NGF is required for proper wiring of the sympathetic nervous system during development. Upon binding to its receptor TrkA, NGF can either signal locally in distal axons or in the cell body from ?signaling endosomes? (SEs) of the postganglionic neuron. Trafficking of the TrkA-SE to the cell body is critical for many developmental processes, including survival and synapse formation. Additionally, presynaptic neuron survival mirrors that of sympathetic ganglia and, by extension, the final target. Improper regulation of these processes has been linked to neurodevelopmental disorders such as mental retardation, and autism. NGF/TrkA internalization and retrograde transport down the axon in SEs is widely studied, however, there is a substantial gap in our knowledge when it comes to the fate of the SE once it gets back to the cell body. We have discovered a dynamic novel trafficking pathway of SEs in the soma and dendrites, retrograde transcytosis (RT). RT consists of exocytosis of SEs in the soma and dendrites and subsequent re-endocytosis of TrkA into long-lasting SEs which evade degradation. The premise for this proposal rests on our recent discovery that TrkA-SE number in the soma declines by 50% after 6 hours, but NGF signaling continues for 12 or more hours. The mechanism underlying this extremely long signal duration is unknown. The disappearance of TrkA-SEs from the soma could be due to degradation, but we now propose a novel alternative hypothesis: RT might lead to exocytosis of TrkA from multivesicular bodies (MVBs), resulting not only in surface appearance of activated TrkA on the soma, but also in secretion of NGF-TrkA in extracellular vesicles (EVs) in dendrites which continue to signal. EV biology is a nascent field, but a range of EV functions have been described mainly in non-neuronal systems. EVs have a demonstrated role in tissue repair, immune surveillance, transportation of miRNAs, and activation of signaling cascades. There have been a handful of recent EV studies focusing on neurons, however EVs have not previously been shown to participate in trophic neurodevelopmental processes. As a first step to ask if TrkA can be secreted in EVs to support long-lasting signaling, we succeeded in purifying EVs from PC12 cells. These EVs contain TrkA and can elicit functional responses in SCG neurons. Since nothing is known about how TrkA-EVs are generated and how they compare in terms of composition to EVs from non-neuronal cells, we propose an exploratory set of experiments to rigorously purify and molecularly define TrkA-EVs from sympathetic neurons, and to determine if signaling downstream of TrkA affects their production. We will use innovative approaches including microfluidic devices, rigorous purification coupled to mass spectrometry, and state-of-the art flow cytometry to fully characterize the molecular constituents of TrkA- EVs. These experiments are an essential first step in determining the form, function and locus of action of this potentially novel mode of trophic signaling. Our long-term goal is to explore a new type of neuron-neuron communication that may be critical for development of a functional circuit: secretion of neurotrophic TrkA-EVs.
Delineating the cellular and molecular mechanisms underlying nerve growth factor-mediated long distance signaling for neuronal survival, axon growth and synapse formation should represent a new way to think about nervous system patterning. The proposed research is significant because the new insights gained will lead to fundamental advances by investigating a novel mode of growth factor long distance signaling, i.e. secretion of extracellular vesicles. This work raises the possibility of new targets for tailoring translational strategies in the future. Moreover, this line of investigation may reveal clues about the etiology and progression of pathologies that remodel the nervous system, such as ALS or Alzheimer?s.
