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Allele-Specific Knockdown of ALS-Associated Mutant TDP-43 in Neural Stem Cells Derived from Induced Pluripotent Stem Cells

Nishimura, Agnes L.; Scotter, Emma L.; Abdelgany, Amr; Shum, Carole; Sardone, Valentina; Wright, Jamie; Lee, Youn-Bok; Chen, Han-Jou; Bilican, Bilada; Carrasco, Monica; Maniatis, Tom; Chandran, Siddharthan; Gallo, Jean-Marc; Rogelj, Boris; Shaw, Christopher E.

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43M337V mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43wt or GFP-TDP-43M337V or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43M337V allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.

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Academic Units
Biochemistry and Molecular Biophysics
Published Here
October 18, 2016
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