Figures and data
Generation and characterization of human CTD brain organoids
A. Intracellular concentration of creatine in CTD iPSCs after 1h incubation with creatine-supplemented media. Healthy control is BJ. n=3 to 4, one-way ANOVA, Tukey’s multiple comparison test.
B-D. Intracellular concentration of creatine in CTD-rescue iPSCs CTD1-4 (B), CTD2-3 (C) and CTD3-7
(D) after 1h of incubation with creatine-supplemented media. Healthy controls are BJ and SP. n=3, two- way ANOVA, Šídák’s multiple comparisons test.
E. Schematic protocol of brain organoid development from iPSCs. Representative images are shown for each stage. Scale bar: 200 µm.
F. Relative mRNA expression of the telencephalon marker SIX3. n=6 to 10
G. Quantification of the diameters of healthy (BJ) and pathological brain organoids at different time points. N=2 to 22 brain organoids per productions, 4 to 6 productions per cell line.
H-I. Intracellular concentration of creatine in brain organoids and CTD-rescue brain organoids CTD1-4
(H) and CTD3-7 (I) after 6h of incubation in creatine-supplemented media. Healthy controls are BJ and SP. n=2 to 4, two-way ANOVA, Šídák’s multiple comparisons test.
CTD brain organoid organization and neurogenesis deficit
A. Representative images of 2 months old brain organoids tissue sections immunostained for PAX6 (radial glial cell and forebrain marker), MAP2 (neuronal marker), PSD95 (post-synaptic marker), TBR1 (immature neuron marker), NeuN (neuronal marker), SOX2 (radial glial cell marker), β-III tubulin (intermediate progenitor marker), GFAP (astrocyte marker). DAPI marks nuclei in blue. Scale bar: 200 µm
B. Relative mRNA expression of PAX6 and SOX2 (radial glial cell markers), GRIA2 and vGluT1 (glutamatergic markers), GABBR1 (GABAergic marker), PSD95 (post-synaptic marker) and CREB. n=3 to 6, one-way ANOVA, Tukey’s multiple comparison test
Unsupervised hierarchical clustering, reproducibility plots, principal component analysis (PCA), volcano plots, and Venn diagram showing overlap of the proteins.
A-R. The degree and quality of the separation of the data between the various groups being compared Healthy (BJ) vs CTD-derived brain organoids (CTD1_4); Healthy (BJ) vs CTD-derived brain organoids (CTD2_3); Healthy (BJ) vs CTD-derived brain organoids (CTD3_7) was assessed using unsupervised hierarchical clustering, reproducibility plots and principal component analysis (PCA), and the differentially expressed proteins were visualized using volcano plots.
S-U. Venn diagram showing overlap of the differentially expressed proteins between healthy and CTD- derived brain organoids identified using modification of the R package for ROTS. (S) Venn Diagrams for all proteins differentially expressed between BJ and CTD organoids. (T) Venn Diagrams for proteins downregulated in CTD compared to BJ organoids. (U) Venn Diagrams for proteins upregulated in CTD compared to BJ organoids.
Representation of heatmaps and graphs obtained by the absolute GSEA for significant pathways with enrichment scores.
A-C. Enrichment score and graphical representation for the GSEA for healthy versus CTD-derived brain organoids obtained across the gene sets c2_cp; c3_tft; c4_cgn; c5_bp; c5_mf; c7.
Heatmap, STRING interaction and GO Molecular Functional analysis
A. Heatmap showing expression levels of the 142 proteins in the top 90 percentile.
B. Heatmap showing expression levels of 32 most altered and abundant proteins were found to be significantly altered in CTD-derived cerebral organoids compared to normal organoids.
C. STRING interaction of the 30 proteins selected.
D. GO Molecular Functional analysis obtained using Enrichr on the 32 proteins.
Relationship between GSK3β and neurogenesis deficit
A-B. Representative western blot of GSK3β and pSer9-GSK3β in CTD vs healthy brain organoids (A) and graph showing analysis of P-GSK3β/GSK3β ratio (B). n=8, one-way ANOVA, Dunnett’s multiple comparison test.
C-D. Representative western blot of SOX2 in CTD vs healthy brain organoids (C) and graph showing analysis of SOX2 relative expression (D). n=8, one-way ANOVA, Tukey’s multiple comparison test.
E-L. Representative western blot of PAK1 (E), MAP1B (G), GSK3β and pSer9-GSK3β (I), and SOX2
(K) in brain organoids obtained from CTD iPSCs and CTD-rescue iPSCs (CTD 1-4), and graphs showing analysis of PAK1 (F), MAP1B (H), P-GSK3β/GSK3β ratio (J), SOX2 (L) relative expression. n=2 to 8, one-way ANOVA, Dunnett’s multiple comparison test. (I and J) the lanes were run on the same gel but were noncontiguous.
Schematic presentation of the main findings of the study.
The decrease in cerebral creatine pool could favor the accumulation of the dephosphorylated form of GSK3β, making it more active. This kinase has many targets in the cell, including transcription factors whose activity it can modulate. This modulation will in turn modify the expression of genes, which can influence several cellular processes: the reorganization of microtubules with the regulation of MAP1B, synaptic function, and neurogenesis thus affecting the functionality of brain cells and consequently cognitive functions. The alteration of neurons could also be explained by a mitochondrial dysfunction due to the decrease of creatine levels and the modulation of GSK3β activity, which is known to regulate mitochondrial activity.
