The anterior insula bidirectionally modulates cost-benefit decision-making on a rodent gambling task.

Deficits in cost-benefit decision-making, as assessed in the Iowa Gambling Task (IGT), are commonly observed in neuropsychiatric disorders such as addiction. There is considerable variation in the maximization of rewards on such tasks, both in the general population and in rodent models, suggesting individual differences in decision-making may represent a key endophenotype for vulnerability to neuropsychiatric disorders. Increasing evidence suggests that the insular cortex, which is involved in interoception and emotional processes in humans, may be a key neural locus in the control of decision-making processes. However, the extent to which the insula contributes to individual differences in cost-benefit decision-making remains unknown. Using male Sprague Dawley rats, we first assessed individual differences in the performance over the course of a single session on a rodent analogue of the IGT (rGT). Rats were matched for their ability to maximize reward and received bilateral excitotoxic or sham lesions of the anterior insula cortex (AIC). Animals were subsequently challenged on a second rGT session with altered contingencies. Finally, animals were also assessed for instrumental conditioning and reversal learning. AIC lesions produced bidirectional alterations on rGT performance; rats that had performed optimally prior to surgery subsequently showed impairments, and animals that had performed poorly showed improvements in comparison with sham-operated controls. These bidirectional effects were not attributable to alterations in behavioural flexibility or in motivation. These data suggest that the recruitment of the AIC during decision-making may be state-dependent and help guide response selection towards subjectively favourable options.

From impulses to maladaptive actions: the insula is a neurobiological gate for the development of compulsive behavior.

Impulsivity is an endophenotype of vulnerability for compulsive behaviors. However, the neural mechanisms whereby impulsivity facilitates the development of compulsive disorders, such as addiction or obsessive compulsive disorder, remain unknown. We first investigated, in rats, anatomical and functional correlates of impulsivity in the anterior insular (AI) cortex by measuring both the thickness of, and cellular plasticity markers in, the AI with magnetic resonance imaging and in situ hybridization of the immediate early gene zif268, respectively. We then investigated the influence of bilateral AI cortex lesions on the high impulsivity trait, as measured in the five-choice serial reaction time task (5-CSRTT), and the associated propensity to develop compulsivity as measured by high drinking levels in a schedule-induced polydipsia procedure (SIP). We demonstrate that the AI cortex causally contributes to individual vulnerability to impulsive-compulsive behavior in rats. Motor impulsivity, as measured by premature responses in the 5-CSRTT, was shown to correlate with the thinness of the anterior region of the insular cortex, in which highly impulsive (HI) rats expressed lower zif268 mRNA levels. Lesions of AI reduced impulsive behavior in HI rats, which were also highly susceptible to develop compulsive behavior as measured in a SIP procedure. AI lesions also attenuated both the development and the expression of SIP. This study thus identifies the AI as a novel neural substrate of maladaptive impulse control mechanisms that may facilitate the development of compulsive disorders.

In search of predictive endophenotypes in addiction: insights from preclinical research.

Drug addiction is widely recognized to afflict some but not all individuals by virtue of underlying risk markers and traits involving multifaceted interactions between polygenic and external factors. Remarkably, only a small proportion of individuals exposed to licit and illicit drugs develop compulsive drug-seeking behavior, maintained in the face of adverse consequences and associated detrimental patterns of drug intake involving extended and repeated bouts of binge intoxication, withdrawal and relapse. As a consequence, research has increasingly endeavored to identify distinctive neurobehavioral mechanisms and endophenotypes that predispose individuals to compulsive drug use. However, research in active drug users is hampered by the difficulty in categorizing putatively causal behavioral traits prior to the initiation of drug use. By contrast, research in experimental animals is often hindered by the validity of approaches used to investigate the neural and psychological mechanisms of compulsive drug-seeking habits in humans. Herein, we survey and discuss the principal findings emanating from preclinical animal research on addiction and highlight how specific behavioral endophenotypes of presumed genetic origin (e.g. trait anxiety, novelty preference and impulsivity) differentially contribute to compulsive forms of drug seeking and taking and, in particular, how these differentiate between different classes of stimulant and non-stimulant drugs of abuse.

Cocaine-induced sensitization is associated with altered dynamics of transcriptional responses of the dopamine transporter, tyrosine hydroxylase, and dopamine D2 receptors in C57Bl/6J mice.

RATIONALE: Behavioural sensitization is a long lasting phenomenon that has been proposed to be involved in drug addiction. Although the expression of cocaine-induced sensitization has been associated with the activity of the mesencephalic dopaminergic neurons, little is known about the transcriptional adaptations of these neurons to a new challenge with cocaine long after cessation of repeated exposure to the drug. OBJECTIVES: We studied the time course of the mRNA levels of three main regulatory elements of dopaminergic transmission after a challenge with cocaine (15 mg/kg) that followed 21 days of withdrawal from a cocaine pretreatment (20 mg/kg, ip, every 2 days for 21 days) in C57Bl/6J mice. MATERIALS AND METHODS: Mice were placed 45 min in activity chambers and were killed 45 min, 2 h or 24 h after the challenge injection. Dopamine transporter (DAT), D2 auto-receptor (D2) and tyrosine hydroxylase (TH) mRNA levels were assessed by in situ hybridization in the ventral tegmental area and the substantia nigra compacta. RESULTS: As compared to vehicle challenge, cocaine challenge in vehicle pretreated mice induced a rapid increase (+208%) in DAT mRNA (45 min) followed by a delayed decrease (-70%) (24 h), while TH and D2 mRNA were both increased (+45%) 24 h after the challenge. In cocaine pretreated mice, cocaine-induced short-term increase and long-term decrease in DAT mRNA levels were amplified (+328%) and reduced (-40%), respectively. CONCLUSIONS: Repeated exposure to cocaine alters the transcriptional response of DA neurons to a new cocaine challenge long after cessation of repeated exposure to the drug. They point to the DAT mRNA as a major responsive element to a new presentation of cocaine.

Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors.

It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2). The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design. We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli. In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E.coli. Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized. Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the "hydrophobic core" of signal sequences. High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation. In unc- cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force. Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery.

Haemolytic onset of Wilson disease in a patient with homozygous truncation of ATP7B at Arg1319.

We describe a 19-year-old woman with haemolytic anaemia and thrombocytopenia as the initial manifestation of Wilson disease (WD). There are two reasons for reporting such an improbable case. First, it emphasizes the importance of recognizing atypical clinical presentations of potentially lethal recessive traits for which therapy is available. Second, it shows that, even in a monogenic disorder like WD, the phenotype cannot be extrapolated from the mutated genotype in a simple fashion; this patient had a relatively late-onset form of WD despite homozygosity for a genetic lesion leading to an apparent complete loss of function of the WD copper transporter.

Human myoblast fusion requires expression of functional inward rectifier Kir2.1 channels.

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through alpha1H T channels.

Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion.

Escherichia coli K-12, the most widely used laboratory bacterium, does not secrete proteins into the extracellular medium under standard growth conditions, despite possessing chromosomal genes encoding a putative type II secretion machinery (secreton). We show that in wild-type E.coli K-12, divergent transcription of the two operons in the main chromosomal gsp locus, encoding the majority of the secreton components, is silenced by the nucleoid-structuring protein H-NS. In mutants lacking H-NS, the secreton genes cloned on a moderate-copy-number plasmid are expressed and promote efficient secretion of the endogenous, co-regulated endochitinase ChiA. This is the first time that secretion of an endogenous extracellular protein has been demonstrated in E.coli K-12.

Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery.

A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG. These mutations slow down the export of PAI2-AP. Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB-AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles.

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.

Membrane topology of CadA homologous P-type ATPase of Helicobacter pylori as determined by expression of phoA fusions in Escherichia coli and the positive inside rule.

The only experimental data available on the membrane topology of transition metal ATPases are from in vitro studies on two distinct P-type ATPases (CadA and CopA) of a gastric bacterium, Helicobacter pylori, both postulated to contain eight transmembrane domains (H1 to H8). In this study, H. pylori CadA ATPase was subjected to analysis of membrane topology in vivo by expression of ATPase-alkaline phosphatase (AP) hybrid proteins in Escherichia coli using a novel vector, pBADphoA. This vector contains an inducible arabinose promoter and unique restriction sites for fusion of DNA fragments to phoA. The phoA gene lacking sequences encoding its N-terminal signal peptide was linked to the C-terminal regions of the postulated five cytoplasmic and four periplasmic segments of the H. pylori pump. The results obtained by heterologous expression of ATPase-AP hybrid proteins showed consistence with a model of eight transmembrane domains. They also demonstrated that the H. pylori ATPase sequences are well assembled in the cytoplasmic membrane of E. coli, a neutralophilic bacterium. Cloning and amino acid sequence analysis of the homologous ATPase of Helicobacter felis further verified the topological model for the H. pylori pump analyzed here, although the degree of amino acid sequence identity varied between the corresponding transmembrane segments, from 25% for H1 up to 100% for H6. It was found that the topology of ATPase follows the 'positive inside rule'. With respect to the bioenergetic capacities of H. pylori, we discuss here the membrane potential as a possible factor directing insertion of ATPases in the cytoplasmic membrane of gastric bacteria.

Urokinase receptor (uPAR, CD87) is a platelet receptor important for kinetics and TNF-induced endothelial adhesion in mice.

BACKGROUND: Urokinase plasminogen activator receptor (uPAR, CD87) is a widely distributed 55-kD, glycoprotein I-anchored surface receptor. On binding of its ligand uPA, it is known to increase leukocyte adhesion and traffic. Using genetically deficient mice, we explored the role of uPAR in platelet kinetics and TNF-induced platelet consumption. METHODS AND RESULTS: Anti-uPAR antibody stained platelets from normal (+/+) but not from uPAR-/- mice, as seen by fluorescence-activated cell sorter analysis. 51Cr-labeled platelets from uPAR-/- donors survived longer than those from +/+ donors when injected into a +/+ recipient. Intratracheal TNF injection induced thrombocytopenia and a platelet pulmonary localization, pronounced in +/+ but absent in uPAR-/- mice. Aprotinin, a plasmin inhibitor, decreased TNF-induced thrombocytopenia. TNF injection markedly reduced the survival and increased the pulmonary localization of 51Cr-labeled platelets from +/+ but not from uPAR-/- donors, indicating that it is the platelet uPAR that is critical for their response to TNF. As seen by electron microscopy, TNF injection increased the number of platelets and polymorphonuclear neutrophils (PMNs) in the alveolar capillaries of +/+ mice, whereas in uPAR-/- mice, platelet trapping was insignificant and PMN trapping was slightly reduced. Platelets within alveolar capillaries of TNF-injected mice were activated, as judged from their shape, and this was evident in +/+ but not in uPAR-/- mice. CONCLUSIONS: These results demonstrate for the first time the critical role of platelet uPAR for kinetics as well as for activation and endothelium adhesion associated with inflammation.

Single stress induces long-lasting elevations in vasopressin mRNA levels in CRF hypophysiotrophic neurones, but repeated stress is required to modify AVP immunoreactivity.

Repeated stress is known to induce an increased vasopressin (AVP) expression in paraventricular corticotrophin-releasing factor (CRF) neurones which is supposed to enhance the ACTH-releasing capacity of these cells. To test the hypothesis that a single stress is sufficient to produce these changes, we used quantitative in-situ hybridization analysis to measure steady state CRF and AVP mRNA. Moreover the colocalized AVP and CRF immunoreactive sites were assessed in the dense core vesicle compartment of CRF axon terminals in the external zone of the median eminence with quantitative immunoelectron microscopy. Acute immobilization produced a significant increase in the average AVP and CRF mRNA levels (145% and 65%, respectively, above control values) in the medial parvocellular subdivisions of the paraventricular nucleus (PVN), and these changes persisted for over 4 days after stress. In contrast to these changes in AVP mRNA levels, there were no concomitant changes in AVP immunostaining in CRF terminals and axons during the 4-day period. However, when immobilization stress was repeated daily, the number of CRF terminals containing AVP increased progressively. Moreover, the ratio of AVP and CRF immunoreactivity in the dense core vesicle compartment was increased. Taken together, these results provide evidence that single stress experience can cause long-lasting changes in AVP and CRF mRNA steady state expression that is not apparently accompanied by changes in peptide levels. They also suggest that repeated stress is required for developing progressive shifts in the neurohormone storage pattern of these neurones.

Transcriptional activation of ydeA, which encodes a member of the major facilitator superfamily, interferes with arabinose accumulation and induction of the Escherichia coli arabinose PBAD promoter.

Induction of genes expressed from the arabinose PBAD promoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to the ydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient -35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). The ydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of ydeA has no detectable effect on cell growth. Thus, ydeA appears to be nonessential under usual laboratory growth conditions. The ydeA gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of ydeA transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.

Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.

Soluble form of complement C3b/C4b receptor (CR1) results from a proteolytic cleavage in the C-terminal region of CR1 transmembrane domain.

The complement C3b/C4b receptor (CR1) is an integral protein, anchored in the plasma membrane through a hydrophobic domain of 25 amino acids, but is also found in the plasma in soluble form (sCR1). A recombinant, soluble form of CR1 has been demonstrated to reduce complement-dependent tissue injury in animal models of ischaemia/reperfusion. In view of the important pathophysiological relevance of sCR1, we have investigated the mechanisms governing CR1 release by using various mutated and chimaeric receptors transiently expressed in COS cells. Pulse-chase experiments revealed that (1) sCR1 is produced by a proteolytic process, (2) the cleavage site lies within the C-terminus of CR1 transmembrane domain, (3) the proteolytic process involves a fully glycosylated CR1 form and (4) this process takes place in late secretory vesicles or at the plasma membrane.

Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice.

AdmATF is a recombinant adenovirus encoding a secreted version of the amino-terminal fragment (ATF) of murine urokinase (uPA). This defective adenovirus was used in three murine models to assess the antitumoral effects associated with local or systemic delivery of ATF, a broad cell invasion inhibitor that antagonizes uPA binding to its cell surface receptor (uPAR). A single intratumoral injection of AdmATF into pre-established MDA-MB-231 human breast xenografts grown in athymic mice, or into pre-established C57/BL6 syngeneic Lewis lung carcinoma resulted in a specific arrest of tumor growth. Neovascularization within and at the vicinity of the injection site was also suppressed, suggesting that AdmATF inhibited primary tumor growth by targeting angiogenesis. AdmATF also interfered with tumor cell establishment at distant sites: (1) lung dissemination of Lewis lung carcinoma cells was significantly reduced following intratumoral injection at the primary site; and (2) systemic administration of AdmATF inhibited subsequent liver metastasis in a LS174T human colon carcinoma xenograft model. These data outline the potential of using a recombinant adenovirus directing the secretion of an antagonist of cell-associated uPA for cancer gene therapy.