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Single molecule and single particle studies of neuronal axonal transport.

机译：神经元轴突运输的单分子和单颗粒研究。

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摘要

Neurons are highly branched cells with numerous axons and dendrites extending from cell body. In humans, some axons can be as long as 1 meter with a cell body less than 50 micrometer in diameter. During early development, sympathetic neurons and certain types of sensory neurons rely on the protein Nerve Growth Factor (NGF) secreted by peripheral tissues for survival. To support neuron survival, NGF binds receptors on axon terminals and traverses the entire length of the axons to reach cell body. In this thesis, we applied single molecule fluorescence microscopy to study this transport process in living neurons.;Using pseudo-Total-Internal-Reflection-Fluorescence (pseudo-TIRF) microscopy and quantum dot (Qdot) labeled NGF, we directly visualized the movement of NGF containing endosomes, one at a time, in the axons of living Dorsal Root Ganglion (DRG) neurons. We observed that endosomes containing Qdot-NGF move only retrogradely in a "stop-and-go" fashion with an average speed of 1.31 +/- 0.03 mum/s. This observed average velocity is consistent with the average of previous measurements. We also discovered that under physiological concentrations of NGF, about 90% of all the endosomes contained only a single NGF dimer, and that the number of NGF molecules in each endosome increased as the NGF concentration rose.;Subsequently, using a dual color imaging technique, we simultaneously monitored the movement of Qdot-NGF and its surface receptor TrkA. We labeled TrkA by engineering a fusion protein with Green Fluorescent Protein (GFP). Even in the absense of NGF, we observed both anterograde and retrograde transport of TrkA-GFP in DRG axons. We discovered that the majority of retrogradely transported TrkA-GFP were in the activated state. One cause for the NGF independent retrograde transport of TrkA-GFP was TrkA-GFP autoactivation following its over-expression. Finally, we found that retrograde TrkA-GFP flux was significantly greater in the presence of the ligand. However, there was no significant colocalization of TrkA-GFP and Qdot-NGF. We concluded that it was likely due to activation and internalization of TrkA in the absence of concurrent NGF internalization.;We then applied our techniques to the Giant Axon Neuropathy (GAN) disease mouse model. We observed that retrograde transport of Qdot-NGF was largely impaired in gigaxonin null neurons compared to the speed in wild type neurons. We also found that overexpression of Map8-GFP protein in wild type DRG neurons could cause similar retrograde transport impairment. From these observations combined with the biochemical evidence that Map8 was accumulated in gigaxonin null neurons, we concluded that the neurodegeneration in GAN mouse model was probably caused by Map8 accumulation, which originates from the loss of gigaxonin, and causes a retrograde transport imparement.
机译：神经元是高度分支的细胞，具有许多从细胞体延伸的轴突和树突。在人类中，某些轴突的直径可能小于50微米，最长可达1米。在早期发育中，交感神经元和某些类型的感觉神经元依赖于周围组织分泌的神经生长因子（NGF）蛋白来维持生存。为了支持神经元的生存，NGF结合轴突末端的受体，并遍历轴突的整个长度到达细胞体。本文采用单分子荧光显微镜研究了活体神经元中的这种转运过程。利用伪全内反射荧光显微镜和量子点标记的NGF直接观察了运动一次在活的背根神经节（DRG）神经元轴突中一次含有NGF的内体。我们观察到，含有Qdot-NGF的内体仅以“走走停停”的方式逆行，平均速度为1.31 +/- 0.03 mum / s。该观察到的平均速度与先前测量的平均值一致。我们还发现，在NGF的生理浓度下，大约90％的内体仅包含一个NGF二聚体，并且随着NGF浓度的增加，每个内体中NGF分子的数量均增加了;随后，采用了双色成像技术，我们同时监测了Qdot-NGF及其表面受体TrkA的运动。我们通过工程改造与绿色荧光蛋白（GFP）的融合蛋白标记了TrkA。即使在NGF缺失的情况下，我们也观察到DRG轴突中TrkA-GFP的顺行和逆行转运。我们发现大多数逆行运输的TrkA-GFP处于激活状态。 TrkA-GFP的NGF独立逆行转运的一个原因是其过度表达后TrkA-GFP自激活。最后，我们发现在配体存在下逆行的TrkA-GFP通量明显更大。但是，TrkA-GFP和Qdot-NGF没有明显的共定位。我们的结论是，这可能是由于在没有同时发生NGF内在化的情况下TrkA的激活和内在化所致。；然后我们将技术应用于巨轴突神经病（GAN）疾病小鼠模型。我们观察到，与野生型神经元的速度相比，在吉格松素无效神经元中Qdot-NGF的逆行运输受到很大的损害。我们还发现，野生型DRG神经元中Map8-GFP蛋白的过度表达可能会引起类似的逆行运输损伤。从这些观察结果和生化证据表明Map8积累在gigaxonin无效神经元中，我们得出结论，GAN小鼠模型中的神经变性可能是由Map8积累引起的，Map8积累起因于gigaxonin的丧失，并导致逆行运输障碍。

著录项

  • 作者

    Chen, Liang.;

  • 作者单位

    Stanford University.;

  • 授予单位 Stanford University.;
  • 学科 Biology Neuroscience.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 128 p.
  • 总页数 128
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 神经科学;生物物理学;
  • 关键词

  • 入库时间 2022-08-17 11:38:25

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