DRAM1 Regulates Autophagy Flux through Lysosomes

Citation: Zhang X-D, Qi L, Wu J-C, Qin Z-H ( DRAM1 Regulates Autophagy Flux through Lysosomes Xing-Ding Zhang. 0 Lin Qi. 0 Jun-Chao Wu 0 Zheng-Hong Qin 0 Arun Rishi, Wayne State University, United States of America 0 Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science , Suzhou , China We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes. - Funding: This work was partially supported by the National Natural Science Foundation of China (No 30930035), by the National Basic Science Key Project (973 project, CB510003), by the Priority Academic Program development of Jiangsu Higher Education Institutes, and by Graduate Training Innovation Project of Jiangsu Province (CX09B_042Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. 3-nitropropionic acid (3-NP), a suicide inhibitor of the mitochondrial respiratory enzyme succinate dehydrogenase (SDH) [1], induces striatal cell death in vivo and in vitro [24]. When intoxicated in vivo, 3-NP produces symptoms and striatal neuronal loss in human brains replicating neuropathology of Huntingtons disease [4], [5]. We previously reported that intrastriatal administration of 3-NP induced TP53-dependent autpophagy activation and apoptosis. The TP53 specific inhibitor pifithrin-a (PFT-a) blocked induction of autophagic proteins including DNA Damage Regulated Autophagy Modulator1 (DRAM1), LC3-II and beclin1 and apoptotic proteins including TP53-upregulated modulator of apoptosis (PUMA) and BAX. Both pharmacological inhibitors of autophagy and caspases effectively inhibited 3-NP-induced cell death [6], [7]. DRAM1, a novel TP53 target gene, is an evolutionarily conserved lysosomal protein and has been reported to play an essential role in TP53-dependent autophagy activation and apoptosis [8]. The mechanism by which DRAM1 promotes autophagy is not clear. It is proposed that DRAM1 may exert its effects on autophagy through lysosomes, given the fact as a lysosomal membrane protein. Uncovering the molecular mechanism by which DRAM1 regulates autophagy would provide a better understanding of the role of TP53 signaling pathway in the regulation of cell death and survival. Autophagy is a pathway delivering cytoplasmic components to lysosomes for degradation [913]. Macroautophagy involves the sequestration of a region of the cytoplasm in a double-membrane structure to form a unique vesicle called the autophagosome. Acidification of lysosomes is crucial for activation of cathepsins, fusion of lysosomes and autophagosomes and effective degradation of autophagic substrates. However, these late digestive steps of autophagy remain largely uncharacterized. Lysosomes are cytoplasmic organelles that contain several enzymes mostly belonging to the hydrolases [14]. Internal pH of lysosomal is characteristically acidic and it is maintained around pH 4.5 by a proton pump, that transport H+ ions into lysosomes [15], [16]. Many autophagy inhibitors including the vinca alkaloids (e.g., vinblastine) and microtubule poisons that inhibit fusion of autophagosomes with lysosomes, inhibitors of lysosomal enzymes (e.g., leupeptin, pepstatin A and E64d), and compounds that elevate lysosomal pH (e.g., inhibitors of vacuolar-type ATPases, such as bafilomycin A1 and weak base amines including ammonia, methyl- or propylamine, chloroquine, and Neutral Red, some of which slow down fusion), act at the fusion and lysosomal degradation steps [17]. Lysosomal enzymes also play a role in activation of certain types of caspases and therefore, are involved in apoptosis [18]. Inhibition of lysosomes or lysosomal enzymes protects neurons against excitotoxicity and ischemic insults [19], [20]. Thus, it is of particularly interest to investigate if DRAM1 modulates autophagy through influencing lysosomal functions. In this study, we report that 3-NP induced DRAM1-dependent stimulation of autophagy in A549 cell lines. DRAM1 promotes autophagy flux by enhancing lysosomal acidification. Materials and Methods Cell Lines and Reagents A549 (TP53+/+) and H1299 (TP532/2) and Hela cell lines were purchased from Shanghai Institute of Biochemistry and Cell Biology in China, and were grown at 37uC in 5% CO2 in RPMI supplemented with 2 mmol/L L-glutamine and 10% FCS. Primary mouse embryonic fibroblasts (MEFs) were derived from p53 wt and p53 KO sibling embryos, and maintained with DMEM supplemented with 10% FCS and antibiotics. 3-NP (N5636), 3-MA (M9281), carbonyl cyanide m-chlorophenylhydrazone (CCCP, C2759), ATP (A6559), chloroquine (C6628), E64d (E8640) and Z-Vad-FMK (V116) were all purchased from Sigma-Aldrich (Saint Louis, MO, USA). LysoTracker Red (L7528) and LysoSentor (L7533) were purchased from Invitrogen-Molecular Probes (Shanghai, China). All cell culture reagents were purchased from Gibco (Gaithersburg, MD, USA) unless otherwise noted. Expression of GFP-LC3 and DRAM1-pEGFP The activation of autophagy was detected following transfection of cells with GFP-LC3 and mRFP-GFP-LC3 expression plasmids (kindly provided by Dr. (...truncated)