Cytotoxic effects of G(M1) ganglioside and amyloid β-peptide on mouse embryonic neural stem cells

Abstract

AD (Alzheimer's disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between G(M1) ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells), but the precise effects of G(M1) and Aβ on NSCs have not yet been clarified. We evaluated the effect of G(M1) and Aβ-(1-40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either G(M1) or Aβ-(1-40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of G(M1) and Aβ-(1-40). The exogenously added G(M1) and Aβ-(1-40) were confirmed to incorporate into NECs. The Ras-MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with G(M1) and Aβ-(1-40), but caspase 3 was activated. NECs treated with G(M1) and Aβ-(1-40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that G(M1) and Aβ-(1-40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1-40) and G(M1) co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.

Keywords: AD, Alzheimer’s disease; Alzheimer’s disease (AD); Aβ, amyloid β-peptide; CCD, charge-coupled device; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular-signal-regulated kinase; FITC-Aβ-(1–40), FITC-conjugated Aβ-(1–40); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM1 ganglioside; IACUC, Institutional Animal Care and Use Committee; IL, interleukin; MAP2, microtubule-associated protein 2; MAPK, mitogen-activated protein kinase; N2-DMEM/F12, N2-supplemented DMEM/Ham’s Nutrient Mixture F12; NEC, neuroepithelial cell; NSC, neural stem cell; RT–PCR, reverse transcription–PCR; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; amyloid β-peptide (Aβ); apoptosis; bFGF, basic fibroblast growth factor; biotin-Ctxb, biotin-conjugated cholera toxin B subunit; glycosphingolipid; neural stem cell.

Figure 1. Effects of low concentrations of G_M1 and Aβ-(1–40) on NECs

The number of NECs cultured in the presence of bFGF (0 or 5 ng/ml), G_M1 (0, 1, 5 or 10 μM) and Aβ-(1–40) (0, 1 or 5 μM) for 4 days was estimated by the WST-8 assay. bFGF was added as a mitogen of NECs. The spectrophotometric attenuance (Abs.) measured at the wavelength of 450 nm (reference: 650 nm) by this assay is highly correlated with the number of living NECs.

Figure 2. Effects of high concentrations of G_M1 and Aβ-(1–40) on NECs

The number of NECs cultured in the presence of bFGF (0 or 5 ng/ml), G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) for four days was estimated by the WST-8 assay. Abs., absorbance.

Figure 3. Incorporation of exogenously added G_M1 and Aβ-(1–40) into NECs

NECs were cultured in the presence of G_M1 (0 or 40 μM) and FITC-Aβ-(1–40) (0 or 10 μM) for 2 days, and then stained with biotin-Ctxb and rhodamine-conjugated streptavidin. Nuclei were stained with Hoechst 33258.

Figure 4. Cell proliferation signalling in NECs treated with G_M1 and Aβ-(1–40)

(A) Activation of ERK (MAPK) in NECs treated with G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) for 2 days and then stimulated with bFGF (0 or 10 ng/ml) for 10 min was analysed by Western blotting. bFGF was used as an inducer of ERK activation. (B) Expression of p27^Kip1, a cyclin-dependent kinase inhibitor up-regulated by ganglioside stimulation and involved in ganglioside-induced inhibition of neural cell proliferation, in NECs treated with G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) for 2 days was analysed by Western blotting.

Figure 5. Neural lineage marker expression in NECs treated with G_M1 and Aβ-(1–40)

Expression of (A) nestin (a marker protein of neural stem cells) and (B) β-III tubulin (a marker protein of mature neurons) in NECs treated with G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) for 3 days was analysed by cell staining. Nuclei were stained with Hoechst 33258. (C) Expression of nestin and MAP2 (a marker gene of immature and mature neurons) in NECs treated with G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) for 2 days was analysed by RT–PCR. ‘G3PDH’ indicates control GAPDH. ‘−RT’ indicates negative controls without reverse transcription.

Figure 6. Apoptosis of NECs treated with G_M1 and Aβ-(1–40)

(A) Activation of caspase 3 (a critical executioner of apoptosis or programmed cell death signalling) in NECs treated with G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) in the presence of bFGF (5 ng/ml) for 2 days was analysed by Western blotting. An inhibitor of N-linked glycosylation, tunicamycin (1 μg/ml for 10 h), was used as a positive control to activate stress-mediated cell death signalling. (B) Apoptotic cells in NECs treated with G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) in the presence of bFGF (5 ng/ml) for 3 days were detected with the TUNEL assay. Nuclei were stained with Hoechst 33258. (C) The proportion of TUNEL-positive cells in NECs treated with or without G_M1 and Aβ-(1–40).

Figure 7. Effects of Aβ-(1–40) pre-incubated with G_M1 on NECs

To evaluate whether the cytotoxic effect of Aβ-(1–40) and G_M1 can be inhibited by sequestering, Aβ-(1–40) (0 or 10 μM) was pre-incubated with G_M1 (0 or 40 μM) at 37°C for 30 min to allow their binding. The number of NECs cultured in N2-DMEM/F12 containing the Aβ-(1–40) pre-incubated with G_M1 in the presence of bFGF (0 or 5 ng/ml) for 4 days was estimated by the WST-8 assay. Abs., absorbance.

Figure 8. Physical interaction of G_M1 and Ctxb (A) or Aβ-(1–40) (B)

(A) G_M1 (0, 1, 10, 100 or 1000 pmol per well) on polystyrene 96-well white plates was incubated with 1 μM of biotin-Ctxb and then 2 μg/ml of Cy2-conjugated streptavidin. (B) G_M1 (0, 1 or 10 nmol per well), cholesterol (0 or 7.5 nmol per well) and sphingomyelin (0 or 7.5 nmol per well) on polystyrene 96-well white plates were incubated with 5 μM of FITC-Aβ-(1–40). The fluorescence intensities, which reflect the physical interaction of G_M1 with Ctxb or FITC-Aβ-(1–40), were measured using a Victor³ V multilabel plate reader equipped with a λ_ex = 485 nm filter and a λ_em = 535 nm filter.

Figure 9. Effects of G_M1 and Aβ-(1–40) on NSCs

(A) NSCs prepared from striata of mouse embryos in the form of neurospheres at 0, 3 and 7 days in vitro (DIV). (B) NSCs stained with subclass control IgG (coIgG) or anti-nestin antibody. Nuclei were stained with Hoechst 33258. (C) Apoptotic cells in NSCs treated with G_M1 (0 or 40 μM) and Aβ-(1–40) (0 or 10 μM) in the presence of bFGF (5 ng/ml) for 3 days were detected with the TUNEL assay. Nuclei were stained with Hoechst 33258. (D) The proportion of TUNEL-positive cells in NSCs treated with or without G_M1 and Aβ-(1–40).