Imaging nerve growth factor signal transduction in live neurons

描述（由申请人提供）： 我们的主要目标是了解神经生长因子（NGF）信号从轴突终末传播到细胞体的机制。NGF逆行信号传导对于某些类型神经元的存活、分化和维持至关重要。据报道，中断的NGF逆行转运导致阿尔茨海默病或唐氏综合征患者脑中基底前脑胆碱能（BFC）神经元的丢失。该项目将使用先进的成像技术直接可视化神经生长因子运输活神经元在真实的时间。我们着重探讨正常和唐氏综合征小鼠神经生长因子转运的动态特征。目标是：1.利用量子点标记的神经生长因子，以纳米分辨率追踪神经内体的运动，研究神经内体在轴突中的运动特征，并确定其暂停机制。2.通过用与TrkA受体C末端融合的可光活化绿色荧光蛋白标记缺乏NGF的内体，确定是否存在缺乏NGF的内体，它们是否与NGF信号传导相关，以及是否存在独立于内体转运的替代信号传导途径。3.通过表征运输动力学的个体特征，包括平均速度、移动速度、暂停持续时间和暂停频率，识别唐氏综合征小鼠神经元中中断的NGF运输的异常特征。4.通过检查淀粉样前体蛋白在唐氏综合征小鼠中的过度表达如何可能导致导致结构或轴突特征缺陷，从而导致NGF转运中断，确定淀粉样前体蛋白过度表达如何导致唐氏综合征小鼠中异常的NGF逆行转运。实现这些目标将增加我们对NGF信号如何在正常和变性神经元中传播的理解。更广泛地说，这些研究将有助于阐明阿尔茨海默病和唐氏综合征的发病机制。

DESCRIPTION (provided by applicant): Our broad objective is to understand the mechanisms by which the nerve growth factor (NGF) signal is propagated from the axon terminal to the cell body. NGF retrograde signaling is critical for the survival, differentiation, and maintenance of certain types neurons. Disrupted NGF retrograde transport was reported to contribute to the loss of the basal forebrain cholinergic (BFC) neurons in the brains of patients with Alzheimer's Disease or Down's Syndrome. This project will use advanced imaging techniques to directly visualize NGF transport in live neurons in real time. We focus on exploring dynamic features of NGF transport in normal and Down's Syndrome mice. The aims are: 1. Characterize the movement of NGF-containing endosomes in axons and define their pausing mechanism(s), by using quantum dot conjugated NGF to track endosomal movements with nanometer resolution. 2. Determine whether NGF-lacking endosomes are present, whether they are relevant for NGF signaling, and whether there are alternative signaling pathways independent of endosomal transport, by marking the NGF-lacking endosomes with photo-activatable green fluorescence proteins that are fused to the C-terminal of TrkA receptor. 3. Identify the abnormal features of disrupted NGF transport in Down Syndrome mouse neurons, by characterizing individual features of transport dynamics, which include the average speed, the moving speed, the pausing duration, and the pausing frequency. 4. Determine how amyloid precursor protein overexpression leads to the abnormal NGF retrograde transport in Down Syndrome mouse by examining how overexpression of amyloid precursor protein in DS mice might cause defective structural or axonal features that lead to disrupted NGF transport. Achieving those aims will increase our understanding of how NGF signal is propagated in normal and degenerative neurons. More broadly, those studies will contribute to elucidate the pathogenesis of Alzheimer's disease and Down syndrome.