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Fig. 8 | Acta Neuropathologica Communications

Fig. 8

From: The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects

Fig. 8

Rescue of endosomal phenotype in BIN1 null mutant neurons by transduction of BIN1 Isoform 1. a–a’’’ Representative images of BIN1 WT and KO hiNs in 6-weeks-old 2D culture immunolabeled with antibodies against tdTomato (tdT, red, a), EEA1 (green, a’), MAP2 (grey, a’’), and stained with Hoechst 33258 (blue, a’’’) (Scale Bar = 10 µm). b Plot showing the cumulative distribution of EEA1+ puncta volumes in transduced BIN1 KO hiNs with tdT lentiviral constructs; BIN1iso1 (dashed red line), BIN1iso9 (dashed red line), and Mock-tdT (solid red line). BIN1 WT hiNs tranduced with Mock-tdT (solid blue line) is indicated to show the rescue effect of the BIN1iso1 construct (orange line) in BIN1-null mutant neurons. Kolmogorov–Smirnov test followed by Bonferroni correction: BIN1 WT + Mock vs BIN1 KO + Mock: ****Padj < 0.0001; BIN1 WT + Mock vs BIN1 KO + iso9: Padj < 0.0001; BIN1 KO + Mock vs BIN1 KO + Iso1: ****Padj < 0.0001; BIN1 WT + Mock vs BIN1 KO + Iso1: Padj = 0.174; BIN1 KO + Mock vs BIN1 KO + Iso9: Padj = 0.207 (N = 3 independent cell cultures). c Plot showing the cumulative distribution of EEA1+ puncta volumes in transduced BIN1 WT hiNs with tdTomato (tdT)-expressing lentiviral constructs; BIN1iso1 (dashed blue line), BIN1iso9 (dotted blue line), and Mock-tdT (solid blue line). Kolmogorov–Smirnov test followed by Bonferroni correction: BIN1 WT + Mock vs BIN1 WT + Iso1: *Padj = 0.04; BIN1 WT + Mock vs BIN1 WT + Iso9: ****Padj < 0.0001 (N = 3 independent cell cultures). d Graph showing the quantification of numbers of EEA1 puncta per cell in cells transduced with tdT-tagged lentiviral constructs (N = 3 independent cell cultures. EEA1 + puncta/neuron: BIN1 WT + Mock: 65.86 ± 12.78; BIN1 WT + Iso1: 59.75 ± 8.895; BIN1 WT + Iso9: 69.82 ± 11.42; BIN1 KO + Mock: 56.47 ± 12.27; BIN1 KO + Iso1: 55.65 ± 14.52; BIN1 KO + Iso9: 77.31 ± 11.04; p = 0.81, ANOVA F(5,90)). e Quantification of MAP2+ /tdTomato+ neurons in BIN1iso1- and BIN1iso9-transduced cells relative to Mock-tdT-transduced cells (N = 3 independent cell cultures; Number of tdT+ cells: Mock = 468; BIN1iso1 = 193; BIN1iso9 = 103; ANOVA followed by Dunnett’s multiple comparisons test, **p = 0.0289)

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