NGF-TRKA ENDOSOME DYNAMICS, SIGNALING AND FUNCTION IN SYMPATHETIC NEURON DENDRITES
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Date
2016-05-26
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Johns Hopkins University
Abstract
Nerve growth factor (NGF) is the prototypical neurotrophin, playing key roles in cell growth, survival, and target innervation as well as dendritic growth and the formation and maintenance of synaptic connections. Sympathetic neurons are dependent on target-derived NGF for survival and as such have become an exemplary model for studying NGF function. NGF signals via retrogradely transported NGF-TrkA endosomes. Recently we have discovered the retrograde transport of TrkA endosomes into the dendritic compartment of sympathetic neurons where they may contribute to the formation and organization of synapses. In this study, we developed a real time imaging paradigm to examine the mobility and transport of TrkA in dendrites, comparing findings to TrkA endosome movement and transport in axons and cell bodies. Using this method we observe that after application of NGF to the distal axon compartment of cultured neurons, TrkA endosomes move in a saltatory manner retrogradely through the axon, slow down or halt in the soma, and move in a bidirectional manner in dendrites. Although TrkA endosomes in dendrites move at a comparable rate to those in axons, their unique dynamics (i.e. more direction changes) result in a smaller net displacement than endosomes in axons. Further, immunocytochemistry using antibodies specific for a TrkA phosphorylated residue (Y785) that supports downstream signaling cascades of the NGF-TrkA complex demonstrates that retrogradely trafficked TrkA endosomes within dendrites are signaling competent and that P-TrkA positive endosomes juxtapose postsynaptic density complexes, in vitro and in vivo. These findings suggest that target-derived NGF-TrkA endosomes signal within dendrites to form and maintain synapses. Functional experiments that combine chemical genetics with drug loaded PLGA microspheres allow for spatially specific inhibition of TrkA kinase activity and have revealed that TrkA activity is necessary in the somatodendritic compartment for both synapse formation (in vitro) and maintenance (in vivo). We have achieved the ability to inhibit TrkA kinase activity in dendrites of cultured neurons, observing that retrogradely transported TrkA endosomes signal within dendrites to maintain PSD clusters. This work reveals a novel mode of NGF-dependent synapse formation and maintenance, and the mechanism by which target fields control circuit assembly.
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NGF, TrkA, signaling endosome, neurotrophins, retrograde transport, synapse maintenance