Endocrine function of neuropeptide Y knockout mice

Among its many proposed functions, neuropeptide Y (NPY) is thought to modulate the hypothalamic-pituitary axis. Specifically, increased hypothalamic NPY signaling may be critical in mediating the neuroendocrine response to fasting. To determine the consequences of NPY deficiency on endocrine physiology, multiple hormones were quantitated in wildtype and NPY-knockout mice under fed and fasted conditions. Serum concentrations of leptin, corticosterone, thyroxine, and testosterone were normal in NPY-knockout males fed ad libitum. A 48-hour fast resulted in a 50% reduction in leptin, a 60% reduction in thyroxine, a 75% reduction in testosterone, and a 12-fold increase in corticosterone in both wildtype and NPY-knockout mice. Fasting also increased the estrous cycle length by 3 days in both wildtype and NPY-deficient female mice. We conclude that NPY is not essential for appropriate function of the gonadotropic, thyrotropic, or corticotropic axes under ad lib fed conditions or in response to acute fasting.     

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.     

Working memory deficits in neuronal nitric oxide synthase knockout mice: potential impairments in prefrontal cortex mediated cognitive function

Neuronal nitric oxide synthase (nNOS) forms nitric oxide (NO), which functions as a signaling molecule via S-nitrosylation of various proteins and regulation of soluble guanylate cyclase (cGC)/cyclic guanosine monophosphate (cGMP) pathway in the central nervous system. nNOS signaling regulates diverse cellular processes during brain development and molecular mechanisms required for higher brain function. Human genetics have identified nNOS and several downstream effectors of nNOS as risk genes for schizophrenia. Besides the disease itself, nNOS has also been associated with prefrontal cortical functioning, including cognition, of which disturbances are a core feature of schizophrenia. Although mice with genetic deletion of nNOS display various behavioral deficits, no studies have investigated prefrontal cortex-associated behaviors. Here, we report that nNOS knockout (KO) mice exhibit hyperactivity and impairments in contextual fear conditioning, results consistent with previous reports. nNOS KO mice also display mild impairments in object recognition memory. Most importantly, we report for the first time working memory deficits, potential impairments in prefrontal cortex mediated cognitive function in nNOS KO mice. Furthermore, we demonstrate Disrupted-in-Schizophrenia 1 (DISC1), another genetic risk factor for schizophrenia that plays roles for cortical development and prefrontal cortex functioning, including working memory, is a novel protein binding partner of nNOS in the developing cerebral cortex. Of note, genetic deletion of nNOS appears to increase the binding of DISC1 to NDEL1, regulating neurite outgrowth as previously reported. These results suggest that nNOS KO mice are useful tools in studying the role of nNOS signaling in cortical development and prefrontal cortical functioning.     

Scrapie-infected mice and PrP knockout mice share abnormal localization and activity of neuronal nitric oxide synthase

PrP(Sc), the only identified component of the scrapie prion, is a conformational isoform of PrPc. The physiological role of PrPc, a glycolipid-anchored glycoprotein, is still unknown. We have shown previously that neuronal nitric oxide synthase (nNOS) activity is impaired in the brains of mice sick with experimental scrapie as well as in scrapie-infected neuroblastoma cells. In this work we investigated the cell localization of nNOS in brains of wild-type and scrapie-infected mice as well as in mice in which the PrP gene was ablated. We now report that whereas in wild-type mice, nNOS, like PrPc, is associated with detergent-insoluble cholesterol-rich membranous microdomains (rafts), this is not the case in brains of scrapie-infected or in those of adult PrP(0/0) mice. Also, adult PrP(0/0), like scrapie-infected mice, show reduced nNOS activity. We suggest that PrPc may play a role in the targeting of nNOS to its proper subcellular localization. The similarities of nNOS properties in PrP(0/0) as compared with scrapie-infected mice suggest that at least this role of PrPc may be impaired in scrapie-infected brains.     

Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin-1 and aquaporin-3

Aquaporin (AQP) water channel AQP3 has been proposed to be the major glycerol and non-AQP1 water transporter in erythrocytes. AQP1 and AQP3 are also expressed in the kidney where their deletion in mice produces distinct forms of nephrogenic diabetes insipidus. Here AQP1/AQP3 double knockout mice were generated and analyzed to investigate the functional role of AQP3 in erythrocytes and kidneys. 53 double knockout mice were born out of 756 pups from breeding double heterozygous mice. The double knockout mice had reduced survival and impaired growth compared with the single knockout mice. Erythrocyte water permeability was 7-fold reduced by AQP1 deletion but not further reduced in AQP1/AQP3 null mice. AQP3 deletion did not affect erythrocyte glycerol permeability or its inhibition by phloretin. Daily urine output in AQP1/AQP3 double knockout mice (15 ml) was 9-fold greater than in wild-type mice, and urine osmolality (194 mosm) was 8.4-fold reduced. The mice remained polyuric after DDAVP administration or water deprivation. The renal medulla in most AQP1/AQP3 null mice by age 4 weeks was atrophic and fluid-filled due to the severe polyuria and hydronephrosis. Our data provide direct evidence that AQP3 is not functionally important in erythrocyte water or glycerol permeability. The renal function studies indicate independent roles of AQP1 and AQP3 in countercurrent exchange and collecting duct osmotic equilibration, respectively.     

Gastric body cholinergic contractile signal transduction in M2 and M3 receptor knockout mice

Abstract

Although most smooth muscles express a greater density of M₂ than M₃ muscarinic receptors, based on the potency of subtype selective muscarinic receptor antagonists, the M₃ subtype predominantly mediates contraction. The effect of inhibitors of putative contractile signal transduction pathway enzymes on carbachol-induced contractions was determined in wild-type (WT) mice and mice lacking either the M₂ (M₂KO) or the M₃ (M₃KO) receptor subtype. Contractile responses to KCl, then increasing carbachol concentrations in the presence and absence of enzyme inhibitors was determined. The KCl-induced contraction was not different between strains. The carbachol response was unaffected in the M₂KO strain but decreased 42% in M₃KO mice (p?<?0.01). Darifenacin potency was high in both WT and M₂KO strains, indicating M₃-mediated contractions, and low in the M₃KO strain, suggesting M₂-mediated contractions. The phosphatidyl inositol-specific phospholipase C (Pi-PLC) inhibitor ET-18-OCH₃ had no effect. Inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) and sphingomyelin synthase with D609 decreased maximal contraction in all strains. M₃-mediated contractions in the M₂KO strain were decreased 54% by the protein kinase C (PKC) inhibitor chelerythrine. M₂-mediated contractions in the M₃KO and WT strains were decreased by the Rho kinase (ROCK) inhibitor Y27632 as well as the ROCK, PKA and PKG inhibitor H89. The M₃ subtype activates PKC and either PC-PLC or sphingomyelin synthase, while the M₂ subtype activates ROCK and either PC-PLC or sphingomyelin synthase. These studies suggest that multiple parallel pathways mediate cholinergic contractions in stomach body smooth muscle.     

Detection of NaCl and KCl in TRPV1 knockout mice

Both amiloride-sensitive and -insensitive mechanisms contribute to NaCl taste transduction. The amiloride-sensitive mechanism relies on the epithelial Na(+) channel ENaC, which is widely expressed on the apical membrane of fungiform taste cells. The amiloride-insensitive mechanism, which predominates in circumvallate and foliate taste buds, was recently reported to involve a variant of the nonselective cation channel TRPV1. We performed 2-bottle preference and threshold experiments with TRPV1 knockout mice and wild-type (C57BL/6J) controls to test for NaCl preference and detection thresholds in the presence and absence of amiloride. Surprisingly, TRPV1 knockout mice not only detected NaCl in the presence of amiloride but they preferred NaCl over water at concentrations avoided by the wild-type mice. NaCl detection thresholds were between 2 and 3 mM for both genotypes. Amiloride increased the detection thresholds of wild-type mice but not knockout mice. The knockout mice also preferred 100 mM KCl compared with wild-type controls, suggesting that TRPV1 receptors may mediate a general aversive response to salts. Analyses of consumption data also revealed that TRPV1 knockout mice ingested more of the NaCl, with and without amiloride, and KCl solutions than the wild-type mice. However, comparisons of preference ratios and consumption volumes indicated that both wild-type and TRPV1 knockout mice avoided citric acid in quite a similar manner, suggesting that TRPV1 receptors do not mediate the detection of citric acid. These data, taken together, suggest that additional mechanisms must contribute to the amiloride-insensitive NaCl response.     

Npp1 promotes atherosclerosis in ApoE knockout mice

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PP(i)), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in human beings. NPP1 and PP(i) have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification. Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high-fat/high-cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) mice demonstrated decreased Opn expression. The 28-week-old ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) had significantly smaller atherosclerotic lesions compared with wild-type congenic ApoE(-/-) mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE(-/-) mice without Npp1 deficiency. We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout 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.     

Study of P450 function using gene knockout and transgenic mice

The xenobiotic-metabolizing P450s have been extensively studied for their ability to metabolize endogenous and exogenous chemicals. The latter include drugs and dietary and environmentally derived toxicants and carcinogens. These enzymes also metabolize endogenous steroids and fatty acids. P450s are thought to be required for efficient removal of most xenobiotics from the body and to be responsible for the hazardous effects of toxicants and carcinogens based on their ability to convert chemicals to electrophilic metabolites that can cause cellular damage and gene mutations. P450 catalytic activities have been extensively studied in vitro and in cell culture, yielding considerable information on their mechanisms of catalysis, substrate specificities, and metabolic products. Targeted gene disruption has been used to determine the roles of P450s in intact animals and their contributions to the mechanisms of toxicity and carcinogenesis. The P450s chosen for study, CYP1A1, CYP1B1, CYP1A2, and CYP2E1, are conserved in mammals and are known to metabolize most toxicants and chemical carcinogens. Mice lacking expression of these enzymes do not differ from wild-type mice, indicating that these P450s are not required for development and physiological homeostasis. However, the P450 null mice have altered responses to the toxic and carcinogenic effects of chemicals as compared with wild-type mice. These studies establish that P450s mediate the adverse effects of drugs and dietary, environmental, and industrial chemicals and serve to validate molecular epidemiology studies that seek to determine links between P450 polymorphisms and susceptibility to chemically associated diseases. More recently, P450 humanized mice have been produced.     

Increased neuronal excitability, synaptic plasticity, and learning in aged Kvbeta1.1 knockout mice

BACKGROUND: Advancing age is typically accompanied by deficits in learning and memory. These deficits occur independently of overt pathology and are often considered to be a part of "normal aging." At the neuronal level, normal aging is known to be associated with numerous cellular and molecular changes, which include a pronounced decrease in neuronal excitability and an altered induction in the threshold for synaptic plasticity. Because both of these mechanisms (neuronal excitability and synaptic plasticity) have been implicated as putative cellular substrates for learning and memory, it is reasonable to propose that age-related changes in these mechanisms may contribute to the general cognitive decline that occurs during aging. RESULTS: To further investigate the relationship between aging, learning and memory, neuronal excitability, and synaptic plasticity, we have carried out experiments with aged mice that lack the auxiliary potassium channel subunit Kvbeta1.1. In aged mice, the deletion of the auxiliary potassium channel subunit Kvbeta1.1 resulted in increased neuronal excitability, as measured by a decrease in the post-burst afterhyperpolarization. In addition, long-term potentiation (LTP) was more readily induced in aged Kvbeta1.1 knockout mice. Finally, the aged Kvbeta1.1 mutants outperformed age-matched controls in the hidden-platform version of the Morris water maze. Interestingly, the enhancements in excitability and learning were both sensitive to genetic background: The enhanced learning was only observed in a genetic background in which the mutants exhibited increased neuronal excitability. CONCLUSIONS: Neuronal excitability is an important determinant of both synaptic plasticity and learning in aged subjects.     

Cardiovascular abnormalities in Folr1 knockout mice and folate rescue

BACKGROUND: Periconceptional folic acid supplementation is widely believed to aid in the prevention of neural tube defects (NTDs), orofacial clefts, and congenital heart defects. Folate-binding proteins or receptors serve to bind folic acid and 5-methyltetrahydrofolate, representing one of the two major mechanisms of cellular folate uptake. METHODS: We herein describe abnormal cardiovascular development in mouse fetuses lacking a functional folate-binding protein gene (Folr1). We also performed a dose-response study with folinic acid and determined the impact of maternal folate supplementation on Folr1 nullizygous cardiac development. RESULTS: Partially rescued preterm Folr1(-/-) (formerly referred to as Folbp1) fetuses were found to have outflow tract defects, aortic arch artery abnormalities, and isolated dextracardia. Maternal supplementation with folinic acid rescued the embryonic lethality and the observed cardiovascular phenotypes in a dose-dependant manner. Maternal genotype exhibited significant impact on the rescue efficiency, suggesting an important role of in utero folate status in embryonic development. Abnormal heart looping was observed during early development of Folr1(-/-) embryos partially rescued by maternal folinic acid supplementation. Migration pattern of cardiac neural crest cells, genetic signals in pharyngeal arches, and the secondary heart field were also found to be affected in the mutant embryos. CONCLUSIONS: Our observations suggest that the beneficial effect of folic acid for congenital heart defects might be mediated via its impact on neural crest cells and by gene regulation of signaling pathways involved in the development of the pharyngeal arches and the secondary heart field.     

Alteration of the neuronal and glial cell profiles in Neu1-deficient zebrafish

Glycoconjugate Journal - Neu1 is a glycosidase that releases sialic acids from the non-reducing ends of glycoconjugates, and its enzymatic properties are conserved among vertebrates. Recently,...     

RAGE potentiates Abeta-induced perturbation of neuronal function in transgenic mice

Receptor for Advanced Glycation Endproducts (RAGE), a multiligand receptor in the immunoglobulin superfamily, functions as a signal-transducing cell surface acceptor for amyloid-beta peptide (Abeta). In view of increased neuronal expression of RAGE in Alzheimer's disease, a murine model was developed to assess the impact of RAGE in an Abeta-rich environment, employing transgenics (Tgs) with targeted neuronal overexpression of RAGE and mutant amyloid precursor protein (APP). Double Tgs (mutant APP (mAPP)/RAGE) displayed early abnormalities in spatial learning/memory, accompanied by altered activation of markers of synaptic plasticity and exaggerated neuropathologic findings, before such changes were found in mAPP mice. In contrast, Tg mice bearing a dominant-negative RAGE construct targeted to neurons crossed with mAPP animals displayed preservation of spatial learning/memory and diminished neuropathologic changes. These data indicate that RAGE is a cofactor for Abeta-induced neuronal perturbation in a model of Alzheimer's-type pathology, and suggest its potential as a therapeutic target to ameliorate cellular dysfunction.     

Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice

In vitro studies indicate a role for the LIM kinase family in the regulation of cofilin phosphorylation and actin dynamics. In addition, abnormal expression of LIMK-1 is associated with Williams syndrome, a mental disorder with profound deficits in visuospatial cognition. However, the in vivo function of this family of kinases remains elusive. Using LIMK-1 knockout mice, we demonstrate a significant role for LIMK-1 in vivo in regulating cofilin and the actin cytoskeleton. Furthermore, we show that the knockout mice exhibited significant abnormalities in spine morphology and in synaptic function, including enhanced hippocampal long-term potentiation. The knockout mice also showed altered fear responses and spatial learning. These results indicate that LIMK-1 plays a critical role in dendritic spine morphogenesis and brain function.     

Novel insights into intermediate-filament function from studies of transgenic and knockout mice

Summary The function of intermediate-filament (IF) proteins has been a matter of speculation for a long time. Now, the analysis of genetically altered mice is contributing to the understanding of their function. While the initial analysis of knockout mice supports the global view that keratins in epidermis and desmin in muscle serve an important structural function by protecting these tissues against mechanical stress, the detailed examination of these and other mice suggests that IF are more than passive cytoskeletal proteins. This is highlighted by mice with deficiencies for keratins in internal epithelia, vimentin, GFAP, or neurofilament proteins. These lack overt phenotypes expected as a result of cytoskeletal deficiency but show defects compatible with a role of IF in protecting tissues against toxic and other forms of stress. Moreover, the first round of gene replacement experiments suggests that keratins from internal epithelia are unable to take the place of their epidermal counterparts. The development of mice with point mutations, paralleled by the mutation analysis of human diseases and the characterization of IF-associated proteins will be instrumental to understand why evolution has produced such a diverse gene family to encode simple 10 nm diameter filaments.     

Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant

The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(I93M) leads to the degeneration of dopaminergic neurons.