Figure 5.

3R to 4R MAPT ASOs result in endolysosomal dysfunction. (A–C) Representative immunocytochemistry of human induced pluripotent stem cell (hiPSC)-derived neurons expressing EEA1 proteins (A) (red = EEA1; green = TUJ1; blue = DAPI). Scale bars = 10 μm. A significant increase in the average size of EEA1+ puncta (B) and frequency (C) of early endosomes with size > 1 μm² in neurons treated with 4R-T antisense oligonucleotides (ASOs) (n = 3 control lines). (D–F) Representative immunocytochemistry of hiPSC-derived neurons expressing LAMP1 proteins (D) (red = LAMP1; green = TUJ1; blue = DAPI). Scale bars = 10 μm. A significant increase in the average size of LAMP1+ puncta (E) and frequency (F) of late endosomes/lysosomes with size > 1 μm² in neurons treated with 4R-T ASOs (n = 3 control lines). (G) Schematic of the lysosomal galectin puncta assay used in this study. (H) Representative immunocytochemistry of hiPSC-derived neurons expressing Galectin 1 proteins (green = Galectin 1; red = MAP2; blue = DAPI). Scale bars = 5μm. (I) A significant increase in the number of Galectin 1+ puncta in neurons treated with 4R-T ASOs compared with scrambled controls (n = 3 control lines).