Reduced anxiety-like and depression-related behavior in neuropeptide Y Y4 receptor knockout mice

Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety- and depression-like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4−/−) mice with control and germ line Y2−/− animals. Anxiety- and depression-like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress-induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4−/− and Y2−/− mice exhibited reduced anxiety-related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4−/− but reduced in Y2−/− mice. The basal rectal temperature exhibited diurnal and genotype-related alterations. Control mice had temperature minima at noon and midnight, whereas Y4−/− and Y2−/− mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4−/− and Y2−/− mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2−/− but not in Y4−/− mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety-like and depression-related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.     

Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.     

Three distinct roles of aquaporin-4 in brain function revealed by knockout mice

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K⁺ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.     

Three distinct roles of aquaporin-4 in brain function revealed by knockout mice

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K+ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.     

Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.     

Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice

Which of Y1-Y5 receptors (Rs) mediate NPY's angiogenic activity was studied using Y2R-null mice and R-specific antagonists. In Y2R-null mice, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were decreased by 50%; Y1R-antagonist blocked the remaining response. NPY-induced sprouting was equally inhibited by Y2R- (and Y5R- but less by Y1R-) antagonists in wild type mice. Spontaneous and NPY-induced revascularization of ischemic gastrocnemius muscles were similarly reduced in Y2R-null mice. Thus, NPY-induced angiogenesis, spontaneous and ischemic, is primarily mediated by Y2Rs. However, Y5Rs and, to a lesser degree Y1Rs, also may play a role in NPY-mediated angiogenesis.     

Neuropeptide Y (NPY) Y2 receptor-selective agonist inhibits food intake and promotes fat metabolism in mice: combined anorectic effects of Y2 and Y4 receptor-selective agonists

Peripheral administration of the endogenous Y(2) and Y(4) receptor selective agonists, PYY(3-36) and PP, have been shown to inhibit food intake and body weight gain in rodents, and to reduce appetite and caloric intake in humans. We have previously developed a long-acting, potent and highly selective Y(2) receptor selective agonist, N-alpha-Ac-[Nle(24,28), Trp(30), Nva(31), Psi(35-36)]PYY(22-36)-NH(2) (BT-48). BT-48 (ip) dose-dependently inhibited ad lib food intake and also decreased the respiratory quotient in mice during both the light and dark periods. The latter observation is indicative of enhanced fat metabolism. Moreover, BT-48 also inhibited food intake in fasted mice. Combined ip administration of BT-48 (50nmol/mouse) with a highly potent and selective Y(4) anorectic peptide, BVD-74D (50nmol/mouse), resulted in a powerful and long lasting inhibitory effect on food intake. As expected, this inhibitory effect on food intake was nearly double that exhibited by either peptide (50nmol/mouse) alone. In summary, BT-48, unlike PYY(3-36), exhibits little or no affinity to other "Y" receptors, and may therefore have a better clinical potential than PYY(3-36) for control of food intake. Moreover, it appears that treatment with a combination of Y(2) and Y(4) receptor selective agonists may constitute a more powerful approach to control food intake than treatment with either of these agonists alone.     

P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation

Nucleotides released upon brain injury signal to astrocytes and microglia playing an important role in astrogliosis, but the participation of microglia in the purinergic modulation of astrogliosis is still unclear. Highly enriched astroglial cultures and co-cultures of astrocytes and microglia were used to investigate the influence of microglia in the modulation of astroglial proliferation mediated by nucleotides. In highly enriched astroglial cultures, adenosine-5’-triphosphate (ATP), adenosine 5’-O-(3-thio)-triphosphate (ATPγS), adenosine 5’-O-(3-thio)-diphosphate (ADPβS; 0.01–1 mM), and adenosine-5’-diphosphate (ADP; 0.1–1 mM) increased proliferation up to 382%, an effect abolished in co-cultures containing 8% of microglia. The loss of ATP proliferative effect in co-cultures is supported by its fast metabolism and reduced ADP accumulation, an agonist of P2Y_1,12 receptors that mediate astroglial proliferation. No differences in ADPβS and ATPγS metabolism or P2Y_1,12 receptors expression were found in co-cultures that could explain the loss of their proliferative effect. However, conditioned medium from microglia cultures or co-cultures treated with ADPβS, when tested in highly enriched astroglial cultures, also prevented ADPβS proliferative effect. None of the uracil nucleotides tested had any effect in proliferation of highly enriched astroglial cultures, but uridine-5′-triphosphate (UTP; 0.1–1 mM) inhibited proliferation up to 66% in co-cultures, an effect that was dependent on uridine-5’-diphosphate (UDP) accumulation, coincident with a co-localization of P2Y₆ receptors in microglia and due to cell apoptosis. The results indicate that microglia control astroglial proliferation by preventing the proliferative response to adenine nucleotides and favouring an inhibitory effect of UTP/UDP. Several microglial P2Y receptors may be involved by inducing the release of messengers that restrain astrogliosis, a beneficial effect for neuronal repair mechanisms following brain injury.     

Cholinergic modulation of narcoleptic attacks in double orexin receptor knockout mice

To investigate how cholinergic systems regulate aspects of the sleep disorder narcolepsy, we video-monitored mice lacking both orexin (hypocretin) receptors (double knockout; DKO mice) while pharmacologically altering cholinergic transmission. Spontaneous behavioral arrests in DKO mice were highly similar to those reported in orexin-deficient mice and were never observed in wild-type (WT) mice. A survival analysis revealed that arrest lifetimes were exponentially distributed indicating that random, Markovian processes determine arrest lifetime. Low doses (0.01, 0.03 mg/kg, i.p.), but not a high dose (0.08 mg/kg, i.p.) of the cholinesterase inhibitor physostigmine increased the number of arrests but did not alter arrest lifetimes. The muscarinic antagonist atropine (0.5 mg/kg, i.p.) decreased the number of arrests, also without altering arrest lifetimes. To determine if muscarinic transmission in pontine areas linked to REM sleep control also influences behavioral arrests, we microinjected neostigmine (50 nl, 62.5 μM) or neostigmine + atropine (62.5 μM and 111 μM respectively) into the nucleus pontis oralis and caudalis. Neostigmine increased the number of arrests in DKO mice without altering arrest lifetimes but did not provoke arrests in WT mice. Co-injection of atropine abolished this effect. Collectively, our findings establish that behavioral arrests in DKO mice are similar to those in orexin deficient mice and that arrests have exponentially distributed lifetimes. We also show, for the first time in a rodent narcolepsy model, that cholinergic systems can regulate arrest dynamics. Since perturbations of muscarinic transmission altered arrest frequency but not lifetime, our findings suggest cholinergic systems influence arrest initiation without influencing circuits that determine arrest duration.     

Oleanolic acid exerts inhibitory effects on the late stage of osteoclastogenesis and prevents bone loss in osteoprotegerin knockout mice

Postmenopausal women undergo rapid bone loss, which caused by the accelerated osteoclastic bone resorption. Receptor activator of nuclear factor kappa-B ligand (RANKL) plays critical and essential roles on varied stages of osteoclastogenesis. Oleanolic acid (OA), a naturally derived small compound, has been found suppress osteoclastogenesis in early stage of bone marrow macrophages (BMMs). However, whether OA also regulates the late stage of osteoclastogenesis remains unclear. Here, the regulatory effect of OA on the late stage of osteoclastogenesis was investigated in vitro using RANKL-pretreated BMMs and in vivo using osteoprotegerin (OPG) knockout mice. Our in vitro studies demonstrate that OA inhibits the late stage of osteoclastogenesis from RANKL-pretreated BMMs. For in vivo animal investigation, OA attenuates the bone loss phenotypes in OPG-knockout mice by decreasing the densities of osteoclast, which are in consistent with the finding with in vitro osteoclastogenesis. Mechanistic investigations found that OA largely inhibit the activity of c-Fos and Nuclear factor of activated T-cells c1 (NFATc1) with RANKL-pretreated BMMs and OPG-knockout mice. Furthermore, OA suppresses the activities of osteoclast genes, such as Tartrate resistant acid phosphatase (TRAP), CathepsinK (Ctsk), and Matrix metalloproteinase 9 (MMP9). Taken together these findings, they have not only defined an inhibitory effect of OA in the late stage of osteoclastogenesis but have also gained new molecular mechanisms underlying the process of osteoclast formation.     

Assessment of ethanol consumption and water drinking by NPY Y(2) receptor knockout mice

In recent years, pharmacological and genetic evidence have emerged suggesting that neuropeptide Y (NPY) and the NPY Y(1) receptor are involved with neurobiological responses to ethanol. Pharmacological data implicate a role for the NPY Y(2) receptor in ethanol self-administration. The purpose of the present study was to determine if genetic mutation of the Y(2) receptor would modulate ethanol consumption and/or ethanol-induced sedation. Here, we report that mutant mice lacking the NPY Y(2) receptor (Y(2)(-/-)), when maintained on a mixed 50% 129/ SvJ x 50 % Balb/cJ background, drink significantly less of solutions containing 3 or 6% (v/v) ethanol relative to wild-type (Y(2)(+/+)) mice. These mice drink normal amounts of solutions containing sucrose or quinine, have normal blood ethanol clearance, and show normal sensitivity to ethanol-induced sedation. However, Y(2)(-/-) mice that are backcrossed to a Balb/cJ background show normal consumption of ethanol, indicating that the contributions of the NPY Y(2) receptor to ethanol consumption are genetic background dependent. Consistent with previous data suggesting that NPY modulates water drinking, Y(2)(-/-) mice of both genetic backgrounds consume significantly more water than Y(2)(+/+) mice. The present results suggest roles for the NPY Y(2) receptor in the modulation of ethanol and water consumption.     

Molecular signatures of neurodegeneration in the cortex of PS1/PS2 double knockout mice

Background

Dimebon is an antihistamine compound with a long history of clinical use in Russia. Recently, Dimebon has been proposed to be useful for treating neurodegenerative disorders. It has demonstrated efficacy in phase II Alzheimer's disease (AD) and Huntington's disease (HD) clinical trials. The mechanisms responsible for the beneficial actions of Dimebon in AD and HD remain unclear. It has been suggested that Dimebon may act by blocking NMDA receptors or voltage-gated Ca²⁺ channels and by preventing mitochondrial permeability pore transition.

Results

We evaluated the effects of Dimebon in experiments with primary striatal neuronal cultures (MSN) from wild type (WT) mice and YAC128 HD transgenic mice. We found that Dimebon acts as an inhibitor of NMDA receptors (IC50 = 10 μM) and voltage-gated calcium channels (IC50 = 50 μM) in WT and YAC128 MSN. We further found that application of 50 μM Dimebon stabilized glutamate-induced Ca²⁺ signals in YAC128 MSN and protected cultured YAC128 MSN from glutamate-induced apoptosis. Lower concentrations of Dimebon (5 μM and 10 μM) did not stabilize glutamate-induced Ca²⁺ signals and did not exert neuroprotective effects in experiments with YAC128 MSN. Evaluation of Dimebon against a set of biochemical targets indicated that Dimebon inhibits α-Adrenergic receptors (α_1A, α_1B, α_1D, and α_2A), Histamine H₁ and H₂ receptors and Serotonin 5-HT_2c, 5-HT_5A, 5-HT₆ receptors with high affinity. Dimebon also had significant effect on a number of additional receptors.

Conclusion

Our results suggest that Ca²⁺ and mitochondria stabilizing effects may, in part, be responsible for beneficial clinical effects of Dimebon. However, the high concentrations of Dimebon required to achieve Ca²⁺ stabilizing and neuroprotective effects in our in vitro studies (50 μM) indicate that properties of Dimebon as cognitive enhancer are most likely due to potent inhibition of H1 histamine receptors. It is also possible that Dimebon acts on novel high affinity targets not present in cultured MSN preparation. Unbiased evaluation of Dimebon against a set of biochemical targets indicated that Dimebon efficiently inhibited a number of additional receptors. Potential interactions with these receptors need to be considered in interpretation of results obtained with Dimebon in clinical trials.     

Flaxseed oil and inflammation-associated bone abnormalities in interleukin-10 knockout mice

Interleukin-10-/- (IL-10) knockout (KO) mice develop an intestinal inflammation that closely mimics human inflammatory bowel disease (IBD) which is accompanied by inflammation-associated bone abnormalities and elevated serum proinflammatory cytokines. The objective of this study was to use the IL-10 KO mouse model to determine whether flaxseed oil (FO) diet, rich in alpha-linolenic acid (ALA), attenuates intestinal inflammation and inflammation-associated bone abnormalities, compared to a corn oil (CO) control diet. Male wild-type (WT) or IL-10 KO mice were fed a 10% CO or 10% FO diet from weaning (postnatal day 28) for 9 weeks. At necropsy, serum, intestine, femurs and lumbar vertebrae were collected and analyzed. IL-10 KO mice fed CO had lower femur bone mineral content (BMC; P<.001), bone mineral density (BMD; P<.001), peak load (P=.033) and lumbar vertebrae BMD (P=.02) compared to WT mice fed either diet. Flaxseed oil had a modest, favorable effect on IL-10 KO mice as femur BMC, BMD and peak load were similar to WT mice fed CO or FO. In addition, lumbar vertebra BMD was similar among IL-10 KO mice fed FO and WT mice fed CO or FO. The fact that FO attenuated serum tumor necrosis factor-alpha (TNF-alpha) among IL-10 KO mice suggests that the positive effects of FO on femur BMC, BMD, peak load and vertebral BMD in IL-10 KO mice may have been partly mediated by changes in serum TNF-alpha. In conclusion, these findings suggest that a dietary level of ALA attainable from a 10% flaxseed oil diet results in modest improvements in some bone outcomes but does not attenuate intestinal inflammation that is characteristic of IL-10 KO mice.     

Distribution and function of prostanoid receptors: studies from knockout mice

Recent developments in the molecular biology of the prostanoid receptors has allowed the investigation of the physiological roles of each individual receptor type and subtype. The following article reports the prostanoid receptor distributions deduced from Northern blot and in situ hybridization analyses, summarizes the phenotypes of each receptor knockout mice, and discusses recent studies investigating the effects of each receptor deficiency on the inflammatory response and female reproductive processes. The combination of expression pattern and knockout analyses enabled us to determine which receptor expressed in a particular cell is important for the maintenance of normal and/or pathological physiology. The results from these analyses may be useful in the development of novel therapeutics that can selectively manipulate prostanoid-mediated actions.     

Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice

The aim of the present studies was to determine the effects of reduced or absent serotonin (5-HT) transporters (5-HTTs) on 5-HT2A and 5-HT2C receptors. The density of 5-HT2C receptors was significantly increased in the amygdala and choroid plexus of 5-HTT knockout mice. On the other hand, the density of 5-HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5-HTT knockout mice. However, 5-HT2A mRNA was not changed in any brain region measured. 5-HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5-HT2A receptors was evaluated by hormonal responses to (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5-HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5-HTTs changes the density of 5-HT2A and 5-HT2C receptors in a brain region-specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5-HTT knockout mice.     

GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice

GABA has been proposed to inhibit insulin secretion through GABAB receptors (GABABRs) in pancreatic beta-cells. We investigated whether GABABRs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABAB1 subunit of the GABABR (GABAB(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABAB1 subunit in islet membranes and immunohistochemistry for insulin and GABAB1 were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABABR agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABAB(-/-) mice showed fasting and fed glucose levels similar to WT. GABAB(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABAB(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABAB(-/-) islets. In GABAB(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABAB(-/-) males. Results demonstrate that GABABRs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABABRs induces alterations in pancreatic histology, physiology, and insulin resistance.