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TrkA mediates developmental sympathetic neuron survival in vivo by silencing an ongoing p75NTR-mediated death signal.

Majdan M, Walsh GS, Aloyz R, Miller FD - J. Cell Biol. (2001)

Bottom Line: A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA.Here we provide evidence for the latter model.These data therefore support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Canada H3A 2B4.

ABSTRACT
Developmental sympathetic neuron death is determined by functional interactions between the TrkA/NGF receptor and the p75 neurotrophin receptor (p75NTR). A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA. Here we provide evidence for the latter model. Specifically, experiments presented here demonstrate that the presence or absence of p75NTR does not alter Trk activity or NGF- and NT-3-mediated downstream survival signaling in primary neurons. Crosses of p75NTR-/- and TrkA-/- mice indicate that the coincident absence of p75NTR substantially rescues TrkA-/- sympathetic neurons from developmental death in vivo. Thus, p75NTR induces death regardless of the presence or absence of TrkA expression. These data therefore support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.

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Levels of Trk receptors, Trk receptor activation, and downstream survival signaling in p75NTR−/− SCG neurons. (A) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ SCG at P7, probed for TrkA (RTA), tyrosine hydroxylase (TH) and ERK1. (B) Western blot analysis of lysates of P7 p75NTR−/− versus p75NTR+/+ SCG that were precipitated with WGA and then probed with an antibody specific for TrkC or the intracellular region of p75NTR. Equal amounts of protein from the same lysates were also probed for ERK1. (C) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ ganglia probed for p75NTR (p75), p53, or for total tubulin. (D) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF, and then were induced with 0 or 50 ng/ml NGF for 10 min. Blots were probed with an antibody specific to phosphotyrosine to detect tyrosine phosphorylated Trk (p-TYR), or with antibodies for the activated phosphorylated forms of Akt (p-AKT) or the ERKs (p-ERK), and then reprobed for TrkA (RTA), total ERKs (ERK), or p75NTR (p75). Note that in the ERK reprobe, a mobility shift is evident in the lysates from NGF-treated neurons, consistent with the increased levels of phosphoERK observed. (E and F) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF and induced either with 50 ng/ml NGF for 1 h (E) or 20 ng/ml NT-3 for 10 min (F). Blots were probed with antibodies specific to phosphorylated Akt (p-AKT) or phosphorylated ERKs (p-ERK) and then reprobed with antibodies for total ERKs or for p75NTR (p75). (G) Western blot analysis for p75NTR in equal amounts of protein from p75NTR+/+ versus p75NTR+/− ganglia at P7. The blot was reprobed for tubulin, scanned, and the ratio of p75NTR to tubulin was plotted on the accompanying bar graph.
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fig2: Levels of Trk receptors, Trk receptor activation, and downstream survival signaling in p75NTR−/− SCG neurons. (A) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ SCG at P7, probed for TrkA (RTA), tyrosine hydroxylase (TH) and ERK1. (B) Western blot analysis of lysates of P7 p75NTR−/− versus p75NTR+/+ SCG that were precipitated with WGA and then probed with an antibody specific for TrkC or the intracellular region of p75NTR. Equal amounts of protein from the same lysates were also probed for ERK1. (C) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ ganglia probed for p75NTR (p75), p53, or for total tubulin. (D) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF, and then were induced with 0 or 50 ng/ml NGF for 10 min. Blots were probed with an antibody specific to phosphotyrosine to detect tyrosine phosphorylated Trk (p-TYR), or with antibodies for the activated phosphorylated forms of Akt (p-AKT) or the ERKs (p-ERK), and then reprobed for TrkA (RTA), total ERKs (ERK), or p75NTR (p75). Note that in the ERK reprobe, a mobility shift is evident in the lysates from NGF-treated neurons, consistent with the increased levels of phosphoERK observed. (E and F) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF and induced either with 50 ng/ml NGF for 1 h (E) or 20 ng/ml NT-3 for 10 min (F). Blots were probed with antibodies specific to phosphorylated Akt (p-AKT) or phosphorylated ERKs (p-ERK) and then reprobed with antibodies for total ERKs or for p75NTR (p75). (G) Western blot analysis for p75NTR in equal amounts of protein from p75NTR+/+ versus p75NTR+/− ganglia at P7. The blot was reprobed for tubulin, scanned, and the ratio of p75NTR to tubulin was plotted on the accompanying bar graph.

Mentions: Three potential explanations for the deficit in apoptosis observed in p75NTR−/− SCG are (1) Trk receptor levels, activation, and subsequent downstream survival signaling are increased in the absence of p75NTR; (2) the absence of p75NTR allows TrkA to respond more robustly to nonpreferred ligands such as NT-3 (Benedetti et al., 1993; Ip et al., 1993); and (3) p75NTR mediates a direct apoptotic signaling cascade that is eliminated in its absence (Aloyz et al., 1998). To examine the first two possibilities, we assayed Trk receptor levels, activation, and downstream survival signaling in p75NTR−/− ganglia and cultured p75NTR−/− neurons. Initially, we examined levels of TrkA and TrkC in p75NTR−/− sympathetic ganglia at P7. SCG lysates were run on SDS-PAGE, transferred to nitrocellulose, and probed with an antibody specific to TrkA (RTA) (Clary et al., 1994). Alternatively, lysates were precipitated with WGA, which precipitates glycosylated proteins, and analyzed on Western blots with an antibody specific to the full-length isoform of TrkC (Belliveau et al., 1997). This analysis revealed that levels of TrkA were slightly but consistently decreased in the p75NTR−/− SCG (Fig. 2 A), whereas TrkC levels were constant (Fig. 2 B). In contrast, levels of ERK1 were unchanged (Figs. 2, A and B). Because full-length Trk receptors are only expressed on sympathetic neurons and not on nonneuronal cells in the ganglia, and neuronal number is increased in the absence of p75NTR, these data indicate that the decreased apoptosis in p75NTR−/− SCG is not due to an increase in Trk receptor levels.


TrkA mediates developmental sympathetic neuron survival in vivo by silencing an ongoing p75NTR-mediated death signal.

Majdan M, Walsh GS, Aloyz R, Miller FD - J. Cell Biol. (2001)

Levels of Trk receptors, Trk receptor activation, and downstream survival signaling in p75NTR−/− SCG neurons. (A) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ SCG at P7, probed for TrkA (RTA), tyrosine hydroxylase (TH) and ERK1. (B) Western blot analysis of lysates of P7 p75NTR−/− versus p75NTR+/+ SCG that were precipitated with WGA and then probed with an antibody specific for TrkC or the intracellular region of p75NTR. Equal amounts of protein from the same lysates were also probed for ERK1. (C) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ ganglia probed for p75NTR (p75), p53, or for total tubulin. (D) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF, and then were induced with 0 or 50 ng/ml NGF for 10 min. Blots were probed with an antibody specific to phosphotyrosine to detect tyrosine phosphorylated Trk (p-TYR), or with antibodies for the activated phosphorylated forms of Akt (p-AKT) or the ERKs (p-ERK), and then reprobed for TrkA (RTA), total ERKs (ERK), or p75NTR (p75). Note that in the ERK reprobe, a mobility shift is evident in the lysates from NGF-treated neurons, consistent with the increased levels of phosphoERK observed. (E and F) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF and induced either with 50 ng/ml NGF for 1 h (E) or 20 ng/ml NT-3 for 10 min (F). Blots were probed with antibodies specific to phosphorylated Akt (p-AKT) or phosphorylated ERKs (p-ERK) and then reprobed with antibodies for total ERKs or for p75NTR (p75). (G) Western blot analysis for p75NTR in equal amounts of protein from p75NTR+/+ versus p75NTR+/− ganglia at P7. The blot was reprobed for tubulin, scanned, and the ratio of p75NTR to tubulin was plotted on the accompanying bar graph.
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fig2: Levels of Trk receptors, Trk receptor activation, and downstream survival signaling in p75NTR−/− SCG neurons. (A) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ SCG at P7, probed for TrkA (RTA), tyrosine hydroxylase (TH) and ERK1. (B) Western blot analysis of lysates of P7 p75NTR−/− versus p75NTR+/+ SCG that were precipitated with WGA and then probed with an antibody specific for TrkC or the intracellular region of p75NTR. Equal amounts of protein from the same lysates were also probed for ERK1. (C) Western blot analysis of equal amounts of protein from p75NTR−/− versus p75NTR+/+ ganglia probed for p75NTR (p75), p53, or for total tubulin. (D) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF, and then were induced with 0 or 50 ng/ml NGF for 10 min. Blots were probed with an antibody specific to phosphotyrosine to detect tyrosine phosphorylated Trk (p-TYR), or with antibodies for the activated phosphorylated forms of Akt (p-AKT) or the ERKs (p-ERK), and then reprobed for TrkA (RTA), total ERKs (ERK), or p75NTR (p75). Note that in the ERK reprobe, a mobility shift is evident in the lysates from NGF-treated neurons, consistent with the increased levels of phosphoERK observed. (E and F) Western blot analysis of equal amounts of protein from cultured p75NTR−/− versus p75NTR+/+ neonatal sympathetic neurons that were washed free of NGF and induced either with 50 ng/ml NGF for 1 h (E) or 20 ng/ml NT-3 for 10 min (F). Blots were probed with antibodies specific to phosphorylated Akt (p-AKT) or phosphorylated ERKs (p-ERK) and then reprobed with antibodies for total ERKs or for p75NTR (p75). (G) Western blot analysis for p75NTR in equal amounts of protein from p75NTR+/+ versus p75NTR+/− ganglia at P7. The blot was reprobed for tubulin, scanned, and the ratio of p75NTR to tubulin was plotted on the accompanying bar graph.
Mentions: Three potential explanations for the deficit in apoptosis observed in p75NTR−/− SCG are (1) Trk receptor levels, activation, and subsequent downstream survival signaling are increased in the absence of p75NTR; (2) the absence of p75NTR allows TrkA to respond more robustly to nonpreferred ligands such as NT-3 (Benedetti et al., 1993; Ip et al., 1993); and (3) p75NTR mediates a direct apoptotic signaling cascade that is eliminated in its absence (Aloyz et al., 1998). To examine the first two possibilities, we assayed Trk receptor levels, activation, and downstream survival signaling in p75NTR−/− ganglia and cultured p75NTR−/− neurons. Initially, we examined levels of TrkA and TrkC in p75NTR−/− sympathetic ganglia at P7. SCG lysates were run on SDS-PAGE, transferred to nitrocellulose, and probed with an antibody specific to TrkA (RTA) (Clary et al., 1994). Alternatively, lysates were precipitated with WGA, which precipitates glycosylated proteins, and analyzed on Western blots with an antibody specific to the full-length isoform of TrkC (Belliveau et al., 1997). This analysis revealed that levels of TrkA were slightly but consistently decreased in the p75NTR−/− SCG (Fig. 2 A), whereas TrkC levels were constant (Fig. 2 B). In contrast, levels of ERK1 were unchanged (Figs. 2, A and B). Because full-length Trk receptors are only expressed on sympathetic neurons and not on nonneuronal cells in the ganglia, and neuronal number is increased in the absence of p75NTR, these data indicate that the decreased apoptosis in p75NTR−/− SCG is not due to an increase in Trk receptor levels.

Bottom Line: A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA.Here we provide evidence for the latter model.These data therefore support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Canada H3A 2B4.

ABSTRACT
Developmental sympathetic neuron death is determined by functional interactions between the TrkA/NGF receptor and the p75 neurotrophin receptor (p75NTR). A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA. Here we provide evidence for the latter model. Specifically, experiments presented here demonstrate that the presence or absence of p75NTR does not alter Trk activity or NGF- and NT-3-mediated downstream survival signaling in primary neurons. Crosses of p75NTR-/- and TrkA-/- mice indicate that the coincident absence of p75NTR substantially rescues TrkA-/- sympathetic neurons from developmental death in vivo. Thus, p75NTR induces death regardless of the presence or absence of TrkA expression. These data therefore support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.

Show MeSH
Related in: MedlinePlus