Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

Abstract

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1(G93A) individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dismutase 1 mutant (SOD1(G93A)) and wild-type (SOD1(WT)) mouse models were exposed to H2O2. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenosine monophosphate-activated protein kinase (AMPK) α-subunit, paired box 3 (Pax3) protein, and p53 in western blot analyses. Compared with SOD1(WT) cells, SOD1(G93A) embryonic neural stem cells were more likely to undergo H2O2-induced apoptosis. Phosphorylation of AMPKα in SOD1(G93A) cells was higher than that in SOD1(WT) cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKα. p53 protein levels were also correlated with AMPKα phosphorylation levels. Compound C, an inhibitor of AMPKα, attenuated the effects of H2O2. These results suggest that embryonic neural stem cells from SOD1(G93A) mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKα pathway.

Keywords: NSFC grants; SOD1G93A mouse; adenosine monophosphate-activated protein kinase α; amyotrophic lateral sclerosis; apoptosis; embryonic neural stem cells; hydrogen peroxide; nerve regeneration; neural regeneration; neuroderegeneration; oxidative stress; p53; paired box 3.

Figure 1

Morphology of neural stem cells prepared from embryos of SOD1^WT and SOD1^G93A mice at 1, 3, and 5 days after culture (D1, 3, and 5) (inverted microscope). Embryonic neural stem cells formed typical neurospheres after in vitro culture. SOD1: Superoxide dismutase 1.

Figure 2

Identification of neural stem cells by double immunofluorescence staining of Nestin (red) and Sox2 (green) (immunocytochemistry, fluorescence microscope). Nestin and Sox2 were expressed in the neural stem cells derived from mouse embryos. Blue: DAPI-labeled nuclei. SOD1: Superoxide dismutase 1.

Figure 3

Neural stem cells (NSCs) in SOD1^G93A mice are more susceptible to oxidative injury induced by H₂O₂ (cell viability and proliferation assays). (A) Effects of H₂O₂ on the cell viability of NSCs. The absorbance value of untreated SOD1^WT NSCs was set as 100%. (B) Effect of H₂O₂ on the pro-liferation of NSCs. *P < 0.05; **P < 0.01. Data are presented as the mean ± SD and analyzed using one-way analysis of variance followed by the least significant difference post-hoc test. Each set of measurements was performed in triplicate in at least five independent experiments. H₂O₂ (–) indicates no treatment; h: hour(s); SOD1: superoxied dismutase 1.

Figure 4

Neural stem cells (NSCs) in SOD1^G93A mice are more susceptible to apoptosis induced by H₂O₂ (flow cytometry). (A) Representative scatter plots showing apoptosis in H₂O₂-treated NSCs by Annexin V-FITC and PI staining and flow cytometry. (I) Late apoptotic cells; (II) early apoptotic cells; (III) intact cells; (IV) necrotic cells. (B) Quantification of apoptotic cells. NSCs were cultured for 36 hours and then treated with H₂O₂ for 12 hours, followed by apoptosis detection. *P < 0.05; **P < 0.01. The values are the mean ± SD of five independent experiments (one-way analysis of variance followed by the least significant difference post-hoc test). H₂O₂ (–) indicates no treatment. SOD1: Superoxide dismutase 1.

Figure 5

Adenosine monophosphate-activated protein kinase (AMPK) phosphorylation levels are increased in SOD1^G93A neural stem cells (NSCs) by H₂O₂ exposure with corresponding changes in Pax3 and p53 levels (western blot analysis). (A) Representative western blots displaying expression or activation of AMPK α-subunit (AMPKα), paired box 3 (Pax3), and p53 in NSCs treated with or without H₂O₂. β-Actin was used as a loading control. Data below each lane represent the relative gray scale compared with β-actin control bands. Values below SOD1^WT NSCs treated without H₂O₂ were set as 1. (B) Phosphorylation levels of AMPKα were quantified relative to the total AMPKα level. The expression level in untreated SOD1^WT NSCs was set at 100%. *P < 0.05; **P < 0.01. Data are presented as the mean ± SD and analyzed using one-way analysis of variance followed by the least significant difference post-hoc test. (–): Untreated; WT: superoxide dismutase 1 wild-type neural stem cells; G93A: SOD1^G93A neural stem cells; p-AMPKα: phosphorylated AMPKα; t-AMPKα: total AMPKα. SOD1: Superoxide dismutase 1.

Figure 6

Quantitative real-time PCR analysis of the mRNA expression of adenosine monophosphate (AMP)-activated protein kinase α1 subunit (AMPKα₁) (A), AMP-activated protein kinase α2 subunit (AMPKα₂) (B), paired box 3 (Pax3) (C), and p53 (D) in neural stem cells under different treatment conditions. H₂O₂ (–): Untreated. *P < 0.05; **P < 0.01. Data are presented as the mean ± SD and analyzed using one-way analysis of variance followed by the least significant difference post-hoc test. SOD1: Superoxide dismutase 1.

Figure 7

Effects of adenosine monophosphate-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound (CC) on the viability and apoptosis of neural stem cells (NSCs). (A) Cell viability was significantly affected by the addition of AICAR or compound C. The absorbance value of untreated superoxide dismutase 1 wild-type (SOD1^WT) NSCs was set at 100%. (B) Quantitative assessment of NSC apoptosis by AnnexinV-FITC and PI staining and flow cytometry under different treatment conditions. H₂O₂ (–): Untreated. *P < 0.05; **P < 0.01. Data are presented as the mean ± SD and analyzed using one-way analysis of variance followed by the least significant difference post-hoc test. FITC: Fluoresscen isothiocyante; PI: propidium iodide.

Figure 8

Effects of adenosine monophosphate-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound (CC) on the phosphorylation of AMPKα and protein levels of Pax3 and p53 in neural stem cells (NSCs) (western blot analysis). (A) Representative western blots displaying the phosphorylation of AMPKα, paired box 3 (Pax3), and p53 protein in NSCs exposed to different fac-tors. β-Actin was used as a loading control. Data below each lane represent the relative gray scale compared with β-actin control bands. (B) Phos-phorylation levels of AMPKα relative to total AMPKα levels. The expression level in untreated superoxide dismutase 1 wild-type (SOD1^WT) NSCs was set at 100%. Data are presented as the mean ± SD and analyzed using one-way analysis of variance followed by the least significant difference post-hoc test. p-AMPKα: Phosphorylated AMPKα; t-AMPKα: total AMPKα; H₂O₂ (–): Untreated; WT: SOD1^WT NSCs; G93A: SOD1^G93A NSCs *P < 0.05; **P < 0.01; SOD1: Superoxide dismutase 1.