Neuronal pentraxins modulate cocaine-induced neuroadaptations.

Neuronal pentraxins (NPs) function in the extracellular matrix to bind alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Three NPs have been described, neuronal activity-regulated pentraxin (Narp), which is regulated as an immediate early gene, NP1, and neuronal pentraxin receptor (NPR). Narp and NP1 enhance synaptogenesis and glutamate signaling by clustering AMPA receptors, whereas NPR contributes to removing AMPA receptors during group I metabotropic glutamate receptor-dependent long-term depression. Here, we examine mice with genetic deletions [knockout (KO)] of each NP to assess their contributions to cocaine-induced neuroplasticity. Consistent with a shared AMPA receptor clustering function for Narp and NP1, deletion of either NP caused similar behavioral alterations. Thus, although both Narp and NP1 deletion promoted cocaine-induced place preference, NPR deletion was without effect. In addition, although Narp and NP1 KO showed reduced time in the center of a novel environment, NPR KO mice spent more time in the center. Finally, although Narp and NP1 KO mice showed blunted locomotion after AMPA microinjection into the accumbens 3 weeks after discontinuing repeated cocaine injections, the AMPA response was augmented in NPR KO. Likewise, endogenous glutamate release elicited less motor activity in Narp KO mice. Consistent with reduced AMPA responsiveness after chronic cocaine in Narp KO mice, glutamate receptor 1 was reduced in the PSD fraction of Narp KO mice withdrawn from cocaine. These data indicate that NPs differentially contribute to cocaine-induced plasticity in a manner that parallels their actions in synaptic plasticity.

NAC1 regulates the recruitment of the proteasome complex into dendritic spines.

Coordinated proteolysis of synaptic proteins is required for synaptic plasticity, but a mechanism for recruiting the ubiquitin-proteasome system (UPS) into dendritic spines is not known. NAC1 is a cocaine-regulated transcriptional protein that was found to complex with proteins in the UPS, including cullins and Mov34. NAC1 and the proteasome were cotranslocated from the nucleus into dendritic spines in cortical neurons in response to proteasome inhibition or disinhibiting synaptic activity with bicuculline. Bicuculline also produced a progressive accumulation of the proteasome and NAC1 in the postsynaptic density. Recruitment of the proteasome into dendrites and postsynaptic density by bicuculline was prevented in neurons from mice harboring an NAC1 gene deletion or in neurons transfected with mutated NAC1 lacking the proteasome binding domain. These experiments show that NAC1 modulates the translocation of the UPS from the nucleus into dendritic spines, thereby suggesting a potential missing link in the recruitment of necessary proteolysis machinery for synaptic remodeling.

Opposing roles for the ventral prefrontal cortex and the basolateral amygdala on the spontaneous recovery of cocaine-seeking in rats.

RATIONALE: The neural circuitry subserving cocaine-seeking after extinction vs abstinence alone requires different constituent brain structures. Spontaneous recovery of cocaine-seeking, a model, which incorporates both extinction and abstinence, depends on an unknown neural circuit. OBJECTIVES: The present study examined the hypothesis that the spontaneous recovery of cocaine-seeking would require overlapping but distinct neural circuits compared to models that incorporate either extinction or abstinence alone. MATERIAL AND METHODS: Rats were trained to self-administer cocaine (0.2 mg/inf), then responding on the cocaine-paired lever was extinguished, followed by an additional period of abstinence in the home cage. Finally, rats were returned to the self-administration context for a test of spontaneous recovery (SR TEST). Just before the SR TEST, discrete brain regions were inactivated with a GABA agonist cocktail (1 mM baclofen + 0.1mM muscimol) to determine the relative importance of these brain regions in the spontaneous recovery of cocaine-seeking. RESULTS: The inactivation of the ventromedial prefrontal cortex (vPFC) enhanced cocaine-seeking, whereas the inactivation of the basolateral amygdala (BLA) attenuated spontaneous recovery. Inactivation of the nucleus accumbens core (Core) resembled the effects of BLA inactivation, but these results were confounded by an inhibitory effect of the vehicle treatment. Finally, the spontaneous recovery of cocaine-seeking was unaltered by manipulations of the dorsomedial prefrontal cortex (dPFC) and the nucleus accumbens shell (Shell). CONCLUSIONS: The neural circuitry subserving cocaine-seeking behavior in a spontaneous recovery model requires the BLA and possibly the Core, like extinction models. In addition, this behavior is subject to regulation by vPFC, in a manner functionally opposite to that of the BLA.

Nrf2 gene deletion fails to alter psychostimulant-induced behavior or neurotoxicity.

The transcription factor NF-E2-related factor (Nrf2) regulates the induction of phase 2 detoxifying enzymes by oxidative stress, including synthesis of the catalytic subunit (xCT) of the heterodimeric cystine-glutamate exchanger (system xc-). Repeated cocaine treatment in rats causes persistent neuroadaptations in glutamate neurotransmission in the nucleus accumbens that result, in part, from reduced activity of system xc-. Since in vitro under- or over-expression of Nrf2 regulates system xc- activity and xCT content, it was hypothesized that in vivo deletion of the Nrf2 gene would: 1) decrease system xc- activity, 2) produce a behavioral phenotype resembling that elicited by chronic cocaine administration, and 3) enhance dopamine depletion after methamphetamine-induced oxidative stress. In all three experiments no genotypic difference was measured between mice sustaining homozygous Nrf2 gene deletion and wild-type littermates. Thus, while Nrf2 is a transcriptional regulator of xCT and capable of protecting cells from oxidative stress, following Nrf2 gene deletion this role can be partially compensated by other mechanisms and methamphetamine-induced oxidative stress and dopamine toxicity does not significantly involve Nrf2.

Expression of nitric oxide synthase-2 in the lungs decreases airway resistance and responsiveness.

Individuals with asthma have increased levels of nitric oxide in their exhaled air. To explore its role, we have developed a regulatable transgenic mouse capable of overexpressing inducible nitric oxide synthase in a lung-specific fashion. The CC10-rtTA-NOS-2 mouse contains two transgenes, a reverse tetracycline transactivator under the control of the Clara cell protein promoter and the mouse nitric oxide synthase-2 (NOS-2) coding region under control of a tetracycline operator. Addition of doxycycline to the drinking water of CC10-rtTA-NOS-2 mice causes an increase in nitric oxide synthase-2 that is largely confined to the airway epithelium. The fraction of expired nitric oxide increases over the first 24 h from approximately 10 parts per billion to a plateau of approximately 20 parts per billion. There were no obvious differences between CC10-rtTA-NOS-2 mice, with or without doxycycline, and wild-type mice in lung histology, bronchoalveolar protein, total cell count, or count differentials. However, airway resistance was lower in CC10-rtTA-NOS-2 mice with doxycycline than in CC10-rtTA-NOS-2 mice without doxycycline or wild-type mice with doxycycline. Moreover, doxycycline-treated CC10-rtTA-NOS-2 mice were hyporesponsive to methacholine compared with other groups. These data suggest that increased nitric oxide in the airways has no proinflammatory effects per se and may have beneficial effects on pulmonary function.

Roles for early response cytokines during Escherichia coli pneumonia revealed by mice with combined deficiencies of all signaling receptors for TNF and IL-1.

During infection, inflammation is essential for host defense, but it can injure tissues and compromise organ function. TNF-alpha and IL-1 (alpha and beta) are early response cytokines that facilitate inflammation. To determine the roles of these cytokines with overlapping functions, we generated mice deficient in all of the three receptors mediating their effects (TNFR1, TNFR2, and IL-1RI). During Escherichia coli pneumonia, receptor deficiency decreased neutrophil recruitment and edema accumulation to half of the levels observed in wild-type mice. Thus these receptors contributed to maximal responses, but substantial inflammation progressed independently of them. Receptor deficiency compromised antibacterial efficacy for some infectious doses. Decreased ventilation during E. coli pneumonia was not affected by receptor deficiency. However, the loss of lung compliance during pneumonia was substantially attenuated by receptor deficiency. Thus during E. coli pneumonia in mice, the lack of signaling from TNF-alpha and IL-1 decreases inflammation and preserves lung compliance.

Corticotropin-releasing hormone deficiency increases allergen-induced airway inflammation in a mouse model of asthma.

BACKGROUND: Corticotropin-releasing hormone (CRH) is a major regulator of adrenocorticotropic hormone and the production of glucocorticoids by the adrenal gland. Abnormal regulation of CRH and endogenous glucocorticoids has been implicated in the pathogenesis of asthma. OBJECTIVE: We postulated that CRH deficiency could increase asthma severity by disrupting hypothalamus-pituitary-adrenal axis function and the induction of glucocorticoids through inflammatory and physiologic stress. However, CRH is expressed by several types of immune cells and might be induced at sites of inflammation, where it has local immunostimulatory actions. Thus CRH deficiency could decrease asthma severity. METHODS: To test these possibilities, we subjected CRH-knockout mice to an ovalbumin-induced airway inflammation protocol that mimics many features of asthma. RESULTS: CRH-knockout mice had an increase in airway inflammation of approximately 80% to 300% and an increase in goblet cell hyperplasia of approximately 70% compared with wild-type mice. In contrast, IgE induction was unaffected by CRH deficiency. The increased inflammation in knockout mice was associated with increased tissue resistance, elastance, and hysteresivity. Levels of IL-4, IL-5, IL-13, RANTES, IFN-gamma, and eotaxin were all increased in knockout mice. Serum corticosterone levels were decreased in knockout mice and might account for some of the differences between knockout and wild-type mice. CONCLUSION: We conclude that CRH deficiency disrupts endogenous glucocorticoid production and enhances allergen-induced airway inflammation and lung mechanical dysfunction in mice. Thus inherited or acquired CRH deficiency could increase asthma severity in human subjects.

Induction of early growth-response factor 1 by platelet-derived growth factor in human airway smooth muscle.

Platelet-derived growth factors (PDGF) may contribute to the activation and growth of smooth muscle that is characteristic of airway remodeling in asthmatic patients. Early growth response 1 (EGR-1) is a transcription factor that is induced in several cell types by PDGF and may mediate some of the effects of PDGF. We show that human airway smooth muscle cells in cell culture express EGR-1 1 h after addition of PDGF. Analysis of the EGR-1 promoter indicates that a serum response element located between 663 and 654 bp 5' to the ATG start site is essential for this induction. Serum response factor, E26 transcription factor-like protein 1, and serum protein 1 bind to this region. PDGF causes phosphorylation of ERK1/2 and is temporally associated with E26 transcription factor-like protein 1 phosphorylation. Finally, the specific ERK1/2 inhibitor U-0126 abolishes PDGF-induced expression of EGR-1 in these cells. On the basis of these data, we speculated that EGR-1 would be increased in airway smooth muscle of asthmatic patients compared with nonasthmatic controls. Using immunohistochemistry, we found that EGR-1 protein was expressed in airway smooth muscle cells and epithelial cells of asthmatic patients and nonasthmatic controls; however, there was no significant difference in the intensity of staining between groups. EGR-1 was similarly expressed in the lungs of mice with and without ovalbumin-induced airway inflammation; however, there was no difference between groups by immunohistochemistry and quantitative PCR. Although EGR-1 is induced by PDGF in human airway smooth muscle cells in cell culture, the role of EGR-1 in airway remodeling and asthma remains to be established.

Transforming growth factor-beta1 promoter polymorphism C-509T is associated with asthma.

Transforming growth factor-beta1 (TGF-beta1) is increased in the lungs of individuals with asthma and may modulate airway inflammation and remodeling. Some genetic studies have found that a C-to-T single-nucleotide polymorphism (C-509T) in the TGF-beta1 gene promoter may be associated with altered gene expression and asthma phenotype. To build on these data, we performed a case-control association study at this locus involving 527 subjects with asthma and 170 control subjects without asthma. All individuals were white. Genotyping at 49 unlinked polymorphisms indicated that a subset of case subjects and all control subjects were well matched and without evidence of population stratification. Logistic regression was used to model the effects of age, sex, and genotype on case-control status. The diagnosis of asthma was positively associated with the T allele and TT genotype under a codominant model (odds ratio, 2.98; 95% confidence interval, 1.45 to 6.25; p = 0.003). Total serum IgE, eosinophil count, and FEV1% predicted levels were not associated with this polymorphism. Furthermore, we show that the C-509T polymorphism alters TGF-beta1 promoter-reporter activity and promoter interactions with the transcription factor Yin Yang 1. We conclude that the T allele of C-509T is associated with the diagnosis of asthma and may enhance TGF-beta1 gene transcription.