| 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. |
