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p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors.

Aloyz RS, Bamji SX, Pozniak CD, Toma JG, Atwal J, Kaplan DR, Miller FD - J. Cell Biol. (1998)

Bottom Line: NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax.Finally, when p53 levels are reduced or absent in p53+/- or p53-/- mice, naturally occurring sympathetic neuron death is inhibited.Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.

View Article: PubMed Central - PubMed

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

ABSTRACT
Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/- or p53-/- mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.

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(A–D) p53 and Bax protein levels increase after activation of the MEKK-JNK pathway in sympathetic neurons. (A)  Western blot analysis for c-myc in equal amounts of protein derived from sympathetic neurons infected with 20 moi myc-tagged  MEKK1 (20 moi MEKK) or β-galactosidase (control) adenovirus  for 48 h. In both cases, neurons were maintained in 20 ng/ml NGF  for the entirety of the experiment. (B) Western blot analysis for  phospho-JNK in equal amounts of protein derived from sympathetic neurons that were infected with 50 moi MEKK1 adenovirus and maintained for 48 h (50 moi MEKK), or from uninfected  sister cultures that were either maintained in 10 ng/ml NGF (10  NGF), or that were withdrawn from NGF for 48 h (0 NGF). Note  that the level of phospho-JNK immunoreactivity is similar in neurons withdrawn from NGF or transduced with activated MEKK1.  (C) Western blot analysis for p53 in equal amounts of protein derived from sympathetic neurons that were infected with 20 moi  MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (20  moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF (10 NGF), or that were withdrawn from  NGF for 48 h (0 NGF). Note that p53 protein levels are increased  by activated MEKK1 as they are by NGF withdrawal. (D) Western blot analysis for Bax in equal amounts of protein derived  from sympathetic neurons that were infected with 50 moi  MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (50  moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF for the same time period. (E and F) Increased expression of p53 in sympathetic neurons causes increased Bax protein, but does not affect phosphorylation of JNK.  (E) Western blot analysis for phospho-JNK in equal amounts of  protein derived from sympathetic neurons infected with 20 moi  p53 adenovirus and maintained in 20 ng/ml NGF for 48 h, or from  uninfected sister cultures that were maintained in 20 ng/ml for the  same time period. (F) Western blot analysis for Bax in equal  amounts of protein derived from sympathetic neurons infected  with 20 moi p53 adenovirus and maintained in 20 ng/ml NGF for  48 h, or from uninfected sister cultures maintained in 20 ng/ml  NGF for the same timeperiod. (G) Western blot analysis for p53  in equal amounts of protein derived from sympathetic neurons  maintained in 20 ng/ml NGF and infected with 20 moi p53 adenovirus or 50 moi activated MEKK adenovirus for 30 h. As a control, neurons were withdrawn from NGF (0 NGF) for 30 h.
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Figure 5: (A–D) p53 and Bax protein levels increase after activation of the MEKK-JNK pathway in sympathetic neurons. (A) Western blot analysis for c-myc in equal amounts of protein derived from sympathetic neurons infected with 20 moi myc-tagged MEKK1 (20 moi MEKK) or β-galactosidase (control) adenovirus for 48 h. In both cases, neurons were maintained in 20 ng/ml NGF for the entirety of the experiment. (B) Western blot analysis for phospho-JNK in equal amounts of protein derived from sympathetic neurons that were infected with 50 moi MEKK1 adenovirus and maintained for 48 h (50 moi MEKK), or from uninfected sister cultures that were either maintained in 10 ng/ml NGF (10 NGF), or that were withdrawn from NGF for 48 h (0 NGF). Note that the level of phospho-JNK immunoreactivity is similar in neurons withdrawn from NGF or transduced with activated MEKK1. (C) Western blot analysis for p53 in equal amounts of protein derived from sympathetic neurons that were infected with 20 moi MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (20 moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF (10 NGF), or that were withdrawn from NGF for 48 h (0 NGF). Note that p53 protein levels are increased by activated MEKK1 as they are by NGF withdrawal. (D) Western blot analysis for Bax in equal amounts of protein derived from sympathetic neurons that were infected with 50 moi MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (50 moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF for the same time period. (E and F) Increased expression of p53 in sympathetic neurons causes increased Bax protein, but does not affect phosphorylation of JNK. (E) Western blot analysis for phospho-JNK in equal amounts of protein derived from sympathetic neurons infected with 20 moi p53 adenovirus and maintained in 20 ng/ml NGF for 48 h, or from uninfected sister cultures that were maintained in 20 ng/ml for the same time period. (F) Western blot analysis for Bax in equal amounts of protein derived from sympathetic neurons infected with 20 moi p53 adenovirus and maintained in 20 ng/ml NGF for 48 h, or from uninfected sister cultures maintained in 20 ng/ml NGF for the same timeperiod. (G) Western blot analysis for p53 in equal amounts of protein derived from sympathetic neurons maintained in 20 ng/ml NGF and infected with 20 moi p53 adenovirus or 50 moi activated MEKK adenovirus for 30 h. As a control, neurons were withdrawn from NGF (0 NGF) for 30 h.

Mentions: These results indicated that the apoptotic cascades originating from NGF withdrawal and p75NTR activation share two common components; hyperphosphorylation of c-jun and p53 elevation. On the basis of these findings, we hypothesized that p53 was downstream of the MEKK-JNK pathway, which leads to c-jun hyperphosphorylation (Derijard et al., 1994; Yan et al., 1994). To test this hypothesis, we generated a recombinant adenovirus expressing an activated form of MEKK1 which has previously been shown to cause sympathetic neuron apoptosis and c-jun hyperphosphorylation (Eilers et al., 1998). We first confirmed that this virus expressed the recombinant myc epitope-tagged MEKK1 protein, and that it was capable of activating the MEKK1 downstream target, JNK (Yan et al., 1994), in sympathetic neurons. Specifically, sympathetic neurons were grown in 20 ng/ml NGF for 4 d, were infected with 20 moi of recombinant virus expressing activated MEKK1 or β-galactosidase, and then were analyzed 2 d later for expression of the myc-tagged MEKK1 on Western blots with anti-myc (Fig. 5 A). Analysis of equal amounts of protein from β-galactosidase versus MEKK1-infected sympathetic neurons revealed the presence of a myc-immunoreactive protein of the appropriate size, 35 kD, in the MEKK1-infected neurons (Fig. 5 A). To confirm that virally expressed MEKK1 was capable of activating JNK, we performed similar experiments and then analyzed the lysates for phosphorylation of JNK using a phospho-JNK antibody (Fig. 5 B). In this experiment, we compared MEKK1-infected sympathetic neurons to sympathetic neurons withdrawn from NGF, where JNK is known to be activated (Eilers et al., 1998). Western blot analysis revealed that the activated MEKK1 adenovirus caused phosphorylation of JNK to the same level as did NGF withdrawal, relative to neurons maintained in 10 ng/ml NGF alone (Fig. 5 B).


p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors.

Aloyz RS, Bamji SX, Pozniak CD, Toma JG, Atwal J, Kaplan DR, Miller FD - J. Cell Biol. (1998)

(A–D) p53 and Bax protein levels increase after activation of the MEKK-JNK pathway in sympathetic neurons. (A)  Western blot analysis for c-myc in equal amounts of protein derived from sympathetic neurons infected with 20 moi myc-tagged  MEKK1 (20 moi MEKK) or β-galactosidase (control) adenovirus  for 48 h. In both cases, neurons were maintained in 20 ng/ml NGF  for the entirety of the experiment. (B) Western blot analysis for  phospho-JNK in equal amounts of protein derived from sympathetic neurons that were infected with 50 moi MEKK1 adenovirus and maintained for 48 h (50 moi MEKK), or from uninfected  sister cultures that were either maintained in 10 ng/ml NGF (10  NGF), or that were withdrawn from NGF for 48 h (0 NGF). Note  that the level of phospho-JNK immunoreactivity is similar in neurons withdrawn from NGF or transduced with activated MEKK1.  (C) Western blot analysis for p53 in equal amounts of protein derived from sympathetic neurons that were infected with 20 moi  MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (20  moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF (10 NGF), or that were withdrawn from  NGF for 48 h (0 NGF). Note that p53 protein levels are increased  by activated MEKK1 as they are by NGF withdrawal. (D) Western blot analysis for Bax in equal amounts of protein derived  from sympathetic neurons that were infected with 50 moi  MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (50  moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF for the same time period. (E and F) Increased expression of p53 in sympathetic neurons causes increased Bax protein, but does not affect phosphorylation of JNK.  (E) Western blot analysis for phospho-JNK in equal amounts of  protein derived from sympathetic neurons infected with 20 moi  p53 adenovirus and maintained in 20 ng/ml NGF for 48 h, or from  uninfected sister cultures that were maintained in 20 ng/ml for the  same time period. (F) Western blot analysis for Bax in equal  amounts of protein derived from sympathetic neurons infected  with 20 moi p53 adenovirus and maintained in 20 ng/ml NGF for  48 h, or from uninfected sister cultures maintained in 20 ng/ml  NGF for the same timeperiod. (G) Western blot analysis for p53  in equal amounts of protein derived from sympathetic neurons  maintained in 20 ng/ml NGF and infected with 20 moi p53 adenovirus or 50 moi activated MEKK adenovirus for 30 h. As a control, neurons were withdrawn from NGF (0 NGF) for 30 h.
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Figure 5: (A–D) p53 and Bax protein levels increase after activation of the MEKK-JNK pathway in sympathetic neurons. (A) Western blot analysis for c-myc in equal amounts of protein derived from sympathetic neurons infected with 20 moi myc-tagged MEKK1 (20 moi MEKK) or β-galactosidase (control) adenovirus for 48 h. In both cases, neurons were maintained in 20 ng/ml NGF for the entirety of the experiment. (B) Western blot analysis for phospho-JNK in equal amounts of protein derived from sympathetic neurons that were infected with 50 moi MEKK1 adenovirus and maintained for 48 h (50 moi MEKK), or from uninfected sister cultures that were either maintained in 10 ng/ml NGF (10 NGF), or that were withdrawn from NGF for 48 h (0 NGF). Note that the level of phospho-JNK immunoreactivity is similar in neurons withdrawn from NGF or transduced with activated MEKK1. (C) Western blot analysis for p53 in equal amounts of protein derived from sympathetic neurons that were infected with 20 moi MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (20 moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF (10 NGF), or that were withdrawn from NGF for 48 h (0 NGF). Note that p53 protein levels are increased by activated MEKK1 as they are by NGF withdrawal. (D) Western blot analysis for Bax in equal amounts of protein derived from sympathetic neurons that were infected with 50 moi MEKK1 adenovirus and maintained in 20 ng/ml NGF for 48 h (50 moi MEKK), or from uninfected sister cultures that were maintained in 10 ng/ml NGF for the same time period. (E and F) Increased expression of p53 in sympathetic neurons causes increased Bax protein, but does not affect phosphorylation of JNK. (E) Western blot analysis for phospho-JNK in equal amounts of protein derived from sympathetic neurons infected with 20 moi p53 adenovirus and maintained in 20 ng/ml NGF for 48 h, or from uninfected sister cultures that were maintained in 20 ng/ml for the same time period. (F) Western blot analysis for Bax in equal amounts of protein derived from sympathetic neurons infected with 20 moi p53 adenovirus and maintained in 20 ng/ml NGF for 48 h, or from uninfected sister cultures maintained in 20 ng/ml NGF for the same timeperiod. (G) Western blot analysis for p53 in equal amounts of protein derived from sympathetic neurons maintained in 20 ng/ml NGF and infected with 20 moi p53 adenovirus or 50 moi activated MEKK adenovirus for 30 h. As a control, neurons were withdrawn from NGF (0 NGF) for 30 h.
Mentions: These results indicated that the apoptotic cascades originating from NGF withdrawal and p75NTR activation share two common components; hyperphosphorylation of c-jun and p53 elevation. On the basis of these findings, we hypothesized that p53 was downstream of the MEKK-JNK pathway, which leads to c-jun hyperphosphorylation (Derijard et al., 1994; Yan et al., 1994). To test this hypothesis, we generated a recombinant adenovirus expressing an activated form of MEKK1 which has previously been shown to cause sympathetic neuron apoptosis and c-jun hyperphosphorylation (Eilers et al., 1998). We first confirmed that this virus expressed the recombinant myc epitope-tagged MEKK1 protein, and that it was capable of activating the MEKK1 downstream target, JNK (Yan et al., 1994), in sympathetic neurons. Specifically, sympathetic neurons were grown in 20 ng/ml NGF for 4 d, were infected with 20 moi of recombinant virus expressing activated MEKK1 or β-galactosidase, and then were analyzed 2 d later for expression of the myc-tagged MEKK1 on Western blots with anti-myc (Fig. 5 A). Analysis of equal amounts of protein from β-galactosidase versus MEKK1-infected sympathetic neurons revealed the presence of a myc-immunoreactive protein of the appropriate size, 35 kD, in the MEKK1-infected neurons (Fig. 5 A). To confirm that virally expressed MEKK1 was capable of activating JNK, we performed similar experiments and then analyzed the lysates for phosphorylation of JNK using a phospho-JNK antibody (Fig. 5 B). In this experiment, we compared MEKK1-infected sympathetic neurons to sympathetic neurons withdrawn from NGF, where JNK is known to be activated (Eilers et al., 1998). Western blot analysis revealed that the activated MEKK1 adenovirus caused phosphorylation of JNK to the same level as did NGF withdrawal, relative to neurons maintained in 10 ng/ml NGF alone (Fig. 5 B).

Bottom Line: NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax.Finally, when p53 levels are reduced or absent in p53+/- or p53-/- mice, naturally occurring sympathetic neuron death is inhibited.Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.

View Article: PubMed Central - PubMed

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

ABSTRACT
Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/- or p53-/- mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.

Show MeSH
Related in: MedlinePlus