title: Lysosome vacuolation disrupts the completion of autophagy during norephedrine exposure in SH-SY5Y human neuroblastoma cells.
abstract: In our current study, we examined the mechanism underlying neuronal cell injuries caused by norephedrine in SH-SY5Y human neuroblastoma cells. Norephedrine was found to induce cytoplasmic vacuolation and a resultant loss of cell viability. In the cells treated with norephedrine also, an autophagic marker LC3 was converted to its LC3-II activated form, suggesting the induction of autophagy. In cells transfected with RFP-LC3 and GFP-LAMP1, a punctate patterning of LC3 expression and colocalization of LAMP1 with the formed vacuoles were observed, highlighting the lysosomal nature of the vacuoles and their association with autophagosomes. An autophagic flux assay using tfLC3 (mRFP-GFP-LC3) indicated the formation of autophagosomes and autolysosomes by norephedrine stimulation at an early timepoint (∼3 h). However, at a later timepoint (∼6 h), both the dilation of autolysosomes/lysosomes and the neutralization of the vacuolar pH were also observed. These results thus indicate that norephedrine induces autophagy at an early timepoint and cell death with lysosomal dysfunction and autophagy disruption at a later timepoint.
authors.affiliation: Tokyo Medical and Dental University, From the Section of Forensic Medicine, Tokyo 113-8519, Japan. funakoshi.legm@tmd.ac.jp
authors.forename: Takeshi
authors.initials: T
authors.lastname: Funakoshi
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Aki
authors.forename: Kana
authors.initials: K
authors.lastname: Unuma
authors.forename: Koichi
authors.initials: K
authors.lastname: Uemura
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D018942:Macrolides / Q000494:pharmacology; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics; D010665:Phenylpropanolamine / Q000494:pharmacology*; D034622:RNA Interference; D017382:Reactive Oxygen Species; D014617:Vacuoles / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D000316:Adrenergic alpha-Agonists; D004791:Enzyme Inhibitors; C477012:MAP1LC3A protein, human; D018942:Macrolides; D008869:Microtubule-Associated Proteins; D017382:Reactive Oxygen Species; D010665:Phenylpropanolamine; C040929:bafilomycin A1