Fig 2: A metabolic shift induces AD-like apoptotic competency in human neurons(A and B) Immunostaining (A) and quantification (B) of cleaved caspase-3 over DAPI of ßIII-tub-positive control and AD iNs (control, n = 9; AD, n = 7). Scale bars, 50 µm. Green arrows point out Casp3-positive neurons.(C) Schematic: induction of neuronal apoptosis by ABT-737 treatment.(D) Cell death assessed by cleaved caspase 3/ßIII-tub-positive cells of control (green) and AD (teal) iNs in response to 0–1 µM ABT-737.(E and F) Immunostaining (E) and quantification (F) of cleaved caspase-3 in ßIII-tub neurons after Bcl2 inhibition in control (n = 10) and AD (n = 8) iNs. Scale bars, 50 µm.(G) Pearson correlation analysis of cleaved caspase-3 immunostaining and glycolytic metabolites (UHPLC-MS).(H and I) Control iNs treated with CoDo for 2 days (H) showed increased lactate secretion (vehicle, n = 5; CoDo, n = 5) (I).(J) Quantification of p-PKM2 FI in the nucleus/cytoplasm comparing vehicle-treated (n = 6) and CoDo-treated control iNs (n = 6).(K) Immunostainings of ßIII-tub and EGFP fluorescence of EGFP::PKM2 transduced iNs. Dotted lines show cytoplasmic ROI. Scale bars, 100 and 25 µm.(L) Longitudinal EGFP::PKM2 localization in vehicle-treated (n = 3) or CoCl2-treated (n = 4) control iNs.(M and N) Immunostaining (M) and quantification (N) of cleaved caspase-3 positive cells/DAPI before and after ABT-737 (vehicle and CoDo, n = 6; one-way ANOVA, DF: 23, F = 21.34, p < 0.0001, Dunnett’s multiple comparison). Scale bars, 50 µm.Dots represent individual donors throughout the figure. Bars, mean; error bars, SD; significance, unpaired t test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: PKM2 inhibition ameliorates PKM2-induced apoptotic competency(A) Schematic: shikonin treatment prevents PKM2 nuclear translocation and increases metabolic enzymatic activity.(B) Longitudinal EGFP::PKM2 localization in vehicle-treated (n = 3), CoCl2-treated (n = 4), and CoCl2+shikonin-treated (n = 4) control iNs.(C–F) Immunostaining and quantification of nuclear p-PKM2 (C and D) and H3T11-P (E and F) in AD iNs with and without shikonin (vehicle, n = 6; shikonin, n = 5). Scale bars, 10 µm.(G) Glycolytic metabolites measured by UHPLC-MS-based metabolomics. Size and color of the circles are indicative of abundance (control, n = 3; AD, n = 4; AD-shikonin, n = 4).(H) PCA-based bulk RNA-seq (control, n = 3; AD and AD-S, n = 8).(I and J) Transcriptomic analysis of AD neuronal fate-loss gene sets (I) and hypo-maturity gene sets (J) in control (n = 3), AD (n = 8), and AD + shikonin iNs (n = 8) after 10 days of treatment.(K) Similarity profiles of control, AD, AD + shikonin iNs to neuronal differentiation trajectory of neural stem cells to neurons (Schafer et al., 2019).(L) Quantification of immunostainings for cleaved caspase-3/DAPI in control (n = 9), AD (n = 8), and shikonin-treated AD (n = 8) iNs (one-way ANOVA, DF: 25, F = 4.027, p = 0.03). Scale bars, 50 µm.(M) Radar plot of described phenotype and rescue with shikonin.(D–L) Dots represent individual donors throughout the figure. Bars, mean; error bars, SD; violin plots, median and quartiles. Significance: unpaired t test, *p < 0.05.

Fig 4: Metabolome reveals Warburg-like metabolic switch in AD iNs(A) Colorimetric assay to determine PKM activity in control (n = 7) and AD (n = 6) iNs (Mann-Whitney test).(B) Colorimetric assay to detect secreted lactate in the supernatant of control (n = 9) and AD (n = 10) iNs.(C) Density plot of PC7 of 160 metabolites measured by UHPLC-MS of control (n = 10) and AD (n = 10) iNs.(D) Integrative transcriptomics and metabolomics (IMPaLa) analysis showing over-represented pathways including gene and metabolites rich factors.(E) UHPLC-MS metabolic landscape in control (left; n = 10) and AD (right; n = 10) iNs. Circles represent Z scores of respective metabolites. Size and color of the font indicate the RNA-seq expression levels of related enzymes.(F) Glucose consumption measured as the drop of extracellular glucose after 6 h in culture.(G) Combined averages of all detected glycolytic metabolites in iNs.(H and I) Tracing of isotope-labeled glucose after 6-h incubation with 13C6-glucose. Fraction of labeled glucose detected in lactate and citrate (n = 10 per group).Dots represent individual donors throughout the figure. Bars, mean; error bars, SD; violin plots, median and quartiles. Significance: unpaired t test, *p < 0.05, **p<0.01.

Fig 5: Nuclear PKM2 activity alters the neuronal epigenetic landscape(A) Schematic: phosphorylated PKM2 translocates to the nucleus to interact with transcription factors to regulate gene expression.(B and C) Immunostaining (B) and quantification (C) of p-PKM2(Ser37) (control, n = 8; AD, n = 5). Scale bars, 10 µm.(D and E) Immunostaining (D) and quantification (E) of phosphorylated histone 3 (T11) of MAP2-positive neurons (control, n = 6; AD, n = 5). Scale bars, 10 µm.(F) ATAC-seq profiles around transcriptional start sites of genes regulated by HIF1a, STAT3, and ß-catenin (CTNNB1), based on ReMap2020 (control, n = 11; AD, n = 9).(G) HOMER motif enrichment analysis of AD differentially open peaks for HIF1a and STAT3, as previously published (Mertens et al., 2021).(H and I) Differential expression (H) and GO term enrichment (I) of significant genes regulated by HIF1a or STAT3.(B–H) Violin plots: median and quartiles. Significance: unpaired t test, *p < 0.05, **p < 0.01.