Fig. 1
From: Autophagy activity contributes to the impairment of social recognition in Epac2−/− mice
The Excessive autophagic activity contributes to autistic behavior in Epac2−/− mice. A Representative confocal images demonstrate HyD-LIR-GFP-positive autophagosome in wild-type cortical and Epac2−/− neurons, in the presence or absence of chloroquine (CQ; 50 μM), for 24 h. Scale bar, 10 μm. B Bar graph represents the number of HyD-LIR-GFP-positive autophagosomes in wild-type cortical and Epac2−/− neurons. C Epac2+/+ or Epac2−/− neurons were incubated in the presence or absence of CQ. Then, the cell lysates were subjected to Western blotting with anti-LC3B, anti-GABARAPL1, or anti-β-actin antibodies. D, E The LC3-II and GABARAPL1-II levels were normalized similarly to that of β-actin. Western blot (F) and (G, H) quantitative analysis indicating the protein levels of mTOR, p70S6 kinase, and phosphorylated p70S6 kinase (p-p70S6 kinase) in the cultured cortical neurons of Epac2+/+ and Epac2−/− mice (n.s, no significance). I All Epac2+/+ and Epac2−/− mice showed a significant preference for exploring a stranger mouse rather than an empty cage. Contrary to Epac2+/+, Epac2−/− mice exhibited no difference in durations exploring a stranger mouse vs. a familiar one. Epac2−/−:Atg5+/− mice showed sociability and preference for social novelty similar to Epac2+/+ mice, suggesting the rescue of deficit in social novelty recognition of Epac2−/− mice (n.s, no significance; Str, stranger mouse; Nov, novel mouse). J Epac2−/− mice can detect and discriminate nonsocial and social olfactory cues with normal dishabituation to novel social odor and habituation to repeated same social odor. K, M Western blotting and quantitative analysis indicating the protein levels of ATG5, ATG7, LC3B, and β-actin in cultured cortical neurons in Epac2+/+ and Epac2−/− mice in the presence or absence of siRNAs against ATG5 or ATG7. (M, N) Representative confocal images and quantitative analysis show p62-positive aggregates in wild-type, Epac2−/−, and Epac2−/−:Atg5+/− cortical neurons. Values are presented as a mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 10 μm