Alcohol induces p53-mediated apoptosis in neural crest by stimulating an AMPK-mediated suppression of TORC1, S6K, and ribosomal biogenesis

Abstract

Prenatal alcohol exposure is a leading cause of permanent neurodevelopmental disability and can feature distinctive craniofacial deficits that partly originate from the apoptotic deletion of craniofacial progenitors, a stem cell lineage called the neural crest (NC). We recently demonstrated that alcohol causes nucleolar stress in NC through its suppression of ribosome biogenesis (RBG) and this suppression is causative in their p53/MDM2-mediated apoptosis. Here, we show that this nucleolar stress originates from alcohol's activation of AMPK, which suppresses TORC1 and the p70/S6K-mediated stimulation of RBG. Alcohol-exposed cells of the pluripotent, primary cranial NC line O9-1 were evaluated with respect to their S6K, TORC1, and AMPK activity. The functional impact of these signals with respect to RBG, p53, and apoptosis were assessed using gain-of-function constructs and small molecule mediators. Alcohol rapidly (<2 hr) increased pAMPK, pTSC2, and pRaptor, and reduced both total and pS6K in NC cells. These changes persisted for at least 12 hr to 18 hr following alcohol exposure. Attenuation of these signals via gain- or loss-of-function approaches that targeted AMPK, S6K, or TORC1 prevented alcohol's suppression of rRNA synthesis and the induction of p53-stimulated apoptosis. We conclude that alcohol induces ribosome dysbiogenesis and activates their p53/MDM2-mediated apoptosis via its activation of pAMPK, which in turn activates TSC2 and Raptor to suppress the TORC1/S6K-mediated promotion of ribosome biogenesis. This represents a novel mechanism underlying alcohol's neurotoxicity and is consistent with findings that TORC1/S6K networks are critical for cranial NC survival.

Keywords: AMPK; Apoptosis; Craniofacial development; Fetal alcohol spectrum disorders; MTOR; Neural crest; Nucleolar stress; O9-1 cells; P53; RPS6K; Ribosome biogenesis.

Figure 1.. Alcohol rapidly represses ribosome biogenesis and causes nucleolar stress and p53-mediated apoptosis in primary neural crest cells.

(A) ITS-1, a product of de novo rRNA synthesis, is reduced by 1hr following exposure to alcohol. (B) Nucleoli in neural crest are reduced and become diffused within 4hr of alcohol exposure, as visualized using immunostain for the nucleolar protein UBF. (C) p53 is stabilized in ALC neural crest within 2hr of exposure and this persists for at least 18hr. (D) Alcohol causes apoptosis in neural crest at 16hr post-exposure. Red signal is TUNEL, blue is DAPI stain to visualize nuclei. All studies use 80mM alcohol. Values are mean ± SD of N=3 independent experiments. * p<0.05, **p<0.01, ***p<0.001 compared with CON by either two-tailed T-test (D) or one-way (C) or two-way ANOVA (A). CON, control; ALC, alcohol-exposed.

Figure 2.. Alcohol reduces the positive effector of ribosome biogenesis S6K.

(A) Western blot analysis for phospho- and total S6K at the indicated times following alcohol exposure. Total S6K is reduced by 2hr post-exposure; the ratio of pS6K to S6K is reduced by 12hr as compared with control (CON). (B-F) Overexpression of gain-of-function caS6K (B) enhances its protein abundance in ALC but not CON neural crest, (C) elevates the abundance of pRPS6 at 12hr post-exposure, (D) normalizes rRNA synthesis in ALC neural crest and reduces it in CON at 2hr post-exposure, (E) prevents the elevated p53 content in ALC neural crest at 12hr, and (F) prevents alcohol-induced apoptosis, as assessed by TUNEL. All studies use 80mM alcohol. Values are mean ± SD with N=3 independent experiments and analyzed using one-way ANOVA compared with Time 0 (A), or two-way ANOVA compared with CON (B-F). * p<0.05, ** p<0.01, *** p<0.001.

Figure 3.. Alcohol represses the upstream S6K effector TORC1 in neural crest.

(A) Alcohol increases the active phospho-forms of the TORC1 upstream negative effectors pRaptor(S792) and pTSC2(S1387) within 2hr to 6hr of alcohol exposure. (B) Alcohol reduces the downstream TORC1 target p4E-BP1 at 12hr post-exposure. (C) Alcohol does not affect the abundance of the TORC2 effectors pRictor, Deptor, or PKCα at 12hr post-exposure. Values are mean ± SD with N=3 independent experiments and analyzed using one-way ANOVA (A) or two-tailed t-test (B, C) compared with CON. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4.. Treatment with the TORC1 inhibitor rapamycin exacerbates alcohol’s impact on rRNA synthesis, p53 stability, and apoptosis in neural crest.

(A) Rapamycin reduces pS6K in otherwise normal neural crest and further reduces it in ALC neural crest at 12hr post-exposure. (B) Rapamycin reduces rRNA synthesis in ALC but not CON neural crest at 2hr post-exposure. (C) Rapamycin stabilizes p53 protein in CON and ALC neural crest at 12hr post-exposure. (D) Rapamycin causes apoptosis in ALC but not CON neural crest at 16hr post-exposure. Values are mean ± SD with N=3 independent experiments and analyzed using two-way ANOVA compared with CON. * p<0.05, ** p<0.01, *** p<0.001.

Figure 5.. Treatment with the TORC1 activator L-leucine attenuates alcohol’s impact on rRNA synthesis, S6K loss, p53 stability, and apoptosis in neural crest.

(A) Pretreatment with L-leucine enhances rRNA synthesis in CON and normalizes it in ALC neural crest at 2hr post-exposure. (B) L-leucine normalizes total S6K in ALC neural crest at 12hr post-exposure. (C) L-leucine reduces p53 protein content in ALC neural crest at 12hr post-exposure. (D) L-Leucine normalizes apoptosis in ALC neural crest at 16hr post-exposure. For all, values are mean ± SD with N=3 independent experiments and analyzed using two-way ANOVA compared with CON. * p<0.05, ** p<0.01, *** p<0.001.

Figure 6.. Alcohol rapidly elevates pAMPK in neural crest, and the AMPK inhibitor dorsomorphin counters alcohol’s effects on AMPK-TORC1 signaling, rRNA synthesis, and p53-mediated apoptosis.

(A) Alcohol exposure increases pAMPK within 2hr of exposure and it remains elevated for at least 18hr. (B) The AMPK inhibitor dorsomorphin normalizes pAMPK, pRaptor, and pS6K in ALC neural crest at 12hr post-exposure. (C-E) Dorsomorphin also normalizes rRNA synthesis at 2hr (C), p53 protein content at 12hr (D), and apoptosis at 16hr (E) in ALC neural crest. Values are mean ± SD with N=3 independent experiments and analyzed using one-way (A) or two-way ANOVA (B-E) compared with CON. * p<0.05, ** p<0.01, *** p<0.001.

Figure 7.. The AMPK activator A769662 further alters AMPK and S6K, suppresses rRNA synthesis and exacerbates p53-mediated apoptosis in both ALC and CON neural crest.

(A) A769662 further exacerbates pAMPK activation and pS6K in ALC neural crest at 2hr post-exposure, and elevates pAMPK and reduces pS6K in otherwise normal neural crest. (B-D) A769662 further impacts rRNA synthesis (B), p53 protein content, (C), and apoptosis (D) in ALC neural crest, and suppresses rRNA synthesis and elevates p53 protein in CON. (E) L-Leucine further elevates pAMPK in ALC but not CON cells at 12hr post-exposure. Values are mean ± SD with N=3 independent experiments and analyzed using two-way ANOVA compared with CON. * p<0.05, ** p<0.01, *** p<0.001.

Figure 8.. Diagram of alcohol’s proposed mechanism of action in mediating neural crest apoptosis.

Data herein and in [24] suggest that alcohol’s activation of pAMPK activates the TORC1 repressors pTSC2 and pRaptor to reduces TORC1’s stimulation of ribosome biogenesis via S6K, resulting in nucleolar stress and the activation of p53/MDM2-mediated apoptosis.