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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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Cultured p75NTR−/− neurons show enhanced survival in the absence of all Trk signaling. (A) Percentage survival of mouse sympathetic neurons switched for 72 h into varying concentrations of K252A in the presence or absence of 10 ng/ml NGF. Results are normalized so that the number of neurons at the time of NGF withdrawal is 100%. Each point represents the values pooled from two to four independent experiments, each repeated in triplicate. Error bars represent the standard error of the mean. (B) Percentage survival of p75NTR−/− versus p75NTR+/+ (wild-type) neurons at various time points after a switch into 0 ng/ml NGF plus or minus 200 nM K252A. Results represent the mean ± standard error of combined data from four separate experiments, each performed in triplicate.
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fig3: Cultured p75NTR−/− neurons show enhanced survival in the absence of all Trk signaling. (A) Percentage survival of mouse sympathetic neurons switched for 72 h into varying concentrations of K252A in the presence or absence of 10 ng/ml NGF. Results are normalized so that the number of neurons at the time of NGF withdrawal is 100%. Each point represents the values pooled from two to four independent experiments, each repeated in triplicate. Error bars represent the standard error of the mean. (B) Percentage survival of p75NTR−/− versus p75NTR+/+ (wild-type) neurons at various time points after a switch into 0 ng/ml NGF plus or minus 200 nM K252A. Results represent the mean ± standard error of combined data from four separate experiments, each performed in triplicate.

Mentions: Initially, we performed experiments to ensure that K252A was capable of blocking Trk-mediated survival in mouse neurons in the presence of exogenous NGF. We have previously performed similar studies with rat sympathetic neurons, and have demonstrated that 200 nM K252A was sufficient to completely eliminate NGF-mediated TrkA activation and block NGF-mediated survival (Vaillant et al., 1999). To perform these experiments, wild-type mouse sympathetic neurons were established in 50 ng/ml NGF for 5 d, and were then switched into 10 ng/ml NGF in the presence of 10–200 nM K252A. Fields of phase-bright neurons were then counted immediately after the switch, and at 24 h intervals thereafter. This analysis (Fig. 3 A) revealed that in the presence of 10 ng/ml NGF, K252A decreased neuronal survival in a concentration-dependent fashion over 72 h, with the maximal effect apparent at 50–200 nM K252A: in 10 nM K252A, 45 ± 8% of neurons were still alive at 72 h, in 20 nM, 33 ± 4% were alive, whereas with 100 nM, only 14 ± 9% were still alive. We then performed similar studies where neurons were withdrawn from NGF, and various concentrations of K252A were added for 72 h to ensure that all Trk-mediated survival signals were eliminated (Fig. 3 A). This analysis confirmed that the addition of 50 or 200 nM K252A eliminated any residual survival signaling that was due to small amounts of NGF present in the cultures after the washout period. Specifically, in 0 NGF 35 ± 6% of neurons were still alive, whereas in 50 and 200 nM K252A, 20 ± 6% and 18 ± 3% were still alive, respectively.


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)

Cultured p75NTR−/− neurons show enhanced survival in the absence of all Trk signaling. (A) Percentage survival of mouse sympathetic neurons switched for 72 h into varying concentrations of K252A in the presence or absence of 10 ng/ml NGF. Results are normalized so that the number of neurons at the time of NGF withdrawal is 100%. Each point represents the values pooled from two to four independent experiments, each repeated in triplicate. Error bars represent the standard error of the mean. (B) Percentage survival of p75NTR−/− versus p75NTR+/+ (wild-type) neurons at various time points after a switch into 0 ng/ml NGF plus or minus 200 nM K252A. Results represent the mean ± standard error of combined data from four separate experiments, each performed in triplicate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199335&req=5

fig3: Cultured p75NTR−/− neurons show enhanced survival in the absence of all Trk signaling. (A) Percentage survival of mouse sympathetic neurons switched for 72 h into varying concentrations of K252A in the presence or absence of 10 ng/ml NGF. Results are normalized so that the number of neurons at the time of NGF withdrawal is 100%. Each point represents the values pooled from two to four independent experiments, each repeated in triplicate. Error bars represent the standard error of the mean. (B) Percentage survival of p75NTR−/− versus p75NTR+/+ (wild-type) neurons at various time points after a switch into 0 ng/ml NGF plus or minus 200 nM K252A. Results represent the mean ± standard error of combined data from four separate experiments, each performed in triplicate.
Mentions: Initially, we performed experiments to ensure that K252A was capable of blocking Trk-mediated survival in mouse neurons in the presence of exogenous NGF. We have previously performed similar studies with rat sympathetic neurons, and have demonstrated that 200 nM K252A was sufficient to completely eliminate NGF-mediated TrkA activation and block NGF-mediated survival (Vaillant et al., 1999). To perform these experiments, wild-type mouse sympathetic neurons were established in 50 ng/ml NGF for 5 d, and were then switched into 10 ng/ml NGF in the presence of 10–200 nM K252A. Fields of phase-bright neurons were then counted immediately after the switch, and at 24 h intervals thereafter. This analysis (Fig. 3 A) revealed that in the presence of 10 ng/ml NGF, K252A decreased neuronal survival in a concentration-dependent fashion over 72 h, with the maximal effect apparent at 50–200 nM K252A: in 10 nM K252A, 45 ± 8% of neurons were still alive at 72 h, in 20 nM, 33 ± 4% were alive, whereas with 100 nM, only 14 ± 9% were still alive. We then performed similar studies where neurons were withdrawn from NGF, and various concentrations of K252A were added for 72 h to ensure that all Trk-mediated survival signals were eliminated (Fig. 3 A). This analysis confirmed that the addition of 50 or 200 nM K252A eliminated any residual survival signaling that was due to small amounts of NGF present in the cultures after the washout period. Specifically, in 0 NGF 35 ± 6% of neurons were still alive, whereas in 50 and 200 nM K252A, 20 ± 6% and 18 ± 3% were still alive, respectively.

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