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Astute control of brain activity states is critical for adaptive behaviours and survival. In mammals and birds, electroencephalographic recordings reveal alternating states of wakefulness, slow wave sleep and paradoxical sleep (or rapid eye movement sleep). This control is profoundly impaired in narcolepsy with cataplexy, a disease resulting from the loss of orexin/hypocretin neurotransmitter signalling in the brain. Narcolepsy with cataplexy is characterized by irresistible bouts of sleep during the day, sleep fragmentation during the night and episodes of cataplexy, a sudden loss of muscle tone while awake and experiencing emotions. The neural mechanisms underlying cataplexy are unknown, but commonly thought to involve those of rapid eye movement-sleep atonia, and cataplexy typically is considered as a rapid eye movement sleep disorder. Here we reassess cataplexy in hypocretin (Hcrt, also known as orexin) gene knockout mice. Using a novel video/electroencephalogram double-blind scoring method, we show that cataplexy is not a state per se, as believed previously, but a dynamic, multi-phased process involving a reproducible progression of states. A knockout-specific state and a stereotypical paroxysmal event were introduced to account for signals and electroencephalogram spectral characteristics not seen in wild-type littermates. Cataplexy almost invariably started with a brief phase of wake-like electroencephalogram, followed by a phase featuring high-amplitude irregular theta oscillations, defining an activity profile distinct from paradoxical sleep, referred to as cataplexy-associated state and in the course of which 1.5-2 s high-amplitude, highly regular, hypersynchronous paroxysmal theta bursts (∼7 Hz) occurred. In contrast to cataplexy onset, exit from cataplexy did not show a predictable sequence of activities. Altogether, these data contradict the hypothesis that cataplexy is a state similar to paradoxical sleep, even if long cataplexies may evolve into paradoxical sleep. Although not exclusive to overt cataplexy, cataplexy-associated state and hypersynchronous paroxysmal theta activities are highly enriched during cataplexy in hypocretin/orexin knockout mice. Their occurrence in an independent narcolepsy mouse model, the orexin/ataxin 3 transgenic mouse, undergoing loss of orexin neurons, was confirmed. Importantly, we document for the first time similar paroxysmal theta hypersynchronies (∼4 Hz) during cataplexy in narcoleptic children. Lastly, we show by deep recordings in mice that the cataplexy-associated state and hypersynchronous paroxysmal theta activities are independent of hippocampal theta and involve the frontal cortex. Cataplexy hypersynchronous paroxysmal theta bursts may represent medial prefrontal activity, associated in humans and rodents with reward-driven motor impulse, planning and conflict monitoring.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aβ-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aβ oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155 -dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

Dopamine D2 receptor (D2R) signalling has been shown to modulate seizure-induced hippocampal cell death. D2R knockout (D2R−/−) mice are more susceptible to kainic acid (KA)-induced excitotoxicity, displaying cell death in the CA3 subfield of the hippocampus at KA doses not damaging in wild-type (WT) animals. Absence of D2R signalling in the hippocampus leads to activation (dephosphorylation) of glycogen synthase kinase 3β (GSK-3β) after KA (20 mg/kg), which is not associated with a change in the phosphorylation of the GSK-3β regulator Akt at the canonical threonine 308 residue. In the present study, we investigated alternative pathways responsible for the activation of GSK-3β in the hippocampus of the D2R−/− mice 24 h following KA-induced seizures. Here, we show that phosphorylation of Akt occurs at serine 473 (Ser473) in the CA3 region of WT but not D2R−/− mice following KA. Moreover, the CA1 subregion, which does not undergo neurodegeneration in either WT or D2R−/− mice, displays a strong induction of Akt (Ser473) phosphorylation after KA. Additionally, the vulnerability in the CA3 is not associated with changes to p38MAPK and Dishevelled activation, and β-catenin does not appear to be a downstream target of the GSK-3β. Thus, we propose that GSK-3β phosphorylation-mediated hippocampal cell survival may depend on Akt (Ser473) phosphorylation; loss of D2R-mediated signalling in the CA3 region of D2R−/− mice leads to reduced Akt (Ser473) phosphorylation rendering neurons more vulnerable to apoptosis. Further investigation is required to fully elucidate the GSK-3β targets involved in D2R-dependent response to excitotoxicity.

Dendritic cells (DC) are highly specialized antigen-presenting cells characterized by the ability to prime T-cell responses. Mesenchymal stem cells (MSC) are adult stromal progenitor cells displaying immunomodulatory activities including inhibition of DC maturation in vitro. However, the specific impact of MSC on DC functions, upon in vivo administration, has never been elucidated. Here we show that murine MSC impair Toll-like receptor-4 induced activation of DC resulting in the inhibition of cytokines secretion, down-regulation of molecules involved in the migration to the lymph nodes, antigen presentation to CD4 + T cells, and cross-presentation to CD8 + T cells. These effects are associated with the inhibition of phosphorylation of intracellular mitogen-activated protein kinases. Intravenous administration of MSC decreased the number of CCR7 and CD49dβ1 expressing CFSE-labeled DC in the draining lymph nodes and hindered local antigen priming of DO11.10 ovalbumin-specific CD4 + T cells. Upon labeling of DC with technetium-99m hexamethylpropylene amine oxime to follow their in vivo biodistribution, we demonstrated that intravenous injection of MSC blocks, almost instantaneously, the migration of subcutaneously administered ovalbumin-pulsed DC to the draining lymph nodes. These findings indicate that MSC significantly affect DC ability to prime T cells in vivo because of their inability to home to the draining lymph nodes and further confirm MSC potentiality as therapy for immune-mediated diseases.

Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimer's disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment. We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.

Akt1 is implicated in cell metabolism, survival migration, and gene expression; however, little is known about the role of specific Akt isoforms during inflammation in vivo. Thus, we directly explored the roles of the isoforms Akt1 and Akt2 in acute inflammation models by using mice deficient in either Akt1 or Akt2. Akt1 −/− mice showed a markedly reduced edema versus Akt2 −/− and WT controls, and the reduced inflammation was associated with a dramatic decrease in neutrophil and monocyte infiltration. The loss of Akt1 did not affect leukocyte functions in vitro, and bone marrow transplant experiments suggest that host Akt1 regulates leukocyte emigration into inflamed tissues. Moreover, carrageenan-induced edema and the direct propermeability actions of bradykinin and histamine were reduced dramatically in Akt1 −/− versus WT mice. These findings are supported by in vitro experiments showing that Akt1 deficiency or blockade of nitric oxide synthase markedly reduces histamine-stimulated changes in transendothelial electrical resistance of microvascular endothelial cells. Collectively, these results suggest that Akt1 is necessary for acute inflammation and exerts its actions primarily via regulation of vascular permeability, leading to edema and leukocyte extravasation.

These experiments examined the interaction of muscarinic and dopaminergic systems in influencing memory for one-trial inhibitory avoidance training in mice of the C57BL/6 and DBA/2 strains. In both strains, immediate post-training systemic administration of the muscarinic cholinergic agonist oxotremorine enhanced retention and the cholinergic antagonist atropine impaired retention. No effects were seen with injections 2 h post-training. Furthermore, the drugs did not affect retention performance of animals that received no footshock on the training trial. These results confirm previous findings indicating that muscarinic cholinergic drugs affect memory by influencing memory consolidation. In C57 mice, pretreatment with selective D1 or D2 dopamine (DA) receptor agonists (SKF 38393 or LY 171555, respectively) in otherwise non-effective doses (5 and 0.25 mg/kg, respectively) potentiated the effects of oxotremorine (0.04 mg/kg). Furthermore, in C57 mice pretreatment with selective D1 or D2 receptor antagonists (SCH 23390 or (−)-sulpiride) in otherwise non-effective doses (0.025 and 6 mg/kg, respectively) blocked the memory enhancing effects of oxotremorine. The memory impairing effects of atropine (3 mg/kg) were blocked by the D1 and D2 selective agonists and potentiated by the selective D1 or D2 antagonists. In contrast, in DBA mice, the D1 and D2 selective agonists antagonised the memory enhancing effects of oxotremorine (0.02 mg/kg) and potentiated the effects of atropine (2 mg/kg). Furthermore, the D1 and D2 antagonists potentiated the effects of oxotremorine and antagonised those of atropine. These findings indicate that although muscarinic cholinergic influences on memory storage are comparable in mice of these two strains, the cholinergic-dopaminergic interactions are opposite in the two strains. These results have implications for hypotheses of cholinergic and dopaminergic regulation of memory storage.

Summary The hepatitis B virus (HBV) is a common human pathogen that causes acute and chronic liver disease. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma. The contribution of the viral regulatory protein HBx in liver oncogenesis has been supported by our recent studies in a transgenic mouse model, showing that HBx cooperates with c-myc by accelerating the onset of primary liver tumors. Here we show that liver expression of HBx is associated with increased rates of spontaneous apoptosis in liver cells from two different transgenic lines. In transient transfection assays, overexpression of HBx in the established hepatocyte cell line MMHD3 and in human hepatoma cells HepG2 was found to induce apoptosis in a dose-dependent manner. These data suggest that HBx might trigger an apoptotic process in HBV-infected hepatocytes, in turn possibly favoring liver regeneration and accumulation of genetic alterations, ultimately leading to liver cell transformation in chronically infected patients.

Tumors frequently induce immature myeloid cells (iMC), which suppress specific and unrelated cytotoxic T lymphocyte (CTL) responses and are termed myeloid-derived suppressor cells (MDSC). Mainly analyzed by in vitro assays in tumor transplantation models, little is known about their function in autochthonous tumor models in vivo. We analyzed iMC in 3 SV40 large T (Tag)-driven conditional autochthonous cancer models with different immune status: (1) Early Tag-specific CTL competence and rare stochastic Tag activation leading to sporadic cancer, which induces an aberrant immune response and CTL tolerance; (2) Cre/LoxP recombinase-mediated hepatocellular carcinoma (HCC) development in neonatal Tag-tolerant mice; and (3) Tag-activation through Cre recombinase-encoding viruses in the liver and HCC development with systemic anti-Tag CTL immunity. In the first but not two latter models, tumors induced CTL hyporesponsiveness to tumor-unrelated antigens. Regardless of the model, tumors produced interleukin-6 and vascular endothelial growth factor but not granulocyte macrophage–colony-stimulating factor (GM-CSF) and induced iMC (CD11b(+)Gr-1(int)) that suppressed CTL responses in vitro. None of the iMC from the different tumor models suppressed CTL responses in adoptive cell transfer experiments unless GM-CSF was provided in vivo. Together, iMC expand independent of the type of antitumor response and are not immunosuppressive in a cell-autonomous fashion.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activity is modulated by xenobiotics as well as physiological ligands. These compounds may modulate inflammatory responses and contribute to the rising prevalence of allergic diseases observed in industrialized countries. Mast cells (MCs), located within tissues at the boundary of the external environment, represent a potential target of AhR ligands. In this study, we report that murine and human MCs constitutively express AhR, and its activation by the high-affinity ligand 6-formylindolo[3,2-b]carbazole (FICZ) determines a boost in degranulation. On the contrary, repeated exposure to FICZ inhibits MC degranulation. Accordingly, histamine release, in an in vivo passive systemic anaphylactic model, is exacerbated by a single dose and is attenuated by repetitive stimulation of AhR. FICZ-exposed MCs produce reactive oxygen species and IL-6 in response to cAMP-dependent signals. Moreover, AhR-activated MCs produce IL-17, a critical player in chronic inflammation and autoimmunity, suggesting a novel pathway for MC activation in the pathogenesis of these diseases. Indeed, histological analysis of patients with chronic obstructive pulmonary disease revealed an enrichment in AhR/IL-6 and AhR/IL-17 double-positive MCs within bronchial lamina propria. Thus, tissue-resident MCs could translate external chemical challenges through AhR by modulating allergic responses and contributing to the generation of inflammation-related diseases.

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer.

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca2+homeostasis, including Ca2+transporters. One of the major players in the regulation of intracellular Ca2+([Ca2+]i) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca2+clearance in the synaptic sites of hippocampal neurons. Because NCX3, one of the three brain isoforms of the NCX family, is highly expressed in the hippocampal subfields involved in LTP, we hypothesized that it might represent a potential candidate for LTP modulation. To test this hypothesis, we first examined the effect ofncx3gene ablation on NCX currents (INCX) and Ca2+homeostasis in hippocampal neurons.ncx3−/−neurons displayed a reducedINCX, a higher basal level of [Ca2+]i, and a significantly delayed clearance of [Ca2+]ifollowing depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed thatncx3−/−mice had an impaired basal synaptic transmission. Moreover, hippocampal slices fromncx3−/−mice exhibited a worsening in LTP compared with congenicncx3+/+. Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus ofncx3−/−mice both Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared withncx3+/+. Interestingly,ncx3−/−mice displayed a reduced spatial learning and memory performance, as revealed by the novel object recognition, Barnes maze, and context-dependent fear conditioning assays. Collectively, our findings demonstrate that the deletion of thencx3gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.

Psoriasis is a chronic inflammatory disorder of the skin characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. T cell-derived cytokines, such as IFN-γ and IL-17A, play a major role in the psoriasis-associated epidermal hyperplasia, even though factors/mechanisms that regulate the production of these cytokines are not fully understood. We have recently shown that IL-21 is synthesized in excess in psoriatic skin lesions and causes epidermal hyperplasia when injected intradermally in mice. Moreover, in the human psoriasis SCID mouse model, neutralization of IL-21 reduces both skin thickening and expression of inflammatory molecules, thus supporting the pathogenic role of IL-21 in psoriasis. However, the basic mechanism by which IL-21 promotes skin pathology remains unknown. In this study, we show that CD4+ cells accumulate early in the dermis of IL-21–treated mice and mediate the development of epidermal hyperplasia. Indeed, IL-21 fails to induce skin damage in RAG1-deficient mice and CD4+ cell-depleted wild-type mice. The majority of CD4+ cells infiltrating the dermis of IL-21–treated mice express IFN-γ and, to a lesser extent, IL-17A. Studies in cytokine knockout mice show that IFN-γ, but not IL-17A, is necessary for IL-21–induced epidermal hyperplasia. Finally, we demonstrate that IFN-γ–producing CD4+ cells infiltrating the human psoriatic plaque express IL-21R, and abrogation of IL-21 signals reduces IFN-γ expression in cultures of psoriatic CD4+ cells. Data indicate that IL-21 induces an IFN-γ–dependent pathogenic response in vivo, thus contributing to elucidate a mechanism by which IL-21 sustains skin-damaging inflammation.

SummaryOur findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

Interleukin 17-producing T helper cells (T(H)-17 cells) are important in experimental autoimmune encephalomyelitis, but their route of entry into the central nervous system (CNS) and their contribution relative to that of other effector T cells remain to be determined. Here we found that mice lacking CCR6, a chemokine receptor characteristic of T(H)-17 cells, developed T(H)-17 responses but were highly resistant to the induction of experimental autoimmune encephalomyelitis. Disease susceptibility was reconstituted by transfer of wild-type T cells that entered into the CNS before disease onset and triggered massive CCR6-independent recruitment of effector T cells across activated parenchymal vessels. The CCR6 ligand CCL20 was constitutively expressed in epithelial cells of choroid plexus in mice and humans. Our results identify distinct molecular requirements and ports of lymphocyte entry into uninflamed versus inflamed CNS and suggest that the CCR6-CCL20 axis in the choroid plexus controls immune surveillance of the CNS.

In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility. In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility. © 2007 Elsevier Inc. All rights reserved.

Huntington disease (HD) is a hereditary neurodegenerative disorder caused by an expanded number of CAG repeats in the huntingtin gene. A hallmark of HD is unintended weight loss, the cause of which is unknown. In order to elucidate the underlying mechanisms of weight loss in HD, we studied its relation to other disease characteristics including motor, cognitive, and behavioral disturbances and CAG repeat number.In 517 patients with early stage HD, we applied mixed-effects model analyses to correlate weight changes over 3 years to CAG repeat number and various components of the Unified Huntington's Disease Rating Scale (UHDRS). We also assessed the relation between CAG repeat number and body weight and caloric intake in the R6/2 mouse model of HD.In patients with HD, mean body mass index decreased with -0.15 units per year (p < 0.001). However, no single UHDRS component, including motor, cognitive, and behavioral scores, was independently associated with the rate of weight loss. Patients with HD with a higher CAG repeat number had a faster rate of weight loss. Similarly, R6/2 mice with a larger CAG repeat length had a lower body weight, whereas caloric intake increased with larger CAG repeat length.Weight loss in Huntington disease (HD) is directly linked to CAG repeat length and is likely to result from a hypermetabolic state. Other signs and symptoms of HD are unlikely to contribute to weight loss in early disease stages. Elucidation of the responsible mechanisms could lead to effective energy-based therapeutics.

Introduction: Les cellules de la crete neurale (NC) se differencient in vitro en neuroblastes precurseurs du systeme nerveux enterique (ENS) quand ils sont stimules par des agents specifiques. Nous developpons une etude chez l'animal pour savoir si les neuroblastes derives de la crete neurale peuvent survivre et coloniser in vivo l'intestin d'une souris. Methodes: Les precurseurs du neuroblastes du ENS sont obtenus de la portion vagale des tubes neuraux d'embryons de souris 296 CD-1 et Gtrosa26. Les cellules embryonnaires de Gtrosa26 sont identifiables par leur activite beta-galactosidase ce qui permet de les reconnaitre par coloration bleue. L'hote utilise dans cette etude etait la souris Dom, un animal modele pour la maladie de Hirschsprung (megacolon aganglionnaire). Les nouveaux-nes de souris Dom (n=43) ont recu des cellules derivees de la crete neurale inoculees dans le plan sero-musculaire de l'intestin (33/ 43) ou directement dans la cavite abdominale (10/43). Resultats: Toutes les souris Dom ont survecu a l'intervention et ont ete sacrifiees apres 7 ou 14 jours. Les etudes histo-chimiques ont recherche les cellules implantees chez toutes les souris. Celles-ci ont montre une colonisation myenterique specifique de l'intestin aganglionnaire et ganglionnaire. Conclusion: Le resultat de cette etude est de montrer le tropisme specifique des cellules derivees de la crete neurale vers les sites cible sous l'action d'agents chemotactiques inconnus. Ce procede experimental peut etre un possible traitement pour les formes d'anomalie du systeme nerveux enterique telles que les aganglionoses totales.

Immunotherapy may provide valid alternative therapy for patients with hormone-refractory metastatic prostate cancer. However, if the tumor environment exerts a suppressive action on antigen-specific tumor-infiltrating lymphocytes (TIL), immunotherapy will achieve little, if any, success. In this study, we analyzed the modulation of TIL responses by the tumor environment using collagen gel matrix–supported organ cultures of human prostate carcinomas. Our results indicate that human prostatic adenocarcinomas are infiltrated by terminally differentiated cytotoxic T lymphocytes that are, however, in an unresponsive status. We demonstrate the presence of high levels of nitrotyrosines in prostatic TIL, suggesting a local production of peroxynitrites. By inhibiting the activity of arginase and nitric oxide synthase, key enzymes of L-arginine metabolism that are highly expressed in malignant but not in normal prostates, reduced tyrosine nitration and restoration of TIL responsiveness to tumor were achieved. The metabolic control exerted by the tumor on TIL function was confirmed in a transgenic mouse prostate model, which exhibits similarities with human prostate cancer. These results identify a novel and dominant mechanism by which cancers induce immunosuppression in situ and suggest novel strategies for tumor immunotherapy.

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.

The glycolipid alpha-galactosylceramide (alpha-GalCer), ligand of NKT cells, has been recently shown to induce antitumor immunity in mice through the induction of IL-12 production by dendritic cells. In the present study we compared alpha-GalCer and rIL-12 antitumor activities in the treatment of hepatic metastases of the C-26 murine colon carcinoma. We show that in immunocompetent mice the two molecules display similar efficacy, whereas in mice knockout (KO) for beta2-microglobulin (beta2m), IFN-gamma or IL-12p40, alpha-GalCer antitumor activity is severely impaired. Conversely,in all such KO mice, rIL-12 retains its efficacy. In this context, the IL-12 effect relies on NK cell function since it is abrogated by antibodies to NK1.1, expressed by both NK and NKT cells, but not in beta2m KO mice that lack NKT and CD8 T cells, but have a perfectly functional NK cell population. Furthermore, in IFN-gamma and IL-12p40 double KO mice, exogenous rIL-12 completely loses antitumor efficacy, suggesting the existence of an IFN-gamma-independent IL-12 effect that does require the presence of endogenous IL-12p40 chain.

Constitutive cyclooxygenase-1 enzyme synthesizes prostaglandins which are thought to play an important role in the functional integrity of the stomach gastric mucosa. Recently, it was shown that cyclooxygenase-1 deficient mutant mice did not develop spontaneous gastric pathology and appear less sensitive to indomethacin-induced gastric damage.To investigate gastric acid secretion in cyclooxygenase-1 deficient mutant mice.The basal and histamine or isobutyl methylxanthine-stimulated acid secretion in stomachs of cyclooxygenase-1 deficient homozygous mice and the effect of indomethacin was compared with that of heterozygous and wild-type mice using isolated lumen perfused mouse stomachs, in organ baths, monitored by pH-electrodes.There was no significant difference in the basal or histamine stimulated gastric acid secretion between wild-type or heterozygous or homozygous mice. However, isobutyl methylxanthine was more potent in the cyclooxygenase-1 deficient and heterozygous mice than in wild-type mice. Indomethacin, at concentrations below 1 mM, had no effect on either basal or histamine stimulated acid secretion in any of the mice populations.Gastric acid secretion is maintained without prostaglandin involvement in cyclooxygenase-1 deficient mice. The finding that basal and histamine-stimulated gastric acid secretion was similar in the cyclooxygenase-1 deficient, compared to wild-type mice is consistent with the lack of spontaneous gastric pathology in the cyclooxygenase-1 deficient mice.

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100\% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.

The interaction between muscarinic-cholinergic and dopaminergic systems in the modulation of memory storage of Y-maze discrimination (YMD) task was examined in C57BL/6 and DBA/2 strains of mice. In C57BL/6 mice, post-training systemic (i.p.) administration of the D 2 -agonist quinpirole facilitated retention and the D 2 -antagonist (−)-sulpiride impaired retention. Opposite effects were observed in DBA/2 strain. The facilitating or impairing effects of quinpirole and (−)-sulpiride were blocked by simultaneous post-training administration of muscarinic-cholinergic agonists and antagonists. The memory enhancing effects of the cholinergic agonist oxotremorine were not blocked by simultaneous administration of sulpiride in C57BL/6 mice or quinpirole in DBA/2 mice. Furthermore, the memory impairing effects of the cholinergic antagonist atropine were not blocked by simultaneous administration of quinpirole in C57BL/6 mice or sulpiride in DBA/2 mice. These findings indicate that the effects of D 2 -receptor agonists and antagonists on retention of YMD task are strain-dependent and mediated through muscarinic-cholinergic mechanisms.

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.

Post-training administration of the inhibitor of cholinesterase enzymes, physostigmine, dose-dependently (0.025-0.4 mg/kg) improved retention of an inhibitory avoidance response in C57BL/6 (C57) as well as in DBA/2 (DBA) mice, the latter being more responsive than C57 mice. The effects on retention performance induced by physostigmine in C57 and DBA mice appeared to be due to an effect on memory consolidation. In fact, they were observed when drugs were given at short, but not long, periods of time after training, which is when the memory trace is susceptible to modulation. Moreover, these effects are not to be ascribed to a rewarding or non-specific action of the drugs on retention performance, as the latencies during the retention test of those mice that had not received a footshock during the training were not affected by the post-training drug administration. Post-training administration of cocaine (1-5 mg/kg) dose-dependently improved retention of an inhibitory avoidance response in C57 mice, while impairing it in the DBA strain, thus confirming previous results (Puglisi-Allegra et al. 1994b). Pretreatment with cocaine at ineffective doses as well as at an effective one potentiated the effects of an ineffective as well as of an effective dose of physostigmine in C57 mice, while it antagonized the effects of the inhibitor of cholinesterase enzymes on memory consolidation in DBA mice. The present results indicate that the indirect DA receptor agonist cocaine affects physostigmine action on memory consolidation in an opposite manner in the two inbred strains, pointing to genotype-dependent interaction between cholinergic and dopaminergic activity in memory consolidation.

RET/PTC1 is a chimeric oncogene created by the fusion of the tyrosine kinase domain of RET to the 5'-terminal region of another gene named H4. So far, this oncogene has been found activated only in human papillary thyroid carcinomas. In order to investigate its transforming properties in vivo, we have produced transgenic mice carrying RET/PTC1 under the control of the H4 promoter. The transgene was expressed in several tissues, consistently with the ubiquitous expression of the wild type H4 gene. Mammary adenocarcinomas and, less frequently, hyperplasia of sebaceous glands and rare benign skin tumors, named pilomatrixomas, developed in these mice. The tumors were shown to express the transgene both at the RNA and protein level. These results demonstrate that the transforming ability of the RET/PTC1 oncogene is not restricted to the thyroid epithelium in vivo. Despite its ubiquitous expression, however, RET/PTC1 was able to induce only a limited number of tumor types; specifically mammary epithelium was affected by transgene expression, thus suggesting that RET/PTC1 is able to couple with transforming pathways specific for these glandular cells.

Post-training administration of cocaine (1–10 mg/kg) or nomifensine (1–10 mg/kg) dose-dependently improves retention of an inhibitory avoidance response in C57BL16 mice, while impairing it in the DBA/2 strain. The effects on retention performance induced by the psychostimulant and the dopamine (DA) reuptake blocker in C57BL/6 and DBA/2 mice appear to be due to an effect on memory consolidation. In fact, they were observed when drugs were given at short, but not long, periods of time after training, i.e. when the memory trace is susceptible to modulation. Moreover, these effects are not to be ascribed to an aversive or a rewarding or non-specific action of the drugs on retention performance, as the latencies during the retention test of those mice that had not received a footshock during the training were not affected by the post-training drug administration. The strain-dependent effects of an intermediate dose (5 mg/kg) of both cocaine and nomifensine were reversed by pretreatment with either selective D1 or D2 DA receptor antagonists SCH 23390 and (-)-sulpiride administered at per se non-effective doses (0.025 and 6 mg/kg, respectively), thus suggesting that D1 and D2 receptor types are similarly involved in modulating memory processes. These results show that the effects of cocaine on memory consolidation are related to its dopaminergic action, since they are similar to those produced by nomifensine and, what is more important, are antagonized by pretreatment with DA receptor antagonists. Moreover, they point to possible genotype-dependent factors in DA mediated effects of cocaine on memory consolidation, indicating inbred mice as an experimental tool to investigate neural mechanisms underlying interindividual susceptibility to drug addiction.

Immobilization stress induced, in mice of both C57BL/6 (C57) and DBA/2 (DBA) strains, an increase in dihydroxyphenylacetic acid (DOPAC)/dopamine (DA) and homovanillic acid (HVA)/DA ratios and a reduction of 3-methoxytyramine (3-MT)/DA ratio in the caudatus putamen (CP) and nucleus accumbens septi (NAS). These effects were already evident after 30 min stress in the NAS, while in the CP 120 min were needed in order to show the effects of stress. Immobilization did not produce any effects on dopaminergic metabolism in the frontal cortex (FC) of the C57 strain either after 30 or after 120 min stress while in mice of the DBA strain a time-dependent effect of stress on the HVA/DA ratio was evident. When B6D2F1 hybrids were considered, the effects produced by 120 min immobilization in the CP and the NAS paralleled those observed in parental strains, while in the FC 120 min stress indcued the same increase of HVA observed in DBA mice, thus suggesting that the pattern of response in the FC that characterizes the DBA strain may be inherited through a dominant pattern of inheritance.

Stress-induced analgesia, assessed in two strains of mice (C57BL/6 and SEC/1Re), presents daily or circadian variations which are related to fluctuations in the release of endogenous opioids. These fluctuations may account for differences in individual reactivity to stressful events during 24 h.

Shock-induced aggressive behavior (SIAB) is absent or very weak in C57BL/6 (C57) mice at the age of 12 weeks while it reaches high levels at the age of 20 weeks. This age-dependent increase of aggressive responses is absent in DBA/2 (DBA) mice. Aggressive C57 mice (20 week old) are characterized by lower GABA levels in amygdala, striatum and substantia nigra than both non-aggressive C57 (12 week old) and DBA mice (12-20 week old). Concerning turnover rate, C57 mice at the age of 20 weeks show lower turnover rate values in cerebellum and raphe and higher values in septum in comparison with 12 week old mice of the same strain. These results are discussed in terms of the role of GABA function in brain areas which are involved in the control of emotionality and aggressive behavior.

The present study aims to assess the protective role of the antioxidant enzyme catalase (CAT) with relation to hydrogen peroxide (H2O2) degradation in oxygen plus water on electrophysiological and fluorescence changes induced by in vitro ischemia and on brain damage produced by transient in vivo ischemia. Neuroprotective effects of CAT were determined by means of electrophysiological recordings and confocal fluorescence microscopy in the hippocampal slice preparation. Ischemia was simulated in vitro by oxygen/glucose deprivation (OGD). In vivo ischemia was produced by transient middle cerebral artery occlusion (MCAo). A protection of the rat CA1 field excitatory postsynaptic potential (fEPSP) loss caused by a prolonged OGD (40 min) was observed after exogenous CAT (500 U/mL) bath-applied before a combined exposure to OGD and H2O2 (3 mM). Of note, neither H2O2 nor exogenous CAT alone had a protective action when OGD lasted for 40 min. The CAT-induced neuroprotection was confirmed in a transgenic mouse model over-expressing human CAT [Tg(CAT)]. In the presence of H2O2, the hippocampus of Tg(CAT) showed an increased resistance against OGD compared to that of wild-type (WT) animals. Moreover, CAT treatment reduced for about 50 min fEPSP depression evoked by repeated applications of H2O2 in normoxia. A lower sensitivity to H2O2-induced depression of fEPSPs was also indicated by the rightward shift of concentration-response curve in Tg(CAT) compared to WT mice. Noteworthy, Tg(CAT) mice had a reduced infarct size after MCAo. Our data suggest new strategies to reduce neuronal damage produced by transient brain ischemia through the manipulation of CAT enzyme.

Our research goal is to better understand the mechanisms controlling the initiation and progression of thyroid diseases. One such disease, papillary thyroid carcinoma (PTC), is the leading endocrine malignancy in the United States. Recently, a family of related fusion proteins, RET/PTC1-5, has been implicated in the early stages of PTC. Although all five members of this family have the c-RET proto-oncogene kinase domain in their COOH terminus, little is known about how these genes alter follicular cell biology. Consequently, to answer questions related to the mechanism of the RET/PTC fusion protein action, we have devised a molecular genetic strategy to study PTC using a mouse model of thyroid disease. A new member of this fusion oncogene family, RET/PTC3, which has been implicated in more cases of solid tumor carcinoma (79\%) than PTC1 or PTC2 and predominates (80\%) in radiation-induced thyroid cancer of children, was investigated in our study. We have generated transgenic mice expressing human RET/PTC3 exclusively in the thyroid. These mice develop thyroid hyperplasia, solid tumor variants of papillary carcinoma and metastatic cancer. This new transgenic line will be useful in deciphering the molecular and biological mechanisms that cause PTC and histological variations in humans.