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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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E1B55K reduces p53 levels in sympathetic neurons  withdrawn from NGF. (A) Western blot analysis for p53 in sympathetic neurons that were withdrawn from NGF for 36 h (0  NGF), that were maintained in 10 ng/ml NGF (10 NGF), or that  were infected with 500 moi of recombinant adenovirus expressing  A262 or E1B55K, and 2 d later were withdrawn from NGF for 24 h.  Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein  levels were assessed using anti-p53. Note that p53 levels increase  after NGF withdrawal, and that expression of E1B55K reverses  this increase, while expression of A262 has no effect. (B) The  same blot as in panel (A) reprobed for the cytoskeletal protein  tubulin. (C) Western blot analysis for E1B55K using anti-E1B55K in equal amounts of protein derived from sympathetic  neurons infected for 36 h with adenoviruses expressing E1B55K  or the E1B55K mutant A262. The antibody used for these studies  recognizes both the wild-type and mutant proteins. (D) Western  blot analysis for p53 in sympathetic neurons that were maintained  in 20 ng/ml NGF, and that were infected with 50 moi of recombinant adenovirus expressing activated MEKK1 (MEKK) with or  without 200 moi of E1B55K for 36 h. Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein levels were assessed using  anti-p53. (E) The same blot as in D reprobed for TrkA.
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Figure 3: E1B55K reduces p53 levels in sympathetic neurons withdrawn from NGF. (A) Western blot analysis for p53 in sympathetic neurons that were withdrawn from NGF for 36 h (0 NGF), that were maintained in 10 ng/ml NGF (10 NGF), or that were infected with 500 moi of recombinant adenovirus expressing A262 or E1B55K, and 2 d later were withdrawn from NGF for 24 h. Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein levels were assessed using anti-p53. Note that p53 levels increase after NGF withdrawal, and that expression of E1B55K reverses this increase, while expression of A262 has no effect. (B) The same blot as in panel (A) reprobed for the cytoskeletal protein tubulin. (C) Western blot analysis for E1B55K using anti-E1B55K in equal amounts of protein derived from sympathetic neurons infected for 36 h with adenoviruses expressing E1B55K or the E1B55K mutant A262. The antibody used for these studies recognizes both the wild-type and mutant proteins. (D) Western blot analysis for p53 in sympathetic neurons that were maintained in 20 ng/ml NGF, and that were infected with 50 moi of recombinant adenovirus expressing activated MEKK1 (MEKK) with or without 200 moi of E1B55K for 36 h. Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein levels were assessed using anti-p53. (E) The same blot as in D reprobed for TrkA.

Mentions: To ensure that the effects of E1B55K were being mediated via p53, we examined levels of p53 in NGF-withdrawn sympathetic neurons after viral infection; the vector used not only ablates p53 through the actions of E1B55K, but also targets p53 for degradation through the adenoviral E4orf6 product (Yew and Berk, 1992; Querido et al., 1997; Teodoro and Branton, 1997). For these experiments, sympathetic neurons were initially selected in 50 ng/ml NGF for 3 d, were infected with the adenoviruses expressing E1B55K or the mutant E1B55K, and 2 d later were withdrawn from NGF. As a control, neurons were not infected or were infected with the β-galactosidase virus. Analysis of p53 protein levels 36 h after this treatment revealed that levels of p53 protein were similarly elevated in NGF-withdrawn neurons that were uninfected, or that were expressing either β-galactosidase or the mutant E1B55K (Fig. 3 A). In contrast, p53 protein levels were significantly reduced in the neurons expressing E1B55K, as predicted. Reprobing of the same blot with an antibody specific for the cytoskeletal protein tubulin confirmed that equal amounts of protein were present in all of the samples (Fig. 3 B). Moreover, the differential effect of E1B55K versus the A262 mutant was not due to a difference in levels of expression of these two proteins, since the A262 protein was expressed at higher levels than E1B55K (Fig. 3 C). These experiments therefore indicate that elevated p53 protein levels are essential for NGF-withdrawal induced apoptosis of sympathetic neurons.


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)

E1B55K reduces p53 levels in sympathetic neurons  withdrawn from NGF. (A) Western blot analysis for p53 in sympathetic neurons that were withdrawn from NGF for 36 h (0  NGF), that were maintained in 10 ng/ml NGF (10 NGF), or that  were infected with 500 moi of recombinant adenovirus expressing  A262 or E1B55K, and 2 d later were withdrawn from NGF for 24 h.  Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein  levels were assessed using anti-p53. Note that p53 levels increase  after NGF withdrawal, and that expression of E1B55K reverses  this increase, while expression of A262 has no effect. (B) The  same blot as in panel (A) reprobed for the cytoskeletal protein  tubulin. (C) Western blot analysis for E1B55K using anti-E1B55K in equal amounts of protein derived from sympathetic  neurons infected for 36 h with adenoviruses expressing E1B55K  or the E1B55K mutant A262. The antibody used for these studies  recognizes both the wild-type and mutant proteins. (D) Western  blot analysis for p53 in sympathetic neurons that were maintained  in 20 ng/ml NGF, and that were infected with 50 moi of recombinant adenovirus expressing activated MEKK1 (MEKK) with or  without 200 moi of E1B55K for 36 h. Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein levels were assessed using  anti-p53. (E) The same blot as in D reprobed for TrkA.
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Figure 3: E1B55K reduces p53 levels in sympathetic neurons withdrawn from NGF. (A) Western blot analysis for p53 in sympathetic neurons that were withdrawn from NGF for 36 h (0 NGF), that were maintained in 10 ng/ml NGF (10 NGF), or that were infected with 500 moi of recombinant adenovirus expressing A262 or E1B55K, and 2 d later were withdrawn from NGF for 24 h. Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein levels were assessed using anti-p53. Note that p53 levels increase after NGF withdrawal, and that expression of E1B55K reverses this increase, while expression of A262 has no effect. (B) The same blot as in panel (A) reprobed for the cytoskeletal protein tubulin. (C) Western blot analysis for E1B55K using anti-E1B55K in equal amounts of protein derived from sympathetic neurons infected for 36 h with adenoviruses expressing E1B55K or the E1B55K mutant A262. The antibody used for these studies recognizes both the wild-type and mutant proteins. (D) Western blot analysis for p53 in sympathetic neurons that were maintained in 20 ng/ml NGF, and that were infected with 50 moi of recombinant adenovirus expressing activated MEKK1 (MEKK) with or without 200 moi of E1B55K for 36 h. Equivalent amounts of protein from lysed neurons were electrophoresed, transferred to nitrocellulose filters, and p53 protein levels were assessed using anti-p53. (E) The same blot as in D reprobed for TrkA.
Mentions: To ensure that the effects of E1B55K were being mediated via p53, we examined levels of p53 in NGF-withdrawn sympathetic neurons after viral infection; the vector used not only ablates p53 through the actions of E1B55K, but also targets p53 for degradation through the adenoviral E4orf6 product (Yew and Berk, 1992; Querido et al., 1997; Teodoro and Branton, 1997). For these experiments, sympathetic neurons were initially selected in 50 ng/ml NGF for 3 d, were infected with the adenoviruses expressing E1B55K or the mutant E1B55K, and 2 d later were withdrawn from NGF. As a control, neurons were not infected or were infected with the β-galactosidase virus. Analysis of p53 protein levels 36 h after this treatment revealed that levels of p53 protein were similarly elevated in NGF-withdrawn neurons that were uninfected, or that were expressing either β-galactosidase or the mutant E1B55K (Fig. 3 A). In contrast, p53 protein levels were significantly reduced in the neurons expressing E1B55K, as predicted. Reprobing of the same blot with an antibody specific for the cytoskeletal protein tubulin confirmed that equal amounts of protein were present in all of the samples (Fig. 3 B). Moreover, the differential effect of E1B55K versus the A262 mutant was not due to a difference in levels of expression of these two proteins, since the A262 protein was expressed at higher levels than E1B55K (Fig. 3 C). These experiments therefore indicate that elevated p53 protein levels are essential for NGF-withdrawal induced apoptosis of sympathetic neurons.

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