Imaging NGF signal transduction in live neurons

我们的主要目标是了解神经生长因子（NGF）信号的机制， 从轴突末端向细胞体增殖。神经生长因子逆行信号传导对于存活至关重要， 分化和维持某些类型的神经元。据报道， 导致脑梗死患者脑中基底前脑胆碱能（BFC）神经元的丢失， 老年痴呆症或唐氏综合症。该项目将使用先进的成像技术， 真实的实时显示活神经元中的NGF运输。我们着重探讨神经生长因子的动态特性 在正常和唐氏综合症小鼠中的转运。其目标是： 1.描述轴突中含有神经生长因子的核内体的运动并定义其暂停 机制，通过使用量子点缀合的NGF来跟踪具有纳米颗粒的内体运动， 分辨率 2.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通路，通过标记 具有融合至C末端的可光激活的绿色荧光蛋白的缺乏NGF的内体 TrkA受体 3.识别唐氏综合征小鼠神经元中破坏的NGF转运的异常特征， 描述运输动力学的各个特征，包括平均速度、移动速度、 速度、暂停持续时间和暂停频率。 4.确定淀粉样前体蛋白过度表达如何导致异常的NGF逆行 通过检测淀粉样前体蛋白在唐氏综合征小鼠中的过度表达 小鼠可能导致结构或轴突特征缺陷，导致NGF转运中断。 实现这些目标将增加我们对NGF信号如何在正常细胞中传播的理解， 退化的神经元更广泛地说，这些研究将有助于阐明 老年痴呆症和唐氏综合症。

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 forebarin 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 neruons 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 inlcude 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.