Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli

Recent studies have shown that caveolin-1 (Cav-1) plays an important role as a regulator of angiogenesis in vitro. Here, we use Cav-1 knockout (KO) mice as a model system to examine the in vivo relevance of these findings. A primary mediator of angiogenesis is basic fibroblast growth factor (bFGF). Thus, we studied bFGF-induced angiogenesis in Cav-1 KO mice using a reconstituted basement membrane system, ie, Matrigel plugs, supplemented with bFGF. In Cav-1 KO mice, implanted Matrigel plugs showed a dramatic reduction in both vessel infiltration and density, as compared with identical plugs implanted in wild-type control mice. We also examined the necessity of Cav-1 to support the angiogenic response of an exogenous tumor by subcutaneously injecting Cav-1 KO mice with the melanoma cell line, B16-F10. We show that tumor weight, volume, and vessel density are all reduced in Cav-1 KO mice, consistent with diminished angiogenesis. Ultrastructural analysis of newly formed capillaries within the exogenous tumors reveals a lack of endothelial caveolae and incomplete capillary formation in Cav-1 KO mice. These results provide novel evidence that Cav-1 and caveolae play an important positive role in the process of pathological angiogenesis in vivo.     

Galanin type 1 receptor knockout mice show altered responses to high-fat diet and glucose challenge

Galanin, a brain and pancreatic peptide with three receptor subtypes (GALR1, GALR2, and GALR3), is hypothesized to participate in energy homeostasis and glucoregulation. Hypothalamic galanin expression is induced by dietary fat, and intra-hypothalamic galanin administration has orexigenic/anabolic properties. Systemic galanin infusion alters glucoregulation in non-human species, partly through direct actions on pancreatic islets. However, the physiologic significance of endogenous galanin-GALR signaling is unclear. The present studies tested the hypotheses that GALR1 deficiency alters food intake and feed efficiency following switches to high-fat diet and that GALR1 deficiency alters whole-body glucose homeostasis. Adult, male GALR1 knockout (-/-), heterozygote (+/-), and C57BL/6J control (+/+) mice were studied. GALR1 deficiency impaired adaptation to a 3-day high-fat diet challenge, leading to increased food intake, feed efficiency and weight gain. However, during the following 2 weeks, GALR1 knockout mice decreased intake, consuming less daily energy than while maintained on low-fat diet and also than heterozygote littermates. Chow-maintained GALR1 knockout mice showed relative hyperglycemia in fed and d-glucose (i.p. 1.5 g/kg)-challenged states. GALR1 knockout mice showed normal food intake, feed efficiency and weight accrual on low-fat diets, normal fasted glucose levels, and normal glucose sensitivity to porcine insulin (i.p. 1 IU/kg) in vivo. The results support the hypotheses that galanin-GALR1 systems help adapt food intake and metabolism to changes in dietary fat and modulate glucose disposition in mice.     

Meal patterns and foraging in melanocortin receptor knockout mice

We report the meal patterns of mice with the deletion of either the melanocortin type 3 or 4 receptors (MC3RKO or MC4RKO) compared with that of the wild type (WT) under conditions of varying foraging costs. Mice lived in two-lever operant chambers; the completion of a designated number of responses (termed procurement fixed ratio or PFR) on the "foraging" lever activated the other lever. On this second lever, the completion of a designated number of responses (termed consumatory fixed ratio or CFR) caused the delivery of a 20-mg food pellet. Animals could complete as many CFRs as they wished to constitute a meal, but whenever 10 min elapsed without pressing on this second lever, the meal was terminated and pressing on the "foraging" lever was again required to initiate a new meal. At lower PFRs, mice of all three genotypes took 5-7 well-defined meals per day of approximately 35 pellets/meal. At the highest PFR, mice of all three groups took about half this number of meals, with some increase in meal size, and total intake was slightly reduced. MC4RKO mice were obese compared with WT or MC3RKO but failed to eat more food in the operant chambers and, as a consequence, lost weight, regardless of PFR. Thus, changes in meal-taking strategies as a function of imposed foraging cost are not critically dependent on either MC3 or MC4 receptors, but these conditions did not allow us to study meal patterns in MC4RKO mice that are hyperphagic.     

Glucose-dependent insulinotropic polypeptide receptor knockout mice are impaired in learning, synaptic plasticity, and neurogenesis

Glucose-dependent insulinotropic polypeptide (GIP) is a key incretin hormone, released from intestine after a meal, producing a glucose-dependent insulin secretion. The GIP receptor (GIPR) is expressed on pyramidal neurons in the cortex and hippocampus, and GIP is synthesized in a subset of neurons in the brain. However, the role of the GIPR in neuronal signaling is not clear. In this study, we used a mouse strain with GIPR gene deletion (GIPR KO) to elucidate the role of the GIPR in neuronal communication and brain function. Compared with C57BL/6 control mice, GIPR KO mice displayed higher locomotor activity in an open-field task. Impairment of recognition and spatial learning and memory of GIPR KO mice were found in the object recognition task and a spatial water maze task, respectively. In an object location task, no impairment was found. GIPR KO mice also showed impaired synaptic plasticity in paired-pulse facilitation and a block of long-term potentiation in area CA1 of the hippocampus. Moreover, a large decrease in the number of neuronal progenitor cells was found in the dentate gyrus of transgenic mice, although the numbers of young neurons was not changed. Together the results suggest that GIP receptors play an important role in cognition, neurotransmission, and cell proliferation.     

Fxr1 knockout mice show a striated muscle phenotype: implications for Fxr1p function in vivo

FXR1 is one of the two known homologues of FMR1. FXR1 shares a high degree of sequence homology with FMR1 and also encodes two KH domains and an RGG domain, conferring RNA-binding capabilities. In comparison with FMRP, very little is known about the function of FXR1P in vivo. Mouse knockout (KO) models exist for both Fmr1 and Fxr2. To study the function of Fxr1 in vivo, we generated an Fxr1 KO mouse model. Homozygous Fxr1 KO neonates die shortly after birth most likely due to cardiac or respiratory failure. Histochemical analyses carried out on both skeletal and cardiac muscles show a disruption of cellular architecture and structure in E19 Fxr1 neonates compared with wild-type (WT) littermates. In WT E19 skeletal and cardiac muscles, Fxr1p is localized to the costameric regions within the muscles. In E19 Fxr1 KO littermates, in addition to the absence of Fxr1p, costameric proteins vinculin, dystrophin and alpha-actinin were found to be delocalized. A second mouse model (Fxr1 + neo), which expresses strongly reduced levels of Fxr1p relative to WT littermates, does not display the neonatal lethal phenotype seen in the Fxr1 KOs but does display a strongly reduced limb musculature and has a reduced life span of approximately 18 weeks. The results presented here point towards a role for Fxr1p in muscle mRNA transport/translation control similar to that seen for Fmrp in neuronal cells.     

Deletion including the oligophrenin-1 gene associated with enlarged cerebral ventricles, cerebellar hypoplasia, seizures and ataxia.

Non-specific X-linked mental retardation is a heterogeneous group of disorders with an incidence of approximately 1 in 500 males. A recently identified gene in Xq12, encoding a Rho-GTPase-activating protein, was found to be mutated in individuals with mental retardation. We describe here two sisters with a 46,XY karyotype and a microdeletion of the oligophrenin-1 gene and 1.1 Mb of flanking DNA. We have characterised the molecular interval defining this microdeletion syndrome with the fibre-FISH technique. A visual physical map of 1.2 Mb was constructed which spans the oligophrenin-1 gene and the androgen receptor gene. The analysis of the patients revealed a deletion which extended from the 5' end of the AR gene to a region approximately 80 kb proximal to the EPLG2 gene. The clinical manifestations of the two sisters include psychomotor retardation, seizures, ataxia, hypotonia and complete androgen insensitivity. Cranial MRI scans show enlargement of the cerebral ventricles and cerebellar hypoplasia. Our findings give further support for the involvement of the oligophrenin-1 gene in specific morphological abnormalities of the brain which is of importance in the investigation of male patients presenting with mental retardation. In combination with our results from physical mapping we suggest that a region around the oligophrenin-1 locus is relatively bereft of vital genes.     

Interleukin 1 receptor antagonist knockout mice show enhanced microglial activation and neuronal damage induced by intracerebroventricular infusion of human β-amyloid

Background  

Interleukin 1 (IL-1) is a key mediator of immune responses in health and disease. Although classically the function of IL-1 has been studied in the systemic immune system, research in the past decade has revealed analogous roles in the CNS where the cytokine can contribute to the neuroinflammation and neuropathology seen in a number of neurodegenerative diseases. In Alzheimer's disease (AD), for example, pre-clinical and clinical studies have implicated IL-1 in the progression of a pathologic, glia-mediated pro-inflammatory state in the CNS. The glia-driven neuroinflammation can lead to neuronal damage, which, in turn, stimulates further glia activation, potentially propagating a detrimental cycle that contributes to progression of pathology. A prediction of this neuroinflammation hypothesis is that increased IL-1 signaling in vivo would correlate with increased severity of AD-relevant neuroinflammation and neuronal damage.     

Anxiety, motor activation, and maternal-infant interactions in 5HT1B knockout mice.

This study describes the development of anxiety and motor activation in mice lacking the serotonin (5HT) 1B receptor and in wild type controls and characterizes their early mother-infant interactions. In the isolation-induced ultrasonic vocalization paradigm, 5HT1B knockout pups vocalized less and were hyperactive, rearing, jumping, and rolling more often than wild type pups. One week postpartum, 5HT1B knockout mothers spent 20% more of their time outside the nest and were also hyperactive, rearing and climbing to the edge of the cage more often than the wild type mothers. There were no genotype effects on pup retrieval. Knockout adults were less anxious in the elevated plus-maze, defecated less, and head-dipped more, although none of the standard measures of anxiety (time and entries in the open arms) were different. 5HT1B knockout mice of both sexes were hyperactive during both the light and the dark phases of the 24-hr cycle. Thus, 5HT1B knockout mice show reduced anxiety and are hyperactive throughout their life.     

COACH syndrome: Report of two brothers with congenital hepatic fibrosis,cerebellar vermis hypoplasia,oligophrenia, ataxia,and mental retardation

Congenital hepatic fibrosis (CHF) is probably the most common cause of non-icteric hepatosplenomegaly and is encountered mainly in children and young adults. We describe here two brothers from healthy, non-consanguineous parents. The patients showed early hepatosplenomegaly, portal hypertension, and no apparent kidney involvement. Clinical and laboratory findings were similar in both patients. Liver biopsies showed the presence of broad septa of fibrous tissue containing abundant bile ducts, portal tracts enlarged by fibrosis, and preserved lobular architecture. The histological findings were suggestive of CHF. Ophthalmological assessment demonstrated visual impairment with mild exotropia, nystagmus, and oculomotor apraxia. Neurological examination showed moderate mental retardation and cerebellar ataxia. Brain MRI confirmed cerebellar malformation with inferior vermis hypoplasia. This pattern of defects is consistent with COACH syndrome (Cerebellar vermis hypoplasia, Oligophrenia, congenital Ataxia, Coloboma, Hepatic fibrocirrhosis) which has previously been reported in five other cases. Our report may contribute to a better delineation of the COACH syndrome phenotype in the spectrum of oculo-encephalo-hepato-renal disorders. © 1996 Wiley-Liss, Inc.     

Mutation theory of aging,assessed in transgenic mice and knockout mice

A vital question in the mutation theory of aging is whether mutation accumulates with age. If it does, what are the causes and consequences of the accumulation of mutation? The recent development of transgenic mice has made it possible to study mutation in different kinds of tissues and at a molecular level. An application of these mice to the study of age-dependent alteration has revealed that mutation does accumulate in the aging process. Studies have also revealed several important characteristics of mutation associated with aging. (1) The rate of age-dependent increase of mutant frequency varies among different types of tissue. (2) The rate is not in parallel with the cell proliferation rate of the tissue. (3) Some types of mutation are unique to specific tissues, suggesting the presence of a mechanism of mutation relative to tissue type. On the other hand, several kinds of knockout mice defective in DNA repair have been shown to exhibit tissue lesions and shortened life span. These characteristics provide a new view on the relationship between aging and the genome maintenance system. Here we review the current status of research on the correlation between mutation and aging undertaken by the use of transgenic and knockout mice.     

The neuroanatomy of Eml1 knockout mice,a model of subcortical heterotopia

The cerebral cortex is a highly organized structure responsible for advanced cognitive functions. Its development relies on a series of steps including neural progenitor cell proliferation, neuronal migration, axonal outgrowth and brain wiring. Disruption of these steps leads to cortical malformations, often associated with intellectual disability and epilepsy. We have generated a new resource to shed further light on subcortical heterotopia, a malformation characterized by abnormal neuronal position. We describe here the generation and characterization of a knockout (KO) mouse model for Eml1, a microtubule‐associated protein showing mutations in human ribbon‐like subcortical heterotopia. As previously reported for a spontaneous mouse mutant showing a mutation in Eml1, we observe severe cortical heterotopia in the KO. We also observe abnormal progenitor cells in early corticogenesis, likely to be the origin of the defects. EML1 KO mice on the C57BL/6N genetic background also appear to present a wider phenotype than the original mouse mutant, showing additional brain anomalies, such as corpus callosum abnormalities. We compare the anatomy of male and female mice and also study heterozygote animals. This new resource will help unravel roles for Eml1 in brain development and tissue architecture, as well as the mechanisms leading to severe subcortical heterotopia.     

Jejunal afferent nerve sensitivity in wild-type and TRPV1 knockout mice

The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine intestine. Multiunit activity was recorded in vitro from mesenteric afferents supplying segments of mouse jejunum taken from wild-type (WT) and TRPV1 knockout (TRPV1^−/−) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so the pressure–response curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase in activity. Afferent response to distension was significantly lower in TRPV1^−/− than in WT mice. Single-unit analysis revealed three functional types of afferent fibres: (1) low-threshold fibres (2) wide dynamic range fibres and (3) high-threshold fibres. There was a marked downward shift of the pressure–response curve for wide dynamic range fibres in the TRPV1^−/− mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 m m ) was also attenuated in the TRPV1^−/− mice. In contrast, the response to bath application of bradykinin (1 μ m , 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μ m ) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1^−/− afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.     

Notch1 and T-cell development: insights from conditional knockout mice

Notch proteins influence cell-fate decisions in many developmental systems. Gain-of-function studies have suggested a crucial role for Notch1 signaling at several stages during lymphocyte development, including the B/T, alphabeta/gammadelta and CD4/CD8 lineage choices. Here, we critically re-evaluate these conclusions in the light of recent studies that describe inducible and tissue-specific targeting of the Notch1 gene.     

Perforin knockout mice,but not mice with MAIDS,show protection against experimental cytomegalovirus retinitis after adoptive transfer of immune cells with a functional perforin cytotoxic pathway

Summary Adoptive transfer studies were performed to test the hypothesis that the perforin cytotoxic pathway is more important than the Fas/FasL cytotoxic pathway in protection against experimental murine cytomegalovirus (MCMV) retinitis. Splenic immune cells from donor MCMV-immunized normal mice or gld mice deficient in Fas/FasL-mediated cytotoxicity significantly reduced the frequency and severity of MCMV retinitis following subretinal MCMV challenge when transferred into recipient PKO mice deficient in perforin-mediated cytotoxicity. In sharp contrast, splenic cells from donor MCMV-immunized PKO mice failed to provide protection against MCMV retinitis when transferred into recipient PKO mice. Protection was not achieved, however, in recipient mice with retrovirus-induced immunodeficiency (MAIDS), even when splenic cells originated from MCMV-immunized normal mice.     

Double knockout mice show BASH and PKCdelta have different epistatic relationships in B cell maturation and CD40-mediated activation

The development and survival of mature B cells requires an antigen-independent signal from the B cell receptor (BCR) through an adaptor protein containing an SH2 domain, BASH (BLNK/SLP-65). It also requires signaling through BAFF and the BAFF receptor (BAFF-R), and is negatively regulated by protein kinase Cdelta (PKCdelta). In PKCdelta-deficient mice, B cell maturation occurs independently of the BAFF receptor (BAFF-R), indicating that BAFF-R signaling promotes maturation by inhibiting the negative function of PKCdelta. To clarify which of the two signaling pathways plays the primary role in B cell maturation, we crossed BASH-deficient mice with PKCdelta-deficient mice to generate BASH/PKCdelta-double knockout (DKO) mice. In the DKO mice, B cell maturation was blocked at the transitional type 1 (T1) stage and B cells were prone to apoptosis, in common with BASH-deficient mice. This indicates that BASH-mediated BCR signaling primarily controls B cell survival and maturation, with BAFF-R signaling and its inhibition of PKCdelta acting as a secondary regulator. By contrast, CD40-mediated proliferation and antibody production, which are low in BASH-deficient mice, were rescued in the DKO mice, indicating that the suppression of CD40-mediated B cell activation by PKCdelta is epistatic to BASH-mediated promotion. The physiological relevance of these opposing hierarchical effects of BASH and PKCdelta in the regulation of B cell maturation and activation is discussed.     

IFN-gamma knockout mice show Th2-associated delayed-type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection

Delayed-type hypersensitivity (DTH) has been demonstrated to be a Th1 type immune response which is important in the host defense against infection with intracellular bacteria, including Chlamydia. In the present study, we surprisingly observed that C. trachomatis mouse pneumonitis MoPn-infected IFN-gamma gene knockout (KO) mice mounted strong DTH responses following foopad challenge with inactivated organisms. The DTH responses in IFN-gamma KO mice were associated with Th2 cytokine production and partially blocked by anti-IL-4 monoclonal antibodies. In addition, the inflammatory cells in IFN-gamma KO mice failed to target the cellular sites of chlamydial inclusions in infected tissues and failed to clear the infection. The data, in conjunction with previous studies, suggest that different types (Th1 and Th2 associated) of DTH responses may function differently in host defense against chlamydial infection and that the functional differences in DTH responses may account for the dual role that DTH is speculated to play in chlamydial protective immunity and immunopathology. Moreover, the data suggest that the IFN-gamma KO mouse is a useful model system for studying chlamydial pathogenesis.     

Normal neutrophil functions in sphingosine kinase type 1 and 2 knockout mice

Sphingosine kinase (SPHK) has been implicated as an important element in neutrophil responses to diverse stimulatory agents. To get more insight into the role of the type 1 and 2 isoforms of SPHK in neutrophil functions, we made use of the respective SPHK knockout mice. Neutrophils isolated from the bone marrow of these mice showed normal increase of intracellular Ca(2+) when stimulated in vitro by fMLP, platelet-activating factor, the anaphylatoxin C5a, or ATP, and normal migration towards fMLP and C5a. Also, recruitment of neutrophils into the peritoneum towards the chemokines KC and MIP-2 or to LPS, and into the peripheral blood after fMLP injection was similar in SPHK knockout strains and wild-type animals. An in vivo model of bacterial lung infection revealed an accelerated progression of disease in SPHK2 (but not SPHK1) knockout mice as compared to wild-type controls. However, effector functions of SPHK-deficient neutrophils, such as superoxide production, beta-glucuronidase release and their capacity to kill bacteria were unchanged as compared to wild-type cells. To conclude, the data derived from SPHK knockout mice do not support the hypothesis that any of the two lipid kinases plays a crucial role in signalling downstream of various neutrophil stimuli; SPHKs appear not to be essential for neutrophil recruitment and effector functions.     

Gastric acid secretion in cholecystokinin-1 receptor, -2 receptor,and -1, -2 receptor gene knockout mice

Gastrin is important for stimulating acid secretion as well as differentiating gastric mucosal cells via cholecystokinin-2 receptors (CCK-2Rs). In turn, CCK acts preferably via CCK-1R to release somatostatin, and somatostatin has been postulated to exhibit a tonic inhibition of gastrin bioactivity. The present study was designed to examine the hypothesis that CCK-1R and 2R may act in opposite directions in gastric acid secretion. Having generated CCK-1R(−/−), 2R(−/−), and 1R(−/−)2R(−/−) mice, we examined the regulation of gastric acid secretion in four genotypes including wild-type mice. Parietal cells possess histamine receptors, muscarinic receptors, and CCK-2Rs. Since histamine increases cAMP and carbachol increases calcium, the responses of gastric acid secretion to graded doses of histamine, carbachol, and a combination of histamine + carbachol were determined. The sensitivity to histamine did not differ among the four genotypes, while the maximal acid secretion was lower in CCK-2R(−/−) mice than in wild-type mice. In addition, sensitivity to carbachol was impaired in mice without CCK-2R. The interaction of histamine and carbachol was conserved in all genotypes. In conclusion, CCK-2R is necessary to respond to carbachol as well as to produce the maximal acid secretion, while the role of CCK-1R in acid secretion is less important.