Fig. 8

Altered autophagy in senescent primary neurons modulates intraneuronal Aβ accumulation. a Illustration depicting the process of autophagy. Chloroquine (CQ) blocks the fusion between the lysosome and the autophagosome. b Schematic for the experimental design followed to measure the autophagic flux in vitro. Primary neuronal cultures were obtained from WT and G3Terc^−/− mice and treated with CQ (25 μM) or vehicle (Veh) at 14 days in vitro (DIV). Cell lysates were collected after 6 h of treatment for subsequent Western blot analysis. c Western blot analysis showing the difference in LC3-II/LC3-I ratio and in p62 relative protein levels after CQ treatment in the cell lysates mentioned in “b”. Actin was used as loading control. Results are expressed as difference between CQ and Veh conditions. *P < 0.05 (two-tailed Student’s t-test, n = 4 cultures/group). d Primary neurons obtained from WT and G3Terc^−/− mice were infected with hAPP3xmut-expressing lentiviruses and treated with rapamycin (RAP) or vehicle (Veh) for 4 days, and intraneuronal Aβ42 accumulation was evaluated by immunostaining analysis of Aβ42 (red) and the neuronal marker MAP2 (green). Relative fluorescence intensity was calculated. **P < 0.01 (Two-way ANOVA with Tukey’s post-hoc analysis, n = 4–5 cultures/group). Scale bar: 50 μm. e MSD Electro-Chemiluminescence Immuno-Assay showing human Aβ40 and Aβ42 levels in WT and G3Terc^−/− neurons overexpressing hAPP3xmut and treated with rapamycin (RAP) or vehicle (Veh) for 4 days. Results were normalized by the amount of hAPP protein levels obtained in previous Western blot analyses, and the levels in the control group were set as 100%. *P < 0.05, **P < 0.01 (Two-way ANOVA with Tukey’s post-hoc analysis, n = 3–4 cultures/group). hAβ signal was not detected in non-infected WT and G3Terc^−/− neurons. All data are presented as the mean ± SEM