Stable expression of short interfering RNA for DT-diaphorase induces neurotoxicity.
DT-Diaphorase has been proposed to play a neuroprotective role in dopaminergic neurons by preventing aminochrome neurotoxicity. There are several studies supporting this idea, but in all studies, we used dicoumarol, an inhibitor of DT-diaphorase. We have designed and developed two siRNA to silence the expression of DT-diaphorase to study its role in aminochrome metabolism. We transduced RCSN-3 cells with retroviral particles containing a pRetroSuper plasmid coding a siRNA for DT-diaphorase. The cells selected in the presence of puromycin generated a stable cell line RCSN-3Nq6 and RCSN-3Nq7 with low expression of DT-diaphorase (27% and 33% of wild type, respectively). A significant cell death was observed in RCSN-3 cells expressing siRNA Nq6 and Nq7 for DT-diaphorase when were incubated with 100 μM aminochrome during 48 (4- and 3.5-fold, respectively; P < 0.01). These results support the protective role of DT-diaphorase against aminochrome neurotoxicity in dopaminergic neurons containing neuromelanin and show that Nq6 and Nq7 siRNA are very useful tools to study the role of DT-diaphorase in aminochrome metabolism.
Lozano
Jorge
J
Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile, Chile, Laboratory Medicine, Clinical Research Centre-Novum, Karolinska Institutet, Sweden.
Muñoz
Patricia
P
Nore
Beston F
BF
Ledoux
Susan
S
Segura-Aguilar
Juan
J
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D045563:Indolequinones / Q000378:metabolism / Q000633:toxicity; D008543:Melanins / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D018696:Neuroprotective Agents / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D051381:Rats
D045563:Indolequinones; D008543:Melanins; D018696:Neuroprotective Agents; D034741:RNA, Small Interfering; C014121:neuromelanin; C091584:aminochrome 1; D016660:NAD(P)H Dehydrogenase (Quinone)