Imaging nerve growth factor signal transduction in live neurons

我们的主要目标是了解神经生长因子(NGF)信号是如何 从轴突末端传播到细胞体。NGF逆行信号对生存至关重要， 某些类型神经元的分化和维持。据报道，NGF逆行运输中断 在慢性阻塞性肺疾病患者脑内基础前列环素胆碱能神经元的丢失 阿尔茨海默病或唐氏综合症。该项目将使用先进的成像技术直接 实时可视化NGF在活神经元中的传输。重点探讨了NGF的动态特性 正常和唐氏综合征小鼠体内的转运。目标是： 1.描述轴突内含神经生长因子的内小体的运动并确定它们的暂停 机制(S)，通过使用量子点共轭神经生长因子来追踪纳米级的内体运动 决议。 2.确定是否存在缺乏NGF的内体，它们是否与NGF信号相关， 以及是否存在独立于内体运输的替代信号通路，通过标记 与C末端融合的具有光激活绿色荧光蛋白的缺乏NGF的内体 TrkA受体。 3.通过以下方法确定唐氏综合征小鼠神经元NGF运输中断的异常特征 表征运输动力学的个别特征，包括平均速度、移动 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NGF异常逆行 唐氏综合征小鼠体内淀粉样前体蛋白过度表达对转运的影响 小鼠可能会导致结构或轴突功能缺陷，从而导致NGF运输中断。 实现这些目标将增加我们对NGF信号如何在正常和 退化的神经元。更广泛地说，这些研究将有助于阐明糖尿病的发病机制。 阿尔茨海默病和唐氏综合症。

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.