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Retrograde Transport Redux
Neuron, Vol. 39, 1–8, July 3, 2003, Copyright 2003 by Cell Press
Previews
Retrograde Transport Redux Trafficking of trophic factors in axons can occur in a retrograde and anterograde direction. Recent findings bring further support for a vesicle-based process for retrograde transport but raise new questions that need to be pursued. Unraveling the exact mechanisms that account for retrograde transport of neurotrophins and their receptors will reveal the cellular requirements for propagating trophic signals over long distances. The study of retrograde transport has continuously provided a framework for understanding the actions of neurotrophic factors. Historically, a remarkable number of discoveries have stemmed from following the fate of endocytosed NGF in neurons. Thirty years ago, Ian Hendry, Hans Thoenen, and Martin Schwab carried out experiments to follow the uptake and transport of 125INGF in the periphery. These findings have offered clues for how peripheral tissues communicate with innervating neurons. They confirmed that neurotrophins are produced and released from the target tissues and then undergo internalization and transport to the cell body, in keeping with the neurotrophic hypothesis set forth by Levi-Montalcini and Hamburger. Ironically, the presence of NGF in the CNS also came as a result of years of effort to detect retrograde transport of NGF in the brain. A number of attempts to find retrograde transport of 125I-NGF in brain initially failed in the early 1980s. The detection of NGF-responsive basal forebrain cholinergic neurons, which participate in uptake and transport of NGF from the cortex, hippocampus, and olfactory bulb, actually came from pursuing the fate of 125I-NGF in the CNS. In fact, the subsequent identification and purification of BDNF by Yves Barde was prompted by the realization at that time that very few neuronal populations in the CNS were capable of responding or transporting NGF. Hence, following the trail of NGF transport has given rise to discoveries that have made an indelible impact upon neurobiology. Decades later, interest in transport mechanisms for NGF has again hit center stage. Two papers in a recent issue of Neuron reflect the growing curiosity of how this protein gains access to the interior of the nerve terminal and winds up in the cell body, literally many miles away. In response to results reported a year ago that NGF internalization and transport are not required for a retrograde response (MacInnis and Campenot, 2002), Ginty and colleagues (Ye et al., 2003 [this issue of Neuron]) argue that NGF and its tyrosine receptor TrkA not only gain access to the internal transport machinery, but also function inside the cell. Using compartmentalized cultures, NGF introduced in the nerve terminals of sympathetic neurons was detected along with TrkA in an intact form in the cell body. Application of neutralizing NGF antibodies in the cell body by a peptide carrier resulted in apoptosis. Many years ago, NGF alone in the cell
body was found to be insufficient to provide a trophic signal. The new experiment indicates that retrogradely transported NGF in the cell body is critical for retrograde survival signaling. Concomitantly, the retrograde appearance of phosphorylated TrkA receptor in the cell body was not affected by a Trk inhibitor, K-252a, administered in the middle of the axon. Therefore, Trk kinase activity in the middle compartment is not required for retrograde survival signaling, as well as for 125I-NGF retrograde transport. Interestingly, TrkA activity appears to be used differently in separate domains of the neuron. How can we reconcile these results with those reported by MacInnis and Campenot (2002), who found that survival signals occur without uptake of NGF in distal compartments? Both utilized rat sympathetic neuron cultures, which are particularly advantageous due to their exclusive expression of TrkA receptors and reliance upon NGF for survival. The cultures from both groups are healthy and exhibit robust responses to NGF. Both based their conclusions on a similar set of techniques and reagents, including compartmentalized cultures, antibodies that recognized TrkA and its targets, the kinase inhibitor K-252a, and NGF covalently crosslinked to beads (Riccio et al., 1997; MacInnis and Campenot, 2002; Ye et al., 2003). However, there existed subtle differences in the culture conditions used by each group to maintain sympathetic neurons. For example, different serum conditions were used. Campenot’s laboratory prepares cultures containing serum only in the cell body compartment, whereas Ginty’s group uses serum in both compartments. Because of these different conditions, the extent of growth and axon arborization in compartmentalized chambers may become quite different (compare Figure 1B in Ye et al. [2003] with Figure 1 from Senger and Campenot [1997]). Exuberant growth of axons may have an impact upon the density, activity, localization, and functions of the Trk receptor or intersecting pathways, such that signaling to the cell body is differentially altered. Whether culture differences contributed to the behavior of NGF at the distal nerve terminal with its receptors has not yet been addressed, but it is possible that different sympathetic neuron culture conditions can produce divergent results. As in the case for many findings that are inconsistent, an elucidation of the reasons that account for a discrepancy may be as illuminating as the actual result itself. The biological effects of trophic factors require that signals are conveyed over long distances from the nerve terminal to the cell body. How is this accomplished? A variety of models have been put forth and extensively discussed (Ginty and Segal, 2002; MacInnis and Campenot, 2002; Miller and Kaplan, 2002). For the neurotrophins, both of their receptors, Trks and p75, undergo retrograde transport. Similarly, there is evidence in sensory neurons that the GDNF family and its binding receptors are also retrogradely transported. Many other proteins, such as TGF-, FGF, IGF, LIF, and CNTF, are similarly taken up and transported. Hence, the mechanisms that account for transport of different ligands and
Neuron 2
their receptors will likely share general features and specialized sorting signals. It has been proposed that motile vesicles containing the ligand and its receptor are delivered to the cell body, where signals are transduced to ensure neuronal survival and differentiation. The term “signaling endosome” has been coined to describe how vesicular transport engages signaling in the cell body of PC12 cells (Grimes et al., 1996). There is considerable support for this model. Endosomes serve as a meeting point for the formation of signaling complexes for a wide variety of ligands and their receptors, including TGF- and EGF. Since the formation of transport vesicles would orient the intracellular domain of the receptors to face the cytoplasm, each receptor would be available for interactions with cytoplasmic proteins and produce many signaling possibilities. This idea has been extended by Delcroix et al. (2003 [this issue of Neuron]), who provide further evidence that endosomes serve as vesicular carriers of NGF and TrkA. In this study, a new system for studying NGF transport from the target of DRG sensory neurons is used. Instead of compartmentalized cultures, a small piece of sciatic nerve was isolated in a chamber, and the retrograde movement of 125I-labeled NGF was tracked biochemically. This sciatic nerve chamber is advantageous, since it avoids local injury or trauma associated with in vivo ligation experiments. Proteins associated with NGF and its receptor could be traced to either retrograde and anterograde pools. Several notable signaling proteins—Erk1/2 and p38—could be detected with Rab5B and EEA1, signature proteins for the early endosome. The unique aspect of this study stems from the ability to follow vesicles and endosomal membranes in a directional manner in an intact nerve preparation. While endosomes are widely accepted as an organelle for signaling, several questions are raised. What is the nature of the signaling vesicles? How are early endosomes linked to the motors that drive the receptor complex during transport? What signals and proteins are linked to intracellular Trk receptors to promote survival? Previous studies indicated that NGF could be localized to multivescular bodies, and other mechanisms, such as pinocytosis, are involved in its uptake in cells. In contrast, uncoated vesicles containing TrkA and Rab5 were detected (Delcroix et al., 2003). There must be a way in which these signaling vesicles are distinguished and passed onto microtubule or actin-based motors. Aside from a link between Trk receptors and dynein subunits (Yano et al., 2001), not much is known about how neurotrophin-Trk vesicles are sorted toward transport versus a degradative or a recycling pathway. There are likely to be connections between actin-based motor and dynein-based motor machinery during the process of retrograde transport. Also, Trk receptors can be activated in the absence of neurotrophin binding, indicating that additional mechanisms can be in play (Lee and Chao, 2001). In addition to motor and signaling proteins, another overlooked component is the p75 receptor, which is internalized slower than TrkA (Bronfman et al., 2003), but nevertheless undergoes retrograde transport (Lalli and Schiavo, 2002). One property of p75 is the ability to induce an apoptotic signal. It is intriguing that proapoptotic members of the Bcl-2 family, namely Bim and
Bmf, are closely associated with the dynein motor complex, and their release to the mitochondria is dependent upon the detachment from dynein light chains (Puthalakath et al., 1999, 2001). It is therefore tempting to speculate that translocation of proteins such as p75, Bim, and Bmf or molecules downstream may be involved in mediating cell death signals from the target. Further insights into the retrograde transport process will explain how neurotrophic signals are transmitted and may account for etiologies associated with neurodegenerative diseases. Specific responses in the nucleus to neurotrophins are required for survival, axonal growth, and changes in long-term synaptic efficacy. The induction of Nef-induced activation of nuclear Factor of Activated T cells (NFAT) transcription factor activity by neurotrophins has been recently linked to axonal outgrowth and not to neuronal survival (Graef et al., 2003). This implies that a segregation of signals is made by neurons, which depends upon how trophic factor receptors are transported and localized. How the many effects of neurotrophins are dictated by the intracellular location of the ligand-receptor complexes represents a challenging problem that future studies of retrograde transport will eventually solve.
Moses V. Chao Molecular Neurobiology Program Skirball Institute of Biomolecular Medicine Department of Cell Biology and Department of Physiology and Neuroscience New York University School of Medicine 540 First Avenue New York, New York 10016 Selected Reading Bronfman, F., Tcherpakov, M., Jovin, T., and Fainzilber, M. (2003). J. Neurosci. 23, 3209–3220. Delcroix, J.-D., Valletta, J.S., Wu, C., Hunt, S.G., Kowal, A.S., and Mobley, W.C. (2003). Neuron 39, this issue, 69–84. Ginty, D., and Segal, R. (2002). Curr. Opin. Neurobiol. 12, 268–274. Graef, I., Wang, F., Charron, F., Chen, L., Neilson, J., Tessier-Lavigne, M., and Crabtree, G. (2003). Cell 113, 657–670. Grimes, M.L., Zhou, J., Beattie, E.C., Yuen, E.C., Hall, D.E., Valletta, J.S., Topp, K.S., LaVail, J.H., Bunnett, N.W., and Mobley, W.C. (1996). J. Neurosci. 16, 7950–7964. Lalli, G., and Schiavo, G. (2002). J. Cell Biol. 156, 233–239. Lee, F.S. and Chao, M.V. (2001). Proc. Natl. Acad. Sci. USA 92, 3555–3560. MacInnis, B., and Campenot, R. (2002). Science 295, 1536–1539. Miller, F., and Kaplan, D. (2002). Science 295, 1471–1473. Puthalakath, H., Huang, D.C., O’Reilly, L.A., King, S.M., and Strasser, A. (1999). Mol. Cell 3, 287–296. Puthalakath, H., Villunger, A., O’Reilly, L., Beaumont, J., Coultas, L., Cheney, R., Huang, D., and Strasser, A. (2001). Science 293, 1929–1933. Riccio, A., Pierchala, B., Ciarallo, C., and Ginty, D. (1997). Science 277, 1097–1100. Senger, D., and Campenot, R. (1997). J. Cell Biol. 138, 411–421. Yano, H., Lee, F., Kong, H., Chuang, J.-Z., Arevalo, J., Perez, P., Sung, C.-H., and Chao, M.V. (2001). J. Neurosci. 21, RC125. Ye, H., Kuruvilla, R., Zweifel, L.S., and Ginty, D.D. (2003). Neuron 39, this issue, 57–68.
Previews
Retrograde Transport Redux Trafficking of trophic factors in axons can occur in a retrograde and anterograde direction. Recent findings bring further support for a vesicle-based process for retrograde transport but raise new questions that need to be pursued. Unraveling the exact mechanisms that account for retrograde transport of neurotrophins and their receptors will reveal the cellular requirements for propagating trophic signals over long distances. The study of retrograde transport has continuously provided a framework for understanding the actions of neurotrophic factors. Historically, a remarkable number of discoveries have stemmed from following the fate of endocytosed NGF in neurons. Thirty years ago, Ian Hendry, Hans Thoenen, and Martin Schwab carried out experiments to follow the uptake and transport of 125INGF in the periphery. These findings have offered clues for how peripheral tissues communicate with innervating neurons. They confirmed that neurotrophins are produced and released from the target tissues and then undergo internalization and transport to the cell body, in keeping with the neurotrophic hypothesis set forth by Levi-Montalcini and Hamburger. Ironically, the presence of NGF in the CNS also came as a result of years of effort to detect retrograde transport of NGF in the brain. A number of attempts to find retrograde transport of 125I-NGF in brain initially failed in the early 1980s. The detection of NGF-responsive basal forebrain cholinergic neurons, which participate in uptake and transport of NGF from the cortex, hippocampus, and olfactory bulb, actually came from pursuing the fate of 125I-NGF in the CNS. In fact, the subsequent identification and purification of BDNF by Yves Barde was prompted by the realization at that time that very few neuronal populations in the CNS were capable of responding or transporting NGF. Hence, following the trail of NGF transport has given rise to discoveries that have made an indelible impact upon neurobiology. Decades later, interest in transport mechanisms for NGF has again hit center stage. Two papers in a recent issue of Neuron reflect the growing curiosity of how this protein gains access to the interior of the nerve terminal and winds up in the cell body, literally many miles away. In response to results reported a year ago that NGF internalization and transport are not required for a retrograde response (MacInnis and Campenot, 2002), Ginty and colleagues (Ye et al., 2003 [this issue of Neuron]) argue that NGF and its tyrosine receptor TrkA not only gain access to the internal transport machinery, but also function inside the cell. Using compartmentalized cultures, NGF introduced in the nerve terminals of sympathetic neurons was detected along with TrkA in an intact form in the cell body. Application of neutralizing NGF antibodies in the cell body by a peptide carrier resulted in apoptosis. Many years ago, NGF alone in the cell
body was found to be insufficient to provide a trophic signal. The new experiment indicates that retrogradely transported NGF in the cell body is critical for retrograde survival signaling. Concomitantly, the retrograde appearance of phosphorylated TrkA receptor in the cell body was not affected by a Trk inhibitor, K-252a, administered in the middle of the axon. Therefore, Trk kinase activity in the middle compartment is not required for retrograde survival signaling, as well as for 125I-NGF retrograde transport. Interestingly, TrkA activity appears to be used differently in separate domains of the neuron. How can we reconcile these results with those reported by MacInnis and Campenot (2002), who found that survival signals occur without uptake of NGF in distal compartments? Both utilized rat sympathetic neuron cultures, which are particularly advantageous due to their exclusive expression of TrkA receptors and reliance upon NGF for survival. The cultures from both groups are healthy and exhibit robust responses to NGF. Both based their conclusions on a similar set of techniques and reagents, including compartmentalized cultures, antibodies that recognized TrkA and its targets, the kinase inhibitor K-252a, and NGF covalently crosslinked to beads (Riccio et al., 1997; MacInnis and Campenot, 2002; Ye et al., 2003). However, there existed subtle differences in the culture conditions used by each group to maintain sympathetic neurons. For example, different serum conditions were used. Campenot’s laboratory prepares cultures containing serum only in the cell body compartment, whereas Ginty’s group uses serum in both compartments. Because of these different conditions, the extent of growth and axon arborization in compartmentalized chambers may become quite different (compare Figure 1B in Ye et al. [2003] with Figure 1 from Senger and Campenot [1997]). Exuberant growth of axons may have an impact upon the density, activity, localization, and functions of the Trk receptor or intersecting pathways, such that signaling to the cell body is differentially altered. Whether culture differences contributed to the behavior of NGF at the distal nerve terminal with its receptors has not yet been addressed, but it is possible that different sympathetic neuron culture conditions can produce divergent results. As in the case for many findings that are inconsistent, an elucidation of the reasons that account for a discrepancy may be as illuminating as the actual result itself. The biological effects of trophic factors require that signals are conveyed over long distances from the nerve terminal to the cell body. How is this accomplished? A variety of models have been put forth and extensively discussed (Ginty and Segal, 2002; MacInnis and Campenot, 2002; Miller and Kaplan, 2002). For the neurotrophins, both of their receptors, Trks and p75, undergo retrograde transport. Similarly, there is evidence in sensory neurons that the GDNF family and its binding receptors are also retrogradely transported. Many other proteins, such as TGF-, FGF, IGF, LIF, and CNTF, are similarly taken up and transported. Hence, the mechanisms that account for transport of different ligands and
Neuron 2
their receptors will likely share general features and specialized sorting signals. It has been proposed that motile vesicles containing the ligand and its receptor are delivered to the cell body, where signals are transduced to ensure neuronal survival and differentiation. The term “signaling endosome” has been coined to describe how vesicular transport engages signaling in the cell body of PC12 cells (Grimes et al., 1996). There is considerable support for this model. Endosomes serve as a meeting point for the formation of signaling complexes for a wide variety of ligands and their receptors, including TGF- and EGF. Since the formation of transport vesicles would orient the intracellular domain of the receptors to face the cytoplasm, each receptor would be available for interactions with cytoplasmic proteins and produce many signaling possibilities. This idea has been extended by Delcroix et al. (2003 [this issue of Neuron]), who provide further evidence that endosomes serve as vesicular carriers of NGF and TrkA. In this study, a new system for studying NGF transport from the target of DRG sensory neurons is used. Instead of compartmentalized cultures, a small piece of sciatic nerve was isolated in a chamber, and the retrograde movement of 125I-labeled NGF was tracked biochemically. This sciatic nerve chamber is advantageous, since it avoids local injury or trauma associated with in vivo ligation experiments. Proteins associated with NGF and its receptor could be traced to either retrograde and anterograde pools. Several notable signaling proteins—Erk1/2 and p38—could be detected with Rab5B and EEA1, signature proteins for the early endosome. The unique aspect of this study stems from the ability to follow vesicles and endosomal membranes in a directional manner in an intact nerve preparation. While endosomes are widely accepted as an organelle for signaling, several questions are raised. What is the nature of the signaling vesicles? How are early endosomes linked to the motors that drive the receptor complex during transport? What signals and proteins are linked to intracellular Trk receptors to promote survival? Previous studies indicated that NGF could be localized to multivescular bodies, and other mechanisms, such as pinocytosis, are involved in its uptake in cells. In contrast, uncoated vesicles containing TrkA and Rab5 were detected (Delcroix et al., 2003). There must be a way in which these signaling vesicles are distinguished and passed onto microtubule or actin-based motors. Aside from a link between Trk receptors and dynein subunits (Yano et al., 2001), not much is known about how neurotrophin-Trk vesicles are sorted toward transport versus a degradative or a recycling pathway. There are likely to be connections between actin-based motor and dynein-based motor machinery during the process of retrograde transport. Also, Trk receptors can be activated in the absence of neurotrophin binding, indicating that additional mechanisms can be in play (Lee and Chao, 2001). In addition to motor and signaling proteins, another overlooked component is the p75 receptor, which is internalized slower than TrkA (Bronfman et al., 2003), but nevertheless undergoes retrograde transport (Lalli and Schiavo, 2002). One property of p75 is the ability to induce an apoptotic signal. It is intriguing that proapoptotic members of the Bcl-2 family, namely Bim and
Bmf, are closely associated with the dynein motor complex, and their release to the mitochondria is dependent upon the detachment from dynein light chains (Puthalakath et al., 1999, 2001). It is therefore tempting to speculate that translocation of proteins such as p75, Bim, and Bmf or molecules downstream may be involved in mediating cell death signals from the target. Further insights into the retrograde transport process will explain how neurotrophic signals are transmitted and may account for etiologies associated with neurodegenerative diseases. Specific responses in the nucleus to neurotrophins are required for survival, axonal growth, and changes in long-term synaptic efficacy. The induction of Nef-induced activation of nuclear Factor of Activated T cells (NFAT) transcription factor activity by neurotrophins has been recently linked to axonal outgrowth and not to neuronal survival (Graef et al., 2003). This implies that a segregation of signals is made by neurons, which depends upon how trophic factor receptors are transported and localized. How the many effects of neurotrophins are dictated by the intracellular location of the ligand-receptor complexes represents a challenging problem that future studies of retrograde transport will eventually solve.
Moses V. Chao Molecular Neurobiology Program Skirball Institute of Biomolecular Medicine Department of Cell Biology and Department of Physiology and Neuroscience New York University School of Medicine 540 First Avenue New York, New York 10016 Selected Reading Bronfman, F., Tcherpakov, M., Jovin, T., and Fainzilber, M. (2003). J. Neurosci. 23, 3209–3220. Delcroix, J.-D., Valletta, J.S., Wu, C., Hunt, S.G., Kowal, A.S., and Mobley, W.C. (2003). Neuron 39, this issue, 69–84. Ginty, D., and Segal, R. (2002). Curr. Opin. Neurobiol. 12, 268–274. Graef, I., Wang, F., Charron, F., Chen, L., Neilson, J., Tessier-Lavigne, M., and Crabtree, G. (2003). Cell 113, 657–670. Grimes, M.L., Zhou, J., Beattie, E.C., Yuen, E.C., Hall, D.E., Valletta, J.S., Topp, K.S., LaVail, J.H., Bunnett, N.W., and Mobley, W.C. (1996). J. Neurosci. 16, 7950–7964. Lalli, G., and Schiavo, G. (2002). J. Cell Biol. 156, 233–239. Lee, F.S. and Chao, M.V. (2001). Proc. Natl. Acad. Sci. USA 92, 3555–3560. MacInnis, B., and Campenot, R. (2002). Science 295, 1536–1539. Miller, F., and Kaplan, D. (2002). Science 295, 1471–1473. Puthalakath, H., Huang, D.C., O’Reilly, L.A., King, S.M., and Strasser, A. (1999). Mol. Cell 3, 287–296. Puthalakath, H., Villunger, A., O’Reilly, L., Beaumont, J., Coultas, L., Cheney, R., Huang, D., and Strasser, A. (2001). Science 293, 1929–1933. Riccio, A., Pierchala, B., Ciarallo, C., and Ginty, D. (1997). Science 277, 1097–1100. Senger, D., and Campenot, R. (1997). J. Cell Biol. 138, 411–421. Yano, H., Lee, F., Kong, H., Chuang, J.-Z., Arevalo, J., Perez, P., Sung, C.-H., and Chao, M.V. (2001). J. Neurosci. 21, RC125. Ye, H., Kuruvilla, R., Zweifel, L.S., and Ginty, D.D. (2003). Neuron 39, this issue, 57–68.