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.

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.

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.

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.

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.

Pre- and postsynaptic N-methyl-D-aspartate receptors are required for sequential printing of fear memory engrams.

The organization of fear memory involves the participation of multiple brain regions. However, it is largely unknown how fear memory is formed, which circuit pathways are used for "printing" memory engrams across brain regions, and the role of identified brain circuits in memory retrieval. With advanced genetic methods, we combinatorially blocked presynaptic output and manipulated N-methyl-D-aspartate receptor (NMDAR) in the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) before and after cued fear conditioning. Further, we tagged fear-activated neurons during associative learning for optogenetic memory recall. We found that presynaptic mPFC and postsynaptic BLA NMDARs are required for fear memory formation, but not expression. Our results provide strong evidence that NMDAR-dependent synaptic plasticity drives multi-trace systems consolidation for the sequential printing of fear memory engrams from BLA to mPFC and, subsequently, to the other regions, for flexible memory retrieval.

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.

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.

Chemical Oral Cancerogenesis Is Impaired in PI3Kγ Knockout and Kinase-Dead Mice.

We investigated the role of PI3Kγ in oral carcinogenesis by using a murine model of oral squamous carcinoma generated by exposure to 4-nitroquinoline 1-oxide (4NQO) and the continuous human cancer cell line HSC-2 and Cal-27. PI3Kγ knockout (not expressing PI3Kγ), PI3Kγ kinase-dead (carrying a mutation in the PI3Kγ gene causing loss of kinase activity) and wild-type (WT) C57Bl/6 mice were administered 4NQO via drinking water to induce oral carcinomas. At sacrifice, lesions were histologically examined and stained for prognostic tumoral markers (EGFR, Neu, cKit, Ki67) and inflammatory infiltrate (CD3, CD4, CD8, CD19 and CD68). Prevalence and incidence of preneoplastic and exophytic lesions were significantly and similarly delayed in both transgenic mice versus the control. The expression of prognostic markers, as well as CD19+ and CD68+ cells, was higher in WT, while T lymphocytes were more abundant in tongues isolated from transgenic mice. HSC-2 and Cal-27 cells were cultured in the presence of the specific PI3Kγ-inhibitor (IPI-549) which significantly impaired cell vitality in a dose-dependent manner, as shown by the MTT test. Here, we highlighted two different mechanisms, namely the modulation of the tumor-infiltrating cells and the direct inhibition of cancer-cell proliferation, which might impair oral cancerogenesis in the absence/inhibition of PI3Kγ.

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.