Figure 5.

Chronic CORT treatment induces hyperactive neuronal autophagy and promotes lysosomal degradation of neuronal BDNF in mice. (A) Representative western blots analyzing protein expression in control and CORT groups. (B) Quantification of LC3II expression. n = 3 mice per group, P = 0.0015. (C) Quantification of SQSTM1/p62 expression. n = 3 mice per group, P = 0.0115. (D) Quantification of Atg5 expression. n = 3 mice per group, P = 0.0106. (E) Quantification of ULK1 expression. n = 3 mice per group, P = 0.9826. (F) Representative images of the control and CORT DG with double immunostaining of LC3⁺ (red) and NeuN⁺ (green) cells. Arrowheads indicate LC3⁺/NeuN⁺ cells. (G) Representative electron microscopic images of autophagosomes (white triangles) and autolysosomes (white arrows) in control and CORT mice. (H) Timeline of the procedure for the AAV-mCherry-GFP-LC3 experiment (top) and schematic diagram illustrating the detection of different autophagic structures by the mCherry-GFP-LC3 fusion protein (bottom). (I) Representative images of AAV-expressed mCherry-GFP-LC3 in control and CORT groups. (J) Quantification of autophagosomes (yellow puncta) in cell soma. n = 4 mice per group, P = 0.0111. (K) Quantification of autolysosomes (red-only puncta) in cell soma. n = 4 mice per group, P < 0.0001. (L) Quantification of autophagic flux. n = 4 mice per group, P = 0.0002. (M) Representative images of the control and CORT DG with triple immunostaining of BDNF⁺ (red), LAMP2⁺ (green), and NeuN⁺ (gray) cells. The dashed circles indicate BDNF⁺ neurons. (N) Quantification of the number of LAMP2 puncta (green) in BDNF⁺NeuN⁺ cell soma. n = 3 mice per group, P < 0.0001. Scale bar = 50 μm (Figure 5F). Scale bar = 5 μm (Figure 5G, 5I and 5M). Data are presented as the mean ± SEM. Two-tailed unpaired t-test was used to identify statistically significant differences between datasets (*P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group). n.s., non-significant difference.