Trafficking and axodendritic transcytosis of BDNF in hippocampal neurons [electronic resource]

Brain derived neurotrophic factor (BDNF) plays a key role in the growth, development and maintenance of the central and peripheral nervous systems. Exogenous BDNF activates its membrane receptors at the axon terminal, and subsequently sends regulation signals to the cell body. To understand how BDNF signal propagates in neurons, it is important to follow the trafficking of BDNF after it is internalized at the axon terminal. Here we labeled BDNF with bright, photostable quantum dot (QD-BDNF) and followed the axonal transport of QD-BDNF in real time in hippocampal neurons. We showed that QD-BDNF was able to bind BDNF receptors and activate downstream signaling pathways. When QD-BDNF was applied to the distal axons of hippocampal neurons, it was observed to be actively transported toward the cell body at an average speed of 1.11 ± 0.05 [mu] m/s. A closer examination revealed that QD-BDNF was transported by both discrete endosomes and multivesicular body-like structures. Our results showed that QD-BDNF could be used to track the movement of exogenous BDNF in neurons over long distances and to study the signaling organelles that contain BDNF. Surprisingly, not all QD-BDNF ended up in lysosomes in the cell body for degradation. Instead, some BDNF was transported to dendrites possibly for signaling purposes. BDNF transcytosis challenges the model that neurotrophic action is terminated by rapid degradation of the neurotrophin molecule after delivery of the trophic signal. It is postulated that BDNF as well as other neurotrophic factors might be recycled and targeted to dendrites as endogenous neurotrophins for the purpose of releasing. To develop a comprehensive understanding of the molecular mechanisms of BDNF transcytosis, it is important to follow BDNF retrograde transport in axons toward the cell body, axodendritic translocation, properties of the BDNF endosomes transcytosed into dendrites, and study whether these processes are activity dependent. Using QD for monitoring BDNF transcytosis through neurons requires that the labeling method does not perturb the biological function of BDNF. Meanwhile, to observe the trafficking of QD-BDNF internalized from the axon terminal, it is necessary to polarize the growth of axons into a fluidic isolated environment and separate from cell body and dendrites. With modifications and coating with neurite guidance molecules in the channels, the microfluidic platform can be improved to fit the purpose of observing BDNF transcytosis in a more controlled environment. By combining fluorescent labeled BDNF, microfluidic neuron culture platform and dual-color imaging, we found that (1) After BDNF internalized from axon terminal transport retrogradely into soma, most goes to degradation but some enters dendrites and ends up at different locations with its receptor TrkB; (2) Cargos transcytosed from axon terminal can be directed into dendrites and transport in similar manner as endogenous protein cargos.

Keywords: Chemistry.
Advisor: Cui Bianxiao primary advisor. Thesis advisor
Boxer Steven G. (Steven George)
Cegelski Lynette advisor. Thesis advisor