Incentive learning underlying cocaine-seeking requires mGluR5 receptors located on dopamine D1 receptor-expressing neurons.

Understanding the psychobiological basis of relapse remains a challenge in developing therapies for drug addiction. Relapse in cocaine addiction often occurs following exposure to environmental stimuli previously associated with drug taking. The metabotropic glutamate receptor, mGluR5, is potentially important in this respect; it plays a central role in several forms of striatal synaptic plasticity proposed to underpin associative learning and memory processes that enable drug-paired stimuli to acquire incentive motivational properties and trigger relapse. Using cell type-specific RNA interference, we have generated a novel mouse line with a selective knock-down of mGluR5 in dopamine D1 receptor-expressing neurons. Although mutant mice self-administer cocaine, we show that reinstatement of cocaine-seeking induced by a cocaine-paired stimulus is impaired. By examining different aspects of associative learning in the mutant mice, we identify deficits in specific incentive learning processes that enable a reward-paired stimulus to directly reinforce behavior and to become attractive, thus eliciting approach toward it. Our findings show that glutamate signaling through mGluR5 located on dopamine D1 receptor-expressing neurons is necessary for incentive learning processes that contribute to cue-induced reinstatement of cocaine-seeking and which may underpin relapse in drug addiction.

CREB has a context-dependent role in activity-regulated transcription and maintains neuronal cholesterol homeostasis.

Induction of specific gene expression patterns in response to activity confers functional plasticity to neurons. A principal role in the regulation of these processes has been ascribed to the cAMP responsive element binding protein (CREB). Using genome-wide expression profiling in mice lacking CREB in the forebrain, accompanied by deletion of the cAMP responsive element modulator gene (CREM), we here show that the role of these proteins in activity-induced gene expression is surprisingly selective and highly context dependent. Thus, only a very restricted subset of activity-induced genes (i.e., Gadd45b or Nr4a2) requires these proteins for their induction in the hippocampus after kainic acid administration, while they are required for most of the cocaine-induced expression changes in the striatum. Interestingly, in the absence of CREB, CREM is able to rescue activity-regulated transcription, which strengthens the notion of overlapping functions of the two proteins. In addition, we show that cholesterol metabolism is dysregulated in the brains of mutant mice, as reflected coordinated expression changes in genes involved in cholesterol synthesis and neuronal accumulation of cholesterol. These findings provide novel insights into the role of CREB and CREM in stimulus-dependent transcription and neuronal homeostasis.

Secretory phospholipase A(2) induces apoptosis via a mechanism involving ceramide generation.

Secretory phospholipase A(2) (sPLA(2)) plays important roles in cellular signaling and various biological events. In this study, we examined the biological effects and the potential signaling mechanism of purified sPLA(2) in MV1Lu cells. Three types of snake venom sPLA(2) were purified and their enzymatic activities were characterized by using various lipid substrates prepared from [3H]-myristate-labeled cells and by determining their effects on the induction of arachidonic acid (AA) release. The purified sPLA(2) induced apoptosis in Mv1Lu cells in a dose- and time-dependent manner, and was associated with a rapid increase in the intracellular ceramide level. Similar apoptotic effects were observed in Mv1Lu cells treated with exogenous ceramide analog, C(2)- and C(8)-ceramide. Moreover, treatment of cells with sphingomyelinase (SMase), which reduced the intracellular SM level, enhanced the apoptotic response to sPLA(2)s. sPLA(2)s also displayed an inhibitory effect on bradykinin-induced phospholipase D (PLD) activity, which can be imitated by exogenous ceramide. Our data indicate that sPLA(2) induces cell apoptosis via a mechanism involving increased ceramide generation.

Regulation of Protein Kinase C in A-549 Cells by Phorbol Ester.

Protein kinase C, a family of serine-threonine protein kinases, mediates a variety of intracellular signaling events. Here, the regulatory effect of phorbol 12-myristate 13-acetate(PMA)on the several PKC isozymes in the human lung carcinoma cells A-549 was studied. The expression of PKC-alpha PKC-betaII PKC-gamma PKC-delta and PKC-epsilon in A-549 cells was examined. No detectable PKC-zeta was observed. Short-term treatment of cells with PMA led to the translocation of these PKC isozymes, to different extent, from cytosol to cell membrane. Whereas, prolonged treatment of cells with PMA pronouncedly reduced the levels of PKC-alpha PKC-gamma PKC-delta and PKC-epsilon, but the intracellular level of PKC-betaII was not affected. Furthermore, PMA was observed to have differential effects on the down-regulation of PKC isozymes located in the cytosol and of those located in the membrane. Prolonged PMA treatment caused extensive decrease in the levels of cytosolic PKC-delta and PKC-gamma, and depleted cytosolic PKC-alpha and PKC-betaII. However, the amount levels of membrane PKC-alpha PKC-betaII PKC-gamma PKC-delta isozymes were not decreased. In contrast, PKC-epsilon in both cytosol and membrane fraction was obviously down-regulated by prolonged PMA treatment. This study provided novel evidence on the PMA-mediated activation and down-regulation of different PKC isozymes, which might be helpful in deepening our understanding on the roles of PKC activation and the alterations of their intracellular levels in processes of chemical carcinogenesis.

Phorbol Ester Induces Formation of A New Phospholipase D Product.

It is demonstrated here that phorbol-12-myristate-13-acetate (PMA) induced the formation of a butanolated-sphingolipids in A-549 cells. In cells labeled with (3)H -serine, the distribution of the radioactivity was easily found inphosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), but not in phosphatidic acid (PA) and its immediate metabolic derivatives, suggesting that the newly detected PLD products were derived from sphingolipids and were different from the conventional product of PLD. Protein kinase C (PKC)inhibitors or PKC down-regulation by prolonged PMA treatment inhibited the PMA-induced generation of this butanolated-sphingolipid, indicating that PKC activation was required in this process.

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone.

The tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. We show here that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo.

Germ line transmission and expression of an RNAi cassette in mice generated by a lentiviral vector system.

We have used a lentiviral delivery system (LentiLox3.7) to generate transgenic mice harbouring RNA interference (RNAi) against the hepatocyte nuclear factor 4 gamma (HNF4gamma). HNF4gamma is a nuclear receptor with unknown function. Our analyses performed on founder (F(0)) and first generation (F(1)) mice revealed mosaicism in F(0) founders and a low efficiency of transgenesis (6%) in F(1) mice. These data, together with the observation of multiple silenced transgenes, do not favour the use of LentiLox3.7 lentivirus for transgenesis. Despite the low efficiency of transgenesis, we achieved a tissue-dependent knockdown of HNF4gamma expression in some mice.

Genetic inactivation of the transcription factor TIF-IA leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis.

Growth-dependent regulation of rRNA synthesis is mediated by TIF-IA, a basal transcription initiation factor for RNA polymerase I. We inactivated the murine TIF-IA gene by homologous recombination in mice and embryonic fibroblasts (MEFs). TIF-IA-/- embryos die before or at embryonic day 9.5 (E9.5), displaying retardation of growth and development. In MEFs, Cre-mediated depletion of TIF-IA leads to disruption of nucleoli, cell cycle arrest, upregulation of p53, and induction of apoptosis. Elevated levels of p53 after TIF-IA depletion are due to increased binding of ribosomal proteins, such as L11, to MDM2 and decreased interaction of MDM2 with p53 and p19(ARF). RNAi-induced loss of p53 overcomes proliferation arrest and apoptosis in response to TIF-IA ablation. The striking correlation between perturbation of nucleolar function, elevated levels of p53, and induction of cell suicide supports the view that the nucleolus is a stress sensor that regulates p53 activity.