred to nonapoptotic processes in other cellular contexts.