Limits...
Poly(ADP-ribosyl)ation of p53 contributes to TPEN-induced neuronal apoptosis.

Kim HL, Ra H, Kim KR, Lee JM, Im H, Kim YH - Mol. Cells (2015)

Bottom Line: Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment.Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1.Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Sejong University, Seoul 143-747, Korea.

ABSTRACT
Depletion of intracellular zinc by N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

No MeSH data available.


Related in: MedlinePlus

Requirement for PARP-1 in TPEN-induced neuronal apoptosis. (A) LDH release or PI-positive cells (mean ± SEM, n = 4 cultures) in mouse cortical neuron cultures after 24-h exposure to TPEN (2 μM) with or without PARP inhibitors nicotinamide (NAM; 10 mM) or 3-aminobenzamide (AB; 10 mM). *p < 0.05 vs. TPEN alone, ANOVA. (B) Phase-contrast (upper) or PI-stained (lower) photomicrographs of identical fields of cultured cortical neurons exposed to sham wash (CTRL) or TPEN with or without NAM or AB for 24 h. Arrows indicate typical apoptotic nuclei. Scale bar = 100 μm. (C) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN with or without NAM or AB. Arrows indicate typical morphology of apoptotic condensed nuclei. *p < 0.05 vs. TPEN alone, ANOVA. (D) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN. Arrows indicate typical morphology of apoptotic condensed nuclei. In PARP−/− neuronal cultures, zinc-depleted neuronal apoptosis was markedly attenuated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400305&req=5

f1-molce-38-4-312: Requirement for PARP-1 in TPEN-induced neuronal apoptosis. (A) LDH release or PI-positive cells (mean ± SEM, n = 4 cultures) in mouse cortical neuron cultures after 24-h exposure to TPEN (2 μM) with or without PARP inhibitors nicotinamide (NAM; 10 mM) or 3-aminobenzamide (AB; 10 mM). *p < 0.05 vs. TPEN alone, ANOVA. (B) Phase-contrast (upper) or PI-stained (lower) photomicrographs of identical fields of cultured cortical neurons exposed to sham wash (CTRL) or TPEN with or without NAM or AB for 24 h. Arrows indicate typical apoptotic nuclei. Scale bar = 100 μm. (C) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN with or without NAM or AB. Arrows indicate typical morphology of apoptotic condensed nuclei. *p < 0.05 vs. TPEN alone, ANOVA. (D) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN. Arrows indicate typical morphology of apoptotic condensed nuclei. In PARP−/− neuronal cultures, zinc-depleted neuronal apoptosis was markedly attenuated.

Mentions: To quantify neuronal apoptosis, we used three different methods. First, we measured the release of LDH from neurons into the bathing media. The chemical inhibition of PARP-1 significantly attenuated TEPN-induced neuronal death (Fig. 1A, left). However, some apoptotic insults such as staurosporine do not result in LDH leakage from cell because the process of apoptosis is followed by plasma membrane blebbing rather than rupture. Therefore, we next used PI staining to confirm TPEN-induced neuronal death and its reduction by PARP-1 inhibition. Consistent with our LDH results, the number of PI-positive neurons was markedly increased by TPEN (Fig. 1A, right; Fig. 1B), and some PI-positive nuclei showed typical shrunken and fragmented morphology (Fig. 1B, arrow). However, this effect was significantly reversed by chemical inhibitors of PARP-1 (Fig. 1A, right; Fig. 1B). To further evaluate apoptotic cell death, we observed the morphology of Hoechst 33342-stained nuclei. Whereas TPEN markedly increased the presence of bright and shrunken nuclei, which is a marker of apoptosis, NAM and AB reversed this change in morphology (Fig. 1C). Consistent with these findings, the genetic deletion of PARP-1 almost completely blocked TPEN-induced neuronal apoptosis (Fig. 1D).


Poly(ADP-ribosyl)ation of p53 contributes to TPEN-induced neuronal apoptosis.

Kim HL, Ra H, Kim KR, Lee JM, Im H, Kim YH - Mol. Cells (2015)

Requirement for PARP-1 in TPEN-induced neuronal apoptosis. (A) LDH release or PI-positive cells (mean ± SEM, n = 4 cultures) in mouse cortical neuron cultures after 24-h exposure to TPEN (2 μM) with or without PARP inhibitors nicotinamide (NAM; 10 mM) or 3-aminobenzamide (AB; 10 mM). *p < 0.05 vs. TPEN alone, ANOVA. (B) Phase-contrast (upper) or PI-stained (lower) photomicrographs of identical fields of cultured cortical neurons exposed to sham wash (CTRL) or TPEN with or without NAM or AB for 24 h. Arrows indicate typical apoptotic nuclei. Scale bar = 100 μm. (C) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN with or without NAM or AB. Arrows indicate typical morphology of apoptotic condensed nuclei. *p < 0.05 vs. TPEN alone, ANOVA. (D) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN. Arrows indicate typical morphology of apoptotic condensed nuclei. In PARP−/− neuronal cultures, zinc-depleted neuronal apoptosis was markedly attenuated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400305&req=5

f1-molce-38-4-312: Requirement for PARP-1 in TPEN-induced neuronal apoptosis. (A) LDH release or PI-positive cells (mean ± SEM, n = 4 cultures) in mouse cortical neuron cultures after 24-h exposure to TPEN (2 μM) with or without PARP inhibitors nicotinamide (NAM; 10 mM) or 3-aminobenzamide (AB; 10 mM). *p < 0.05 vs. TPEN alone, ANOVA. (B) Phase-contrast (upper) or PI-stained (lower) photomicrographs of identical fields of cultured cortical neurons exposed to sham wash (CTRL) or TPEN with or without NAM or AB for 24 h. Arrows indicate typical apoptotic nuclei. Scale bar = 100 μm. (C) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN with or without NAM or AB. Arrows indicate typical morphology of apoptotic condensed nuclei. *p < 0.05 vs. TPEN alone, ANOVA. (D) Photomicrographs (left) and quantitative analysis (right; n = 4 cultures) of Hoechst 33342-positive apoptotic cells in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures after 24-h exposure to sham wash (CTRL) or TPEN. Arrows indicate typical morphology of apoptotic condensed nuclei. In PARP−/− neuronal cultures, zinc-depleted neuronal apoptosis was markedly attenuated.
Mentions: To quantify neuronal apoptosis, we used three different methods. First, we measured the release of LDH from neurons into the bathing media. The chemical inhibition of PARP-1 significantly attenuated TEPN-induced neuronal death (Fig. 1A, left). However, some apoptotic insults such as staurosporine do not result in LDH leakage from cell because the process of apoptosis is followed by plasma membrane blebbing rather than rupture. Therefore, we next used PI staining to confirm TPEN-induced neuronal death and its reduction by PARP-1 inhibition. Consistent with our LDH results, the number of PI-positive neurons was markedly increased by TPEN (Fig. 1A, right; Fig. 1B), and some PI-positive nuclei showed typical shrunken and fragmented morphology (Fig. 1B, arrow). However, this effect was significantly reversed by chemical inhibitors of PARP-1 (Fig. 1A, right; Fig. 1B). To further evaluate apoptotic cell death, we observed the morphology of Hoechst 33342-stained nuclei. Whereas TPEN markedly increased the presence of bright and shrunken nuclei, which is a marker of apoptosis, NAM and AB reversed this change in morphology (Fig. 1C). Consistent with these findings, the genetic deletion of PARP-1 almost completely blocked TPEN-induced neuronal apoptosis (Fig. 1D).

Bottom Line: Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment.Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1.Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Sejong University, Seoul 143-747, Korea.

ABSTRACT
Depletion of intracellular zinc by N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

No MeSH data available.


Related in: MedlinePlus