Biochemistry

Cocaine and many other psychostimulants strongly induce urokinase-type plasminogen activator (uPA) expression in the mesolimbic dopaminergic pathway, which plays a major role in drug-mediated behavioral plasticity [Bahi A, Boyer F, Gumy C, Kafri T, Dreyer JL. In vivo gene delivery of urokinase-type plasminogen activator with regulatable lentivirus induces behavioral changes in chronic cocaine administration. Eur J Neurosci 2004;20:3473-88; Bahi A, Boyer F, Kafri T, Dreyer JL. Silencing urokinase in the ventral tegmental area in vivo induces changes in cocaine-induced hyperlocomotion. J Neurochem 2006;98:1619-31; Bahi A, Dreyer JL. Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine-and morphine-induced reward and behavioral sensitization. Genes Brain Behav 2007]. In this study, the role of mesolimbic dopamine (DA) pathways in the development of cocaine reward was examined by conditioned-place preference in rats with bilateral intra-accumbens injections of uPA-expressing lentiviral vectors. We show that overexpression of uPA in the Nac significantly augments cocaine-induced place preference. Furthermore, while this did not affect the ability of preference to be extinguished, reinstatement with a low dose of cocaine produced significantly greater preference to the cocaine-associated context. Once CPP had been established, and the preference extinguished, reinstatement induced by a priming dose of cocaine was facilitated by uPA. Inhibition of this serine protease expression using doxycycline abolished the augmented acquisition produced by overexpression of uPA but not the expression of the cocaine-induced CPP. When uPA is inhibited during the acquisition phase, animals no longer demonstrate place preference for the environment previously paired with cocaine. B428, a specific uPA inhibitor does not affect drug reinstatement after extinction if uPA has been activated during acquisition, a clear indication that uPA is involved in the acquisition phase of conditioned-place preference. Our results suggest that that increased uPA expression with repeated drug exposure produces conditions for enhanced acquisition of cocaine-induced CPP, indicating that cocaine-induced CPP and reinstatement may be dependent on active extracellular uPA.

CD81, a tetraspanin transmembrane protein involved in cell adhesion, is up-regulated in the mesolimbic dopaminergic pathway 24 h following acute administration of high doses of cocaine [Brenz-Verca et al., (2001) Mol. Cell. Neurosci., 17, 303±316]. Further evidence consecutive with this observation and based on microarray analysis are presented here. In addition, a regulatable lentivirus was developed bearing the rat CD81 gene under the control of a tetracycline inducible system. This lentivirus vector was stereotaxically injected into the ventral tegmental area (VTA) of two groups of animals, one fed water (expressing CD81) and the other Doxycycline solution (which down-regulates CD81 expression) and locomotor activity after chronic cocaine administration (10 mg/kg daily) was monitored. After 2 weeks, the groups were inverted, animals receiving water were placed on Doxycycline and the second group was placed on water. In all cases highly a signi®cant increase (3.2-fold) in locomotor activity was observed in animals expressing CD81 in the VTA vs. animals placed on Doxycycline. Similar studies where CD81 was delivered into the nucleus accumbens (NAcc) resulted in signi®cantly higher effects (30%), in accordance with microarray data and our previous reports, yielding a 4.2-fold increase in locomotor activity. No change was observed under similar conditions in control animals, which were injected a regulatable lentivirus expressing GFP. These ®ndings suggest that CD81 expression in the mesolimbic dopaminergic pathway contributes to behavioural changes associated with cocaine sensitization. This study provides a powerful approach for evaluating a gene function in vivo in a single animal under various paradigms, even on gene candidates, which display small changes of expression.

Alcohol abuse is a major health, economic and social concern in modern societies, but the exact molecular mechanisms underlying ethanol addiction remain elusive. Recent findings show that small non-coding microRNA (miRNA) signaling contributes to complex behavioral disorders including drug addiction. However, the role of miRNAs in ethanol-induced conditioned-place preference (CPP) and voluntary alcohol consumption has not yet been directly addressed. Here, we assessed the expression profile of miR124a in the dorsal striatum of rats upon ethanol intake. The results show that miR124a was downregulated in the dorso-lateral striatum (DLS) following alcohol drinking. Then, we identified brain-derived neurotrophic factor (BDNF) as a direct target of miR124a. In fact, BDNF mRNA was upregulated following ethanol drinking. We used lentiviral vector (LV) gene transfer technology to further address the role of miR124a and its direct target BDNF in ethanol-induced CPP and alcohol consumption. Results reveal that stereotaxic injection of LV-miR124a in the DLS enhances ethanol-induced CPP as well as voluntary alcohol consumption in a two-bottle choice drinking paradigm. Moreover, miR124a-silencer (LV-siR124a) as well as LV-BDNF infusion in the DLS attenuates ethanol-induced CPP as well as voluntary alcohol consumption. Importantly, LV-miR124a, LV-siR124a and LV-BDNF have no effect on saccharin and quinine intake. Our findings indicate that striatal miR124a and BDNF signaling have crucial roles in alcohol consumption and ethanol conditioned reward.

Extracellular serine proteases of the plasminogen activator family (tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) may modulate synaptic adhesion and associate with learning behavior. Psychostimulants strongly induce their expression in the mesolimbic dopaminergic pathway, but cocaine preferentially induces uPA, whereas morphine and amphetamine preferentially induce tPA. tPA-expressing animals displayed enhanced conditional place preference (CPP) for amphetamine compared with uPA-overexpressing animals. Thus, modulation of the plasminogen system in the brain might be a potential target against drugs of abuse. In the present study, we aim to identify whether tPA is involved in the acquisition/learning phase or in the expression/retrieval phase of conditioned drug preference. For this purpose, animals were injected with lentiviruses expressing or silencing tPA in the NAc and place preference was assessed. We found that tPA expression is associated with acquisition of place preference and animals overexpressing tPA spend 487% of the time in the drug-associated compartment, compared with 60% for control animals. When ectopic expression of tPA has been inhibited by doxycycline during acquisition, animals do no more associate the environment with the drug. Suppression of endogenous tPA expression in animals treated with LV-siRNA fully suppresses place preference, and these animals appear to avoid the drug-associated box. tPA overexpression delays extinction, but priming with low doses of amphetamine reinstates place preference even after full extinction. Together, these data clearly indicate that tPA plays an important role in acquisition of amphetamine-induced CPP, but its role in CPP expression does not seem important.

Molecular adaptations underlying drug seeking and relapse remain largely unknown. Studies highlight post-transcriptional modifications mediated by microRNAs (miRNAs) in addiction and other neurological disorders. We have previously shown that chronic cocaine suppresses miR-124 and let-7d and induces the expression of miR-181a in mesolimbic pathway. To further address the role and target gene regulation network of these miRNAs in vivo in cocaine addiction, we developed lentiviral vector (LV)-expressing miRNAs and their corresponding silencers for stable and regulatable miRNA expression. We tested in vivo miRNA gain and loss of function on cocaineinduced conditioned place preference (CPP) by localized LV-miRNA regulation in the nucleus accumbens (NAc). LV-miR-124 and let-7d expression in the NAc attenuates cocaine CPP, whereas LV-miR-181a enhances it. Silencing miRNAs by corresponding LV-miRNA silencers expressing perfect miRNA target sequences inversed this effect on cocaine CPP. Doxycycline treatment for switching off silencer expression abolished the observed behavioral changes. Behavioral changes mediated by LV-miRNA regulation resulted in dynamic alterations in transcription factors, receptors, and other effector genes involved in cocaine-induced plasticity. Our results describe a complex regulatory pathway mediated by miRNAs in cocaine-mediated neuronal adaptations.

Background Brain-derived neurotrophic factor (BDNF) is involved in the survival and function of midbrain DA neurons. BDNF action is mediated by the TrkB receptortyrosine kinase, and both BDNF and TrkB transcripts are widely expressed in the rat mesolimbic pathway, including the nucleus accumbens (NAc) and the ventral tegmentum area (VTA).

Synucleins have attracted much attention because of their involvement in several neurodegenerative disorders. In a screening for genes differentially expressed after high-dose cocaine exposure, we found g-synuclein as a major upregulated candidate in the tegmentum. Overexpression of both a-and g-synuclein after drug treatment was con®rmed by means of microarrays, yielding an increase in the hippocampus, the striatum and the tegmentum (2.65 Â, 1.96 Â and 3.5 Â, respectively, for a-synuclein vs. 2.7 Â, 1.96 Â and 7.16 Â for g-synuclein), but no change in the nucleus accumbens. Investigation of the distribution of mRNA (by in situ hybridization) and of the proteins (by immunocytochemistry) shows in both cases a clearly distinct pattern of expression for a-and g-synuclein. a-synuclein displays a very characteristic distribution, con®ned to speci®c nuclei, whereas g-synuclein is more widely expressed throughout the brain. mRNA of both a-and g-synucleins display a complementary pattern of expression all over the cortex. In contrast to g-synuclein, a-synuclein is neuronal, being only found in NeuN-expressing cells, and is expressed in the basal ganglia (faintly) and in the substantia nigra compacta where it is highly correlated with tyrosine hydroxylase. Immunocytochemistry shows that g-synuclein generally colocalizes with glial ®brillary acidic protein-expressing cells and is abundant in the red nucleus, the substantia nigra reticulata and the anterior commissure, while g-synuclein mRNA labels the matrix compartments of the caudate-putamen. The role of synucleins in relation to cocaine-induced plasticity or neurotoxicity is discussed.

Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens/olfactory tubercle, respectively, constituting mesostriatal and mesolimbic pathways. The molecular signals that confer target specificity of different dopaminergic neurons are not known. We now report that EphB1 and ephrin-B2, a receptor and ligand of the Eph family, are candidate guidance molecules for the development of these distinct pathways. EphB1 and ephrin-B2 are expressed in complementary patterns in the midbrain dopaminergic neurons and their targets, and the ligand specifically inhibits the growth of neurites and induces the cell loss of substantia nigra, but not ventral tegmental, dopaminergic neurons. These studies suggest that the ligand-receptor pair may contribute to the establishment of distinct neural pathways by selectively inhibiting the neurite outgrowth and cell survival of mistargeted neurons. In addition, we show that ephrin-B2 expressio...

Brain-specifi c neural-zinc-fi nger transcription factor-2b (NZF2b/7ZFMyt1) is induced in the mesolimbic dopaminergic region after chronic cocaine exposure and lentiviral-mediated expression of NZF2b/7ZFMyt1 in the nucleus accumbens results in decreased locomotor activity . In this study the role of NZF2b/7ZFMyt1 in active cocaine seeking and of its interaction with histone deacetylase on the altered behavior has been observed. Localized expression of NZF2b/7ZFMyt1 in the nucleus accumbens resulted in attenuated cocaine self-administration, whereas silencing this transcription factor with lentiviruses expressing siRNAs increased the animal′s motivation to self-infuse cocaine. Low doses of sodium butyrate, a potent inhibitor of histone deacetylase, were suffi cient to reverse the NZF2b/7ZFMyt1-mediated decrease in cocaine self-administration. NZF2b/7ZFMyt1 expression resulted in strong induction of transcription factors REST1 and NAC1 and of the dopamine D2 receptor, with concomitant inhibition of BDNF and its receptor TrkB. We show that NZF2b/7ZFMyt1 colocalizes with histone deacetylase-2 (HDAC2), probably overcoming the suppression of transcriptional activity caused by Lingo1. These fi ndings show that molecular adaptations mediated by NZF2b/7ZFMyt1 expression possibly lead to decreased responsiveness to the reinforcing properties of cocaine and play a prominent role in affecting the behavioral changes induced by the drug.

Plasma membrane oxidoreductases have been described in all cells and use extracellular impermeant electron acceptors (DCIP, Ferricyanide) that are reduced by NADH. They appear to regulate the overall cell activity in response to oxidative stress from the cellular environment. An NADH-DCIP reductase has been described at the plasma membrane of NB41A3, a neuroblastoma cell line Biochim. Biophys. Acta 1183, 513-520) whose activation with extracellular impermeant substrates promotes cell growth. Elutriation was performed to separate cells and the various fractions were analysed for enzyme activity on intact cells combined with flow cytometry. These studies showed that the enzyme is mostly induced and activated during the G1 and during the G2/M-phases. These observations were further corroborated with specific inhibitors of the cell cycle. A three-fold increase in enzyme activity was observed in the presence of c~-amanitin, a specific cell cycle inhibitor of the Gl-phase. Taxol, a specific inhibitor of the M-phase, also induces a significant increase in enzyme activity. FACS analysis of taxol-treated and a-amanitin-treated cells corroborated these data. The cells have been synchronized and the enzyme activity was measured at different time intervals. An activity increase was observed after ca. 2-3 h, that corresponds to a raise in the M-phase, according to FACS data. Furthermore, NTera-2 cells -a human neuroblastoma cell line that differentiates into fully mature neurones in the presence of retinoic acid -exhibit a 50% decrease in the enzyme activity during the G0-phase upon differentiation, compared to undifferentiated cells. Together the data presented in this paper show that this plasma membrane NADH-diaphorase affects cell growth and differentiation and is strongly modulated at various phases of the cell cycle.

It has been reported by Biochem. Biophys. Res. Commun. 74,[384][385][386][387][388][389] that phenacyl bromide reacts with a single reactive sulfhydryl group of aconitase, abolishing enzyme activity. Substrate or analogs have a protective effect. This group is therefore at the catalytic site of the enzyme. Aconitase is also known to be an Fe-S protein, paramagnetic as obtained on purification (Ruzicka and Beinert (1978) J. Biol. Chem. 253, 2514-2517). We have attempted to obtain information on the location of the Fe-S cluster of aconituse with respect to the catalytically active site by attaching nitroxide-labelled sulfhydryl reagents of the bromoacyl and nudeimide type to the sensitive sulfhydryl group. The EPR signals of those spin-labelled sulfhydhryl reagents that abolish enzyme activity disappear during reaction with aconitase. EPR spectra at 13 K of the product obtained by reaction of three spin labels (two maleimides and one bromoacyl) with aconitase included a half-field transition at g ---4.0 which is characteristic of spin-spin interaction. On the basis of calculations of the dependence of the intensity of the half-field transition on the distance between two interaOin~ unpaired electrons (Eaton and Eaton, (1982) J. Am. Chem. Soc. 104, 5002-5003) the distances between the nitmxide N-O bond and the center of the Fe-S duster for the three spin labels were calculated to be 10.5, 11 and 13 P,. Combined distance and orientation data for the three spin labels indicate that the reactive suifhydryl group is about 12 h, from the center of the Fe-S cluster.

It is known that aconitase from mammalian mitochondria is only partially active as isolated but may be activated by incubation with iron, ascorbate, and a thiol, or with dithionite. It has been suggested that the added Fe in the activation mixture is essential for activation and that it is incorporated in the enzyme [Villafranca, J. . However, it is shown in this paper that, when the enzyme has a full complement of 3Fe and 3S, full activation is reached coulometrically, without iron or other chemical reducing agents. It is clear, therefore, that the role of activators is to reduce the ironsulfur cluster of the enzyme.

CD81, also known as target of the antiproliferative antibody, is known to be expressed in astrocytes and involved in cell adhesion and, recently, we demonstrated its induction exclusively in the accumbens following cocaine. In the present study, the sensitivity of CD81-deficient mice to behavioral effects of cocaine was evaluated. It was found that CD81-deficient mice exhibited altered sensitivity to cocaine as assessed in the place preference conditioning paradigm and locomotor activity. This deficit in place preference conditioning was not accompanied by a deficit in acquisition or retention of water maze behavior. In addition, CD81 knockout mice exhibited higher levels of nucleus accumbens dopamine as compared to their controls. These observations are discussed in the context of the role of CD81 in cocaine-mediated behaviors.

Iron-sulfur clusters in proteins are now recognized as among the main types of electron-transferring groups in biological systems, besides heme and flavins. Recent developments have brought forth a better understanding about the ways the protein environment modulates the potential of the cluster by placing the cluster in a more or less hydrophobic surrounding. Refinement in models, extensive studies on the kinetics of electron transfer (e.g. by measurement of the electronic spin lattice relaxation time) and the introduction of novel spectroscopic methods (EXAFS, magnetic CD and others) in the elucidation of structures in various systems are among the main developments. Other advances include EPR studies of the spatial orientation of Fe-S centers in complex membraneous systems (e.g. in mitochondria) and the recent elucidation of the nature of center X in photosystem I by M6ssbauer-spectroscopy. M6ssbauer studies have also been described on a number of Fe-S proteins (nitrogenase, aconitase, some ferredoxins, etc.) and revealed the existence of novel structures that enlarged the number of known basic units of Fe-S centers. These advances include: 1. the discovery of a novel non-heme Fe-protein (called desulforedoxin) of the rebredoxin type, 2. the elucidation of the nitrogenase Fe-S centers and the nitrogenase cofactor and 3. the discovery of a three-iron cluster in several enzyme s and some ferredoxins. The latter 3-Fe cluster seems capable of being converted into a classical 4-Fe cluster under appropriate conditions, a phenomenon that plays a role in activation-deactivation of some enzymes (e.g. aconitase). It is now recognized that some iron-sulfur clusters may be involved in systems devoided of any oxydation-reduction reaction and may act as sensors of the surrounding redox potential, triggering the activation/deactivation of an enzyme (cf. e.g. aconitase).