title: Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak.
abstract: Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.
authors.affiliation: Department of Biology, Yonsei University College of Life Science and Biotechnology, 134 Shinchon-dong Seodaemoon-gu, Seoul 120-749, Korea.
authors.forename: Junghyun
authors.initials: J
authors.lastname: Lim
authors.forename: Yunsu
authors.initials: Y
authors.lastname: Lee
authors.forename: Hyun-Wook
authors.initials: HW
authors.lastname: Kim
authors.forename: Im Joo
authors.initials: IJ
authors.lastname: Rhyu
authors.forename: Myung Sook
authors.initials: MS
authors.lastname: Oh
authors.forename: Moussa B H
authors.initials: MB
authors.lastname: Youdim
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Yue
authors.forename: Young J
authors.initials: YJ
authors.lastname: Oh
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009550:Nigericin / Q000494:pharmacology*; D061209:Proton Ionophores / Q000494:pharmacology; D034622:RNA Interference; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D061209:Proton Ionophores; D050998:bcl-2 Homologous Antagonist-Killer Protein; D002738:Chloroquine; D008985:Monensin; D009550:Nigericin