DSCAM/PAK1 pathway suppression reverses neurogenesis deficits in iPSC-derived cerebral organoids from patients with Down syndrome
Xiao-Yan Tang, Lei Xu, Jingshen Wang, Yuan Hong, Yuanyuan Wang, Qian Zhu, Da Wang, Xin-Yue Zhang, Chun-Yue Liu, Kai-Heng Fang, Xiao Han, Shihua Wang, Xin Wang, Min Xu, Anita Bhattacharyya, Xing Guo, Mingyan Lin, Yan Liu
Xiao-Yan Tang, Lei Xu, Jingshen Wang, Yuan Hong, Yuanyuan Wang, Qian Zhu, Da Wang, Xin-Yue Zhang, Chun-Yue Liu, Kai-Heng Fang, Xiao Han, Shihua Wang, Xin Wang, Min Xu, Anita Bhattacharyya, Xing Guo, Mingyan Lin, Yan Liu
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Research Article Neuroscience

DSCAM/PAK1 pathway suppression reverses neurogenesis deficits in iPSC-derived cerebral organoids from patients with Down syndrome

Abstract

Down syndrome (DS), caused by trisomy of chromosome 21, occurs in 1 of every 800 live births. Early defects in cortical development likely account for the cognitive impairments in DS, although the underlying molecular mechanism remains elusive. Here, we performed histological assays and unbiased single-cell RNA-Seq (scRNA-Seq) analysis on cerebral organoids derived from 4 euploid cell lines and from induced pluripotent stem cells (iPSCs) from 3 individuals with trisomy 21 to explore cell-type–specific abnormalities associated with DS during early brain development. We found that neurogenesis was significantly affected, given the diminished proliferation and decreased expression of layer II and IV markers in cortical neurons in the subcortical regions; this may have been responsible for the reduced size of the organoids. Furthermore, suppression of the DSCAM/PAK1 pathway, which showed enhanced activity in DS, using CRISPR/Cas9, CRISPR interference (CRISPRi), or small-molecule inhibitor treatment reversed abnormal neurogenesis, thereby increasing the size of organoids derived from DS iPSCs. Our study demonstrates that 3D cortical organoids developed in vitro are a valuable model of DS and provide a direct link between dysregulation of the DSCAM/PAK1 pathway and developmental brain defects in DS.

Authors

Xiao-Yan Tang, Lei Xu, Jingshen Wang, Yuan Hong, Yuanyuan Wang, Qian Zhu, Da Wang, Xin-Yue Zhang, Chun-Yue Liu, Kai-Heng Fang, Xiao Han, Shihua Wang, Xin Wang, Min Xu, Anita Bhattacharyya, Xing Guo, Mingyan Lin, Yan Liu

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Figure 7

Knocking down DSCAM rescues abnormal neurogenesis in DS-derived cortical cultures.

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Knocking down DSCAM rescues abnormal neurogenesis in DS-derived cortical...
(A) Representative images of day-30 organoids stained with Hoechst, which show the quantitation of the different parameters. Scale bar: 50 μm. (B and C) Quantitation of the basal membrane length and loop tissue area in the neuroepithelial loops of trisomy 21 and euploid organoids after 30 days of differentiation. Organoids (n ≥15) from 3 independent biologic replicate experiments were analyzed for each cell line. Data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by 1-way ANOVA followed by Dunnett’s multiple-comparison test. (D) Representative images of day-30 trisomy 21 and DSCAM-KD organoids stained for Ki67, SOX2, PAX6, MAP2, CTIP2, and TUJ1 expression. Scale bar, 50 μm. (E) Quantification of the proportion of Ki67+, SOX2+, and PAX6+ cells in day-30 trisomy 21 and DSCAM-KD organoids. n = 17–33 VZ-like regions in at least 10 organoids per cell line. Data represent the mean ± SEM. **P < 0.01 and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s multiple-comparison test. (F and G) Immunocytochemical staining and quantification of CTIP2+ cells in both trisomy 21 and DSCAM-KD organoids after 50 days of differentiation. n = 13–15 neural tube–like regions in at least 7 organoids per cell line. Data represent the mean ± SEM. ***P < 0.001, by 1-way ANOVA followed by Dunnett’s multiple-comparison test. Scale bar: 35 μm. (H and I) Immunocytochemical staining and quantification of the proportion of SATB2+ cells in both trisomy and DSCAM-KD organoids on day 70. n = 11 neural tube–like regions in at least 6 organoids per cell line. Data represent the mean ± SEM. ***P < 0.001, by Student’s t test. Scale bar: 35 μm.