title: Lysosome dysfunction triggers Atg7-dependent neural apoptosis.
abstract: Macroautophagy (autophagy) is a process wherein bulk cytosolic proteins and damaged organelles are sequestered and degraded via the lysosome. Alterations in autophagy-associated proteins have been shown to cause neural tube closure defects, neurodegeneration, and tumor formation. Normal lysosome function is critical for autophagy completion and when altered may lead to an accumulation of autophagic vacuoles (AVs) and caspase activation. The tumor suppressor p53 is highly expressed in neural precursor cells (NPCs) and has an important role in the regulation of both autophagy and apoptosis. We hypothesized that altered lysosome function would lead to NPC death via an interaction between autophagy- and apoptosis-associated proteins. To test our hypothesis, we utilized FGF2-expanded NPCs and the neural stem cell line, C17.2, in combination with the lysosomotropic agent chloroquine (CQ) and the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1). Both CQ and Baf A1 caused concentration- and time-dependent AV accumulation, p53 phosphorylation, increased damage regulator autophagy modulator levels, caspase-3 activation, and cell death. Short hairpin RNA knockdown of Atg7, but not Beclin1, expression significantly inhibited CQ- and Baf A1-induced cell death, indicating that Atg7 is an upstream mediator of lysosome dysfunction-induced cell death. Cell death and/or caspase-3 activation was also attenuated by protein synthesis inhibition, p53 deficiency, or Bax deficiency, indicating involvement of the intrinsic apoptotic death pathway. In contrast to lysosome dysfunction, starvation-induced AV accumulation was inhibited by either Atg7 or Beclin1 knockdown, and Atg7 knockdown had no effect on starvation-induced death. These findings indicate that Atg7- and Beclin1-induced autophagy plays a cytoprotective role during starvation but that Atg7 has a unique pro-apoptotic function in response to lysosome dysfunction.
authors.affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017, USA. kcwalls@uab.edu
authors.forename: Ken C
authors.initials: KC
authors.lastname: Walls
authors.forename: Arindam P
authors.initials: AP
authors.lastname: Ghosh
authors.forename: Aimee V
authors.initials: AV
authors.lastname: Franklin
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Klocke
authors.forename: Mary
authors.initials: M
authors.lastname: Ballestas
authors.forename: John J
authors.initials: JJ
authors.lastname: Shacka
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Roth
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology; D017209:Apoptosis; D001343:Autophagy; D000071193:Autophagy-Related Protein 7; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2; D005455:Fluorescent Antibody Technique; D008247:Lysosomes / Q000378:metabolism* / Q000473:pathology; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000502:physiology
chemical_list: D000935:Antifungal Agents; D000962:Antimalarials; C502661:Atg7 protein, mouse; D018942:Macrolides; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D016222:Fibroblast Growth Factor 2; D002738:Chloroquine; C040929:bafilomycin A1; D020169:Caspases; D000071193:Autophagy-Related Protein 7