Patient iPSC-derived neural progenitor cells display aberrant cell cycle control, p53, and DNA damage response protein expression in schizophrenia

Abstract

Background: Schizophrenia (SCZ) is a severe psychiatric disorder associated with alterations in early brain development. Details of underlying pathomechanisms remain unclear, despite genome and transcriptome studies providing evidence for aberrant cellular phenotypes and pathway deregulation in developing neuronal cells. However, mechanistic insight at the protein level is limited.

Methods: Here, we investigate SCZ-specific protein expression signatures of neuronal progenitor cells (NPC) derived from patient iPSC in comparison to healthy controls using high-throughput Western Blotting (DigiWest) in a targeted proteomics approach.

Results: SCZ neural progenitors displayed altered expression and phosphorylation patterns related to Wnt and MAPK signaling, protein synthesis, cell cycle regulation and DNA damage response. Consistent with impaired cell cycle control, SCZ NPCs also showed accumulation in the G2/M cell phase and reduced differentiation capacity. Furthermore, we correlated these findings with elevated p53 expression and phosphorylation levels in SCZ patient-derived cells, indicating a potential implication of p53 in hampering cell cycle progression and efficient neurodevelopment in SCZ.

Conclusions: Through targeted proteomics we demonstrate that SCZ NPC display coherent mechanistic alterations in regulation of DNA damage response, cell cycle control and p53 expression. These findings highlight the suitability of iPSC-based approaches for modeling psychiatric disorders and contribute to a better understanding of the disease mechanisms underlying SCZ, particularly during early development.

Keywords: Cell cycle; Cellular signaling; DigiWest; IPSC; Neural progenitors; Proteomics; Schizophrenia; p53.

Fig. 1

Marker expression in iPSC and NPC. A: DigiWest data (AFI = accumulated fluorescent intensity) of cell-type specific marker proteins in iPSC (n = 21) and NPC (n = 21); Mann–Whitney test. B: Western Blot mimic (gray-scale image) of markers displayed in A (exemplarily shown for one differentiation only). C: Example ICC images of marker expression in iPSC (left) and NPC (right) obtained by high-content microscopy. Scale bars: 50 µm. CTR and SCZ cells will be addressed separately at a later stage. *p < 0.05, ****p < 0.0001. Error bars: S.E.M

Fig. 2

Expression signatures and pathway upregulation during differentiation. A: Heatmap and Hierarchical Cluster analysis of analytes significantly different between iPSC (n = 21) and NPC (n = 21) samples (Wilcoxon test, p < 0.001). B: DigiWest data (AFI = accumulated fluorescent intensity) for a subset of proteins with differential expression in iPSC (n = 21) and NPC (n = 21). Proteins are grouped according to their pathway allocation; Mann–Whitney test. C: Example ICC images of beta-catenin, LEF1 and p21 expression in iPSC (top) and NPC (bottom). Scale bars: 50 µm. D: Quantified ICC signals of proteins exemplarily shown in C (iPSC n = 21, NPC n = 21) obtained by high-content microscopy. Data are shown relative to mean iPSC signal; Mann–Whitney test. E–F: Volcano plot of separate iPSC vs NPC comparison for CTR (E—iPSC n = 9, NPC n = 9) and SCZ (F—iPSC n = 12, NPC n = 12) samples (Wilcoxon-Test, p < 0.01). Significantly upregulated proteins are shown in red, downregulated proteins in blue (analytes with FCs < I1I are excluded). Analytes with a significant interaction effect between cell type and disease allocation (p < 0.05, 2-Way-ANOVA) are highlighted (also see Additional File 5 – Figure S6 and Additional File 1—Table S3). *p < 0.05, ***p < 0.001, ****p < 0.0001. Error bars: S.E.M

Fig. 3

SCZ-specific alterations in iPSC. A: Heatmap with Hierarchical Cluster analysis (HCL) of analytes significantly different (Wilcoxon Test, p < 0.05) between CTR (n = 9) and SCZ (n = 12) iPSC. Log2-transformed data is shown relative to mean signal across CTR lines of the respective differentiation. B: Volcano plot of comparison shown in A. C: DigiWest data (relative to control mean) of p53 and p53 – pS15 expression comparing CTR (n = 9) and SCZ (n = 12) iPSC; Mann–Whitney test. D: Example images of total p53 ICC staining in iPSC obtained by high-content microscopy. Scale bars: 50 µm. E: Quantified ICC signal of total p53 expression (relative to CTR mean) in CTR (n = 9) and SCZ (n = 12) iPSC (unpaired t-test). *p < 0.05, ***p < 0.001. Error bars: S.E.M

Fig. 4

SCZ-specific alterations in NPC. A: Heatmap with Hierarchical Cluster analysis (HCL) of analytes significantly different (Wilcoxon Test, p < 0.05) between CTR (n = 9) and SCZ (n = 12) NPC. Log2-transformed data is shown relative to mean signal across CTR lines of the respective differentiation. B: Volcano plot of comparison shown in A. C: DigiWest data (relative to CTR mean) of Oct4, MAP2, NCAM and Sox1 expression in CTR (n = 9) and SCZ (n = 12) iPSC and NPC, respectively; Mann–Whitney test. D: Venn diagram showing the number of analytes differentially regulated between SCZ and CTR in the respective cell type. E: DigiWest data (relative to CTR mean) of p53 – pS15 and p53 (total) expression; CTR n = 9, SCZ n = 12, Mann–Whitney test. F: Quantified Western Blot signals of p53 – pS15 and p53 (total) expression in NPC (relative to CTR mean). Intensities were normalized to beta-Actin signal; CTR n = 6, SCZ n = 8, Mann–Whitney test. G: Western Blot images corresponding to quantification shown in F. H: Quantified ICC signal of total p53 expression (relative to CTR mean) in CTR (n = 9) and SCZ (n = 12) NPC (unpaired t-test). I: example images of total p53 ICC staining as obtained by high-content microscopy. Scale bars: 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars: S.E.M

Fig. 5

Pathway allocations of SCZ-specific proteins. A-E: DigiWest data (relative to CTR mean) of select proteins differentially expressed between CTR (n = 9) and SCZ (n = 12) in NPC only (see Fig. 4D), shown in direct comparison with iPSC (CTR n = 9, SCZ n = 12). Analytes are grouped based on pathway/cellular function. A: Cell cycle regulation, specifically G2/M phase transition, B: DNA damage response, C: Protein synthesis/translation, D: Wnt signaling, E: MAPK/Erk signaling. A complete list of all differentially expressed SCZ-specific analytes can be found in Additional file 1—Table S5. Either the Mann–Whitney test or unpaired t-test was used depending on data distribution. *p < 0.05, **p < 0.01, ***p < 0.001 or as indicated. Error bars: S.E.M

Fig. 6

Phenotypic cell cycle alterations in SCZ NPC correlate with p53 levels. A: Example images of G2/M regulatory proteins Aurora A and Cyclin B1 ICC staining in CTR and SCZ iPSC and NPC obtained by high-content microscopy. Scale bars: 50 µm. B: Quantified ICC signal of Aurora A and Cyclin B1 expression (relative to CTR mean) in CTR (n = 9) and SCZ (n = 12) in iPSC and NPC, respectively (Mann–Whitney test). C: Cell phase distribution of CTR and SCZ NPC (n = 5–8) in percent; 2-Way ANOVA with Tukey’s multiple comparisons test. D: Flow-cytometry cell cycle analysis of CTR and SCZ NPC. Nocodazole was used as a positive control. **p < 0.01 or as indicated. Error bars: S.E.M. E–G: Correlations between p53 – pS15 and E: p53 (total), F: differentiation markers and G: G2/M cell phase regulators. Spearman´s r; **p < 0.01, ***p < 0.001, ****p < 0.0001. The dashed red line indicates a simple linear regression applied to each XY correlation