Radiprodil, a selective GluN2B negative allosteric modulator, rescues audiogenic seizures in mice carrying the GluN2A(N615S) mutation.

BACKGROUND AND PURPOSE: GRIN-related disorders are neurodevelopmental disorders caused by mutations in N-methyl-D-aspartate receptor (NMDAR) subunit genes. A large fraction of these mutations lead to a 'gain of function' (GoF) of the NMDAR. Patients present with a range of symptoms including epilepsy, intellectual disability, behavioural and motor. Controlling seizures is a significant unmet medical need in most patients with GRIN-related disorders. Although several hundred GRIN mutations have been identified in humans, until recently none of the mouse models carrying Grin mutations/deletions showed an epileptic phenotype. The two recent exceptions both carry mutations of GluN2A. The aim of this study was to assess the efficacy of radiprodil, a selective negative allosteric modulator of GluN2B-containing NMDARs, in counteracting audiogenic seizures (AGS) in a murine model carrying the GluN2A(N615S) homozygous mutation (Grin2aS/S mice). EXPERIMENTAL APPROACH: Grin2aS/S mice were acutely treated with radiprodil at different doses before the presentation of a high-frequency acoustic stimulus commonly used for AGS induction. KEY RESULTS: Radiprodil significantly and dose-dependently reduced the onset and severity of AGS in Grin2aS/S mice. Surprisingly, the results revealed a sex-dependent difference in AGS susceptibility and in the dose-dependent protection of radiprodil in the two genders. Specifically, radiprodil was more effective in female versus male mice. CONCLUSION AND IMPLICATIONS: Overall, our data clearly show that radiprodil, a GluN2B selective negative allosteric modulator, may have the potential to control seizures in patients with GRIN2A GoF mutations. Further studies are warranted to better understand the sex-dependent effects observed in this study.

Dietary Intake of Fructooligosaccharides Protects against Metabolic Derangements Evoked by Chronic Exposure to Fructose or Galactose in Rats.

SCOPE: Diets rich in fat and sugars evoke chronic low-grade inflammation, leading to metabolic derangements. This study investigates the impact of fructose and galactose, two commonly consumed simple sugars, on exacerbation of the harmful effects caused by high fat intake. Additionally, the potential efficacy of fructooligosaccharides (FOS), a fermentable dietary fiber, in counteracting these effects is examined. METHODS AND RESULTS: Male Sprague-Dawley rats (six/group) are fed 8 weeks as follows: control 5% fat diet (CNT), 20% fat diet (FAT), FAT+10% FOS diet (FAT+FOS), FAT+25% galactose diet (FAT+GAL), FAT+GAL+10% FOS diet (FAT+GAL+FOS), FAT+25% fructose diet (FAT+FRU), FAT+FRU+10% FOS diet (FAT+FRU+FOS). The dietary manipulations tested do not affect body weight gain, blood glucose, or markers of systemic inflammation whereas significant increases in plasma concentrations of triacylglycerols, cholesterol, aspartate aminotransferase, and alanine aminotrasferase are detected in both FAT+FRU and FAT+GAL compared to CNT. In the liver and skeletal muscle, both sugars induce significant accumulation of lipids and advanced glycation end-products (AGEs). FOS supplementation prevents these impairments. CONCLUSION: This study extends the understanding of the deleterious effects of a chronic intake of simple sugars and demonstrates the beneficial role of the prebiotic FOS in dampening the sugar-induced metabolic impairments by prevention of lipid and AGEs accumulation.

Hypothalamic NPY-Y1R Interacts with Gonadal Hormones in Protecting Female Mice against Obesity and Neuroinflammation.

We previously demonstrated that Npy1rrfb mice, which carry the conditional inactivation of the Npy1r gene in forebrain principal neurons, display a sexually dimorphic phenotype, with male mice showing metabolic, hormonal and behavioral effects and females being only marginally affected. Moreover, exposure of Npy1rrfb male mice to a high-fat diet (HFD) increased body weight growth, adipose tissue, blood glucose levels and caloric intake compared to Npy1r2lox male controls. We used conditional knockout Npy1rrfb and Npy1r2lox control mice to examine whether forebrain disruption of the Npy1r gene affects susceptibility to obesity and associated disorders of cycling and ovariectomized (ovx) female mice in a standard diet (SD) regimen or exposed to an HFD for 3 months. The conditional deletion of the Npy1r gene increased body weight and subcutaneous white adipose tissue weight in both SD- and HFD-fed ovx females but not in cycling females. Moreover, compared with ovx control females on the same diet regimen, Npy1rrfb females displayed increased microglia number and activation, increased expression of Neuropeptide Y (NPY)-immunoreactivity (IR) and decreased expression of proopiomelanocortin-IR in the hypothalamic arcuate nucleus (ARC). These results suggest that in the ARC NPY-Y1R reduces the susceptibility to obesity of female mice with low levels of gonadal hormones and that this effect may be mediated via NPY-Y1R ability to protect the brain against neuroinflammation.

Effect of Placenta-Derived Mesenchymal Stromal Cells Conditioned Media on an LPS-Induced Mouse Model of Preeclampsia.

We tested the pro-angiogenic and anti-inflammatory effects of human placenta-derived mesenchymal stromal cells (hPDMSCs)-derived conditioned media (CM) on a mouse model of preeclampsia (PE), a severe human pregnancy-related syndrome characterized by maternal hypertension, proteinuria, endothelial damage, inflammation, often associated with fetal growth restriction (FGR). At d11 of pregnancy, PE was induced in pregnant C57BL/6N mice by bacterial lipopolysaccharide (LPS) intravenous injection. At d12, 300 µL of unconditioned media (control group) or 300 µL PDMSCs-CM (CM group) were injected. Maternal systolic blood pressure was measured from 9 to 18 days of pregnancy. Urine protein content were analyzed at days 12, 13, and 17 of pregnancy. At d19, mice were sacrificed. Number of fetuses, FGR, fetal reabsorption, and placental weight were evaluated. Placentae were analyzed for sFlt-1, IL-6, and TNF-α gene and protein expressions. No FGR and/or reabsorbed fetuses were delivered by PDMSCs-CM-treated PE mice, while five FGR fetuses were found in the control group accompanied by a lower placental weight. PDMSCs-CM injection significantly decreased maternal systolic blood pressure, proteinuria, sFlt-1, IL-6, and TNF-α levels in PE mice. Our data indicate that hPDMSCs-CM can reverse PE-like features during pregnancy, suggesting a therapeutic role for hPDMSCs for the treatment of preeclampsia.

Conditional Inactivation of Limbic Neuropeptide Y-1 Receptors Increases Vulnerability to Diet-Induced Obesity in Male Mice.

NPY and its Y1 cognate receptor (Y1R) have been shown to be involved in the regulation of stress, anxiety, depression and energy homeostasis. We previously demonstrated that conditional knockout of Npy1r gene in the excitatory neurons of the forebrain of adolescent male mice (Npy1rrfb mice) decreased body weight growth and adipose tissue and increased anxiety. In the present study, we used the same conditional system to examine whether the targeted disruption of the Npy1r gene in limbic areas might affect susceptibility to obesity and associated disorders during adulthood in response to a 3-week high-fat diet (HFD) regimen. We demonstrated that following HFD exposure, Npy1rrfb male mice showed increased body weight, visceral adipose tissue, and blood glucose levels, hyperphagia and a dysregulation of calory intake as compared to control Npy1r2lox mice. These results suggest that low expression of Npy1r in limbic areas impairs habituation to high caloric food and causes high susceptibility to diet-induced obesity and glucose intolerance in male mice, uncovering a specific contribution of the limbic Npy1r gene in the dysregulation of the eating/satiety balance.

NPY-Y1 receptor signaling controls spatial learning and perineuronal net expression.

Perineuronal nets (PNNs) are extracellular matrix structures that form around some types of neurons at the end of critical periods, limiting neuronal plasticity. In the adult brain, PNNs play a crucial role in the regulation of learning and cognitive processes. Neuropeptide Y (NPY) is involved in the regulation of many physiological functions, including learning and memory abilities, via activation of Y1 receptors (Y1Rs). Here we demonstrated that the conditional depletion of the gene encoding the Y1R for NPY in adult forebrain excitatory neurons (Npy1rrfb mutant mice), induces a significant slowdown in spatial learning, which is associated with a robust intensification of PNN expression and an increase in the number of c-Fos expressing cells in the cornus ammonis 1 (CA1) of the dorsal hippocampus. Importantly, the enzymatic digestion of PNNs in CA1 normalizes c-Fos activity and completely rescues learning abilities of Npy1rrfb mice. These data highlight a previously unknown functional link between NPY-Y1R transmission and PNNs, which may play a role in the control of dorsal hippocampal excitability and related cognitive functions.

Sex differences in behavioral and metabolic effects of gene inactivation: The neuropeptide Y and Y receptors in the brain.

Brain and gonadal hormones interplay controls metabolic and behavioral functions in a sex-related manner. However, most translational neuroscience research related to animal models of endocrine and psychiatric disorders are often carried out in male animals only. The Neuropeptide Y (NPY) system shows sex-dependent differences and is sensitive to gonadal steroids. Based on published data from our and other laboratories, in this review we will discuss the sex related differences of NPY action on energy balance, bone homeostasis and behavior in rodents with the genetic manipulation of genes encoding NPY and its Y1, Y2 and Y5 cognate receptors. Comparative analyses of the phenotype of transgenic and knockout NPY and Y receptor rodents unravels sex dependent differences in the functions of this neurotransmission system, potentially helping to develop therapeutics for a variety of sex-related disorders including metabolic syndrome, osteoporosis and ethanol addiction.

The hidden role of NLRP3 inflammasome in obesity-related COVID-19 exacerbations: Lessons for drug repurposing.

COVID-19, the illness caused by SARS-CoV-2, has a wide-ranging clinical spectrum that, in the worst-case scenario, involves a rapid progression to severe acute respiratory syndrome and death. Epidemiological data show that obesity and diabetes are among the main risk factors associated with high morbidity and mortality. The increased susceptibility to SARS-CoV-2 infection documented in obesity-related metabolic derangements argues for initial defects in defence mechanisms, most likely due to an elevated systemic metabolic inflammation ("metaflammation"). The NLRP3 inflammasome is a master regulator of metaflammation and has a pivotal role in the pathophysiology of either obesity or diabetes. Here, we discuss the most recent findings suggesting contribution of NLRP3 inflammasome to the increase in complications in COVID-19 patients with diabesity. We also review current pharmacological strategies for COVID-19, focusing on treatments whose efficacy could be due, at least in part, to interference with the activation of the NLRP3 inflammasome. LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.

Conditional inactivation of Npy1r gene in mice induces sex-related differences of metabolic and behavioral functions.

Sex hormone-driven differences in gene expression have been identified in experimental animals, highlighting brain neuronal populations implicated in dimorphism of metabolic and behavioral functions. Neuropeptide Y-Y1 receptor (NPY-Y1R) system is sexually dimorphic and sensitive to gonadal steroids. In the present study we compared the phenotype of male and female conditional knockout mice (Npy1rrfb mice), carrying the inactivation of Npy1r gene in excitatory neurons of the brain limbic system. Compared to their male control (Npy1r2lox) littermates, male Npy1rrfb mice exhibited hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis that is associated with anxiety and executive dysfunction, reduced body weight growth, after-fasting refeeding, white adipose tissue (WAT) mass and plasma leptin levels. Conversely, female Npy1rrfb mice displayed an anxious-like behavior but no differences in HPA axis activity, executive function and body weight, compared to control females. Moreover, conditional inactivation of Npy1r gene induced an increase of subcutaneous and gonadal WAT weight and plasma leptin levels and a compensatory decrease of Agouti-related protein immunoreactivity in the hypothalamic arcuate (ARC) nucleus in females, compared to their respective control littermates. Interestingly, Npy1r mRNA expression was reduced in the ARC and in the paraventricular hypothalamic nuclei of female, but not male mice. These results demonstrated that female mice are resilient to hormonal and metabolic effects of limbic Npy1r gene inactivation, suggesting the existence of an estrogen-dependent relay necessary to ensure the maintenance of the homeostasis, that can be mediated by hypothalamic Y1R.

Spinal Neuropeptide Y1 Receptor-Expressing Neurons Form an Essential Excitatory Pathway for Mechanical Itch.

Acute itch can be generated by either chemical or mechanical stimuli, which activate separate pathways in the periphery and spinal cord. While substantial progress has been made in mapping the transmission pathway for chemical itch, the central pathway for mechanical itch remains obscure. Using complementary genetic and pharmacological manipulations, we show that excitatory neurons marked by the expression of the neuropeptide Y1 receptor (Y1Cre neurons) form an essential pathway in the dorsal spinal cord for the transmission of mechanical but not chemical itch. Ablating or silencing the Y1Cre neurons abrogates mechanical itch, while chemogenetic activation induces scratching. Moreover, using Y1 conditional knockout mice, we demonstrate that endogenous neuropeptide Y (NPY) acts via dorsal-horn Y1-expressing neurons to suppress light punctate touch and mechanical itch stimuli. NPY-Y1 signaling thus regulates the transmission of innocuous tactile information by establishing biologically relevant thresholds for touch discrimination and mechanical itch reflexes.

Conditional inactivation of Npy1r gene in mice induces behavioural inflexibility and orbitofrontal cortex hyperactivity that are reversed by escitalopram.

Cognitive flexibility is the ability to rapidly adapt established patterns of behaviour in the face of changing circumstance and depends critically on the orbitofrontal cortex (OFC). Impaired flexibility also results from altered serotonin transmission in the OFC. The Y1 (Y1R) and Y5 (Y5R) receptors for neuropeptide Y (NPY) colocalize in several brain regions and have overlapping functions in regulating cognition and emotional behaviour. The targeted disruption of gene encoding Y1R (Npy1r gene) in Y5R containing neurons (Npy1rY5R-/- mice) increases anxiety-like behaviour and spatial reference memory. Here we used the same conditional system to analyse whether the coordinated expression of the Y1R and Y5R might be required for behavioural flexibility in reversal learning tasks, OFC serotoninergic tone and OFC neural activity, as detected by immunohistochemical quantification of the immediate-early gene, c-Fos. In addition, we investigated whether the acute treatment of Npy1rY5R-/- mice with the selective serotonin reuptake inhibitor escitalopram affected behavioural flexibility and OFC c-Fos expression. Npy1rY5R-/- male mice exhibit an impairment in performing the reversal task of the Morris water maze and the water T-maze but normal spatial learning, working memory and sociability, compared to their control siblings. Furthermore, Npy1rY5R-/- male mice display decreased 5-hydroxytriptamine (5-HT) positive fibres and increased baseline neural activity in OFC. Importantly, escitalopram normalizes OFC neural activity and restores behavioural flexibility of Npy1rY5R-/- male mice. These findings suggest that the inactivation of Y1R in Y5R containing neurons increases pyramidal neuron activity and dysregulates serotoninergic tone in OFC, whereby contributing to reversal learning impairment.

Reduced NPY Y1 receptor hippocampal expression and signs of decreased vagal modulation of heart rate in mice.

Central neuropeptide Y (NPY) signaling participates in the regulation of cardiac autonomic outflow, particularly via activation of NPY-Y1 receptors (Y1Rs). However, the specific brain areas and neural pathways involved have not been completely identified yet. Here, we evaluate the role of hippocampal Y1Rs in the modulation of the autonomic control of cardiac function using a conditional knockout mouse model. Radiotelemetric transmitters were implanted in 4-month-old male mice exhibiting reduced forebrain expression (rfb) of the Y1R (Npy1rrfb, n=10) and their corresponding controls (Npy1r2lox, n=8). ECG signals were recorded (i) during resting conditions, (ii) under selective pharmacological manipulation of cardiac vagal activity, and (iii) during acute and chronic psychosocial stress challenges, and analyzed via time- and frequency-domain analysis of heart rate variability. Npy1rrfb mice showed a lower Npy1r mRNA density in the dentate gyrus and in the CA1 region of the hippocampus. Under resting undisturbed conditions, Npy1rrfb mice exhibited (i) a higher heart rate, (ii) a reduced overall heart rate variability, and (iii) lower values of the indices of vagal modulation compared to Npy1r2lox counterparts. Following pharmacological vagal inhibition, heart rate was higher in control but not in Npy1rrfb mice compared to their respective baseline values, suggesting that tonic vagal influences on heart rate were reduced in Npy1rrfb mice. The magnitude of the heart rate response to acute stressors was smaller in Npy1rrfb mice compared to Npy1r2lox counterparts, likely due to a concurrent lower vagal withdrawal. These findings suggest that reduced Y1R expression leads to a decrease in resting vagal modulation and heart rate variability, which, in turn, may determine a reduced cardiac autonomic responsiveness to acute stress challenges.

Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise.

Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16INK4A, p18INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16INK4A, p18INK4C, CDK4 and CDK6 expressions in physiological term villous explants. PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16INK4A, p18INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay. We reported significantly increased p16INK4A and p18INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16INK4 and p18INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast. We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise.

Sex-dependent regulation of hypothalamic neuropeptide Y-Y1 receptor gene expression in leptin treated obese (ob/ob) or lean mice.

Pharmacological and genetic studies have shown that the Y1 receptor (Y1R) for Neuropeptide Y (NPY) plays a crucial role in the control of feeding behavior under metabolic conditions of low leptin levels or leptin deficiency. In this study, we investigated the effect of leptin deficiency and leptin replacement on Y1R gene expression in the hypothalamus of lean and obese Y1R/LacZ transgenic mice (TgY1R/LacZ) carrying the murine Y1R promoter linked to the LacZ gene that induces the expression of ß-galactosidase. Two daily intraperitoneal injections with leptin (1µg/g of body weight for one week) of male and female lean (TgY1R/LacZ+/+) and obese (TgY1R/LacZob/ob) mice induced a significant decrease of body weight in both sexes and genotypes. In males, leptin administration decreased ß-galactosidase activity in the PVN and DMH of lean mice, and increased transgene expression in the same hypothalamic nuclei of obese mice. Sex-related differences were also observed in both genotypes, since leptin treatment failed to affect transgene expression in hypothalamus of lean and obese female mice. These results provide further evidence for a sexual dimorphism of the hypothalamic NPY-Y1R-mediated pathway in response to changes in leptin circulating levels.

Estrogen replacement therapy regulation of energy metabolism in female mouse hypothalamus.

Estrogens play an important role in the regulation of energy homeostasis in female mammals and a reduced ovarian function, due to natural aging or surgery, is associated with body weight increase and fat redistribution. This disruption of energy homeostasis may constitute a trigger for several pathologies known to be associated with climacterium; however, so far, limited attention has been devoted to the ability of estrogen replacement therapies (ERT) to reinstate the balanced energy metabolism characteristic of cycling female mammals. The purpose of the present study was to compare the efficacy of selected ERTs in reversing the ovariectomy-induced gain in body weight. To this aim female ERE-Luc mice were ovariectomized and, after 3 weeks, treated per os for 21 days with: conjugated estrogens, two selective estrogen receptor modulators (bazedoxifene and raloxifene), and the combination of bazedoxifene plus conjugated estrogens (tissue-selective estrogen complex, TSEC). The study shows that the therapy based on TSEC was the most efficacious in reducing the body weight accrued by ovariectomy (OVX). In addition, by means of in vivo imaging, the TSEC treatment was shown to increase estrogen receptor (ER) transcriptional activity selectively in the arcuate nucleus, which is a key area for the control of energy homeostasis. Finally, quantitative analysis of the mRNAs encoding orexigenic and anorexigenic peptides indicated that following ERT with TSEC there was a significant change in Agrp, NPY, and Kiss-1 mRNA accumulation in the whole hypothalamus. Considering that prior studies showed that ERT with TSEC was able to mimic the rhythm of ER oscillatory activity during the reproductive cycle and that such fluctuations were relevant for energy metabolism, the present observations further point to the ER tetradian oscillation as an important component of the ER signaling necessary for the full hormone action and therefore for an efficacious ERT.

Conditional inactivation of neuropeptide Y Y1 receptors unravels the role of Y1 and Y5 receptors coexpressing neurons in anxiety.

BACKGROUND: The Y1 receptor (Y1R) and Y5 receptor (Y5R) for neuropeptide Y share similar actions in the regulation of anxiety. Previously demonstrated that conditional removal of the Y1R during postnatal development in the forebrain excitatory neurons leads to higher anxiety, increased hypothalamus-pituitary-adrenocortical axis activity, and decreased body growth rate in male mice raised by foster mothers that exhibit high levels of maternal care. In the present study, we used the same conditional system to analyze the specific contribution to emotional behavior and stress response of the Y1R coexpressed with the Y5R. METHODS: Using the Cre-loxP recombination system, we investigated anxious behavior, spatial memory, and metabolic functions of conditional knockout mice in which the inactivation of the Npy1r gene was induced in the Y5Rs expressing neurons of juvenile mice (Npy1r(Y5R-/-) ). RESULTS: Npy1r(Y5R-/-) mice show increased anxiety-related behavior but no changes in hypothalamus-pituitary-adrenocortical axis activity or in body weight growth, independently of gender and mouse strain used as foster mothers. Also, Npy1r(Y5R-/-) mice of both genders display increased spatial reference memory in the Morris water maze test. CONCLUSIONS: The results suggest that neuropeptide Y Y1R differentially expressed in the limbic system regulates anxiety and stress responses via distinct neurochemical circuits. In addition, we provide the first experimental genetic evidence that the Y1Rs coexpressed with the Y5R are involved in retention of spatial memory in male and female mice.

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care.

Neuropeptide Y (NPY) plays an important role in stress, anxiety, obesity, and energy homeostasis via activation of NPY-Y1 receptors (Y1Rs) in the brain. However, global knockout of the Npy1r gene has low or no impact on anxiety and body weight. To uncover the role of limbic Y1Rs, we generated conditional knockout mice in which the inactivation of the Npy1r gene was restricted to excitatory neurons of the forebrain, starting from juvenile stages (Npy1r(rfb)). Npy1r(rfb) mice exhibited increased anxiety and reduced body weight, less adipose tissue, and lower serum leptin levels. Npy1r(rfb) mutants also had a hyperactive hypothalamic-pituitary-adrenocortical axis, as indicated by higher peripheral corticosterone and higher density of NPY immunoreactive fibers and corticotropin releasing hormone immunoreactive cell bodies in the paraventricular hypothalamic nucleus. Importantly, through fostering experiments, we determined that differences in phenotype between Npy1r(rfb) and Npy1r(2lox) mice became apparent when both genotypes were raised by FVB/J but not by C57BL/6J dams, suggesting that limbic Y1Rs are key targets of maternal care-induced programming of anxiety and energy homeostasis.

Treatment of newborn G6pc(-/-) mice with bone marrow-derived myelomonocytes induces liver repair.

BACKGROUND & AIMS: Several studies have shown that bone marrow-derived committed myelomonocytic cells can repopulate diseased livers by fusing with host hepatocytes and can restore normal liver function. These data suggest that myelomonocyte transplantation could be a promising approach for targeted and well-tolerated cell therapy aimed at liver regeneration. We sought to determine whether bone marrow-derived myelomonocytic cells could be effective for liver reconstitution in newborn mice knock-out for glucose-6-phosphatase-α. METHODS: Bone marrow-derived myelomonocytic cells obtained from adult wild type mice were transplanted in newborn knock-out mice. Tissues of control and treated mice were frozen for histochemical analysis, or paraffin-embedded and stained with hematoxylin and eosin for histological examination or analyzed by immunohistochemistry or fluorescent in situ hybridization. RESULTS: Histological sections of livers of treated knock-out mice revealed areas of regenerating tissue consisting of hepatocytes of normal appearance and partial recovery of normal architecture as early as 1 week after myelomonocytic cells transplant. FISH analysis with X and Y chromosome paints indicated fusion between infused cells and host hepatocytes. Glucose-6-phosphatase activity was detected in treated mice with improved profiles of liver functional parameters. CONCLUSIONS: Our data indicate that bone marrow-derived myelomonocytic cell transplant may represent an effective way to achieve liver reconstitution of highly degenerated livers in newborn animals.

Modulation of neuropeptide Y and Y1 receptor expression in the amygdala by fluctuations in the brain content of neuroactive steroids during ethanol drinking discontinuation in Y1R/LacZ transgenic mice.

Previous studies have shown that GABAergic neuroactive steroids increase Y1 receptor (Y1R) gene expression in the amygdala of Y1R/LacZ transgenic mice, harbouring the murine Y1R gene promoter linked to a LacZ reporter gene. As ethanol is known to increase GABAergic neuroactive steroids, we investigated the relationship between fluctuations in the brain content of neuroactive steroids induced by chronic voluntary ethanol consumption or ethanol discontinuation and both the level of neuropeptide Y (NPY) immunoreactivity and Y1R gene expression in the amygdala of Y1R/LacZ transgenic mice. Ethanol discontinuation (48 h) after voluntary consumption of consecutive solutions of 3%, 6%, 10% and 20% (v/v) ethanol over 4 weeks produced an anxiety-like behaviour as measured by elevated plus maze. Voluntary ethanol intake increased the cerebrocortical concentration of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) that returned to control level 48 h after discontinuation of ethanol intake. Ethanol discontinuation significantly decreased NPY immunoreactivity and concomitantly increased Y1R/LacZ transgene expression in the amygdala, whereas chronic ethanol intake failed to affect these parameters. The 5alpha-reductase inhibitor finasteride prevented both the increase in the cerebrocortical concentration of 3alpha,5alpha-TH PROG apparent after 4 weeks of ethanol intake and the changes in NPY immunoreactivity and transgene expression induced by ethanol discontinuation. Data suggest that 3alpha,5alpha-TH PROG plays an important role in the changes in NPY-Y1R signalling in the amygdala during ethanol discontinuation.

Expression patterns of promoters for NPY Y(1) and Y(5) receptors in Y(5)RitTA and Y(1)RVenus BAC-transgenic mice.

In the rat brain, neuropeptide Y (NPY) Y(1) and Y(5) receptors are coexpressed in various forebrain regions where they mediate several NPY-activated functions, including feeding behaviour, anxiety, neuronal excitability and hormone secretion. We studied the distribution pattern and cellular colocalization of the Y(1) and the Y(5) receptor gene expression in the mouse brain by using transgenic mice with genomically integrated BAC clones, where the coding regions of the Y(1) and Y(5) receptor genes were replaced by Venus and the synthetic transcription factor itTA reporter genes, respectively (Tg(Y5RitTA/Y1RVenus) mice). Analysis of Venus fluorescence and itTA-mediated activation of Cre recombinase revealed copy number-dependent expression levels, between the lines, but similar expression patterns. In three transgenic lines the BAC encoded Y(5) receptor promoter induced strong Cre expression in the olfactory system, cerebral cortex, hippocampus and basal ganglia. Weaker expression was found in most of the hypothalamic nuclei of line 25, the highest-expressing transgenic line. Activation of Cre was itTA-dependent and could be regulated by doxycycline. The Y(1) receptor promoter-induced Venus fluorescence was intense, widely present through the brain and colocalized with Cre immunostaining in neurons of distinct brain regions, including the cerebral cortex, basolateral amygdala, dentate gyrus and paraventricular nucleus. These data provide a detailed and comparative mapping of Y(1) and Y(5) receptor promoter activity within cells of the mouse brain. The Tg(Y5RitTA/Y1RVenus)-transgenic mice generated here also represent a genetic tool for conditional mutagenesis via the Cre lox system, particularly of genes involved in feeding behaviour, neuronal excitability and hormone secretion.