- Email: [email protected]
NRAGE and the cycling side of the neurotrophin receptor p75
RE S E A R C H
NEWS
NRAGE and the cycling side of the neurotrophin receptor p75 Following the discovery of nerve growth factor (NGF) in 19601, it was not long before its capacity to support the development of selective neuronal populations was demonstrated. Rapidly, NGF became considered as the founder member of a family of growth factors as other neurotrophins, BDNF, NT3 and subsequently NT4/5, were identified. However, it was not until the 1980s that the first receptor for NGF was described2. This receptor was initially thought to be specific for NGF but as other members of the neurotrophin family were described it became clear that it could bind all neurotrophins and as such it became known as the neurotrophic receptor p75 (p75NTR). The transcendence of the identification of p75NTR was somehow devalued by the lack of any obvious catalytic motif within its cytoplasmic domain. Indeed, when the Trk family of receptor tyrosine kinases was shown to specifically bind and transduce the trophic signals initiated by the neurotrophins3, p75NTR became a secondary player in studies of the neurotrophin family. A resurgence in the interest in p75NTR was sparked by the discovery of several homologs, many of which were able to induce apoptosis upon ligand binding (i.e. Fas and the TNF receptor among others). All the members of the p75NTR family contain cysteine-rich motifs in their extracellular domains, a single transmembrane domain, and many contain a motif in their intracellular sequence that is referred to as the death domain (Fig. 1). Based on these structural homologies, several laboratories have tried to find evidence that p75NTR was capable of inducing cell death. The first indication of this came to light in 1993 when the group of Bredesen4 described p75NTR-induced apoptosis, independent of neurotrophin binding in immortalized rat nigral neural cells. Such a ligand-independent effect was confirmed in a few other experimental paradigms but these studies did not rule out an autocrine source of neurotrophins as the cause of this apoptosis. Indeed, ligand-mediated apoptosis through p75NTR is now widely accepted as a common cause of physiological cell death. Since then, an increasing number of examples of p75NTR-induced cell death dependent on NGF and other neurotrophins has been shown during the past five years5–7. During this time, a tremendous effort has been made to identify the machinery involved in the apoptotic signalling of p75NTR. As a result, signalling and effector molecules like ceramide, c-Jun kinase, NF-κB, Bax, some caspases, different members of the
tumour necrosis factor receptor-associated factor (TRAF) family, and Fas-associated phosphatase-1 are all now known to participate in the apoptotic programme initiated by p75NTR (Refs 5,8). However, a more recent advance in the analysis of the apoptotic signalling of p75NTR has demonstrated that p75NTR can interact with molecules involved in the regulation of the cell cycle (Fig. 1). This raises the intriguing possibility that the apoptotic effect of p75NTR may be secondary to conflicting signals for cell division and growth arrest9. In support of this hypothesis, when young retinal neurons cultured in vitro are treated with NGF, they over-express cyclin B2 and re-enter the cell cycle before any signs of cell death are evident10. The pharmacological block of cell cycle progression abolishes this effect of NGF. A possible molecular link between the upregulation of cyclin B2 and cell death could be the tumour suppressor gene p53, which has been shown to mediate p75NTR-induced apoptosis in sympathetic neurons11. Indeed, the inappropriate activation of cell cycle regulatory molecules is known to induce the expression of p19ARF, which subsequently activates p53 and leads to apoptosis via a pathway independent of DNA damage12. The molecular links between p75NTR and the perturbations in the cell cycle are becoming clearer. As such, the zinc finger transcription factor SC-1 has been shown to interact with the intracellular domain of p75NTR. Following the binding of NGF, SC-1 is translocated to the nucleus13, an event that is associated with an absence of BrdU incorporation, although unfortunately, no effects on cell death were described in this study. Another molecule that interacts with the intracellular domain of p75NTR is the neurotrophin receptor interacting factor (NRIF), a zinc finger protein of the C2H2 type that has been shown to actively participate in cell death14. Whether or not NRIF participates in cell cycle regulation is unknown, but Zac1, a tumour suppressor gene that belongs to the same category of zinc finger proteins, can regulate apoptosis and induce cell cycle arrest15. Recently, Philip Barker’s group identified and characterized a neurotrophin-receptorinteracting MAGE homolog (NRAGE)16, a molecule that interacts with p75NTR and that is involved in the regulation of the cell cycle. This protein is a member of the melanoma antigen (MAGE) family of proteins, initially characterized as precursors of a number of cell surface antigens expressed by tumour cells. Although the normal intracellular role of most of the MAGE proteins remains
0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
Neurotrophin
p75NTR
SC-1 NRAGE
NRIF
Cell cycle perturbations
Cyclin B2
p53
Cell death
trends in Neurosciences
Fig. 1. p75NTR apoptotic pathways related to cell cycle regulation. Schematic representation of p75NTR showing the four extracellular cysteine repeats (ovals), the single transmembrane domain (small rectangle), the conserved juxtamembrane domain (black line), and the death domain (big rectangle). p75NTR binds SC-1, NRIF and NRAGE, proteins that all play roles in cell cycle regulation and apoptosis (see text). Their activation by NGF could produce cell-cycle perturbations, such as the upregulation of cyclin B2 and the re-entry into the cell cycle in postmitotic neurons. Under such conditions, the tumour suppressor gene p53, which participates in p75NTR-induced apoptosis, could induce cell death (see text). Unbroken arrows indicate experimentally demonstrated pathways. Broken arrows indicate hypothetical pathways.
unknown, Necdin17,18, a MAGE protein expressed predominantly in postmitotic neurons is able to induce growth arrest of proliferative cells, probably by interacting with the transcription factors E2F1 and p53. NRAGE is structurally homologous to Necdin and also facilitates cell cycle arrest when over-expressed in human embryonic kidney 293 cells. Within the developing CNS, NRAGE colocalizes with p75NTR in the mantle zone, a region where neurons are born, suggesting that NRAGE might participate in the mechanisms that control the arrest of growth that takes place during the genesis of neurons. Moreover, the mantle zone is a region where p75NTR dependent apoptosis has been observed during development19, raising the possibility that NRAGE could be part of the apoptotic signalling cascade. NRAGE was more directly implicated in p75NTR-dependent apoptosis in sympathoadrenal cells. These cells are induced to die in
PII: S0166-2236(00)01704-5
TINS Vol. 23, No. 12, 2000
José María Frade Instituto Cajal, CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain.
591
RESEARCH
NEWS
Acknowledgements The author is grateful to M. Sefton for grammatical corrections of the manuscript.
J
J.M. Frade – NRAGE and p75NTR
the presence of NGF only when NRAGE and p75NTR are co-expressed. The interaction of NGF with p75NTR was responsible for the translocation of NRAGE from the cytoplasm to the cell membrane. One explanation for this phenomenon would be that the diminution of the cytoplasmic pool of NRAGE induced by NGF in cells committed to withdraw from the cell cycle might favour the upregulation of cell cycle progression factors like cyclin B2 and the corresponding activation of p5312. The expression of the NGFspecific receptor TrkA abrogated the apoptotic effect mediated by NRAGE, confirming previous reports regarding the effects of the Trk receptors on the apoptotic cascade initiated by p75NTR (Refs 5,8). The novelty of Barker’s study is that this phenomenon relies, at least in part, in the competition of TrkA and NRAGE for the same p75NTR binding site. Thus, the over-expression of NRAGE blocks the physical association of p75NTR with TrkA. These new data predict an exciting new phase of research for those involved with p75NTR, focused on the interplay between apoptosis and cell cycle regulation. Important are also the consequences for our understanding of neurogenesis, that critical stage when neural cells shift from the mitotic to the postmitotic state.
Selected references 1 Levi-Montalcini, R. (1987) The nerve growth factor: thirty-five years later. EMBO J. 6, 1145−1154 2 Johnson, D. et al. (1986) Expression and structure of the human NGF receptor. Cell 47, 545−554 3 Barbacid, M. (1995) Neurotrophic factors and their receptors. Curr. Opin. Cell Biol. 7, 148−155 4 Rabizadeh, S. et al. (1993) Induction of apoptosis by the low-affinity NGF receptor. Science 261, 345−348 5 Kaplan, D.R. and Miller, F.D. (2000) Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10, 381–391 6 Bamji, S.X. et al. (1998) The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J. Cell Biol. 140, 911–923 7 Friedman, W.J. (2000) Neurotrophins induce death of hippocampal neurons via the p75 receptor. J. Neurosci. 20, 6340–6346 8 Dobrowsky, R.T. and Carter, B.D. (2000) p75 neurotrophin receptor signalling: mechanisms for neurotrophic modulation of cell stress? J. Neurosci. Res. 61, 237–243 9 O’Connor, L. et al. (2000) Apoptosis and cell division. Curr. Opin. Cell Biol. 12, 257−263 10 Frade, J.M. (2000) Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones. J. Cell Sci. 113, 1139−1148 11 Aloyz, R.S. et al. (1998) P53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors. J. Cell Biol. 143, 1691−1703
12 Sherr, C.J. (1998) Tumor surveillance via the ARF-p53 pathway. Genes Dev. 12, 2984–2991 13 Chittka, A. and Chao, M.V. (1999) Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor. Proc. Natl. Acad. Sci. U. S. A. 96, 10705−10710 14 Casademunt, E. et al. (1999) The zinc finger protein NRIF interacts with the neurotrophin receptor p75NTR and participates in programmed cell death. EMBO J. 18, 6050−6061 15 Spengler, D. et al. (1997) Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain. EMBO J. 16, 2814–2825 16 Salehi, A.H. et al. (2000) NRAGE, a novel MAGE protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis. Neuron 27, 279−288 17 Taniura, H. et al. (1998) Necdin, a postmitotic neuron-specific growth suppressor, interacts with viral transforming proteins and cellular transcription factor E2F1. J. Biol. Chem. 273, 720−728 18 Taniura, H. et al. (1999) Physical and functional interactions of neuronal growth suppressor necdin with p53. J. Biol. Chem. 274, 16242−16248 19 Frade, J.M. and Barde, Y-A. (1999) Genetic evidence for cell death mediated by nerve growth factor and the neurotrophin receptor p75 in the developing mouse retina and spinal cord. Development 126, 683−690
OURNAL CLUB Ephrin cleavage: a missing link in axon guidance The wiring up of the nervous system that occurs during development requires axons to navigate with precision to their targets. In recent years, attempts to unravel this problem have focussed on several important families of axon-guidance molecules, including the ephrins and their corresponding Eph receptors. Ephrins are membraneassociated ligands that bind to Eph receptors on adjacent cells, leading to receptor clustering and downstream signalling events. Prominent roles for the ephrins have been uncovered in axon navigation and topographic mapping onto target structures, including the wiring of retinal axons on to the optic tectum. In this and other systems, ephrins have been proposed to act via a repulsive mechanism, causing the collapse of growth cones and retraction of axons from inappropriate territory. This hypothesis contains a paradox, however: how can two membrane-associated molecules that are known to bind each other cause de-adhesion of cells and axon withdrawal? Hattori et al.1 now present a possible solution to this problem, by proposing that ephrin-mediated interactions become 592
TINS Vol. 23, No. 12, 2000
repulsive owing to cleavage of ephrins and shedding from the cell surface. In this study, ephrins were found to be cleaved from the surface of cultured cells upon treatment with clustered Eph receptors. The cleavage is likely to be initiated by the metalloprotease Kuzbanian (Kuz), which was shown to form a stable complex with ephrin-A2. Coexpression of ephrin-A2 and Kuz in cultured cells resulted in ephrin cleavage in response to exposure to Eph receptors. Conversely, expression of a dominantnegative version of Kuz inhibited the ephrin cleavage and shedding. The possible relevance of these findings in vivo was investigated by confronting hippocampal neurons with cells expressing either the normal ephrin-A2 protein, or a version containing a mutation that blocked the cleavage. In both cases, axons that contacted the ephrincontaining cells showed growth cone collapse. But in the absence of ephrin cleavage, axons were delayed in their retraction, or remained stuck to the target cell. This study therefore points to an important role for ephrin cleavage in modulating ephrin function in vivo, leading to the
retraction of axons and targeting to their correct site of termination. These functions could depend critically on the actions of metalloproteases such as Kuz, which has already been shown to play a role in axon guidance and in proteolytic processing of ligands and receptors in other contexts. As these experiments on ephrin cleavage were done in vitro, an evaluation of their implications will await more precise tests in vivo. A good place to start such studies could be the retinotectal system, as Kuz is expressed in the midbrain, which includes the optic tectum. A broader significance for the cleavage of axon guidance molecule ectodomains is already indicated by another paper in the same issue of Science. In this case, Galko and Tessier-Lavigne2 highlight a role for cleavage of the DCC receptor in modulating neuronal responses to the axon guidance molecule netrin. References 1 Hattori, M. et al. (2000) Regulated cleavage of a contact-mediated axon repellent. Science 289, 1360–1365 2 Galko, M.J. and Tessier-Lavigne, M. (2000) Function of an axonal chemoattractant modulated by metalloprotease activity Science 289, 1365–1367
Sarah Guthrie Dept of Anatomy, Guy’s Hospital, London Bridge, London, UK SE1 9RT.
NEWS
NRAGE and the cycling side of the neurotrophin receptor p75 Following the discovery of nerve growth factor (NGF) in 19601, it was not long before its capacity to support the development of selective neuronal populations was demonstrated. Rapidly, NGF became considered as the founder member of a family of growth factors as other neurotrophins, BDNF, NT3 and subsequently NT4/5, were identified. However, it was not until the 1980s that the first receptor for NGF was described2. This receptor was initially thought to be specific for NGF but as other members of the neurotrophin family were described it became clear that it could bind all neurotrophins and as such it became known as the neurotrophic receptor p75 (p75NTR). The transcendence of the identification of p75NTR was somehow devalued by the lack of any obvious catalytic motif within its cytoplasmic domain. Indeed, when the Trk family of receptor tyrosine kinases was shown to specifically bind and transduce the trophic signals initiated by the neurotrophins3, p75NTR became a secondary player in studies of the neurotrophin family. A resurgence in the interest in p75NTR was sparked by the discovery of several homologs, many of which were able to induce apoptosis upon ligand binding (i.e. Fas and the TNF receptor among others). All the members of the p75NTR family contain cysteine-rich motifs in their extracellular domains, a single transmembrane domain, and many contain a motif in their intracellular sequence that is referred to as the death domain (Fig. 1). Based on these structural homologies, several laboratories have tried to find evidence that p75NTR was capable of inducing cell death. The first indication of this came to light in 1993 when the group of Bredesen4 described p75NTR-induced apoptosis, independent of neurotrophin binding in immortalized rat nigral neural cells. Such a ligand-independent effect was confirmed in a few other experimental paradigms but these studies did not rule out an autocrine source of neurotrophins as the cause of this apoptosis. Indeed, ligand-mediated apoptosis through p75NTR is now widely accepted as a common cause of physiological cell death. Since then, an increasing number of examples of p75NTR-induced cell death dependent on NGF and other neurotrophins has been shown during the past five years5–7. During this time, a tremendous effort has been made to identify the machinery involved in the apoptotic signalling of p75NTR. As a result, signalling and effector molecules like ceramide, c-Jun kinase, NF-κB, Bax, some caspases, different members of the
tumour necrosis factor receptor-associated factor (TRAF) family, and Fas-associated phosphatase-1 are all now known to participate in the apoptotic programme initiated by p75NTR (Refs 5,8). However, a more recent advance in the analysis of the apoptotic signalling of p75NTR has demonstrated that p75NTR can interact with molecules involved in the regulation of the cell cycle (Fig. 1). This raises the intriguing possibility that the apoptotic effect of p75NTR may be secondary to conflicting signals for cell division and growth arrest9. In support of this hypothesis, when young retinal neurons cultured in vitro are treated with NGF, they over-express cyclin B2 and re-enter the cell cycle before any signs of cell death are evident10. The pharmacological block of cell cycle progression abolishes this effect of NGF. A possible molecular link between the upregulation of cyclin B2 and cell death could be the tumour suppressor gene p53, which has been shown to mediate p75NTR-induced apoptosis in sympathetic neurons11. Indeed, the inappropriate activation of cell cycle regulatory molecules is known to induce the expression of p19ARF, which subsequently activates p53 and leads to apoptosis via a pathway independent of DNA damage12. The molecular links between p75NTR and the perturbations in the cell cycle are becoming clearer. As such, the zinc finger transcription factor SC-1 has been shown to interact with the intracellular domain of p75NTR. Following the binding of NGF, SC-1 is translocated to the nucleus13, an event that is associated with an absence of BrdU incorporation, although unfortunately, no effects on cell death were described in this study. Another molecule that interacts with the intracellular domain of p75NTR is the neurotrophin receptor interacting factor (NRIF), a zinc finger protein of the C2H2 type that has been shown to actively participate in cell death14. Whether or not NRIF participates in cell cycle regulation is unknown, but Zac1, a tumour suppressor gene that belongs to the same category of zinc finger proteins, can regulate apoptosis and induce cell cycle arrest15. Recently, Philip Barker’s group identified and characterized a neurotrophin-receptorinteracting MAGE homolog (NRAGE)16, a molecule that interacts with p75NTR and that is involved in the regulation of the cell cycle. This protein is a member of the melanoma antigen (MAGE) family of proteins, initially characterized as precursors of a number of cell surface antigens expressed by tumour cells. Although the normal intracellular role of most of the MAGE proteins remains
0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
Neurotrophin
p75NTR
SC-1 NRAGE
NRIF
Cell cycle perturbations
Cyclin B2
p53
Cell death
trends in Neurosciences
Fig. 1. p75NTR apoptotic pathways related to cell cycle regulation. Schematic representation of p75NTR showing the four extracellular cysteine repeats (ovals), the single transmembrane domain (small rectangle), the conserved juxtamembrane domain (black line), and the death domain (big rectangle). p75NTR binds SC-1, NRIF and NRAGE, proteins that all play roles in cell cycle regulation and apoptosis (see text). Their activation by NGF could produce cell-cycle perturbations, such as the upregulation of cyclin B2 and the re-entry into the cell cycle in postmitotic neurons. Under such conditions, the tumour suppressor gene p53, which participates in p75NTR-induced apoptosis, could induce cell death (see text). Unbroken arrows indicate experimentally demonstrated pathways. Broken arrows indicate hypothetical pathways.
unknown, Necdin17,18, a MAGE protein expressed predominantly in postmitotic neurons is able to induce growth arrest of proliferative cells, probably by interacting with the transcription factors E2F1 and p53. NRAGE is structurally homologous to Necdin and also facilitates cell cycle arrest when over-expressed in human embryonic kidney 293 cells. Within the developing CNS, NRAGE colocalizes with p75NTR in the mantle zone, a region where neurons are born, suggesting that NRAGE might participate in the mechanisms that control the arrest of growth that takes place during the genesis of neurons. Moreover, the mantle zone is a region where p75NTR dependent apoptosis has been observed during development19, raising the possibility that NRAGE could be part of the apoptotic signalling cascade. NRAGE was more directly implicated in p75NTR-dependent apoptosis in sympathoadrenal cells. These cells are induced to die in
PII: S0166-2236(00)01704-5
TINS Vol. 23, No. 12, 2000
José María Frade Instituto Cajal, CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain.
591
RESEARCH
NEWS
Acknowledgements The author is grateful to M. Sefton for grammatical corrections of the manuscript.
J
J.M. Frade – NRAGE and p75NTR
the presence of NGF only when NRAGE and p75NTR are co-expressed. The interaction of NGF with p75NTR was responsible for the translocation of NRAGE from the cytoplasm to the cell membrane. One explanation for this phenomenon would be that the diminution of the cytoplasmic pool of NRAGE induced by NGF in cells committed to withdraw from the cell cycle might favour the upregulation of cell cycle progression factors like cyclin B2 and the corresponding activation of p5312. The expression of the NGFspecific receptor TrkA abrogated the apoptotic effect mediated by NRAGE, confirming previous reports regarding the effects of the Trk receptors on the apoptotic cascade initiated by p75NTR (Refs 5,8). The novelty of Barker’s study is that this phenomenon relies, at least in part, in the competition of TrkA and NRAGE for the same p75NTR binding site. Thus, the over-expression of NRAGE blocks the physical association of p75NTR with TrkA. These new data predict an exciting new phase of research for those involved with p75NTR, focused on the interplay between apoptosis and cell cycle regulation. Important are also the consequences for our understanding of neurogenesis, that critical stage when neural cells shift from the mitotic to the postmitotic state.
Selected references 1 Levi-Montalcini, R. (1987) The nerve growth factor: thirty-five years later. EMBO J. 6, 1145−1154 2 Johnson, D. et al. (1986) Expression and structure of the human NGF receptor. Cell 47, 545−554 3 Barbacid, M. (1995) Neurotrophic factors and their receptors. Curr. Opin. Cell Biol. 7, 148−155 4 Rabizadeh, S. et al. (1993) Induction of apoptosis by the low-affinity NGF receptor. Science 261, 345−348 5 Kaplan, D.R. and Miller, F.D. (2000) Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10, 381–391 6 Bamji, S.X. et al. (1998) The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J. Cell Biol. 140, 911–923 7 Friedman, W.J. (2000) Neurotrophins induce death of hippocampal neurons via the p75 receptor. J. Neurosci. 20, 6340–6346 8 Dobrowsky, R.T. and Carter, B.D. (2000) p75 neurotrophin receptor signalling: mechanisms for neurotrophic modulation of cell stress? J. Neurosci. Res. 61, 237–243 9 O’Connor, L. et al. (2000) Apoptosis and cell division. Curr. Opin. Cell Biol. 12, 257−263 10 Frade, J.M. (2000) Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones. J. Cell Sci. 113, 1139−1148 11 Aloyz, R.S. et al. (1998) P53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors. J. Cell Biol. 143, 1691−1703
12 Sherr, C.J. (1998) Tumor surveillance via the ARF-p53 pathway. Genes Dev. 12, 2984–2991 13 Chittka, A. and Chao, M.V. (1999) Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor. Proc. Natl. Acad. Sci. U. S. A. 96, 10705−10710 14 Casademunt, E. et al. (1999) The zinc finger protein NRIF interacts with the neurotrophin receptor p75NTR and participates in programmed cell death. EMBO J. 18, 6050−6061 15 Spengler, D. et al. (1997) Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain. EMBO J. 16, 2814–2825 16 Salehi, A.H. et al. (2000) NRAGE, a novel MAGE protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis. Neuron 27, 279−288 17 Taniura, H. et al. (1998) Necdin, a postmitotic neuron-specific growth suppressor, interacts with viral transforming proteins and cellular transcription factor E2F1. J. Biol. Chem. 273, 720−728 18 Taniura, H. et al. (1999) Physical and functional interactions of neuronal growth suppressor necdin with p53. J. Biol. Chem. 274, 16242−16248 19 Frade, J.M. and Barde, Y-A. (1999) Genetic evidence for cell death mediated by nerve growth factor and the neurotrophin receptor p75 in the developing mouse retina and spinal cord. Development 126, 683−690
OURNAL CLUB Ephrin cleavage: a missing link in axon guidance The wiring up of the nervous system that occurs during development requires axons to navigate with precision to their targets. In recent years, attempts to unravel this problem have focussed on several important families of axon-guidance molecules, including the ephrins and their corresponding Eph receptors. Ephrins are membraneassociated ligands that bind to Eph receptors on adjacent cells, leading to receptor clustering and downstream signalling events. Prominent roles for the ephrins have been uncovered in axon navigation and topographic mapping onto target structures, including the wiring of retinal axons on to the optic tectum. In this and other systems, ephrins have been proposed to act via a repulsive mechanism, causing the collapse of growth cones and retraction of axons from inappropriate territory. This hypothesis contains a paradox, however: how can two membrane-associated molecules that are known to bind each other cause de-adhesion of cells and axon withdrawal? Hattori et al.1 now present a possible solution to this problem, by proposing that ephrin-mediated interactions become 592
TINS Vol. 23, No. 12, 2000
repulsive owing to cleavage of ephrins and shedding from the cell surface. In this study, ephrins were found to be cleaved from the surface of cultured cells upon treatment with clustered Eph receptors. The cleavage is likely to be initiated by the metalloprotease Kuzbanian (Kuz), which was shown to form a stable complex with ephrin-A2. Coexpression of ephrin-A2 and Kuz in cultured cells resulted in ephrin cleavage in response to exposure to Eph receptors. Conversely, expression of a dominantnegative version of Kuz inhibited the ephrin cleavage and shedding. The possible relevance of these findings in vivo was investigated by confronting hippocampal neurons with cells expressing either the normal ephrin-A2 protein, or a version containing a mutation that blocked the cleavage. In both cases, axons that contacted the ephrincontaining cells showed growth cone collapse. But in the absence of ephrin cleavage, axons were delayed in their retraction, or remained stuck to the target cell. This study therefore points to an important role for ephrin cleavage in modulating ephrin function in vivo, leading to the
retraction of axons and targeting to their correct site of termination. These functions could depend critically on the actions of metalloproteases such as Kuz, which has already been shown to play a role in axon guidance and in proteolytic processing of ligands and receptors in other contexts. As these experiments on ephrin cleavage were done in vitro, an evaluation of their implications will await more precise tests in vivo. A good place to start such studies could be the retinotectal system, as Kuz is expressed in the midbrain, which includes the optic tectum. A broader significance for the cleavage of axon guidance molecule ectodomains is already indicated by another paper in the same issue of Science. In this case, Galko and Tessier-Lavigne2 highlight a role for cleavage of the DCC receptor in modulating neuronal responses to the axon guidance molecule netrin. References 1 Hattori, M. et al. (2000) Regulated cleavage of a contact-mediated axon repellent. Science 289, 1360–1365 2 Galko, M.J. and Tessier-Lavigne, M. (2000) Function of an axonal chemoattractant modulated by metalloprotease activity Science 289, 1365–1367
Sarah Guthrie Dept of Anatomy, Guy’s Hospital, London Bridge, London, UK SE1 9RT.