Fig. 3

IHT enhances autophagy and Aβ degradation by autophagosomes in PAM in vivo and in vitro. A, B LAMP1, p62, and LC3 in the hippocampus were detected via western blot. n = 3 (Student's t test). C–E Mice brain sections were co-stained with anti-LAMP1, anti-Iba1, and anti-LC3 antibodies. Scale bar = 10 μm. LC3 accumulation, and colocalization ratio of LC3 with LAMP were quantified in Iba1⁺ cells. n > 20 (Student's t test). F–H Brain sections were co-stained with anti-Iba1, anti-6E10, and anti-LC3 antibodies. Scale bar = 10 μm. The amount of internalized Aβ and Aβ colocalized to LC3 puncta was quantified in Iba1⁺ cells. n > 40 (Student's t test). I Primary microglia were treated with 1 μM oAβ for the indicated durations. Aβ concentration in the medium was measured using ELISA. n = 4 (one-way ANOVA). J Primary microglia were treated with 1 μM oAβ for 24 h, followed by a single day of IHT (10 cycles of 21% O₂ and 8% O₂). Aβ concentration in the medium was determined using ELISA. n = 7 (Student's t test). K–N After oAβ and IHT co-treatments, primary microglia were incubated with 1 μM of Aβ-555 for 30 min (K and L), or further washed with fresh medium (Chase) for 30 min (M and N). Scale bar = 20 μm. The intracellular Aβ-555 was quantified using Image J. n > 40 (two-way ANOVA). O–R Microglia were probed with anti-LC3 or anti LAMP 1 antibodies. Scale bar = 5 μm. The ratio of Aβ-555 colocalization with LC3 or LAMP1 in a single cell was determined via Manders' colocalization coefficients. n > 50 (two-way ANOVA). *P < 0.05, **P < 0.01, and ***P < 0.00. IHT intermittent hypoxia therapy, TG APP/PS1 transgenic mice