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Neurotrophin receptors: mediators of life and death1
Brain Research Reviews 26 Ž1998. 295–301
Neurotrophin receptors: mediators of life and death
1
Moses Chao ) , Patrizia Casaccia-Bonnefil, Bruce Carter, Alexandra Chittka, Haeyoung Kong, Sung Ok Yoon Department of Cell Biology and Anatomy, Cornell UniÕersity Medical College, 1300 York AÕenue, New York, NY 10021, USA
Abstract The mechanism of action of NGF has continued to provide a challenging and formidable problem in signal transduction. NGF can bind independently to two different receptors, the trkA tyrosine kinase receptor and the p75 neurotrophin receptor, which are involved in many different signaling events. In addition to promoting cell differentiation survival, NGF can paradoxically be an inducer of cell death. Several receptor mediated mechanisms are proposed to explain how NGF might act as a trophic factor and as a cell killer. The survival and cell death properties of the receptors are dependent upon the relative ratio of receptors and the persistent nature of the signaling events. q 1998 Elsevier Science B.V. All rights reserved. Keywords: NGF; Neurotrophic; Signal transduction; Death; Receptor
Contents 1. Introduction .
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2. Models of receptor action .
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3. Cell death signaling by neurotrophins 4. Conclusions . References
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1. Introduction The decision of a cell to undergo cell division as opposed to terminal differentiation and cell suicide is dependent upon a delicate balance between many opposing biochemical interactions. On one level, these decisions are dictated by gene regulatory events, which determine the levels of trophic factors and their receptors; essential enzymatic functions, such as phosphatidylinositol 3-kinase and )
Corresponding author. Fax: q1 Ž212. 746-8961; E-mail: [email protected] 1 Published on the World Wide Web on 21 October 1997. 0165-0173r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 1 7 3 Ž 9 7 . 0 0 0 3 6 - 2
MAP kinases; and mitochondrial proteins, such as Bcl-2 family members, which dictate susceptibility to cell death ŽFig. 1.. Protein–protein interactions together with protein phosphorylation serve as the primary regulatory mechanisms for determining cell survival. Cellular responses are dependent upon the strength of the binding interactions from ligand–receptor interactions, transcription factor binding to target DNA sequences, and essential enzymatic complexes, such as cyclinrCDK proteins. The development of the vertebrate nervous system is characterized by considerable programmed cell death. Neurotrophic factors, such as NGF, are synthesized by target tissues and support the survival of afferent neurons
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Fig. 1. A balance of signaling pathways determines the propensity of a cell to undergo cell suicide or survival. Extracellular signals act to initiate these enzymatic activities ŽNFk B, PI3 kinase, MAP kinases, JNK kinases, caspases. or second messengers Žceramide. or Bcl-2 Žced-9. and other family members depending upon the stage of cell commitment.
and promote axonal and dendritic branching. Competition among neurons for limiting amounts of neurotrophin molecules produced by target cells is a principal mechanism for accounting for cell survival during development. An important prediction from this hypothesis is that the efficacy of neurotrophic action will depend upon the concentrations of trophic factors and upon the binding requirements of receptors expressed in responsive cell populations. This article will focus upon how NGF receptors interact to produce alternative signaling outcomes that influence cell viability. The trkA, trkB, and trkC tyrosine kinases serve as the receptors for NGF, BDNF, and NT-3, respectively, and trkA and trkB may also function as receptors for NT-3 and NT-4r5 w1x. In addition, the p75 receptor can serve as a receptor for NGF, BDNF, NT-3 and NT-4, although it exhibits slightly different binding characteristics for each factor. The p75 receptor is the founding member of the TNF superfamily w2x. For NGF responsive neuronal populations, p75 and trkA are frequently co-expressed, while p75 is expressed widely during development in non-neuronal cells and also following nerve injury or trauma. The assignment of p75 functionally as well as structurally to the TNF family is apt, since many receptors in this family serve to modulate the effects of cytokines following inflammation, infection or injury.
2. Models of receptor action Trophic factors exemplified by NGF and its family members, ciliary neurotrophic factor ŽCNTF. and glial derived neurotrophic factor ŽGDNF. all utilize increased tyrosine phosphorylation of cellular substrates to mediate neuronal cell survival ŽFig. 2.. CNTF acts through an a CNTF receptor component, gp130 and LIF subunits, which are linked to the JAKrSTAT signaling molecules w3x, whereas the GDNF receptor complex consists of the c-ret receptor tyrosine kinase and a separate a-subunit w4x.
Actions of the NGF family of neurotrophins are dictated by ras activation through the trk family of receptor tyrosine kinases. Another survival pathway has been defined by phosphatidylinositol-3-kinase w5x, which gives rise to phosphoinositide intermediates that activate Akt w6x. Induction of the serine–threonine kinase Akt is critical for cell survival, as well as cell proliferation. Hence, for many trophic factors, multiple subunits constitute a functional transmembrane complex which activates ras-dependent and ras-independent intracellular signaling. A number of common substrates, including phospholipase C-g , phosphatidylinositol-3-kinase ŽPI-3 kinase. and SHC and Grb2 adaptor proteins are utilized by many tyrosine kinase receptors. This raises the question of how these phosphorylation events lead to different biological outcomes. There are several possibilities. First, the strength and duration of the receptor autophosphorylation events may determine downstream signaling. Second, differential signaling may be controlled by specific dephosphorylation events. Third, there may be unique second messengers or substrates which determine the specific nature of the response. For neurotrophic factors, the duration of signaling has provided an important insight into the differential signaling. In PC12 cells, NGF induces a prolonged activation of ras and MAP kinase activity, lasting for several hours, while EGF-mediated MAP kinase activation is transient in nature w7x. The time course of downstream signaling is therefore one of the major differences that accounts for the differentiation program elicited by NGF versus the action of other mitogenic growth factors, such as EGF. However, the strength of receptor action alone is not sufficient, since the ligand-dependent autophosphorylation of the EGF receptor is actually higher than for the trkA NGF receptor in PC12 cells w8x, so that the level of receptor activation cannot solely account for the difference in trophic factor action. Other novel receptor substrates, such as docking or adaptor proteins w9x, may be recruited by a subset of tyrosine kinase receptors. These interactions appear to provide additional specificity. The p75 receptor, when co-expressed with trk, provides a positive modulatory influence upon trk function w10,11x. In neuronal cell lines that express both trkA and p75 receptors, trkA autophosphorylation is increased, leading to a faster differentiative response, as assayed by more rapid growth arrest and neuronal maturation w11x. In addition, binding studies with 125 I-NGF indicated that p75 increased the number of high affinity sites by increasing the on-rate for trkA. The requirement for trkA and p75 co-expression for high affinity site formation provides an explanation for how the receptors cooperate to increase neurotrophin responsiveness during cell survival and programmed cell death. On the other hand, each receptor can also signal independently, implying that the actions of neurotrophins depend upon whether trk alone, trk plus p75, or p75 alone is expressed.
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Fig. 2. Examples of multisubunit receptors with actions in the nervous system. They include, from left to right, the NGF receptors, p75 and trkA w1,13x; the FGF receptor and proteoglycan w14x; Glial Derived Neurotrophic Factor ŽGDNF. binds to a GPI-anchored a-GNDF receptor and the ret tyrosine kinase receptor w4x; and the CNTF receptor complex, which consists of an a GPI-anchored protein, a , together with gp130 and the LIF receptor subunits w3x. The trk receptors are distinguished by an extracellular domain with multiple repeats of leucine-rich domains and IgG-related domains, and a cytoplasmic region distinguished by a catalytic tyrosine kinase domain, a small insert, and a short cytoplasmic tail. The p75 receptor has a distinctive extracellular domain sequence different from trkA.
Cell survival is enhanced by a high ratio of p75 to trk receptors. Primary culture experiments using sensory neurons from mice carrying a homozygous mutation in the p75 gene indicated that nearly four-fold more NGF was necessary to achieve comparable levels of survival as wild type neurons w12x. Sensory neurons are fully capable of signaling through the trkA receptor in the absence of p75, but with a lower sensitivity. The increased dose requirement for NGF is observed in specific neuronal populations at times when high levels of p75 were expressed. Correspondingly, sympathetic neurons from neonatal p75-null mice require higher concentrations of NGF to survive than neurons from normal mice at earlier developmental stages. A high ratio of p75 to trk is correlated with increased cell survival w13x. Thus, the responsiveness to NGF and the ability to form high affinity sites is dependent upon relative levels of p75 and trk. A favored model of receptors with auxiliary subunits is ligand presentation by the accessory subunit to the tyrosine kinase receptor. This model is applicable to the FGF receptor which utilizes a lower affinity proteoglycan molecule ŽFig. 2. to facilitate high affinity site formation w14x. This mechanism is also applicable to neurotrophin receptors, in which p75 may bind NGF rapidly and facilitates the interaction of NGF with trkA by increasing the local concentration of the NGF ligand. Indeed, co-expression of p75 with trkA receptors increases the binding affinities for NGF w15x. Also, a decrease in p75 levels or inhibition of NGF binding to p75 leads to a reduction of both high and low affinity NGF binding sites w16x. However, recent experiments indicate the presentation model is
not sufficient to explain how p75 can increase trk signaling ŽB.L. Hempstead, unpublished results.. A second mechanism that explains how two receptor components can give rise to a higher affinity is that a conformational change occurs between trk and p75 receptors that gives a more favored binding interaction. Such a conformation change could occur in the absence of ligand. Coexpression of p75 may result in a conformational change in trk, leading to an accelerated rate of association, which leads to a higher affinity site. This model would be compatible with much of the existing data, including biophysical measurements of fluorescence recovery after photobleaching which indicate that trkA immobilizes p75 molecules in the absence of NGF w17x. Furthermore, an association of p75 and trk receptors requires an intact cytoplasmic domain of p75, as well as a kinase-active trk receptor, again suggestive of the importance of conformation. Copatching of the receptors is observed using fluorescent antibodies w18x. Since interactions between p75 or p140 trk can be detected after immunoprecipitation w19x, p75 and trk receptors probably exist in membrane complexes consisting of other signaling, cytoskeletal or adaptor proteins, which may influence the conformational states of the receptors. An important role of p75 is to provide more specificity to the interactions of neurotrophins with trk family members. One interpretation of these results, in terms of a conformational model, is that a high level of p75 may result in a conformational change in the trk molecule which enhances trkA–NGF interactions. A prediction from these observations is that trk family members may respond
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much more selectively to neurotrophins when the p75 neurotrophin receptor is co-expressed.
3. Cell death signaling by neurotrophins A remarkable finding in the past year is that NGF can initiate apoptotic cell death. NGF treatment of medulloblastoma cells expressing transfected trkA receptors resulted in cell death that was directly inhibited by anti-NGF antibodies w20x. Furthermore, this action was only dependent upon trkA autophosphorylation. Although the reason that accounts for this killing activity through trkA is not known, one explanation is that an alteration in cell cycle control subverted medulloblastoma cells to this fate after NGF treatment. The similarity in the intracellular domain of the p75 receptor with other family members, such as the Fas antigen and the p55 TNF receptor w21x suggests that p75 might also function as a cell death molecule. The first report of such an activity was made in immortalized cerebellar neuronal cell lines, which undergo a faster rate of cell death after transfection of p75 w22x. Binding of NGF and agonist antibodies rescued cells from apoptotic cell death. Moreover, sensory neuronal survival experiments indicated that expression of p75 is detrimental for cell survival, particularly during the postnatal periods w23x. In contrast, earlier in development, sensory neurons actually depend upon co-expression of trk and p75 receptors w23x. These results indicated that p75 can signal through a death pathway, similar to its family members, Fas and TNF receptors but that the receptor was also capable of supporting cell survival. Other examples supporting a cell death function of p75 have been documented ŽTable 1.. The number of dying cholinergic neurons in the basal forebrain is higher in wild type animals compared to p75-deficient animals, suggesting that the expression of p75 was detrimental to viability in this cell population. This property was traced to cells that expressed p75, but were deficient in trkA w24x. Also, differentiated PC12 cells undergoing NGF deprivation seem to die more rapidly in cells expressing high levels of p75. These combined studies have suggested that p75 promotes cell death in the absence of NGF binding. Table 1 Examples of neurotrophin-related apoptosis Cell type
Ref.
Immortalized neural cell lines Postnatal sensory neurons Chick retina precursors Isthmo-optic nucleus neurons Basal forebrain cholinergic neurons PC12 cells after NGF withdrawal Mature oligodendrocytes Medulloblastoma
w22x w23x w25x w38x w24x w37x w26x w20x
Fig. 3. NGF causes apoptosis in fully differentiated oligodendrocytes. DNA fragmentation was measured after 4 h treatment of oligodendrocytes isolated from P1 cerebral cortex as described w26x. Cells were treated with 100 ngrml of NGF Žq. or left untreated Žy. in oligodendrocyte differentiation media, including 50 m grml transferrin, 5 m grml putrescine, 3 ngrml progesterone, 2.6 ngrml selenium, 12.5 m grml insulin, 0.4 m grml throxine, 0.3% glucose and 6 mM glutamine.
In contrast to a ligand-independent mechanism, cell death induced by binding of NGF to p75 also has been observed. A clear example of the in vivo consequences of p75-mediated cell death was detected in developing chick retina w25x. Neuronal precursor cells at embryonic day 5 underwent apoptotic cell death, which could be effectively blocked by antibodies to either p75 or NGF. This indicated that endogenous NGF caused the death of retinal neurons at an early developmental age. Similarly, NGF treatment of fully differentiated oligodendrocytes resulted in cell death through binding to p75 receptors w26x. Apoptosis was detected by fluorescent TUNEL labeling and increased DNA fragmentation ŽFig. 3.. Interestingly, the effects of NGF on oligodendrocyte cultures were only observed during terminal differentiation and required high concentrations Ž100 ngrml. and could not be reproduced by similar concentrations of BDNF or NT-3. Progenitor oligodendrocytes ŽO-2A. did not express p75 and were not susceptible to death by neurotrophins. Instead, these cells can also use neurotrophins as trophic factors w27x. Although the majority of oligodendrocytes do not express p75 in vivo in physiological conditions, the oligodendrocyte cultures have provided an experimental system to study signal transduction events following NGF treatment. Glial cells are highly susceptible to injury and inflammation. During traumatic lesion, oligodendroglial cells become more reactive to growth factors and cytokines which are released at the site of injury. Recent evidence indicates that p75-positive oligodendrocytes can be detected in white matter plaques from cases of multiple sclerosis ŽP. Dowling, personal communication.. Also, cultured oligodendro-
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cytes have been found to express trk receptors w27x in the presence of mitogens. In the absence of trkA expression, binding of NGF to p75 receptors can increase intracellular ceramide levels through increased sphingomyelin hydrolysis w28x. When the levels of intracellular ceramide were measured in differentiated oligodendrocytes only expressing p75 receptors, NGF led to an elevation of ceramide levels that was sustained for a period of several hours, instead of minutes. This time course is reminiscent of the activation of ras and MAP kinase activities through trkA. A sustained vs. transient duration in signaling may determine which pathway is dominant. Interestingly, other neurotrophins such as BDNF and NT-3, which also bind to p75, did not induce long-term ceramide production in mature oligodendrocytes. Moreover, cell death in oligodendrocytes through increased intracellular ceramide levels could also be mimicked by the application of exogenous ceramide analogs or bacterial sphingomyelinase or by inhibitors of the glucosylceramide synthase. These data suggest that the high sustained ceramide level elicited by the p75 receptor may contribute to NGF-dependent death. Another criteria for the dose-dependent cell death of primary cortical oligodendrocytes is the activation of c-jun amino-terminal kinase ŽJNK.. The JNK enzymatic activity, a downstream effector of the stress-activated protein kinase family, has been implicated in cell death progression in PC12 cells. In addition, ceramide is a potential activator of JNK. Significantly, incubation of mature oligodendro-
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cytes with 100 ngrml NGF for three hours led to a 3-fold induction of JNK activation. The activation of JNK activity by NGF was similar in magnitude to the effects by ceramide and was not observed in cells whose viability was not affected by NGF, such as NIH3T3 fibroblasts expressing p75. The stress-activated protein kinase, JNK, can therefore be regulated by p75 in oligodendrocytes. When both NGF receptors are co-expressed as in the case of PC12 cells, trkA exerts a suppressive effect upon p75, as assayed by ceramide production w29x. In support of this model ŽFig. 4., introduction of trkA receptors into oligodendrocytes results in a reduction of JNK but leaves NFk B activation through p75 unaffected. While JNK activities are suppressed by co-expression of trkA, MAP kinase activity is increased by NGF binding to trkA ŽYoon, unpublished results.. From these experiments, trkA signaling can selectively down-regulate certain responses from p75. Although p75 can modulate high affinity binding by NGF and trkA signaling, p75 can also display independent signaling properties through ceramide production w28,29x and increased NFk B activity w30x. The activation of JNK by NGF binding to the p75 receptor and the sustained level of ceramide has only been observed in limited cases, such as end-stage oligodendrocytes. Strikingly, other cell types which express p75, such as 3T3 cells, respond to all neurotrophins with a transient increase in ceramide levels w29x, but do not undergo cell death after neurotrophin treatment. Indeed, Schwann cells and melanoma cells
Fig. 4. Two different mechanisms to account for potential interactions between p75 and trk family members. Co-expression of p75 with trkA results in an increase in the affinity of NGF binding and enhance the autophosphorylation and signaling functions of the trkA receptor w10,11,13x. Expression of the trkA receptor suppresses the signal transduction abilities of p75, including ceramide production w29x and cell death ŽYoon, unpublished..
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which express high levels of p75 do not undergo neurotrophin-dependent cell death. These data indicate that p75 produces a dichotomy of effects, some of which are extremely cell specific. Thus, neurotrophin treatment of p75-3T3 cells resulted in a transient elevation of ceramide levels, but a persistent, long-lasting increase in ceramide and JNK activity levels was observed in mature oligodendrocytes. Similar to findings in Schwann cells, signaling by p75 appears to be exclusive to NGF, as BDNF and NT-3 are ineffective w30x. These differences in ligand specificity and time courses may result in separate signaling cascades and different biological responses, depending on the cell type and differentiative stage. A critical requirement for the death pathway is the terminal nature of cell differentiation.
4. Conclusions The NGF receptors provide a clear example of bidirectional crosstalk ŽFig. 4.. NGF binding to p75 can result in independent signaling in the absence of trkA expression. These activities include the induction of NFk B w30x; the hydrolysis of sphingomyelin to ceramide w29x; and the pro-apoptotic functions attributed to p75. An important finding is that trk tyrosine kinase action appears to negate the signaling properties of p75, particularly with respect to ceramide production w29x and programmed cell death activities. Neurotrophin receptors are generally drawn and viewed as isolated integral membrane proteins which span the lipid bilayer, with signal transduction proceeding in a linear step-wise fashion. There are now numerous examples which indicate that each receptor acts not only in a linear, independent manner, but can also influence the activity of other cell surface receptors, either directly or through signaling intermediates. Which step and which intermediates are being utilized for crosstalk between the receptors is an on-going question. Biologically, the cooperative actions of two receptors imply that the possibilities for extracellular signaling are greatly expanded. Cell differentiation is determined by biochemical reactions representing the combined effects of many growth factors and cytokines. This diversity may reflect inherent differences in receptor structure and substrate specificities. Regulation of cellular differentiation and proliferation decisions is likely to be determined by the additive effects of multiple receptors and the duration of second messengers and phosphorylation events. Unlike studies carried out in vitro where cell lines are treated with single factors, growth and survival of cells in vivo are subject to the simultaneous action of multiple polypeptide growth factors. There are several explanations for the cell death ability of neurotrophins. Since the majority of cases involve elevated levels of p75 receptor expression, the responses may
involve signaling intermediates used by TNF and Fas receptors. This includes adaptor proteins FADD and TRADD and downstream effectors, such as caspase-8, the FLICErMACH interacting enzyme w31x. It remains to be seen whether p75 overexpression might co-op the use of these proteins, or other interacting proteins of different specificity. Also, a new family of cytokine associated signaling intermediates, the TNF-associated factors ŽTRAFs., have been described. Originally discovered as molecules associated with the p75 TNF receptor w32x, it is now apparent the TRAF proteins represent a large family of molecules, which carry potential signaling capabilities through NFk B activation. Moreover, these molecules have been found to be associated with receptors required for different biological activities. These receptors include CD30, CD40, and lymphotoxin b w33–35x and even the p55 TNF receptor w36x. It seems likely that similar TRAF proteins will be utilized by p75, since neurotrophins share similar NFk B responses as IL-1 and the TNF family. The goals will be to define the mechanisms which dictate the action of macromolecular receptor complexes during transmembrane signaling. This will likely address the long standing question of how cells make decisions determining growth vs. differentiation and death. The NGF receptor system has provided a remarkable mechanism for controlling a wide gamut of cellular activities, including synaptic plasticity and cell suicide. Defining which proteins interact with each receptor will provide insights into the mechanisms responsible for regulating cell growth, survival, cell differentiation and cell death. It would not be surprising that the same molecules will be found to participate in key decision-making events that lead to these diverse biological outcomes.
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Neurotrophin receptors: mediators of life and death
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Moses Chao ) , Patrizia Casaccia-Bonnefil, Bruce Carter, Alexandra Chittka, Haeyoung Kong, Sung Ok Yoon Department of Cell Biology and Anatomy, Cornell UniÕersity Medical College, 1300 York AÕenue, New York, NY 10021, USA
Abstract The mechanism of action of NGF has continued to provide a challenging and formidable problem in signal transduction. NGF can bind independently to two different receptors, the trkA tyrosine kinase receptor and the p75 neurotrophin receptor, which are involved in many different signaling events. In addition to promoting cell differentiation survival, NGF can paradoxically be an inducer of cell death. Several receptor mediated mechanisms are proposed to explain how NGF might act as a trophic factor and as a cell killer. The survival and cell death properties of the receptors are dependent upon the relative ratio of receptors and the persistent nature of the signaling events. q 1998 Elsevier Science B.V. All rights reserved. Keywords: NGF; Neurotrophic; Signal transduction; Death; Receptor
Contents 1. Introduction .
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2. Models of receptor action .
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3. Cell death signaling by neurotrophins 4. Conclusions . References
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1. Introduction The decision of a cell to undergo cell division as opposed to terminal differentiation and cell suicide is dependent upon a delicate balance between many opposing biochemical interactions. On one level, these decisions are dictated by gene regulatory events, which determine the levels of trophic factors and their receptors; essential enzymatic functions, such as phosphatidylinositol 3-kinase and )
Corresponding author. Fax: q1 Ž212. 746-8961; E-mail: [email protected] 1 Published on the World Wide Web on 21 October 1997. 0165-0173r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 1 7 3 Ž 9 7 . 0 0 0 3 6 - 2
MAP kinases; and mitochondrial proteins, such as Bcl-2 family members, which dictate susceptibility to cell death ŽFig. 1.. Protein–protein interactions together with protein phosphorylation serve as the primary regulatory mechanisms for determining cell survival. Cellular responses are dependent upon the strength of the binding interactions from ligand–receptor interactions, transcription factor binding to target DNA sequences, and essential enzymatic complexes, such as cyclinrCDK proteins. The development of the vertebrate nervous system is characterized by considerable programmed cell death. Neurotrophic factors, such as NGF, are synthesized by target tissues and support the survival of afferent neurons
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Fig. 1. A balance of signaling pathways determines the propensity of a cell to undergo cell suicide or survival. Extracellular signals act to initiate these enzymatic activities ŽNFk B, PI3 kinase, MAP kinases, JNK kinases, caspases. or second messengers Žceramide. or Bcl-2 Žced-9. and other family members depending upon the stage of cell commitment.
and promote axonal and dendritic branching. Competition among neurons for limiting amounts of neurotrophin molecules produced by target cells is a principal mechanism for accounting for cell survival during development. An important prediction from this hypothesis is that the efficacy of neurotrophic action will depend upon the concentrations of trophic factors and upon the binding requirements of receptors expressed in responsive cell populations. This article will focus upon how NGF receptors interact to produce alternative signaling outcomes that influence cell viability. The trkA, trkB, and trkC tyrosine kinases serve as the receptors for NGF, BDNF, and NT-3, respectively, and trkA and trkB may also function as receptors for NT-3 and NT-4r5 w1x. In addition, the p75 receptor can serve as a receptor for NGF, BDNF, NT-3 and NT-4, although it exhibits slightly different binding characteristics for each factor. The p75 receptor is the founding member of the TNF superfamily w2x. For NGF responsive neuronal populations, p75 and trkA are frequently co-expressed, while p75 is expressed widely during development in non-neuronal cells and also following nerve injury or trauma. The assignment of p75 functionally as well as structurally to the TNF family is apt, since many receptors in this family serve to modulate the effects of cytokines following inflammation, infection or injury.
2. Models of receptor action Trophic factors exemplified by NGF and its family members, ciliary neurotrophic factor ŽCNTF. and glial derived neurotrophic factor ŽGDNF. all utilize increased tyrosine phosphorylation of cellular substrates to mediate neuronal cell survival ŽFig. 2.. CNTF acts through an a CNTF receptor component, gp130 and LIF subunits, which are linked to the JAKrSTAT signaling molecules w3x, whereas the GDNF receptor complex consists of the c-ret receptor tyrosine kinase and a separate a-subunit w4x.
Actions of the NGF family of neurotrophins are dictated by ras activation through the trk family of receptor tyrosine kinases. Another survival pathway has been defined by phosphatidylinositol-3-kinase w5x, which gives rise to phosphoinositide intermediates that activate Akt w6x. Induction of the serine–threonine kinase Akt is critical for cell survival, as well as cell proliferation. Hence, for many trophic factors, multiple subunits constitute a functional transmembrane complex which activates ras-dependent and ras-independent intracellular signaling. A number of common substrates, including phospholipase C-g , phosphatidylinositol-3-kinase ŽPI-3 kinase. and SHC and Grb2 adaptor proteins are utilized by many tyrosine kinase receptors. This raises the question of how these phosphorylation events lead to different biological outcomes. There are several possibilities. First, the strength and duration of the receptor autophosphorylation events may determine downstream signaling. Second, differential signaling may be controlled by specific dephosphorylation events. Third, there may be unique second messengers or substrates which determine the specific nature of the response. For neurotrophic factors, the duration of signaling has provided an important insight into the differential signaling. In PC12 cells, NGF induces a prolonged activation of ras and MAP kinase activity, lasting for several hours, while EGF-mediated MAP kinase activation is transient in nature w7x. The time course of downstream signaling is therefore one of the major differences that accounts for the differentiation program elicited by NGF versus the action of other mitogenic growth factors, such as EGF. However, the strength of receptor action alone is not sufficient, since the ligand-dependent autophosphorylation of the EGF receptor is actually higher than for the trkA NGF receptor in PC12 cells w8x, so that the level of receptor activation cannot solely account for the difference in trophic factor action. Other novel receptor substrates, such as docking or adaptor proteins w9x, may be recruited by a subset of tyrosine kinase receptors. These interactions appear to provide additional specificity. The p75 receptor, when co-expressed with trk, provides a positive modulatory influence upon trk function w10,11x. In neuronal cell lines that express both trkA and p75 receptors, trkA autophosphorylation is increased, leading to a faster differentiative response, as assayed by more rapid growth arrest and neuronal maturation w11x. In addition, binding studies with 125 I-NGF indicated that p75 increased the number of high affinity sites by increasing the on-rate for trkA. The requirement for trkA and p75 co-expression for high affinity site formation provides an explanation for how the receptors cooperate to increase neurotrophin responsiveness during cell survival and programmed cell death. On the other hand, each receptor can also signal independently, implying that the actions of neurotrophins depend upon whether trk alone, trk plus p75, or p75 alone is expressed.
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Fig. 2. Examples of multisubunit receptors with actions in the nervous system. They include, from left to right, the NGF receptors, p75 and trkA w1,13x; the FGF receptor and proteoglycan w14x; Glial Derived Neurotrophic Factor ŽGDNF. binds to a GPI-anchored a-GNDF receptor and the ret tyrosine kinase receptor w4x; and the CNTF receptor complex, which consists of an a GPI-anchored protein, a , together with gp130 and the LIF receptor subunits w3x. The trk receptors are distinguished by an extracellular domain with multiple repeats of leucine-rich domains and IgG-related domains, and a cytoplasmic region distinguished by a catalytic tyrosine kinase domain, a small insert, and a short cytoplasmic tail. The p75 receptor has a distinctive extracellular domain sequence different from trkA.
Cell survival is enhanced by a high ratio of p75 to trk receptors. Primary culture experiments using sensory neurons from mice carrying a homozygous mutation in the p75 gene indicated that nearly four-fold more NGF was necessary to achieve comparable levels of survival as wild type neurons w12x. Sensory neurons are fully capable of signaling through the trkA receptor in the absence of p75, but with a lower sensitivity. The increased dose requirement for NGF is observed in specific neuronal populations at times when high levels of p75 were expressed. Correspondingly, sympathetic neurons from neonatal p75-null mice require higher concentrations of NGF to survive than neurons from normal mice at earlier developmental stages. A high ratio of p75 to trk is correlated with increased cell survival w13x. Thus, the responsiveness to NGF and the ability to form high affinity sites is dependent upon relative levels of p75 and trk. A favored model of receptors with auxiliary subunits is ligand presentation by the accessory subunit to the tyrosine kinase receptor. This model is applicable to the FGF receptor which utilizes a lower affinity proteoglycan molecule ŽFig. 2. to facilitate high affinity site formation w14x. This mechanism is also applicable to neurotrophin receptors, in which p75 may bind NGF rapidly and facilitates the interaction of NGF with trkA by increasing the local concentration of the NGF ligand. Indeed, co-expression of p75 with trkA receptors increases the binding affinities for NGF w15x. Also, a decrease in p75 levels or inhibition of NGF binding to p75 leads to a reduction of both high and low affinity NGF binding sites w16x. However, recent experiments indicate the presentation model is
not sufficient to explain how p75 can increase trk signaling ŽB.L. Hempstead, unpublished results.. A second mechanism that explains how two receptor components can give rise to a higher affinity is that a conformational change occurs between trk and p75 receptors that gives a more favored binding interaction. Such a conformation change could occur in the absence of ligand. Coexpression of p75 may result in a conformational change in trk, leading to an accelerated rate of association, which leads to a higher affinity site. This model would be compatible with much of the existing data, including biophysical measurements of fluorescence recovery after photobleaching which indicate that trkA immobilizes p75 molecules in the absence of NGF w17x. Furthermore, an association of p75 and trk receptors requires an intact cytoplasmic domain of p75, as well as a kinase-active trk receptor, again suggestive of the importance of conformation. Copatching of the receptors is observed using fluorescent antibodies w18x. Since interactions between p75 or p140 trk can be detected after immunoprecipitation w19x, p75 and trk receptors probably exist in membrane complexes consisting of other signaling, cytoskeletal or adaptor proteins, which may influence the conformational states of the receptors. An important role of p75 is to provide more specificity to the interactions of neurotrophins with trk family members. One interpretation of these results, in terms of a conformational model, is that a high level of p75 may result in a conformational change in the trk molecule which enhances trkA–NGF interactions. A prediction from these observations is that trk family members may respond
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much more selectively to neurotrophins when the p75 neurotrophin receptor is co-expressed.
3. Cell death signaling by neurotrophins A remarkable finding in the past year is that NGF can initiate apoptotic cell death. NGF treatment of medulloblastoma cells expressing transfected trkA receptors resulted in cell death that was directly inhibited by anti-NGF antibodies w20x. Furthermore, this action was only dependent upon trkA autophosphorylation. Although the reason that accounts for this killing activity through trkA is not known, one explanation is that an alteration in cell cycle control subverted medulloblastoma cells to this fate after NGF treatment. The similarity in the intracellular domain of the p75 receptor with other family members, such as the Fas antigen and the p55 TNF receptor w21x suggests that p75 might also function as a cell death molecule. The first report of such an activity was made in immortalized cerebellar neuronal cell lines, which undergo a faster rate of cell death after transfection of p75 w22x. Binding of NGF and agonist antibodies rescued cells from apoptotic cell death. Moreover, sensory neuronal survival experiments indicated that expression of p75 is detrimental for cell survival, particularly during the postnatal periods w23x. In contrast, earlier in development, sensory neurons actually depend upon co-expression of trk and p75 receptors w23x. These results indicated that p75 can signal through a death pathway, similar to its family members, Fas and TNF receptors but that the receptor was also capable of supporting cell survival. Other examples supporting a cell death function of p75 have been documented ŽTable 1.. The number of dying cholinergic neurons in the basal forebrain is higher in wild type animals compared to p75-deficient animals, suggesting that the expression of p75 was detrimental to viability in this cell population. This property was traced to cells that expressed p75, but were deficient in trkA w24x. Also, differentiated PC12 cells undergoing NGF deprivation seem to die more rapidly in cells expressing high levels of p75. These combined studies have suggested that p75 promotes cell death in the absence of NGF binding. Table 1 Examples of neurotrophin-related apoptosis Cell type
Ref.
Immortalized neural cell lines Postnatal sensory neurons Chick retina precursors Isthmo-optic nucleus neurons Basal forebrain cholinergic neurons PC12 cells after NGF withdrawal Mature oligodendrocytes Medulloblastoma
w22x w23x w25x w38x w24x w37x w26x w20x
Fig. 3. NGF causes apoptosis in fully differentiated oligodendrocytes. DNA fragmentation was measured after 4 h treatment of oligodendrocytes isolated from P1 cerebral cortex as described w26x. Cells were treated with 100 ngrml of NGF Žq. or left untreated Žy. in oligodendrocyte differentiation media, including 50 m grml transferrin, 5 m grml putrescine, 3 ngrml progesterone, 2.6 ngrml selenium, 12.5 m grml insulin, 0.4 m grml throxine, 0.3% glucose and 6 mM glutamine.
In contrast to a ligand-independent mechanism, cell death induced by binding of NGF to p75 also has been observed. A clear example of the in vivo consequences of p75-mediated cell death was detected in developing chick retina w25x. Neuronal precursor cells at embryonic day 5 underwent apoptotic cell death, which could be effectively blocked by antibodies to either p75 or NGF. This indicated that endogenous NGF caused the death of retinal neurons at an early developmental age. Similarly, NGF treatment of fully differentiated oligodendrocytes resulted in cell death through binding to p75 receptors w26x. Apoptosis was detected by fluorescent TUNEL labeling and increased DNA fragmentation ŽFig. 3.. Interestingly, the effects of NGF on oligodendrocyte cultures were only observed during terminal differentiation and required high concentrations Ž100 ngrml. and could not be reproduced by similar concentrations of BDNF or NT-3. Progenitor oligodendrocytes ŽO-2A. did not express p75 and were not susceptible to death by neurotrophins. Instead, these cells can also use neurotrophins as trophic factors w27x. Although the majority of oligodendrocytes do not express p75 in vivo in physiological conditions, the oligodendrocyte cultures have provided an experimental system to study signal transduction events following NGF treatment. Glial cells are highly susceptible to injury and inflammation. During traumatic lesion, oligodendroglial cells become more reactive to growth factors and cytokines which are released at the site of injury. Recent evidence indicates that p75-positive oligodendrocytes can be detected in white matter plaques from cases of multiple sclerosis ŽP. Dowling, personal communication.. Also, cultured oligodendro-
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cytes have been found to express trk receptors w27x in the presence of mitogens. In the absence of trkA expression, binding of NGF to p75 receptors can increase intracellular ceramide levels through increased sphingomyelin hydrolysis w28x. When the levels of intracellular ceramide were measured in differentiated oligodendrocytes only expressing p75 receptors, NGF led to an elevation of ceramide levels that was sustained for a period of several hours, instead of minutes. This time course is reminiscent of the activation of ras and MAP kinase activities through trkA. A sustained vs. transient duration in signaling may determine which pathway is dominant. Interestingly, other neurotrophins such as BDNF and NT-3, which also bind to p75, did not induce long-term ceramide production in mature oligodendrocytes. Moreover, cell death in oligodendrocytes through increased intracellular ceramide levels could also be mimicked by the application of exogenous ceramide analogs or bacterial sphingomyelinase or by inhibitors of the glucosylceramide synthase. These data suggest that the high sustained ceramide level elicited by the p75 receptor may contribute to NGF-dependent death. Another criteria for the dose-dependent cell death of primary cortical oligodendrocytes is the activation of c-jun amino-terminal kinase ŽJNK.. The JNK enzymatic activity, a downstream effector of the stress-activated protein kinase family, has been implicated in cell death progression in PC12 cells. In addition, ceramide is a potential activator of JNK. Significantly, incubation of mature oligodendro-
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cytes with 100 ngrml NGF for three hours led to a 3-fold induction of JNK activation. The activation of JNK activity by NGF was similar in magnitude to the effects by ceramide and was not observed in cells whose viability was not affected by NGF, such as NIH3T3 fibroblasts expressing p75. The stress-activated protein kinase, JNK, can therefore be regulated by p75 in oligodendrocytes. When both NGF receptors are co-expressed as in the case of PC12 cells, trkA exerts a suppressive effect upon p75, as assayed by ceramide production w29x. In support of this model ŽFig. 4., introduction of trkA receptors into oligodendrocytes results in a reduction of JNK but leaves NFk B activation through p75 unaffected. While JNK activities are suppressed by co-expression of trkA, MAP kinase activity is increased by NGF binding to trkA ŽYoon, unpublished results.. From these experiments, trkA signaling can selectively down-regulate certain responses from p75. Although p75 can modulate high affinity binding by NGF and trkA signaling, p75 can also display independent signaling properties through ceramide production w28,29x and increased NFk B activity w30x. The activation of JNK by NGF binding to the p75 receptor and the sustained level of ceramide has only been observed in limited cases, such as end-stage oligodendrocytes. Strikingly, other cell types which express p75, such as 3T3 cells, respond to all neurotrophins with a transient increase in ceramide levels w29x, but do not undergo cell death after neurotrophin treatment. Indeed, Schwann cells and melanoma cells
Fig. 4. Two different mechanisms to account for potential interactions between p75 and trk family members. Co-expression of p75 with trkA results in an increase in the affinity of NGF binding and enhance the autophosphorylation and signaling functions of the trkA receptor w10,11,13x. Expression of the trkA receptor suppresses the signal transduction abilities of p75, including ceramide production w29x and cell death ŽYoon, unpublished..
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which express high levels of p75 do not undergo neurotrophin-dependent cell death. These data indicate that p75 produces a dichotomy of effects, some of which are extremely cell specific. Thus, neurotrophin treatment of p75-3T3 cells resulted in a transient elevation of ceramide levels, but a persistent, long-lasting increase in ceramide and JNK activity levels was observed in mature oligodendrocytes. Similar to findings in Schwann cells, signaling by p75 appears to be exclusive to NGF, as BDNF and NT-3 are ineffective w30x. These differences in ligand specificity and time courses may result in separate signaling cascades and different biological responses, depending on the cell type and differentiative stage. A critical requirement for the death pathway is the terminal nature of cell differentiation.
4. Conclusions The NGF receptors provide a clear example of bidirectional crosstalk ŽFig. 4.. NGF binding to p75 can result in independent signaling in the absence of trkA expression. These activities include the induction of NFk B w30x; the hydrolysis of sphingomyelin to ceramide w29x; and the pro-apoptotic functions attributed to p75. An important finding is that trk tyrosine kinase action appears to negate the signaling properties of p75, particularly with respect to ceramide production w29x and programmed cell death activities. Neurotrophin receptors are generally drawn and viewed as isolated integral membrane proteins which span the lipid bilayer, with signal transduction proceeding in a linear step-wise fashion. There are now numerous examples which indicate that each receptor acts not only in a linear, independent manner, but can also influence the activity of other cell surface receptors, either directly or through signaling intermediates. Which step and which intermediates are being utilized for crosstalk between the receptors is an on-going question. Biologically, the cooperative actions of two receptors imply that the possibilities for extracellular signaling are greatly expanded. Cell differentiation is determined by biochemical reactions representing the combined effects of many growth factors and cytokines. This diversity may reflect inherent differences in receptor structure and substrate specificities. Regulation of cellular differentiation and proliferation decisions is likely to be determined by the additive effects of multiple receptors and the duration of second messengers and phosphorylation events. Unlike studies carried out in vitro where cell lines are treated with single factors, growth and survival of cells in vivo are subject to the simultaneous action of multiple polypeptide growth factors. There are several explanations for the cell death ability of neurotrophins. Since the majority of cases involve elevated levels of p75 receptor expression, the responses may
involve signaling intermediates used by TNF and Fas receptors. This includes adaptor proteins FADD and TRADD and downstream effectors, such as caspase-8, the FLICErMACH interacting enzyme w31x. It remains to be seen whether p75 overexpression might co-op the use of these proteins, or other interacting proteins of different specificity. Also, a new family of cytokine associated signaling intermediates, the TNF-associated factors ŽTRAFs., have been described. Originally discovered as molecules associated with the p75 TNF receptor w32x, it is now apparent the TRAF proteins represent a large family of molecules, which carry potential signaling capabilities through NFk B activation. Moreover, these molecules have been found to be associated with receptors required for different biological activities. These receptors include CD30, CD40, and lymphotoxin b w33–35x and even the p55 TNF receptor w36x. It seems likely that similar TRAF proteins will be utilized by p75, since neurotrophins share similar NFk B responses as IL-1 and the TNF family. The goals will be to define the mechanisms which dictate the action of macromolecular receptor complexes during transmembrane signaling. This will likely address the long standing question of how cells make decisions determining growth vs. differentiation and death. The NGF receptor system has provided a remarkable mechanism for controlling a wide gamut of cellular activities, including synaptic plasticity and cell suicide. Defining which proteins interact with each receptor will provide insights into the mechanisms responsible for regulating cell growth, survival, cell differentiation and cell death. It would not be surprising that the same molecules will be found to participate in key decision-making events that lead to these diverse biological outcomes.
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