title: A phosphomimetic mutant TDP-43 (S409/410E) induces Drosha instability and cytotoxicity in Neuro 2A cells.
abstract: Two DNA/RNA binding proteins, TDP-43 and FUS/TLSU, are involved in RNA processing, and their aberrant mutations induce inherited amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. Wild type TDP-43 and FUS (wtTDP-43 and wtFUS) are mainly localized in the nucleus and biochemically interact with the microRNA processing enzyme Drosha. In this study, we investigated Drosha stability in Neuro 2A cells by gain and loss of function studies of wtTDP-43 and wtFUS and cycloheximide mediated protein degradation assay. We also generated three different phosphomimetic mutants of TDP-43 (S379E, S403/404E and S409/410E) by using a site-directed mutagenesis method and examined Drosha stability to elucidate a correlation between the phosphorylated TDP-43 mutants and Drosha stability. Overexpression of wtTDP-43 and/or wtFUS increased Drosha stability in Neuro 2A cells and double knockdown of wtTDP-43 and wtFUS reduced its stability. However, knockdown of wtTDP-43 or wtFUS did not affect Drosha stability in Neuro 2A cells. Interestingly, a phosphomimetic mutant TDP-43 (S409/410E) significantly reduced Drosha stability via prevention of protein-protein interactions between wtFUS and Drosha, and induced cytotoxicity in Neuro 2A cells. Our findings suggest that TDP-43 and FUS controls Drosha stability in Neuro 2A cells and that a phosphomimetic mutant TDP-43 (S409/410E) which is associated with Drosha instability can induce neuronal toxicity.
authors.affiliation: WCU Neurocytomics Group, Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, South Korea; Department of Neurology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea.
authors.forename: Ki Yoon
authors.initials: KY
authors.lastname: Kim
authors.affiliation: Department of Neurology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea.
authors.forename: Hee-Woo
authors.initials: HW
authors.lastname: Lee
authors.affiliation: Department of Neurology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea.
authors.forename: Yu-Mi
authors.initials: YM
authors.lastname: Shim
authors.affiliation: WCU Neurocytomics Group, Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, South Korea.
authors.forename: Inhee
authors.initials: I
authors.lastname: Mook-Jung
authors.affiliation: Department of Neurology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea. Electronic address: gyesun@daum.net.
authors.forename: Gye Sun
authors.initials: GS
authors.lastname: Jeon
authors.affiliation: Department of Neurology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea. Electronic address: jjsaint@snu.ac.kr.
authors.forename: Jung-Joon
authors.initials: JJ
authors.lastname: Sung
mesh_terms: D000818:Animals; D016923:Cell Death / Q000235:genetics; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034702:RNA-Binding Protein FUS / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism
chemical_list: D003513:Cycloheximide; D004268:DNA-Binding Proteins; D035683:MicroRNAs; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D034702:RNA-Binding Protein FUS; D043244:Ribonuclease III; C000599551:FUS protein, mouse; C578077:Tardbp protein, mouse; C513400:Drosha protein, mouse
keywords: Drosha; FUS; Phosphomimetic mutant TDP-43 (S409/410E); TDP-43