Subcellular Pathways of Internalized Neurotrophics

神经营养因子是神经元间通讯的信使。它们调节神经元的分化和功能。内化、细胞内转运、信号转导和最终降解是营养信号转导的重要步骤。最近的研究表明，内化的神经营养因子可以分为降解途径和循环途径。这项应用的中心目标是阐明内化的神经营养因子是如何被分类到不同的细胞内途径的。这项研究将主要在波兰的Nencki研究所进行，作为NIH#RO 1 EY 12841年度补助金的延伸。这项拟议的研究将利用鸡胚胎发育中的视觉系统作为一个独特的体内模型系统，该系统允许将放射性标记的营养因子引入一个隔室并量化细胞内途径。实验将侧重于比较神经营养因子被树突(视网膜神经节细胞)内化和仅被轴突终末(峡部视神经元)内化后的不同亚细胞通路。这些实验将展示摄取途径(树突和轴突)如何影响随后的信号和降解途径。超微结构水平的放射自显影将被用来确定神经营养因子的亚细胞分布。将比较不同神经营养因子的分布情况。体内转运的神经营养因子的受体结合将通过与受体特异性抗体的交联和免疫沉淀来确定。通过与异源神经营养因子的竞争和酪氨酸激酶受体的失活，对内化神经营养因子积累的细胞器及其途径和亚细胞目的地进行比较和实验操作。分子、药理学和超微结构方法的结合将使我们能够回答有关神经营养素运输的问题，这些问题对于理解这些因素如何调节神经元存活和突触可塑性等各种事件至关重要。神经退行性疾病与营养支持不足有关。了解神经营养因子的正常运输、分类和循环将有助于我们了解病理情况以及外源性神经营养因子如何被用作治疗剂。

Neurotrophic factors are messengers in the communication between neurons. They regulate neuronal differentiation and function. Internalization, intracellular transport, signal transduction and eventual degradation are essential steps in trophic signaling. Recent studies have shown that internalized neurotrophins can be sorted into either degradative or recycling pathways. The application's central aim is to elucidate how internalized neurotrophins are sorted into distinct intracellular pathways. This research will be done primarily at the Nencki Institute in Poland as an extension of NIH grant # RO 1 EY 12841. The proposed studies will utilize the developing visual system of chick embryos as a unique invivo model system which allows the introduction of radiolabeled trophic factors into a compartment and the quantification of intracellular pathways. Experiments will focus on the comparison of different subcellular pathways after internalization of neurotrophins by dendrites (retinal ganglion cells) and after internalization exclusively by axon terminals (isthmo-optic neurons). These experiments will show how the route of uptake (dendritic vs. axonal) influences the subsequent signaling and degradation pathways. Autoradiography at the ultrastructural level will be used to identify the subcellular distribution of neurotrophins. Distribution profiles will be compared between neurotrophins. Receptor binding of invivo- transported neurotrophins will be determined by crosslinking and immunoprecipitation with receptor-specific antibodies. The organelles in which internalized neurotrophins accumulate and their pathways and subcellular destinations will be compared and experimentally manipulated by competition with heterologous neurotrophins and inactivation of tyrosine kinase receptors. The combination of molecular, pharmacological and ultrastructural approaches will allow us to answer questions about trafficking of neurotrophins which are crucial to an understanding of how these factors may regulate events as diverse as neuronal survival and synaptic plasticity. Neurodegenerative diseases have been related to deficits in trophic support. Knowledge about the normal trafficking, sorting and recycling of neurotrophic factors will help us to understand pathologic conditions and how exogenous neurotrophins may be used as therapeutic agents.