Suppression of ROS and expression of Bcl-2 lead to decrease in the levels of autophagy and the type III PI3 kinase activity. (A) Quantification of average type III PI3 kinase activity following knock-down of genes able (yes) and unable (no) to induce autophagy in the presence of NAC. H4 FYVE-dsRed cells were transfected with hit siRNA for 72 h, followed by fixation and imaging on a high-throughput fluorescent microscope at 10× magnification. Average z-scores as compared with nontargeting siRNA are shown. (B) Comparison of the relative average viability of WT or pBabe-Bcl-2–expressing H4 cells transfected with hit gene siRNAs for 72 h. (C) Comparison of average type III PI3 kinase activity in WT or pBabe-Bcl-2–expressing H4 FYVE-dsRed cells following hit siRNA transfection for 72 h. (D) Subdivision of hits whose knock-down was able to induce autophagy under conditions of low PtdIns3P into functional categories based on their ability to up-regulate type III PI3 kinase activity or to alter lysosomal processing. (E) Subdivision of genes whose knock-down led to the induction of autophagy into functional categories based on their dependence on ROS and elevated levels of PtdIns3P (PI3P). *P < 0.05, **P < 0.01 based on two-tailed t test with equal variance. All error bars indicate SEM.

Differential gene expression leads to transcriptional up-regulation of autophagy in Alzheimer's disease. Forrest plots of NES estimates with SD for the screen hit gene sets are shown. (A) GSEA analysis of overall screen hit gene expression in different regions of AD brain as compared with unaffected age-matched controls. (B and C) GSEA analysis of hit genes determined to function as negative (B) or positive (C) regulators of autophagy flux. Blue squares indicate enrichment signals with P ≤ 0.05 in an individual comparison. The size of a square is inversely proportional to the respective SD.

Knock-down of the mitochondrial gene Cox5a leads to generation of ROS and induction of autophagy. (A) Induction of ROS in H4 cells transfected with two independent siRNAs against Cox5a or nontargeting siRNA for 72 h. Cells were stained in 25 mM carboxy-H2DCFDA and Hoechst and imaged at 40×. (B) Induction of autophagy in cells transfected with siRNAs against Cox5a or controls for 72 h; autophagy levels were assessed with antibodies against p62, Atg5 (Atg12-Atg5 complex is shown), and LC3. (C) Induction of autophagy by Cox5a knock-down is suppressed in the presence of the antioxidant NAC. Cells were prepared as in B, except that, where indicated, 10 mM NAC was added to the culture media 24 h after transfection. (D) Knock-down of Cox5a leads to the accumulation of PtdIns3P in a manner dependent on the function of the type III PI3 kinase. Quantification of FYVE-dsRed reporter is shown. Cells were transfected as in B; where indicated, the type III PI3 kinase inhibitor 3MA (10 mM) was added for 8 h before fixation and imaging. (E) Induction of autophagy following Cox5a knock-down depends on the function of the type III PI3 kinase. GFP-LC3 H4 cells were prepared and imaged as in D. (F) Levels of autophagy induced following knock-down of Cox5a were assessed in H4 cells transfected with control siRNA (nt, nontargeting; Tor, mTOR) or siRNA against Vps34 for 72 h by Western blot. *P < 0.05, **P < 0.01 based on two-tailed t test with equal variance. All error bars indicate SEM.

Amyloid β up-regulates autophagy by inducing accumulation or ROS and the type III PI3 kinase activity. (A) Comparison of levels of LC3-II accumulation in the presence or absence of 10 μM E64d following treatment of H4 cells with 5 μM Aβ. (B) Expansion of the lysosomal compartment following Aβ treatment based on the Lamp-1-RFP lysosomal reporter. Cells were treated as in A, then fixed and imaged at 10×. (C) Treatment with Aβ leads to generation of ROS. Cells were stained in 25 mM carboxy-H2DCFDA and imaged at 40× magnification. (D) Induction of autophagy by Aβ is suppressed in the presence of the antioxidant NAC. GFP-LC3 cells were prepared as in B except that, where indicated, 10 mM NAC was added. (E) Aβ induces accumulation of PtdIns3P. FYVE-dsRed cells were prepared and imaged as in B; where indicated, the type III PI3 kinase inhibitor 3MA (10mM) was added for 8 h before fixation. (F) Induction of the type III PI3 kinase activity by Aβ is suppressed in the presence of antioxidant. Cells were prepared as in D. (G) Induction of autophagy by Aβ is dependent on the type III PI3 kinase activity. H4 GFP-LC3 cells were treated and imaged as in E. **P < 0.01 based on two-tailed t test with equal variance. All error bars represent SEM.

Drugs against AD suppress autophagy. H4 GFP-LC3 cells were treated with indicated concentrations of GSHR ligand Ghrelin (A) or CHRND agonist Galanthamine (B) for 24 h and imaged at 10×. *P < 0.05, ** P < 0.01 based on two-tailed t test with equal variance. All error bars represent SEM.

Expression of autophagy screen hit genes in normal human aging. Clustering analysis (dChip) of mRNA expression levels of select autophagy hit genes in younger (≤40 y old) versus older (≥70 y old) human brain samples, based on (i) minimum 1.2-fold change between the average expression, and (ii) P value <0.05 using unpaired t test.