In vivo gene silencing of CD81 by lentiviral expression of small interference RNAs suppresses cocaine-induced behaviour.
The tetraspanin CD81 is induced in the mesolimbic dopaminergic pathway after cocaine administration. To further investigate its role, a regulatable lentivirus (Lenti-CD81) bearing the CD81 gene under the control of a tetracycline-inducible promoter and lentiviruses expressing short hairpin RNA (shRNA) targeted against CD81 (Lenti-CD81-shRNAs) have been prepared. Infection of HEK293T cells in vitro with Lenti-CD81-shRNAs resulted in 96.5% gene silencing (from quantitative real-time PCR(qRT-PCR) and immunocytochemistry). In vivo delivery of Lenti-CD81-shRNA into the nucleus accumbens or ventral tegmental area resulted in 91.3 and 94% silencing of endogenous CD81, respectively. Stereotaxic injection of Lenti-CD81 into these regions, resulting in CD81 overexpression, induced a four- to fivefold increase in locomotor activity after chronic cocaine administration, which returned to basal levels when Lenti-CD81-shRNA had been coinjected or when CD81 expression was blocked by doxycycline. Furthermore, silencing endogenous CD81 in vivo resulted in a significant decrease in locomotor activity over controls, again suppressing cocaine-induced behaviour.
Bahi
Amine
A
Institute of Biochemistry, University of Fribourg, Fribourg, Switzerland.
Boyer
Frederic
F
Kolira
Manoj
M
Dreyer
Jean-Luc
JL
D000779:Anesthetics, Local / Q000633:toxicity; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001522:Behavior, Animal; D015152:Blotting, Northern / Q000379:methods; D002452:Cell Count / Q000379:methods; D002460:Cell Line; D003042:Cocaine / Q000633:toxicity; D019970:Cocaine-Related Disorders / Q000628:therapy; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects* / Q000502:physiology; D005822:Genetic Vectors / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D016086:Lentivirus / Q000235:genetics; D008297:Male; D009043:Motor Activity / Q000187:drug effects / Q000502:physiology; D009714:Nucleus Accumbens / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000494:pharmacology* / Q000627:therapeutic use; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D060225:Tetraspanin 28; D014162:Transfection / Q000379:methods; D017557:Ventral Tegmental Area / Q000187:drug effects / Q000378:metabolism / Q000821:virology
D000779:Anesthetics, Local; D015703:Antigens, CD; C554798:CD81 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D060225:Tetraspanin 28; D003042:Cocaine