Molecular Mechanisms of Neuron Motility and Axon Guidance

大脑的功能依赖于一种精确而复杂的神经元连接模式。该项目的长期目标是了解在发育过程中建立这种连接模式的分子机制，以及这些机制的异常如何导致晚年的阿尔茨海默病(AD)。该项目特别关注基于RNA的调控机制。通过RNA结合蛋白(RBP)调控mRNA翻译的主要优点是：(1)允许在需要蛋白质的特定亚细胞区域局部调节蛋白质合成；(2)协调调节功能相关的大型mRNAs网络的表达。为了了解轴突引导的基本原理，一个主要的模型系统是中线的脊髓连合轴突引导。导航这个中间目标需要轴突被吸引，然后被排斥，这一经典的机制是‘Robo开关’，在交叉后的轴突中，排斥的Robo受体上调；然而，细胞外信号及其触发这种开关的机制仍然未知。我们现在已经发现了一种非常新的Robo开关机制，涉及细胞外配体与跨膜淀粉样前体蛋白(APP)结合，APP与RBP CPEB4在细胞内相互作用，以调节交叉轴突片段中Robo的局部翻译。在确定了这一新的APP-CPEB4途径后，拟议的研究旨在扩大我们对该途径的分子机制和功能的理解。在连合轴突引导中，已知许多蛋白质的表达在中线的轴突节段受到局部调控；拟议中的APP-CPEB4通路研究有望确定一个大型基因网络的协调调节，将许多不同的过去观察结果结合成一个统一的模型，用于这一轴突引导的主要范式。除了轴突引导外，初步研究还揭示了在另一个主要的发育模型系统-皮质神经元迁移中，同一分子途径的一系列重叠但不同的功能。新的APP-CPEB4通路与疾病也有很高的相关性：除了参与导致自闭症谱系障碍(ASD)的发育过程外，APP在我们的通路中的关键作用使其与AD密切相关。关于自闭症，皮质神经元迁移阶段的异常被认为是ASD的主要原因，而小鼠皮质中CPEB4在这一特定阶段的破坏会导致ASD样行为。此外，我们从配体到下游靶点的所有通路组件都与ASD有关，尽管以前没有在统一的模型中联系在一起。关于AD，APP的跨膜结构长期以来一直导致人们认为它是细胞表面受体的想法，然而尽管数十年的密集研究，APP还没有被确定为具有指导性的受体角色--其中配体的时空模式调节下游的发育或生理功能。现在，确定APP的受体角色--包括从配体到信号通路再到功能读数的途径--为理解APP打开了一个质的新层次的大门，这一点尤其重要，因为治疗靶向Aβ的挑战越来越强调理解APP本身的作用。对我们的APP-CPEB4通路的研究将揭示新的生物学原理，同时加深对神经发育和神经退行性疾病潜在机制的理解。方法包括全基因组靶向信使核糖核酸鉴定，以及体外和体内的功能细胞和发育研究。此外，研究APP-CPEB4新通路的信号转导机制对于了解APP-CPEB4通路的运作及其治疗干预的潜力是必不可少的。

The brain relies for its function on a precise and complex pattern of neuronal connections. The broad long-term goal of this project is to understand molecular mechanisms that set up this pattern of connections during development, and how aberrations of these mechanisms lead to Alzheimer’s Disease (AD) later in life. This project focuses particularly on RNA-based regulatory mechanisms. Key advantages of regulating mRNA translation via RNA-binding proteins (RBPs) are: (1) allowing protein synthesis to be locally regulated in specific subcellular regions where the proteins are needed, and (2) coordinately regulating expression of large networks of functionally related mRNAs. To understand the basic principles of axon guidance, a major model system has been spinal commissural axon guidance at the midline. Navigating this intermediate target requires axons to be attracted and then repelled, and the classic mechanism for this is the ‘Robo switch’ where repellent Robo receptors are upregulated in post-crossing axons; however, the extracellular signal and the mechanism by which it triggers this switch have remained unknown. We have now identified a highly novel mechanism for the Robo switch, involving extracellular ligand binding to the transmembrane Amyloid Precursor Protein (APP), which interacts intracellularly with the RBP CPEB4, to regulate Robo local translation in post-crossing axon segments. Having identified this novel APP-CPEB4 pathway, the proposed studies are designed to expand our understanding of the pathway’s molecular mechanisms and functions. In commissural axon guidance, expression of many proteins is known to be locally regulated in axon segments at the midline; the proposed studies of the APP-CPEB4 pathway are expected to identify coordinate regulation of a large gene network, bringing together many disparate past observations into a unifying model for this premier paradigm of axon guidance. In addition to axon guidance, preliminary studies reveal an overlapping yet distinct set of functions for the same molecular pathway in another major developmental model system, cortical neuron migration. The novel APP-CPEB4 pathway also has high relevance to disease: in addition to its involvement in developmental processes that lead to Autism Spectrum Disorder (ASD), the pivotal role of APP in our pathway gives it key relevance to AD. Regarding autism, abnormalities at the cortical neuron migration stage are believed to be a leading cause of ASD, and CPEB4 disruption in mouse cortex at this specific stage causes ASD-like behaviors. Moreover, all the components of our pathway from ligands to downstream targets have been implicated in ASD, though not previously linked in a unifying model. Regarding AD, the transmembrane structure of APP has long led to the idea that it is a cell surface receptor, yet despite decades of intensive work no instructive receptor role – where the spatiotemporal pattern of a ligand regulates a downstream developmental or physiological function – has yet been identified for APP. Now identifying a receptor role for APP – including a pathway from ligands through a signaling pathway to functional readouts – opens the door to a qualitatively new level of understanding APP, which is especially important since the challenges of therapeutically targeting Aβ place increased emphasis on understanding the roles of APP itself. Studies of our APP-CPEB4 pathway will uncover novel biological principles, while leading to enhanced understanding of mechanisms underlying neurodevelopmental and neurodegenerative disorders. Approaches include genome-wide target mRNA identification, and functional cellular and developmental studies in vitro and in vivo. Additionally, studies of signal transduction mechanisms in the novel APP-CPEB4 pathway will be essential to understand its operation and its potential for therapeutic intervention.