The Pluripotency Factor Nanog Protects against Neuronal Amyloid β-Induced Toxicity and Oxidative Stress through Insulin Sensitivity Restoration

Abstract

Amyloid β (Aβ) is a peptide fragment of the amyloid precursor protein that triggers the progression of Alzheimer's Disease (AD). It is believed that Aβ contributes to neurodegeneration in several ways, including mitochondria dysfunction, oxidative stress and brain insulin resistance. Therefore, protecting neurons from Aβ-induced neurotoxicity is an effective strategy for attenuating AD pathogenesis. Recently, applications of stem cell-based therapies have demonstrated the ability to reduce the progression and outcome of neurodegenerative diseases. Particularly, Nanog is recognized as a stem cell-related pluripotency factor that enhances self-renewing capacities and helps reduce the senescent phenotypes of aged neuronal cells. However, whether the upregulation of Nanog can be an effective approach to alleviate Aβ-induced neurotoxicity and senescence is not yet understood. In the present study, we transiently overexpressed Nanog-both in vitro and in vivo-and investigated the protective effects and underlying mechanisms against Aβ. We found that overexpression of Nanog is responsible for attenuating Aβ-triggered neuronal insulin resistance, which restores cell survival through reducing intracellular mitochondrial superoxide accumulation and cellular senescence. In addition, upregulation of Nanog expression appears to increase secretion of neurotrophic factors through activation of the Nrf2 antioxidant defense pathway. Furthermore, improvement of memory and learning were also observed in rat model of Aβ neurotoxicity mediated by upregulation of Nanog in the brain. Taken together, our study suggests a potential role for Nanog in attenuating the neurotoxic effects of Aβ, which in turn, suggests that strategies to enhance Nanog expression may be used as a novel intervention for reducing Aβ neurotoxicity in the AD brain.

Keywords: Nanog; amyloid β; insulin signaling; oxidative stress; senescence.

Figure 1

Overexpression of Nanog suppresses Aβ-induced apoptosis in human neuronal cells (SK-N-MC). Quantification of Nanog expression using real-time PCR (a) and western blot (b). At 24 h post-transfection, both mRNA and protein levels of Nanog were significantly upregulated, indicating the successful overexpression of Nanog; (c) treatment with 2.5 µM of Aβ_1–42 for 24 h markedly induced morphologic changes and cell death and overexpression of Nanog prevented these effects by Aβ; (d) tetrazolium salt methyl-thiazol-tetrazolium (MTT) assays demonstrated that overexpression of Nanog significantly protected against Aβ-induced cytotoxicity; (e) overexpression of Nanog markedly reduced Aβ-induced nucleus fragmentation, determined using 4’,6-diamidino-2-phenylindole (DAPI) staining. The percentage of apoptotic cells was calculated from five random fields; (f) western blotting results revealed that overexpression of Nanog inhibited Aβ-induced caspase-3 and polymerase (PARP) cleavages, two hallmarks of apoptosis. Values were expressed as means ± SEM from at least three independent experiments. The significance of differences was determined through unpaired Student’s t-test or multiple comparisons with Dunnett’s post hoc test at * p < 0.05 and ** p < 0.01 compared with the indicated groups. Scale bar represents 20 μm.

Figure 2

Overexpression of Nanog inhibits Aβ-induced tau phosphorylation and cytotoxicity by restoring impaired insulin signaling; (a) western blots showed that treatment with 2.5 µM of Aβ for 24 h induces a marked increase of insulin receptor substrate 1 (IRS-1) Ser³⁰⁷ phosphorylation, a major marker of insulin resistance. However, overexpression of Nanog greatly inhibited this phosphorylation; (b) western blot analysis of Ser⁴⁷³ phosphorylated Akt confirmed that overexpression of Nanog reverses Aβ-induced insulin signaling blockade; (c) western blot results indicated that overexpression of Nanog inhibits Aβ-induced tau phosphorylation at Thr²³¹ and increases the phosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser⁹; (d) Bright field images showed that Nanog-induced protection is abolished by the co-treatment with LY294002 (20 µM), a specific inhibitor of PI3-kinase; (e) MTT assays showed the LY294002 co-treatment inhibits the protective effect of Nanog; (f) LY294002 markedly reduced Nanog-induced anti-nucleus fragmentation effects determined by using DAPI staining. The arrows indicated apoptotic cells with nuclear fragmentation; (g) western blots showed that LY294002 markedly prevents the reduction of caspase-3 and PARP cleavages, suggesting the protection by Nanog is dependent on insulin signaling. Values were expressed as means ± SEM from at least three independent experiments. The significance of differences was determined through multiple comparisons with Dunnett’s post hoc test at * p < 0.05 and ** p < 0.01 compared with the indicated groups. Scale bar represents 20 μm.

Figure 3

Nanog overexpression ameliorates Aβ-induced mitochondrial superoxide accumulation and cellular senescence. (a) Dihydroethidium (DHE) staining results showed that overexpression of Nanog reduces Aβ-induced intracellular superoxide accumulation; (b) levels of two antioxidant signaling-related proteins nuclear factor erythroid 2-related factor 2 (Nrf2) and superoxide dismutase 1 (SOD1) were analyzed by western blotting; (c) double fluorescence staining of mitochondrial membrane potential by JC-1 was used for detection of mitochondrial membrane potential. Green fluorescence indicated the decreased membrane potential in 2.5 µM of Aβ-treated SK-N-MC cells after 24 h. Red fluorescence indicated that overexpression of Nanog effectively preserves the mitochondrial membrane potential; (d) mRNA levels of neurotrophic factors insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF) were measured by performing qPCR. mRNA levels of IGF-1 and BDNF were significantly increased in Aβ treated Nanog-overexpressed cells; (e) representative results of cytochemical detection of SA-β-galactosidase, a common biomarker used in detecting senescent cells. Senescent cells (red arrows) are blue stained under a bright-field microscope; (f) levels of sirtuin-1 (Sirt1) protein in Aβ-treated cells using western blotting. Values were expressed as means ± SEM from at least three independent experiments. The significance of differences was determined through multiple comparisons with Dunnett’s post hoc test at * p < 0.05 and ** p < 0.01 compared with the indicated groups. LY294002, a PI3K inhibitor for reducing insulin signaling.

Figure 4

Upregulation of Nanog in rat brains improves working and recognition memory deficits induced by Aβ. (a) The experimental protocol of the behavioral tests; (b) six weeks after stereotaxical injection, mRNA levels of Nanog were measured by performing qPCR. Nanog mRNA levels in hippocampus and cortex were significantly increased in Nanog-overexpressed group; (c) western blotting revealed that overexpression of Nanog markedly inhibited IRS-1 Ser³⁰⁷ and restored Akt Ser⁴⁷³ phosphorylation in rat hippocampus; (d) behavioral testing was performed at 6 weeks after stereotaxical surgery. Results showed the percentage of correct responses to select the arm with the reward and time latency to finish each session for the T-maze test; (e) percentage of time spent exploring new or old objects in object recognition test. Upregulation of Nanog in the brain significantly increased the ratio of novel/familiar object exploring time compared to Aβ-injected group. Values were expressed as means ± SEM from at least three independent experiments. The significance of differences was determined through unpaired Student’s t-test or multiple comparisons with Dunnett’s post hoc test at * p < 0.05 and ** p < 0.01 compared with the indicated groups. N.S.: not significant.