Figure 5.

Mitochondrial GSH depletion in APP-PSEN1-SREBF2 mice stimulates autophagosome formation via enhancing the inhibitory effect of Aβ on ATG4B. WT and APP-PSEN1-SREBF2 mice (7-mo-old) were treated with GSHee at 1.25 mmol/kg/d every 12 h for 2 wk. Lysates from ML fractions (for LC3 analysis) or brain homogenates were subjected to western blot analysis. (A) Representative immunoblots showing levels of lipidated LC3B (LC3B-II) and SQSTM1. (B) Representative photomicrographs of hippocampal sections labeled with anti-LC3B and counterstained with DRAQ5 (blue). Scale bar: 50 μm. Graph depicts quantification of the average number of LC3B puncta per cell measured using ImageJ software (80 to 90 cells analyzed per genotype and experimental condition from a pool of at least 4 images). (C) ATG4B activity of brain homogenates from 7-mo-old WT and mutant mice. Lysates were incubated with recombinant HA-GABARAPL2 AMC at 37ºC for 45 min and ATG4B activity was assessed by means of AMC fluorescence. (D) Western blot analysis of ATG4B expression in brain homogenates from 7-mo-old WT and mutant mice. (E) ATG4B activity of neuronal-enriched cultures incubated with 5 μM Aβ for 24 h with or without 30 min preincubation with 2 mM GSHee. Cell lysates were incubated with HA-GABARAPL2 AMC as in C and AMC fluorescence was analyzed. DTT (1 mM) was added to the reaction buffer to assess ATG4B maximum activity. In western blot analyses densitometric values of the bands representing the specific protein immunoreactivity were normalized with the values of the corresponding ACTB bands and expressed as relative intensity values. *P< 0.05 and **P< 0.01; n=3. See Figure S17 for uncropped blots.