Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities

Abstract

Essential E3 ubiquitin ligase HUWE1 (HECT, UBA, and WWE domain containing 1) regulates key factors, such as p53. Although mutations in HUWE1 cause heterogenous neurodevelopmental X-linked intellectual disabilities (XLIDs), the disease mechanisms common to these syndromes remain unknown. In this work, we identify p53 signaling as the central process altered in HUWE1-promoted XLID syndromes. By focusing on Juberg-Marsidi syndrome (JMS), one of the severest XLIDs, we show that increased p53 signaling results from p53 accumulation caused by HUWE1 p.G4310R destabilization. This further alters cell-cycle progression and proliferation in JMS cells. Modeling of JMS neurodevelopment reveals majorly impaired neural differentiation accompanied by increased p53 signaling. The neural differentiation defects can be successfully rescued by reducing p53 levels and restoring the expression of p53 target genes, in particular CDKN1A/p21. In summary, our findings suggest that increased p53 signaling underlies HUWE1-promoted syndromes and impairs XLID JMS neural differentiation.

Keywords: E3 ubiquitin ligase; HUWE1; X-linked intellectual disability; neurodevelopment; p53.

Graphical abstract

Figure 1

p53 signaling is hyperactivated in cells from XLID patients with mutated HUWE1 (A) Schematic representation of XLID-causative HUWE1 mutations analyzed in this study (p.R2981H, p.R4187C, and JMS-p.G4310R; HUWE1 duplication: 2×). Depicted HUWE1 domains: ARLD1/2, Armadillo repeat-like domains 1/2; UBA, ubiquitin-association domain; WWE, tryptophan-tryptophan-glutamate domain; BH3, Bcl-2 homology 3 domain; HECT, homologous to E6-AP carboxyl terminus domain. (B) Top five most significant KEGG pathway terms as determined by gene set enrichment analysis (GSEA) of common differentially expressed genes in XLID patient-derived lymphoblastoid cells (LCs) compared with healthy individual cells (Benjamini corrected p < 0.05). (C) Heatmap of common differentially expressed genes in XLID compared with healthy LCs belonging to the KEGG p53 signaling term. (D–F) mRNA levels of p53 target genes CDKN1A/p21 (D), GADD45α (E), and BBC3/PUMA (F) determined by qRT-PCR analysis of four healthy (GM03798, GM07535, GM16113, and GM16119) and five XLID LCs with mutated HUWE1 (p.R2981H, p.R4187C, JMS-p.G4310R, and HUWE1 duplication). All error bars indicate mean ± SEM (n = 3, biological replicates). Two-tailed unpaired t test; ∗∗p ≤ 0.01, ∗∗∗∗p ≤ 0.0001.

Figure 2

p53 accumulation and activation, caused by HUWE1 p.G4310R, perturb the cell cycle and impair proliferation in JMS patient-derived cells (A) Immunoblot analysis of the HUWE1, p53, and p21 protein levels in healthy control LCs and LCs from two JMS patients (JMS1 and JMS2). p53 protein levels relative to tubulin, serving as loading control, are indicated. (B) In vitro ubiquitination of purified recombinant p53 with increasing amounts of wild-type (WT) and p.G4310R HECT proteins. (C) In silico analysis of difference in the folding free energy change (ΔΔG) of WT and G4310R HUWE1 using the indicated prediction tools. (D) Cell-cycle distribution determined by flow cytometry of healthy control, JMS1, and JMS2 LCs. (E) Fraction of annexin V-positive apoptotic cells measured by flow cytometry. (F) Proliferation rate of healthy control, JMS1, and JMS2 LCs. All error bars indicate mean ± SEM (n ≥ 3, biological replicates). Statistical significance was determined by two-way ANOVA with Tukey post-test (D) and Bonferroni post-test (F); one-way ANOVA with Bonferroni post-test; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, n.s. ≥ 0.05. See also Figure S1.

Figure 3

Neural differentiation of JMS patient hiPSCs is impaired and accompanied by activation of p53 signaling (A) Representative bright-field images of neural differentiation of healthy control hiPSCs (WT-DYS0100 and WT-CRL(S)23) and two hiPSC clones from a JMS patient expressing HUWE1 p.G4310R (JMS-cl.1 and JMS-cl.2) at day 11; neural rosette structures are visible. (B) Relative number of rosettes formed in WT and JMS clones (n ≥ 3, biological replicates). (C–F) qRT-PCR analysis of gene expression of OCT4 (C), NES/NESTIN (D), TUBB3/TUJ1 (E), and DCX (F) in WT and JMS hiPSCs and neural cells (collected at day 13) (n = 3, biological replicates). (G) Immunofluorescence analysis of TUJ1 in WT and JMS hiPSCs at day 13 of neural differentiation. (H) Relative intensity of the TUJ1 signal in WT and JMS clones from experiments like the one in (G) (n ≥ 2, biological replicates). (I–L) qRT-PCR analysis of mRNA levels of the p53 target genes CDKN1A/p21 (I), GADD45α (J), BBC3/PUMA (K), and BAX (L) in WT and JMS hiPSCs and neural cells (collected at day 13) (n = 3, biological replicates). All error bars indicate mean ± SEM; one-way ANOVA followed by Tukey post-test (B); two-way ANOVA followed by Tukey post-test (C–F and I–L); ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, n.s. ≥ 0.05. Scale bar: 400 μm. See also Figures S2 and S3.

Figure 4

p53 downregulation rescues neurodevelopmental defects in XLID JMS patient hiPSCs (A) Relative number of rosettes upon neural differentiation of WT-DYS0100, JMS-cl.1, and JMS-cl.1-expressing shRNA control (shCtrl) or shRNA targeting p53 (shP53a and shP53b) hiPSCs (n ≥ 3, biological replicates). JMS cells harbor HUWE1 p.G4310R. (B–D) mRNA expression levels of NES/NESTIN (B), TUBB3/TUJ1 (C), and DCX (D) analyzed by qRT-PCR in WT and JMS neural cells (collected at day 13) addressed by qRT-PCR. (E) Immunofluorescence analysis of the TUJ1 signal in WT-DYS0100, JMS-cl.1, JMS-cl.1 shCtrl, and JMS-cl.1 shP53a and shP53b at day 13 of neural differentiation. (F–H) qRT-PCR analysis of CDKN1A/p21 (F), GADD45α (G), and BAX (H) expression in WT and JMS neural cells (collected at day 13). All error bars indicate mean ± SEM (n = 3, biological replicates); one-way ANOVA followed by Dunnett’s post-test (A); one-tailed t test (B–D and F–H); ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, n.s. ≥ 0.05. See also Figures S4 and S5.