A phosphomimetic mutant TDP-43 (S409/410E) induces Drosha instability and cytotoxicity in Neuro 2A cells

• Published in: Biochemical and biophysical research communications 2015-08, Vol.464 (1), p.236-243
• Main Authors: Kim, Ki Yoon, Lee, Hee-Woo, Shim, Yu-mi, Mook-Jung, Inhee, Jeon, Gye Sun, Sung, Jung-Joon
• Format: Article
• Language: English
• Subjects: Animals; Cell Death - genetics; Cell Line, Tumor; Cycloheximide - pharmacology; DNA-Binding Proteins - antagonists & inhibitors; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drosha; Enzyme Stability - genetics; FUS; Mice; MicroRNAs - genetics; MicroRNAs - metabolism; Molecular Mimicry; Mutagenesis, Site-Directed; Neurons - drug effects; Neurons - metabolism; Neurons - pathology; Phosphomimetic mutant TDP-43 (S409/410E); Phosphoproteins - genetics; Phosphoproteins - metabolism; Protein Binding; Ribonuclease III - genetics; Ribonuclease III - metabolism; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; RNA-Binding Protein FUS - antagonists & inhibitors; RNA-Binding Protein FUS - genetics; RNA-Binding Protein FUS - metabolism; TDP-43
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2015.06.125

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. •Drosha protein stability is changed by TDP-43 and FUS.•We generate a phosphomimetic mutant of TDP-43 (S409/410E).•Mutant TDP-43 (S409/410E) decreases Drosha protein stability.•Mutant TDP-43 (S409/410E) disturbs interaction of Drosha and FUS.•A phosphomimetic mutant of TDP-43 (S409/410E) induces cell death.