In vivo gene delivery of urokinase‐type plasminogen activator with regulatable lentivirus induces behavioural changes in chronic cocaine administration

Abstract

Serine proteases play a key function in extracellular processes affecting central nervous system plasticity. Recently, the role of extracellular proteolytic processes in regulating synaptic structure and function has been described. However, to date direct evidence linking extracellular serine protease activity with drug-related behavioural changes has not been documented. Importantly, in a screening for genes induced after drug treatment we found that urokinase plasminogen-type activator (uPA) was strongly regulated by cocaine in several protocols of drug administration. Cocaine-induced up-regulation could be verified on microarray analysis under several protocols of drug administration, then further fully confirmed by means of qRT-PCR. As a result, we chose to investigate further uPA function in the mesolimbic dopaminergic pathway, a major target area of cocaine and drugs of misuse. Our approach was based on the characterization of cocaine-induced behavioural changes following lentiviral vector delivery of a doxycycline-regulated uPA expression cassette (or of its mutated form), into specific rat brain areas (the hippocampus, the nucleus accumbens and the ventral tegmental area). We show that doxycycline-dependent over-expression of uPA in these regions yields a 10-to 12.3-fold increase in locomotor activity after cocaine administration. These behavioural effects were completely abolished when the active site of the protease was point-mutated and used as a dominant negative. The physiological relevance of these drastic behavioural changes is discussed.

Viral-Mediated Knockdown of mGluR7 in the Nucleus Accumbens Mediates Excessive Alcohol Drinking and Increased Ethanol-Elicited Conditioned Place Preference in Rats

“…The cloning of the mGlu7 receptor shRNA-expressing viral vectors was performed as described previously (Bahi et al, 2004a(Bahi et al, , 2005(Bahi et al, , 2006. Briefly, a 21-nucleotide mGluR7 shRNA sequence (NCBI Reference Sequence: NM_031040; 1041-1061: (5 0 -GCTTACTTCACATCCCGGACA-3 0 ) or the scrambled control shRNA (5 0 -GTTGGCAGCAGTAACGCAGCA-3 0 ) were chosen.…”

Spontaneous caloric restriction associated with increased leptin levels in obesity-resistant αMUPA mice

“…18 uPA is an extracellular serine protease that has a role in fibrinolysis and tissue remodeling 19 and has been implicated in brain development and plasticity. [20][21][22][23][24][25] aMUPA mice show transgenic expression in the ocular lens as expected from the promoter specificity, however, and also show ectopic expression specifically in the brain and developing teeth. 26,27 aMUPA mice have been used as a model for caloric restriction (CR), as these mice spontaneously consume 20-30% less food when fed ad libitum and live longer (B20%) compared with wild-type (WT) control FVB/N mice.…”

The Role of Tissue-Type Plasminogen Activator System in Amphetamine-Induced Conditional Place Preference Extinction and Reinstatement

“…Vesicular stomatitis virus G-pseudotyped lentiviruses were produced by the transient calcium phosphate cotransfection of human embryonic kidney 293T (HEK293T) cells with pTKs vectors together with pMDG-VSV-G and pDNRF as described previously (Naldini et al, 1996;Bahi et al, 2004aBahi et al, , b, 2005aBahi et al, , b, 2006Bahi and Dreyer, 2007). Lentiviral vector quantifications were performed according to the p24 ELISA kit (KPL, USA) in accordance with the manufacturer's instructions.…”

“…The cDNA was then digested with BamHI and XhoI and cloned into similar sites in the lentiviral system transfer vector pTK431. A control vector construct, in which green fluorescent protein (GFP) expression is regulated by a tetracycline-inducible promoter, was generated by cloning a BamHI/BglII DNA fragment containing the GFP gene into a BamHI site in pTK431 (Bahi et al, 2004a(Bahi et al, , b, 2005a(Bahi et al, , b, 2006Bahi and Dreyer, 2007).…”

“…Previous studies have shown that psychostimulants strongly induce urokinase-type plasminogen activator (uPA) expression in the mesolimbic dopaminergic pathway, which has a major role in drug-mediated behavior and plasticity changes (Bahi et al, 2004a(Bahi et al, , 2006Bahi and Dreyer, 2007). Extracellular serine proteases of the plasminogen activator family may modulate synaptic adhesion and associate with long-term potentiation and learning behavior.…”