Asymptomatic neurotoxicity of amyloid β-peptides (Aβ1-42 and Aβ25-35) on mouse embryonic stem cell-derived neural cells

Abstract

One of the strategies in the establishment of in vitro oxidative stress models for neurodegenerative diseases, such as Alzheimer's disease (AD), is to induce neurotoxicity by amyloid beta (Aβ) peptides in suitable neural cells. Presently, data on the neurotoxicity of Aβ in neural cells differentiated from stem cells are limited. In this study, we attempted to induce oxidative stress in transgenic 46C mouse embryonic stem cell-derived neurons via treatment with Aβ peptides (Aβ1-42 and Aβ25-35). 46C neural cells were generated by promoting the formation of multicellular aggregates, embryoid bodies in the absence of leukemia inhibitory factor, followed by the addition of all-trans retinoic acid as the neural inducer. Mature neuronal cells were exposed to different concentrations of Aβ1-42 and Aβ25-35 for 24 h. Morphological changes, cell viability, and intracellular reactive oxygen species (ROS) production were assessed. We found that 100 µM Aβ1-42 and 50 µM Aβ25-35 only promoted 40% and 10%, respectively, of cell injury and death in the 46C-derived neuronal cells. Interestingly, treatment with each of the Aβ peptides resulted in a significant increase of intracellular ROS activity, as compared to untreated neurons. These findings indicate the potential of using neurons derived from stem cells and Aβ peptides in generating oxidative stress for the establishment of an in vitro AD model that could be useful for drug screening and natural product studies.

FIGURE 1

(A) Morphological characteristics and population doubling time of undifferentiated 46C cells. Good quality 46C cells exhibit a high nucleus-to-cytoplasm ratio. The doubling time for 46C cells was 20.0 ± 2.07 h and is representative of randomly chosen passages (n = 12). (B) Immunostaining and flow cytometry analysis of pluripotency-associated protein markers: Oct4, SSEA1, and Nanog. Blue shows nuclear counterstain with DAPI, while green shows expression of Oct4 and SSEA1; red shows expression of Nanog. Flow cytometry analysis of 46C cells for Oct4, SSEA1, and Nanog. The percentage of fluorescent intensity of gated treated cells is shown on the histograms. (C) 46C cell line was able to generate good quality EBs that presented with cavitation process (center), smoothness of the boundary, and acceptable diameter (308.18 ± 28.16 mm; and is representative of randomly chosen passages; n = 12). The scale bars represent 100 mm (A) and (C); and 200 mm (B) for micrographs. Oct4: Octamer-binding transcription factor 4; SSEA1: Stage-specific embryonic antigen 1.

FIGURE 2

Expression of Sox1^eGFP during neural differentiation of 46C cells in vitro on D4, D6, D8, and D10 EBs. A1, B1, C1, and D1 show phase-contrast pictures of EBs and A2, B2, C2, and D2 fluorescence images of EBs with a clear demonstration of the highest Sox1^eGFP expression on D8. A3, B3, C3, and D3 demonstrate the merge. A4, B4, C4, and D4 demonstrate the flow cytometry analysis for Sox1^eGFP expression during neural differentiation of 46C cells showing that D8 EBs have the highest eGFP expression, which is an indicator of neural differentiation success. The scale bars represent 100 mm for micrographs. D: Day; EBs: Embryoid bodies; GFP: Green fluorescent protein.

FIGURE 3

Immunocytochemical analysis was performed to evaluate the expression of neural and glial cell proteins after neural differentiation of 46C cells on day 5 neuron post-plating on PDL/laminin-coated plate. The expression of (A) post-mitotic neurons (class III β-tubulin); (B) mature neurons (MAP2); (C) neurofilament; and (D) astrocytes (GFAP) was observed in the neuron cultures. Green indicates the expression of protein markers; meanwhile, nuclei were counterstained with PI (red) or DAPI (blue). Scale bar is 100 mm. PDL: Poly-D-Lysine; MAP2: Microtubule-associated protein 2; GFAP: Glial fibrillary acidic protein; PI: Propidium iodide.

FIGURE 4

Morphological appearance of amyloid beta (Aβ) fibrils under transmission electron microscope. Aβ fibril aggregates formed from Aβ_1-42 peptide demonstrating a “striated ribbon” morphology, with a diameter range from 6 to 15 nm (A), while, Aβ_25-35 peptide formed tiny and short Aβ aggregates (B). Scale bar is 200 nm.

FIGURE 5

(A) Neural-like cells derived from 46C cells were exposed to different concentrations of Aβ_1-42 and Aβ_25-35 fibrils for 24 h. About 25–100 μM of Aβ_1-42 fibrils induced a significant decrease in cell survival in a dose-dependent manner as compared to the control, while 50 μM of Aβ_25-35 fibrils decreased the cell viability significantly to 85%, though the IC₅₀ was not obtained. All data are expressed as mean ± SD (n = 3), where *p < 0.05 and ***p < 0.001 vs. untreated group (one-way ANOVA and post hoc Dunnett’s test). (B) Neural cells were not easily damaged by Aβ peptides, and neural cells remained intact when compared to untreated neural cells. Scale bar is 50 μm. (C) About 100 μM of Aβ_1-42 and 50 μM of Aβ_25-35 induced a significant increase in reactive oxygen species (ROS) levels as compared to untreated control. All data are expressed as mean ± SEM (n = 12), where **p < 0.01 and ***p < 0.001 vs. untreated group (one-way ANOVA and post hoc Bonferroni test).