Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner

Abstract

Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer's disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.

Keywords: Astrocytic differentiation; Developmental neurotoxicity; Idebenone; Mitochondrial biogenesis; Neural progenitors; Neuronal; hiPSC; mtDNA copy number.

Fig. 1

Immunocytochemical confirmation of: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). NSC culture exhibit dense and packed morphology, expressing the high level of early neural marker Nestin, proliferation marker Ki67 and early neuronal marker Doublecortin (DCX). During differentiation process, the morphology of cells changed to more elongated, branched, with decreasing Ki67 expression and increasing more advanced neuronal (β-TUBULIN3, MAP2, NF200) and astrocytic (GFAP) markers. Scale bar 50 µm

Fig. 2

The cells at three different stages of neural differentiation: NSC, eNP and NP after 5 days of exposure to the different concentrations of idebenone were tested for (a) viability measured by Alamar Blue assay; (b) ROS level measured by DCFH-DA assay; (c) the mitochondrial membrane potential measured by MitoTracker Red CMXRos staining. After normalization to total cell number results are shown the mean (SEM) of fluorescencee intensity (%) of treated group versus control. Brackets show statistical significance between samples versus control (one way ANOVA, Tukey’s post-test): *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 and comparison between groups (two way ANOVA, Bonferroni post-test): ^#p < 0.05; ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001

Fig. 3

In the developmental stages: NSC, eNP and NP after 5 days exposure to the different concentrations of idebenone. a The Succinate Dehydrogenase Complex Flavoprotein Subunit A (SDHA) level. b The Cyclooxygenase (COX-1) level; c) Total cell number obtained from standard curve (Janus green staining) was measured. Results are shown as mean (SEM) relative percent (%) of absorbance versus control. The SDHA and COX-1 protein level were normalized to cell number with Janus green staining kit. Brackets show statistical significance between samples versus control (one way ANOVA, Tukey’s post-test): *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; and comparison between groups (two way ANOVA, Bonferroni post-test): ^#p < 0.05; ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001

Fig. 4

The relative mtDNA copy number estimated with qPCR measurement of ND1, ND5, SLCO2B1 and SERPINA1 level in NSC, eNP and NP after 5 days of exposure to the 0.5 µM idebenone: a ND1/SLCO2B1 ratio and b ND5/SERPINA1 ratio. Data are presented as mean (SEM). Brackets show statistical significance between samples versus control, (t-Student): *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 and comparison between groups (two way ANOVA, Bonferroni post-test): ^#p < 0.05; ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001

Fig. 5

Real-time (RT-qPCR) evaluation of expression of genes involved in mitochondrial biogenesis and neural differentiation at the NSC, eNP, NP developmental stages. Relative expression of a NRF1, b PPARGC1A, c TFAM, d GFAP, e MAP2 was measured after 5 days of cell treatment with 0.5 µM idebenone. Data are presented as the mean (SEM) from three independent experiments, each in four replicates. Brackets show statistical significance comparison between groups (one way ANOVA, Tukey’s post-test): *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001