Neurotrophic factor trafficking and signaling in development and disease

神经元细胞生物学中的一个基本问题是膜蛋白是如何 在细胞体中生物合成后长距离运输到轴突。轴突靶向 膜蛋白对于神经元连接的形成和维持至关重要 和正常的神经系统。然而，大多数膜蛋白是如何被运送到 轴突仍不明确。 神经生物学中一个长期持有的观点是，信号受体是组成性的， 通过分泌运输传递到轴突。相反，我们发现TrkA神经营养因子 受体是神经元存活、轴突生长和炎症的重要调节因子， 疼痛通过转胞吞作用（一种基于内吞作用的机制）被主动募集到轴突 其中包埋在索马表面的受体被内化并顺行 运输到轴突。引人注目的是，顺行TrkA转胞吞作用由配体触发， 神经生长因子（NGF），作用于轴突终末，提示正反馈 一种机制，用于动态地扩大轴突中受体的可用性， 的需要。此外，我们确定TrkA转胞吞作用是由以下活性引发的： PTP 1B，一种ER驻留蛋白酪氨酸磷酸酶，在细胞体中。的总目标 本申请是为了阐明一种 配体触发受体靶向轴突的表征不佳的模式。在目标1中， 我们将定义在细胞体中启动转胞吞作用的NGF介导的机制， 阐明受体转胞吞的运输路线和运输动力学，和 研究体内TrkA转胞吞作用。在目标2中，我们将测试ER- 锚定的PTP 1B磷酸酶通过控制TrkA生物学功能的获得来促进TrkA生物学功能的获得 受体的长距离转胞吞作用。我们将采用实时成像，生化， 在区室化神经元培养物中的功能分析结合体内 分析转基因小鼠来实现这些目标。这些研究将 解决了一个基本的，但研究甚少的细胞生物学问题， 受体被定向到轴突，并将提供对专门机制的深入了解， 增强神经元对空间作用的外部线索的反应性。

A fundamental question in neuronal cell biology is how membrane proteins are transported long-distance to axons after biosynthesis in cell bodies. Axon targeting of membrane proteins is critical for the formation and maintenance of neuronal connections and for a functional nervous system. Yet, how most membrane proteins are delivered to axons remains undefined. A long-held view in neurobiology is that signaling receptors are constitutively delivered to axons via secretory trafficking. In contrast, we found that TrkA neurotrophin receptors that are essential regulators of neuron survival, axon growth, and inflammatory pain are actively recruited to axons via transcytosis, an endocytosis-based mechanism where receptors embedded in soma surfaces are internalized and anterogradely transported to axons. Strikingly, anterograde TrkA transcytosis is triggered by the ligand, Nerve Growth Factor (NGF), acting on axon terminals, suggesting a positive feedback mechanism that serves to dynamically scale up receptor availability in axons during times of need. Furthermore, we identified that TrkA transcytosis is primed by the activity of PTP1B, an ER-resident protein tyrosine phosphatase, in cell bodies. The overall goal of this application is to elucidate the signaling and trafficking mechanisms underlying a poorly characterized mode of ligand-triggered targeting of receptors to axons. In Aim 1, we will define NGF-mediated mechanisms that initiate transcytosis in cell bodies, elucidate the trafficking itinerary and transport kinetics of receptor transcytosis, and investigate TrkA transcytosis in vivo. In Aim 2, we will test the hypothesis that ER- anchored PTP1B phosphatase promotes a gain of TrkA biological function by controlling the long-distance transcytosis of receptors. We will employ live imaging, biochemical, and functional analyses in compartmentalized neuron cultures in combination with in vivo analyses of genetically modified mice to accomplish these goals. These studies will address a fundamental, yet poorly studied, cell biological question of how signaling receptors are directed to axons, and will provide insight into specialized mechanisms that enhance neuronal responsiveness to spatially acting extrinsic cues.