Extended Data Fig. 1: Early emergence of autolysosomal acidification deficits in Tg2576/TRGL mice brain.
a. Representative fluorescence images from neocortical layer V neurons of TRGL and Tg2576/TRGL mice at two different ages. Neuronal perikarya of 1.6-month-old mice appeared normal in both genotypes, but yellow tfLC3 puncta accumulated in the perikarya of Tg2576/TRGL by 5 months of age (arrows). Scale bar 20 μm (left) or 50 μm (right). Experiment was repeated 3 times independently with similar results. b. Representative fluorescence images of tfLC3, co-labeled with CTSB or LAMP1, in neocortical neurons of 5-month-old TRGL and Tg2576/TRGL mouse brains. pa-AL exhibit a white signal depending on lysosome markers co-label (arrow). Scale bar 20 μm. c. Lysosome enriched fractions for lysosomal vATPase activity assay were isolated using a 25% OptiPrep gradient. Lysosome enriched fractions (grey box; #15~#18) were validated with various organelle markers. Experiment was repeated 3 times independently with similar results. d. Lysosomal vATPase activity of Tg2576/TRGL, 5xFAD/TRGL, and APP51/TRGL mouse cortex compared with littermate control neocortex. WT, M 6 mo (100±5.3 %), Tg2576, M 6mo (65.6±4.1 %), WT, F 6mo (100±0.8 %), Tg2576, F 6mo (56.4±1.9 %), TRGL, F 6mo (100±4.2 %), Tg2576/TRGL, F 6mo (56.4±5.2 %), WT, M 12mo (100±3.7 %), Tg2576, M 12mo (46.8±2.8 %), TRGL, M 2.7mo (100±1.8 %), 5xFAD/TRGL, M 2.7mo (68.4±3.2 %), TRGL, F 6mo (100±9.2 %), 5xFAD/TRGL, F 6mo (49.8±4.1 %), TRGL, F 12-15mo (100±5.0 %), APP51/TRGL, F 12-15mo (54.5±10.3 %). Number denotes mean value. n=3-5 mice. Quantitative data are presented as means ±S.E.M. unpaired t-test, two-tailed P value as indicated.
