1State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. 2South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China. 3Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China.
Induced pluripotent stem cells (iPSCs)-derived dopaminergic neurons might be reset back to the fetal state due to reprogramming. Thus, it is a compelling challenge to reliably and efficiently induce disease phenotypes of iPSCs-derived dopaminergic neurons to model late-onset Parkinson’s disease (PD). Here, we applied a small molecule, hydroxyurea (HU), to promote the manifestation of disease relevant phenotypes in iPSCs-based modeling of PD. We established two iPS cell lines derived from two sporadic PD patients. Both patients-iPSCs-derived dopaminergic neurons did not display PD relevant phenotypes after 6 weeks culture. HU treatment remarkably induced ER stress on patients-iPSCs-derived dopaminergic neurons. Moreover, HU treatment significantly reduced neurite outgrowth, decreased the expression of p-AKT and its downstream targets (p-4EBP1 and p-ULK1), and increased the expression level of cleaved-Caspase 3 in patients-iPSCs-derived dopaminergic neurons. The findings of the present study suggest that HU administration could be a convenient and reliable approach to induce disease relevant phenotypes in PD-iPSCs-based models, facilitating to study disease mechanisms and test drug effects.
Tan Yuan,Ke Minjing,Huang Zhijian, et al. Hydroxyurea Facilitates Manifestation of Disease Relevant Phenotypes in Patients-Derived IPSCs-Based Modeling of Late-Onset Parkinson’s Disease[J]. Aging and disease,
2019, 10(5): 1037-1048.
Figure 1. Hyroxyurea (HU) induced senescence-associated features in fibroblasts. (A) representative images of cell morphology and growth of human fibroblasts after treatment of HU at various concentrations for 4 days. (B) Cell number quantification after treatment of HU for 4 days. The cell number was represented by the percentage of survived cells after HU treatment compared to the total cells in the control group without HU treatment. The results were obtained from 3 independent experiments. (C) Representative images of the fibroblasts with 8 mM HU treatment for 4 days. (D) The number of cells in the visual field of phase contrast microscope. E: Representative images of the fibroblasts stained with Ki67, γH2AX, and SA-β-Gal. F-H: Quantification of positive staining and comparisons between the control group without HU treatment (Ctrl group) and the HU treatment group. The results were presented with averages ± SEM from 3 independent experiments, *p < 0.05, **p <0.01. Scale bar: 50 μm for A and E, 200 μm for C.
Figure 2. The dopaminergic differentiation of UC-12-iPSCs. (A) The workflow used dopaminergic differentiation of UC-12-iPSCs. (B) A representative phase contrast image of the UC-12-iPSCs. (C) A representative phase contrast image of UC-12-iPSCs under differentiation. (D) A representative image of LMX1A-positive midbrain floorplate precursors derived from the UC-12-iPSCs. E: A representative image of FOXA2-positive midbrain floorplate precursors derived from the UC-12-iPSCs. (F-I) Representative images of the dopaminergic neurons TH (F), Girk2 (G), Nurr1 (H), and DAT (I). Scale bar: 75 μm.
Figure 3. ER stress was induced by HU treatment in the UC-12-iPSCs-derived dopaminergic neurons. (A, B) The neurite length in the UC-12-iPSCs-derived dopaminergic neurons was dramatically reduced after 4 days of the HU treatment. The neurite was revealed by MAP2 staining. (C, D) Western blot analysis showed that the HU treatment decreased the expression of Tuj1 in UC-12-iPSCs-derived dopaminergic neurons. (E) The number of differentially expressed genes between the control group and the HU treatment group is shown with a Venn diagram. The middle circle indicated the number of mutual expressed genes between the control group and the HU treatment group. (F) The statistics of pathway enrichment analysis for the HU-induced aging in the UC-12-iPSCs-derived dopaminergic neurons. (G) Altered expression levels of genes related to the ER stress pathway. (H) The ER stress related genes were verified by qPCR. (I-J) The expression level of the key proteins in the ER stress pathways was further verified and quantified by western blot analysis. Scale bar: 45 μm for A and 120 μm for C.
Figure 4. Dopaminergic neurons differentiated from the SPD-1 and the SPD-2 iPSCs. (A) Representative immunostaining images on differentiated neurons derived from the SPD-1 and the SPD-2 iPSCs. (B-D) The western blot analysis demonstrated that a lengthy culture did not enhanced the expression of key molecules related to PD-specific phenotypes in either the SPD-1 or the SPD-2 iPSCs-derived dopaminergic neurons. Scale bar: 80 μm.
Figure 5. The PD-iPSCs-based disease phenotypes were induced via the HU treatment. (A) The HU treatment significantly reduced the neurite length in both the SPD-1 and the SPD-2 iPSCs-derived dopaminergic neurons. (B, C) Western blot analysis demonstrated that the HU treatment enhanced the expression level of the PD-associated proteins in the SPD-1 iPSCs-derived dopaminergic neurons. (D, E) Western blot analysis demonstrats that the HU treatment enhanced the expression level of the PD-associated proteins in SPD-2 iPSCs-derived dopaminergic neurons. (F, G) The ER stress related genes were verified by the qPCR in the HU-treated PD iPSC-derived dopaminergic neurons. (H, I) The expression level of the key proteins in the ER stress pathways in the HU-treated SPD-1 and the SPD-2 iPSCs-derived dopaminergic neurons were further verified and quantified by western blot analysis. J: The expression of the genes related to the ER stress pathways were further verified by the qPCR in the HU-treated PD iPSC-derived dopaminergic neurons. (K) Simplified scheme of the unfolded protein response (UPR) signaling pathway. Three different branches of the UPR were activated by ER stress: IRE1, PERK, and ATF6. The IRE1 spliced the cytosolic XBP1 mRNA to obtain the XBP1s transcription factor, which helped to resolve the ER stress [34, 35]. The PERK initiated the UPR by phosphorylation of eIF2a, which attenuated the global protein synthesis and contributed to restoring the ER homeostasis . Yellow highlighted arrows indicate the two branches of the UPR signaling pathways that could take part in HU-induced cellular senescence. Scale bar: 80 μm.
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