11674 • The Journal of Neuroscience, September 16, 2009 • 29(37):11674–11685 Development/Plasticity/Repair Axonal Targeting of Trk Receptors via Transcytosis Regulates Sensitivity to Neurotrophin Responses Maria Ascan˜o, Alissa Richmond, Philip Borden, and Rejji Kuruvilla Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218 Axonal targeting of trophic receptors is critical for neuronal responses to extracellular developmental cues, yet the underlying trafficking mechanisms remain unclear. Here, we report that tropomyosin-related kinase (Trk) receptors for target-derived neurotrophins are anterogradely trafficked to axons via transcytosis in sympathetic neurons. Using compartmentalized cultures, we show that mature receptors on neuronal soma surfaces are endocytosed and remobilized via Rab11-positive recycling endosomes into axons. Inhibition of dynamin-dependent endocytosis disrupted anterograde transport and localization of TrkA receptors in axons. Anterograde TrkA deliv- ery and exocytosis into axon growth cones is enhanced by nerve growth factor (NGF), acting locally on distal axons. Perturbing endocytic recycling attenuated NGF-dependent signaling and axon growth while enhancing recycling conferred increased neuronal sensitivity to NGF. Our results reveal regulated transcytosis as an unexpected mode of Trk trafficking that serves to rapidly mobilize ready-synthesized receptors to growth cones, thus providing a positive feedback mechanism by which limiting concentrations of target-derived neurotro- phins enhance neuronal sensitivity. Introduction in the rough endoplasmic reticulum (ER) and undergoing mat- Polarized membrane trafficking of signaling receptors is critical uration in the ER–Golgi network in neuronal cell bodies, are for developing neurons to respond to extracellular stimuli medi- shipped directly to axons in Golgi-derived vesicles. However, in ating growth, guidance, and neuronal survival. The family of some cases, axonal targeting is accomplished by a more circuitous tropomyosin-related kinases (Trk) receptors provides a promi- endocytosis-dependent mechanism called transcytosis in which nent example of trophic factor receptors that undergo long- proteins are initially delivered to somatodendritic compartments distance axonal trafficking to regulate growth and survival of and then endocytosed and transported anterogradely for inser- developing neurons (Zweifel et al., 2005). A key mechanism by tion into axon terminals (Horton and Ehlers, 2003). Transcytosis which the prototypical neurotrophin, nerve growth factor has been best characterized in polarized epithelial cells, including (NGF), secreted by peripheral target tissues, provides trophic hepatocytes and enterocytes, in which endocytic trafficking from support to sympathetic neurons is by regulating endocytosis and basolateral to apical surfaces is the primary mode for delivery of retrograde trafficking of NGF:TrkA complexes in signaling endo- newly synthesized apical membrane proteins (Bastaki et al., 2002; somes from axon terminals to cell bodies (Delcroix et al., 2003; Ye Tuma and Hubbard, 2003). So far, transcytotic delivery from et al., 2003; Heerssen et al., 2004; Zweifel et al., 2005). However, neuronal soma surfaces to axons has been demonstrated for only the neurotrophin-mediated developmental processes of axon a limited number of membrane proteins, including L1/neuron– growth, guidance, and neuronal survival must also place an enor- glia cell adhesion molecule (NgCAM) (Wisco et al., 2003), a cell mous demand on rapid axonal targeting of Trk receptors to en- adhesion molecule important for axon guidance, transferrin (Tf) sure sensitive functional responses. Currently, little is known receptor (He´mar et al., 1997), which regulates cellular iron ho- about the membrane trafficking mechanisms regulating proper meostasis, and the type 1 cannabinoid receptor CB1R (Leterrier et localization of Trk receptors in axons. al., 2006), a highly abundant G-protein-coupled receptor in the Membrane proteins are often delivered to axons via the secre- nervous system implicated in modulating synaptic plasticity. tory pathway (Horton and Ehlers, 2003). Proteins, after synthesis However, the implications of transcytosis on receptor signaling and function have remained unclear. Received March 31, 2009; revised July 31, 2009; accepted Aug. 11, 2009. Here, we show that Trk receptors undergo transcytosis in de- ThisworkwassupportedwithfundsfromtheNationalInstitutesofHealthGrantR01MH080738andaWhitehall veloping sympathetic neurons, which involves the endocytic re- Foundation award (R.K.). We are grateful to D. D. Ginty, A. Riccio, B. Wendland, E. Lei, and H. Zhao for insightful moval of biochemically mature receptors from neuronal cell comments on this manuscript and to all members of the Kuruvilla, Hattar, and Zhao laboratories for helpful discus- bodies and retargeting them to axons through recycling endo- sions on this study. We thank D. D. Ginty for providing TrkA–GFP and TrkB:TrkA plasmids, R. Y. Tsien for mcherry plasmid, M. Ehlers for EGFP–Rab11a–WT and EGFP–Rab11a–S25N plasmids, and J. E. Pessin for the Dyn–K44A somes. We provide evidence that neurotrophins acting on axon adenovirus. We also thank Francis Lee for help with the FLAG antibody feeding assays and the Integrated Imaging terminals of compartmentalized cultures can recruit their Trk Center (IIC) Hopkins Biology for technical support in imaging analyses. receptors to their sites of action via transcytosis. Perturbing Correspondence should be addressed to Rejji Kuruvilla, Department of Biology, Johns Hopkins University, endocytic recycling attenuated NGF-dependent signaling and Baltimore, MD 21218. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.1542-09.2009 functional responses, whereas enhancing recycling increased Copyright © 2009 Society for Neuroscience 0270-6474/09/2911674-12$15.00/0 neuronal responsiveness to NGF. Together, our results suggest Ascan˜o et al. • Regulated Anterograde Trafficking of Trk Receptors J. Neurosci., September 16, 2009 • 29(37):11674–11685 • 11675 that this previously uncharacterized mode of TrkA trafficking immunoblotting with a TrkA antibody (Millipore Bioscience Research allows neurons to adjust their sensitivity to the limiting concen- Reagents). trations of NGF found in vivo. Surface TrkA levels. Neurons were briefly treated with a 5 min pulse of NGF (100 ng/ml) in the presence or absence of monensin (10 ␮M) at 37°C and then quickly moved to 4°C. Surface levels of TrkA were assessed by Materials and Methods cell-surface biotinylation at 4°C for 30 min, neutravidin precipitation, DNA and adenoviral constructs and TrkA immunoblotting. Enhanced green fluorescent protein (EGFP)–Rab11a–wild-type (WT) Transcytosis of TrkA receptors in compartmentalized cultures. Approxi- and EGFP–Rab11a–S25N plasmids were a gift from Dr. Michael Ehlers mately 250,000–300,000 sympathetic neurons were grown per compart- (Duke University, Durham, NC). EGFP–Rab11a–Q70L was generated mentalized culture chamber for 7–10 d in vitro (DIV) until axonal from EGFP–Rab11a–WT using site-directed mutagenesis (QuikChange; projections into the side chambers were evident. Neurons were starved of Stratagene). All EGFP–Rab11a constructs were subcloned into pshuttle– NGF for 2 d, and surface TrkA receptors in cell body compartments were cytomegalovirus (CMV) vector (Stratagene). The mcherry–Rab11a con- biotinylated with a membrane-impermeable biotin (EZ-Link NHS-S-S- struct was generated by subcloning mcherry (kindly provided by Dr. biotin) at 4°C for 25 min. After incubating the cultures at 37°C for4hin Roger Tsien, University of California San Diego, La Jolla, CA) with the presence or absence of NGF (100 ng/ml) added only to axons, lysates Rab11a construct into pshuttle–CMV vector. The N-terminal FLAG tag were harvested from cell body and axonal compartments separately and was placed on the 5Ј end of TrkB:TrkA chimeric receptor (provided by subjected to neutravidin precipitation and TrkA immunoblotting. Dr. David Ginty, Johns Hopkins University, Baltimore, MD) by PCR. TrkB:A chimeric receptor has amino acid residues 1-423 belonging to the Live-cell antibody feeding assay extracellular domain of TrkB and residues 424-809 belonging to the Trk receptor internalization. Sympathetic neurons were infected with ad- transmembrane and cytoplasmic domains of TrkA. FLAG–TrkB:A was enoviral vectors expressing GFP or FLAG–TrkB:A chimeric receptors. subcloned into pAdTrack–CMV shuttle vector (Stratagene), which also Infected neurons were identified by GFP expression. NGF was with- expresses GFP. TrkA–GFP (gift from Dr. David Ginty) and farnesylated drawn from the cultures without adding the anti-NGF antibody, and EGFP (Clontech) were cloned into pshuttle–CMV expression vectors. cells were incubated with mouse anti-FLAG antibody (M2, 4.2 ␮g/ml; Replication-incompetent recombinant adenoviral constructs were gen- Sigma) or Fab fragments derived from the FLAG antibody (10 ␮g/ml; erated for EGFP–Rab11a constructs and the FLAG–TrkB:A chimeric re- prepared according to the instructions from Pierce Chemical) for 30 min ceptor using the AdEasy adenoviral vector system (Stratagene). at 4°C in PBS. Excess antibody was washed off, and cells were moved to Recombinant viral backbones including the genes of interest were 37°C for 30 min under various conditions for internalization. Cells were transfected into HEK 293 cells (American Type Culture Collection) using then washed quickly with PBS and immediately