Extended Data Fig. 6: Aberrant response to lysosomal damage at basal conditions and during LLOME treatment are prominent in aged/sAD tNeurons.
From: Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer’s disease
(a) Schematic for describing the ESCRT- and Galectin-mediated lysosomal quality control (LQC) machinery. When undergoing lysosomal damage stress, lysosomal membrane is subjected to rupture. To avoid the leakage of lysosomal contents and activation of cell death pathways, the compromised lysosomes recruit ESCRT proteins (ESCRTs) to repair the small membrane wounds and Galectins to target the massively damaged lysosomes for lysophagy. (b) Representative images for immunostaining of CHMP2B (magenta), LAMP2 (green) and Tuj1 (blue) in young, aged and aged/sAD tNeurons, related to Fig. 3c. Arrowhead: high-density accumulations of CHMP2B in the vicinity of plasma membrane (solid) and within neurites (hollow). Scale bar: 10 μm. (c) Representative images for immunostaining of Galectin-3 (magenta), LAMP2 (green) and Tuj1 (blue) in young, aged and aged/sAD tNeurons, related to Fig. 3c. Scale bar: 10 μm. (d) Measurement of spontaneous cell death, indicated by apoptosis markers (for example active Caspase-3/7), in young, aged, aged/sAD and fAD-PSEN1 tNeurons during in vitro culture at PID 35, 38 and 42. PID 35: n = 162 (young), 158 (aged), 148 (aged/sAD) and 136 (fAD-PSEN1) cells; PID 38: n = 163 (young), 159 (aged), 147 (aged/sAD) and 156 (fAD-PSEN1) cells; PID 42: n = 159 (young), 151 (aged), 142 (aged/sAD) and 145 (fAD-PSEN1) cells. (e) Immunostaining of TDP-43 and Hsp70 (magenta), LAMP1 (green) and Tuj1 (blue) during DMSO Ctrl or 0.25 mM LLOME treatment for 30 min in young, aged, aged/sAD tNeurons at PID 35 (scale bar: 10 μm). Insert: higher magnification view of protein co-localization within individual neuron (scale bar: 2 μm). Arrowhead: protein co-localization. (f) Changes in lysosomal acidification by detecting the intensity of preloaded pH-sensitive FITC-conjugated Dextran in tNeurons at basal conditions and during 0.25 mM LLOME treatment for 30 min. Ctrl: n = 145 (young), 135 (aged) and 148 (aged/sAD) cells; LLOME: n = 142 (young), 137 (aged) and 155 (aged/sAD) cells. (g) IF analysis of co-localization of APP-CTF with LAMP1 in aged/sAD tNeurons (scale bar: 10 μm). Insert: higher magnification view of APP-CTF and LAMP1 (scale bar: 2 μm). (h) IF analysis of co-localization of Aβ42 with LC3B in aged/sAD tNeurons (scale bar: 10 μm). Insert: higher magnification view of Aβ42 and LC3B (scale bar: 2 μm). In panels d and f, the boxes show median and 1st and 3rd quartile and the whiskers extending 1.5 times the interquartile range from the boxes. Data show box-and-whisker plots of three independent experiments and cell lines from independent HC and AD donors. Statistical analysis is performed using Two-Way ANOVA followed by Bonferroni post-hoc analysis. Source numerical data are provided.
