Fig. 4

Blockage of autophagy pathway decreases survival of astrocytes in response to Aβ oligomer. a, Aβ monomer or oligomer induces astrocytic cell death. Treatment conditions in human astrocytes: Aβ monomer, 1 µM; Aβ oligomer, 1 µM. Cell viability was measured by MTT assay. b, Inhibition of autophagy function exacerbates cell death in Aβ monomer or oligomer-treated human astrocytes. Treatment conditions: Aβ monomer or oligomer, 1 µM; E64D/Pepstatin A (E/P), acidic protease inhibitors, 10 µg/ml; time, 24 h. Bar graphs represent mean ± SEM from three separate experiments. c, 3MA, a PI3-kinase and autophagosome formation inhibitor, exacerbates cell death of Aβ oligomer-treated primary mouse astrocytes. Treatment conditions: Aβ oligomer, 1 µM; 3MA, 1 mM; time, 24 h. d, Representative images of astrocyte morphology, apoptotic cell death signals (green), and necrosis cell death signals (red) with or without autophagy inhibitors [E/P or chloroquine (CQ)] in human astrocytes under Aβ oligomer treatment. Treatment conditions: Aβ oligomer, 1 µM; E64D, 10 µg/ml; Pepstatin A, 10 µg/ml; CQ, 20 µM; time, 24 h. Scale bars (white): 10 μm. e, Ratio change of cell death patterns (apoptosis, necrosis, and late apoptosis) in astrocytes with or without autophagy inhibitors. f, Working mechanism of autophagy sensor, RFP-GFP-LC3B. Green dots indicate autophagosomes and red dots indicate autolysosomes. g, Representative images of RFP-GFP-LC3B-expressing astrocytes in the presence or absence of autophagy inhibitors (CQ or E/P) in Aβ oligomer-treated human astrocytes. Scale bars: 10 μm. h, Quantification of the size of autophagosome (green dots) and autolysosome (red dots). Cell count: control, n = 8, CQ, n = 10; E/P, n = 10; Aβ, n = 10; Aβ + CQ, n = 7; Aβ + E/P, n = 9. i, Representative transmission electron microscopy (TEM) images representing autophagosome (double membrane vesicles) and autolysosome (dark and dense vesicles) in Aβ oligomer-treated cultured astrocytes. Scale bars (black): upper, 5 μm; middle and bottom, 0.5 μm. Av, autophagy vesicles; N, nucleus. j, Quantification of size and number of autophagic vesicles. Data are presented as mean ± SEM. Measurement and count of ROI (100 µm²)/cell. Significantly different at *, p < 0.05; **, p < 0.01