Compartmentalized neuronal cell culture system. This in vitro model system is used to study axonal trafficking of neurotrophins and their receptors, as well as neurotrophin signaling in axon terminals and in cell bodies. Compartmented cultures of sympathetic and sensory neurons are established by placing Teflon dividers onto a thin layer of vacuum grease, which allows diffusion-limited compartmentalization of neuronal cell bodies and distal axons.

The proper functioning of the nervous system relies on the establishment of precise neuronal circuits. These neuronal circuits are largely formed during early development. To form functional neuronal circuits, neurons receive specific information in the form of extracellular cues from the target tissues that they innervate. To date, the family of neurotrophins provides the best example of these target-derived instructive cues that regulate diverse developmental events in the vertebrate nervous system, including survival, axonal and dendritic growth and synapse formation. Using a combination of cell biological, biochemical and imaging techniques as well as mouse genetics, we are actively pursuing two lines of research:

1. How neurotrophic factors coordinate neuronal development by regulating the neuronal endocytic machinery, and
2. Growth factor signaling pathways underlying axonal growth, morphology and innervation of target tissues during development.

Regulation of neuronal development by trafficking of neurotrophins and their receptors

For decades, it has been known that neurotrophins and their receptors undergo long-range trafficking in neurons, but how neurotrophins utilize the trafficking machinery to regulate distinct aspects of neuronal development remains poorly characterized. Our long-term goal is to gain insight into how neurotrophins coordinate neuronal development by regulating the cell’s endocytic machinery. Using a combination of fluorescent, biochemical and electron microscopic assays, we are investigating molecular mechanisms of endocytosis, recycling and axonal transport of neurotrophins and their receptors in developing neurons. We are employing structure-function analyses to identify endocytic motifs in the neurotrophin receptors, the Trk receptor tyrosine kinases, that mediate distinct aspects of receptor trafficking. Finally, we are assessing the role of distinct modes of Trk trafficking on neurotrophin-dependent survival, axonal growth and neuronal morphology.

We recently found that neuronal TrkA receptors undergo constitutive cycling between the plasma membrane and intracellular endosomes. Constitutive trafficking of TrkA receptors maintains a dynamic intracellular pool of TrkA receptors that are rapidly mobilized to developing growth cones via Rab-11-containing recycling endosomes, upon neurotrophin stimulation. Developing neurons exhibit decreased responsiveness to NGF when recycling pathways are impaired, whereas enhanced recycling conferred neuronal sensitivity to lower concentrations of NGF. Our results suggest that constitutive trafficking of TrkA receptors might facilitate rapid and sensitive neuronal responses to limiting concentrations of neurotrophins, found in vivo.

Identification of local and retrograde signaling mechanisms underlying neurotrophin-mediated axonal growth

We are also investigating the intracellular signaling pathways by which target-derived neurotrophins regulate multiple stages of axonal growth including axon initiation, projection along intermediate targets and innervation of final target tissues. We are employing in vitro and in vivo approaches to dissect the local signaling pathways activated in axon terminals, as well as transcriptional programs activated in remote neuronal soma by target-derived neurotrophins, that mediate unique aspects of axonal growth and morphology.

We recently identified a member of the Wnt family of growth factors, Wnt5a, as being a transcriptional target of NGF signaling. Employing a unique culture system of compartmentalized neurons, and genetically modified mouse models, we are testing the hypothesis that interactions between Wnts expressed in neurons and neurotrophins secreted by their target tissues influence axonal growth events during target innervation. These studies will define whether complexity of neurotrophin actions in the nervous system can be accounted for, by regulation of expression of other growth factors.

A fundamental question in neuronal development is how signals at the growth cones of projecting axons are conveyed to the nucleus to control transcriptional programs important for axon growth, guidance and innervation of target tissues. Recently, we identified a local requirement for calcineurin/nuclear factor of activated T-cells (NFAT) signaling within axons to support target derived neurotrophin mediated axon growth. We are currently investigating the molecular mechanisms by which Calcineurin/NFAT signaling in developing axons is propagated to the nucleus to activate transcriptional programs important for neurotrophin mediated axon growth.