RNA interference-mediated inhibition of wild-type Torsin A expression increases apoptosis caused by oxidative stress in cultured cells.
To assess RNAi mediated inhibition of the expression of wt-DYT1 on H(2)O(2)-induced toxicity in NIH 3T3 cells and primary cortical neurons. To detect the function of wild-type Torsin A and the effect of SiRNA on the wt-DYT1 gene. The shRNA expression vector was constructed by ligating annealed complementary shRNA oligonucleotides into the down-stream of the human U6 promoter (PU6) of the RNAi-ready pSIREN-Shuttle vector. Then, the pSIREN-Shuttle-DYT1-shRNA cassette was ligated to Adeno-X Viral DNA to construct the recombinant adenoviral vector pAd-DYT1-shRNA. Cultured cerebral cortical neurons and NIH 3T3 cells were transfected with pAd-DYT1-shRNA and pSIREN-Shuttle-DYT1-shRNA. We evaluated NIH 3T3 cells and neurons in the presence of oxidative stress using a TUNEL assay under different conditions. The knockdown efficacy of the DYT1 was confirmed by real-time RT-PCR and Western Blot analysis. After exposure to H(2)O(2,) the quantity of NIH 3T3 cells transfected with pSIREN-Shuttle-DYT1-shRNA, which stained positively in the TUNEL assay, was significantly higher than the cells transfected with pSIREN-Shuttle-negative control-shRNA. (44.85 +/- 1.81% vs. 8.98 +/- 2.73%, t = 26.168). There were significantly more apoptotic neurons infected with pAd-DYT1-shRNA (45.63 +/- 7.53%) than neurons infected with pAd-X-negative control-shRNA (17.33 +/- 2.43%) (t = 9.816). The observed silencing of wild-type Torsin A expression by DYT1-shRNA was sequence-specific. RNAi-mediated inhibition of the expression of wild-type Torsin A increases apoptosis caused by oxidative stress. It is reasonable to consider that wild-type Torsin A has the capacity to protect cortical neurons against oxidative stress, and in the development of DYT1-delta GAG-dystonia the neuroprotective function of wild-type Torsin A may be compromised.
Chen
Xue-Ping
XP
Department of Neurology, West China Hospital, SiChuan University, Chengdu, Sichuan, China.
Hu
Xiao-Hui
XH
Wu
Shu-Hui
SH
Zhang
Yang-Wei
YW
Xiao
Bo
B
Shang
Hui-Fang
HF
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D006801:Humans; D016547:Kinesins; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018384:Oxidative Stress; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
C491548:Dyt1 protein, mouse; D008869:Microtubule-Associated Proteins; D018832:Molecular Chaperones; D034741:RNA, Small Interfering; D016547:Kinesins
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