Figure 8. Dysfunctional autophagy contributes to neuronal senescence. (A) There was an accumulation of enlarged lysosomes and undigested p62/SQSTM1 in senescent neurons. Top row, lysosomes were detected with Lysotracker® and nuclei with Hoechst in primary culture of cortical cells incubated during the indicated DIV. Bottom rows, immunofluorescence to detect the indicated proteins in cortical cells cultured during 6 or 26 days. Nuclei were stained with DAPI. Notice that even though lysosomes and p62/SQSTM1 accumulated at 26 DIV, their intracellular distribution did not overlap. mTOR distribution did not change. Scale bars represent 25 μm. (B) The autophagic flux was impaired in senescent neurons. Western blot of total protein extracts from cortical cells cultured at 6 or 26 days, without (C) or with (CQ) 20 μM Chloroquine for 4 hr. Graphs represent the mean of densitometry analysis of four independent experiments. Bars represent SEM. Two-way RM ANOVA followed by Sidak´s multiple comparison test. *p<0.001.