The vasoconstrictor neuropeptide Y (NPY) has been shown to down-regulate tyrosine hydroxylase expression in cultured adrenal chromaffin cells, which probably accounts for the higher plasma resting norepinephrine (NE) and epinephrine (E) concentrations observed in Y1 knock-out mice (Y1-/-) than in wild-type mice (Y1+/+). The aim of this work was to study the hemodynamic response of Y1-/- mice to an acute stimulation of the sympathetic nervous system (cold pressor test, CPT). Plasma catecholamine concentrations were higher in Y1-/- mice than in wild-type animals at the end of the CPT. The CPT-induced increase in mean arterial blood pressure (MAP) and heart rate (HR) was similar in both genotypes. Independently of the genotype, females had significantly slower HR than males throughout the 15 min duration of the CPT. There was no difference in the sensitivity of the baroreceptor reflex, as reflected by the change in HR divided by the concurrent change in MBP between Y1-/- and Y1+/+ mice. In conclusion, mice lacking the Y1 receptor can maintain normal hemodynamic response to an acute activation of the sympathetic system, albeit at the expense of increased catecholamine discharge.; http://www.sciencedirect.com/science/article/B6T0M-4MT54Y4-3/1/f346666c9ec021eeffb80529f17474da
The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene 'targeting'). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions...
The liver is the major organ responsible for the uptake of modified low-density lipoprotein (LDL) from the blood circulation, with endothelial and Kupffer cells as major cellular uptake sites. Scavenger-receptors, which include various classes, are held responsible for this uptake. Mice deficient in scavenger-receptor class A types I and II were created and the fate of acetylated LDL (Ac-LDL) in vivo and its interaction with liver endothelial, Kupffer and peritoneal macrophages was characterized. Surprisingly, the decay in vivo (t12 < 2 min), tissue distribution and liver uptake (at 5 min it was 77.4 +/- 4.6% of the injected dose) of Ac-LDL in the knock-out mice were not significantly different from control mice (t12 < 2 min and liver uptake 79.1 +/- 4.6% of the injected dose). A separation of mice liver cells into parenchymal, endothelial and Kupffer cells 10 min after injection of Ac-LDL indicated that in both control and knock-out mice the liver endothelial cells were responsible for more than 70% of the liver uptake. Both in control and knock-out mice, preinjection of polyinosinic acid (poly I, 200 microg) completely blocked the liver uptake, indicating that both in control and knock-out mice the scavenger-receptors are sensitive to poly I. Preinjection of suboptimal poly I concentrations (20 and 50 microg) provided evidence that the serum decay and liver uptake of Ac-LDL is more readily inhibited in the knock-out mice as compared with the control mice...
ALDH3A1 (aldehyde dehydrogenase 3A1) is abundant in the mouse cornea but undetectable in the lens, and ALDH1A1 is present at lower (catalytic) levels in the cornea and lens. To test the hypothesis that ALDH3A1 and ALDH1A1 protect the anterior segment of the eye against environmentally induced oxidative damage, Aldh1a1(−/−)/Aldh3a1(−/−) double knock-out and Aldh1a1(−/−) and Aldh3a1(−/−) single knock-out mice were evaluated for biochemical changes and cataract formation (lens opacification). The Aldh1a1/Aldh3a1- and Aldh3a1-null mice develop cataracts in the anterior and posterior subcapsular regions as well as punctate opacities in the cortex by 1 month of age. The Aldh1a1-null mice also develop cataracts later in life (6–9 months of age). One- to three-month-old Aldh-null mice exposed to UVB exhibited accelerated anterior lens subcapsular opacification, which was more pronounced in Aldh3a1(−/−) and Aldh3a1(−/−)/Aldh1a1(−/−) mice compared with Aldh1a1(−/−) and wild type animals. Cataract formation was associated with decreased proteasomal activity, increased protein oxidation, increased GSH levels, and increased levels of 4-hydroxy-2-nonenal- and malondialdehyde-protein adducts. In conclusion, these findings support the hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via nonenzymatic (light filtering) and enzymatic (detoxification) functions.
The anti-inflammatory cytokine interleukin (IL)-10 plays an important role in the regulation of host-immune responses. Here we studied the role IL-10 plays in host responses to cytomegalovirus (CMV) infection. We demonstrate that manifestations of murine CMV (MCMV) disease are more severe in IL-10 knock-out mice, despite significantly reduced levels of viral replication. Cytokine analysis of serum revealed increased levels of interferon (IFN)-γ, monocyte chemotactic protein 1 (MCP-1) and IL-6, all of which are potent stimulators of inflammatory responses. Depletion of IFN-γ by monoclonal antibodies in IL-10 knock-out mice failed to improve the physical condition of the mice, while increasing viral replication. In contrast, serum levels of IL-6 in the knock-out animals were unaffected by IFN-γ depletion and remained significantly elevated early in the course of infection. These data suggest that increased weight loss observed in IL-10 knock-out mice may be attributed to the uncontrolled production of proinflammatory cytokines, including IL-6.
Protein oxidation has been linked to accelerated aging and is a
contributing factor to many diseases. Methionine residues are particularly
susceptible to oxidation, but the resulting mixture of methionine
R-sulfoxide (Met-RO) and methionine S-sulfoxide (Met-SO) can
be repaired by thioredoxin-dependent enzymes MsrB and MsrA, respectively.
Here, we describe a knock-out mouse deficient in selenoprotein MsrB1, the main
mammalian MsrB located in the cytosol and nucleus. In these mice, in addition
to the deletion of 14-kDa MsrB1, a 5-kDa selenoprotein form was specifically
removed. Further studies revealed that the 5-kDa protein occurred in both
mouse tissues and human HEK 293 cells; was down-regulated by MsrB1
small interfering RNA, selenium deficiency, and selenocysteine tRNA mutations;
and was immunoprecipitated and recognized by MsrB1 antibodies. Specific
labeling with 75Se and mass spectrometry analyses revealed that the
5-kDa selenoprotein corresponded to the C-terminal sequence of MsrB1. The
MsrB1 knock-out mice lacked both 5- and 14-kDa MsrB1 forms and showed
reduced MsrB activity, with the strongest effect seen in liver and kidney. In
addition, MsrA activity was decreased by MsrB1 deficiency. Liver and kidney of
the MsrB1 knock-out mice also showed increased levels of
Evidence suggests a critical role for dopamine in the reinforcing effects of cocaine in rats and primates. However, self-administration has been less often studied in the mouse species, and, to date, “knock-out” of individual dopamine-related genes in mice has not been reported to reduce the reinforcing effects of cocaine. We studied the dopamine D1 receptor and cocaine self-administration in mice using a combination of gene-targeted mutation and pharmacological tools. Two cohorts with varied breeding and experimental histories were tested, and, in both cohorts, there was a significant decrease in the number of D1 receptor knock-out mice that met criteria for acquisition of cocaine self-administration (2 of 23) relative to wild-type mice (27 of 32). After extinction of responding with saline self-administration, dose–response studies showed that cocaine reliably and dose dependently maintained responding greater than saline in all wild-type mice but in none of the D1 receptor knock-out mice. The D1-like agonist SKF 82958 (2,3,4,5,-tetrahydro-6-chloro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine hydrobromide) and the D2-like agonist quinelorane both functioned as positive reinforcers in wild-type mice but not in D1 receptor mutant mice...
A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(−/−) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(−/−) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase Cδ, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus...
Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, coding for a β2-microglobulin (β2m)–associated major histocompatibility complex class I-like protein. However, iron accumulation in patients with HH can be highly variable. Previously, analysis of β2mRag1−/− double-deficient mice, lacking all β2m-dependent molecules and lymphocytes, demonstrated increased iron accumulation in the pancreas and heart compared with β2m single knock-out mice. To evaluate whether the observed phenotype in β2mRag1−/− mice was due solely to the absence of Hfe or to other β2m-dependent molecules, we generated HfeRag1−/− double-deficient mice. Our studies revealed that introduction of Rag1 deficiency in Hfe knock-out mice leads to heightened iron overload, mainly in the liver, whereas the heart and pancreas are relatively spared compared with β2mRag1−/− mice. These results suggest that other β2m-interacting protein(s) may be involved in iron regulation and that in the absence of functional Hfe molecules lymphocyte numbers may influence iron overload severity.
FEDDE, KENTON N.; BLAIR, LIBBY; SILVERSTEIN, JULIE; COBURN, STEPHEN P.; RYAN, LAWRENCE M.; WEINSTEIN, ROBERT S.; WAYMIRE, KATRINA; NARISAWA, SONOKO; MILLÁN, JOSÉ L.; MACGREGOR, GRANT R.; WHYTE, MICHAEL P.
Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (−/−) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of ~50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at ~10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.
Type 2 diabetes results from an impairment of insulin action. The first demonstrable abnormality of insulin signaling is a decrease of insulin-dependent glucose disposal followed by an increase in hepatic glucose production. In an attempt to dissect the relative importance of these two changes in disease progression, we have employed genetic knock-outs/knock-ins of the insulin receptor. Previously, we demonstrated that insulin receptor knock-out mice (Insr−/−) could be rescued from diabetes by reconstitution of insulin signaling in liver, brain, and pancreatic β cells (L1 mice). In this study, we used a similar approach to reconstitute insulin signaling in tissues that display insulin-dependent glucose uptake. Using GLUT4-Cre mice, we restored InsR expression in muscle, fat, and brain of Insr−/− mice (GIRKI (Glut4-insulin receptor knock-in line 1) mice). Unlike L1 mice, GIRKI mice failed to thrive and developed diabetes, although their survival was modestly extended when compared with Insr−/−. The data underscore the role of developmental factors in the presentation of murine diabetes. The broader implication of our findings is that diabetes treatment should not necessarily target the same tissues that are responsible for disease pathogenesis.
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms...
The ε4 allele of apolipoprotein E (APOE) is currently the major genetic risk factor identified for Alzheimer’s disease (AD). Previous in vivo data from our laboratory has demonstrated that amyloid-β (Aβ) is rapidly removed from the plasma by the liver and kidney and that the rate of its clearance is affected by ApoE in C57BL/6J and APOE−/− mice. To expand upon these findings, we assessed the peripheral clearance of human synthetic Aβ42 in APOE ε2, ε3, and ε4 knock-in and APOE knock-out mice injected with lipidated recombinant apoE2, E3, and E4 protein. Our results show that APOE does influence the rate at which the mice are able to clear Aβ42 from their bloodstream. Both APOE ε4 mice and APOE knock-out mice treated with lipidated recombinant apoE4 demonstrated increased retention of plasma Aβ42 over time compared to APOE ε2/APOE knock-out rE2 and APOE ε3/APOE knock-out rE3 mice. These findings suggest that the peripheral clearance of Aβ42 is significantly altered by APOE genotype. Given that APOE ε4 is a risk factor for AD, then these novel findings provide some insight into the role of ApoE isoforms on the peripheral clearance of Aβ which may impact on clearance from the brain.
The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono--lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni3S2), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni3S2 carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni3S2. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni3S2 in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion...
The trefoil peptide family, consisting in mammals of three members namely TFF1, 2 and 3, plays a cytoprotective role in epithelial cells of various tissues, mainly in the digestive tract. Tff1, Tff2 or Tff3 knock-out mouse models developed various kinds of gastrointestinal impairment. microRNAs are known to be novel gene regulators. We aimed to investigate the physiological role of such miRNAs in Tff2 knock-out mice. Whole miRNome profiling and in silico analysis were performed for Tff2-KO and WT mice. Our latest data explored the role of miRNAs in the regulatory cascades and molecular processes of Tff2−/− mice. As much as 6% of the Tff2-KO mice miRNome was significantly dys-regulated. Further in silico analysis suggests that the respective dys-regulated part of the miRNome is involved in human pathological processes, including pancreatic, colorectal and basal cell cancer. Additionally, the dys-regulated miRNome targets pathways involved in carbohydrate metabolism and adipocytokine signaling. The latter links deficient caloric maintenance in Tff2 and previous observation in Tff3-KO mice with miRNAs. In summary, our proof-of-concept study indicates that miRNAs may play an important role in the regulatory processes of the trefoil peptide family...
Why antidepressants vary in terms of efficacy is currently unclear. Despite the leadership of selective serotonin reuptake inhibitors (SSRIs) in the treatment of depression, the precise neurobiological mechanisms involved in their therapeutic action are poorly understood. A better knowledge of molecular interactions between monoaminergic system, pre- and post-synaptic partners, brain neuronal circuits and regions involved may help to overcome limitations of current treatments and identify new therapeutic targets. Intracerebral in vivo microdialysis (ICM) already provided important information about the brain mechanism of action of antidepressants first in anesthetized rats in the early 1990s, and since then in conscious wild-type or knock-out mice. The principle of ICM is based on the balance between release of neurotransmitters (e.g., monoamines) and reuptake by selective transporters [e.g., serotonin transporter for serotonin 5-hydroxytryptamine (5-HT)]. Complementary to electrophysiology, this technique reflects pre-synaptic monoamines release and intrasynaptic events corresponding to ≈80% of whole brain tissue content. The inhibitory role of serotonergic autoreceptors infers that they limit somatodendritic and nerve terminal 5-HT release. It has been proposed that activation of 5-HT1A and 5-HT1B receptor sub-types limits the antidepressant-like activity of SSRIs. This hypothesis is based partially on results obtained in ICM experiments performed in naïve...
Mice lacking the serotonin receptor 1A [Htr1aknock-out (Htr1aKO)] display increased innate and conditioned anxiety-related behavior. Expression of the receptor in the mouse forebrain during development is sufficient to restore normal anxiety-related behavior to knock-out mice, demonstrating a role for serotonin in the developmental programming of anxiety circuits. However, the precise developmental period as well as the signaling pathways and neural substrates involved in this phenomenon are unknown. Here, we show that pharmacological blockade of the receptor from postnatal day 13 (P13)–P34 is sufficient to reproduce the knock-out phenotype in adulthood, thus defining a role for serotonin in the maturation and refinement of anxiety circuits during a limited postnatal period. Furthermore, we identify increases in the phosphorylation of α-Ca2+/calmodulin-dependent protein kinase II (αCaMKII) at threonine 286 in the hippocampus of young Htr1aKO mice under anxiety-provoking conditions. Increases in αCaMKII phosphorylation were most pronounced in the CA1 region of the hippocampus and were localized to the extrasynaptic compartment, consistent with a tissue-specific effect of the receptor. No changes in αCaMKII phosphorylation were found in adult knock-out mice...
Fragile X Syndrome is the most common inherited intellectual disability, and Fragile X Syndrome patients often exhibit motor and learning deficits. It was previously shown that the fmr1 knock-out mice, a common mouse model of Fragile X Syndrome, recapitulates this motor learning deficit and that the deficit is associated with altered plasticity of dendritic spines. Here, we investigated the motor learning-induced turnover, stabilization and clustering of dendritic spines in the fmr1 knock-out mouse using a single forelimb reaching task and in vivo multiphoton imaging. We report that fmr1 knock-out mice have deficits in motor learning-induced changes in dendritic spine turnover and new dendritic spine clustering, but not the motor learning-induced long-term stabilization of new dendritic spines. These results suggest that a failure to establish the proper synaptic connections in both number and location, but not the stabilization of the connections that are formed, contributes to the motor learning deficit seen in the fmr1 knock-out mouse.
Der erste Teil der Arbeit beschäftigt sich mit dem bereits von anderen Spezies bekannten altersabhängigen Veränderungen der Nierenfunktion. Untersucht wurden junge und ältere CD-1-Mäuse. An wachen Tieren im Stoffwechselkäfigversuch zeigte sich, dass die älteren CD-1-Mäuse im Vergleich zu den jungen CD-1-Mäusen einen grösseren Flüssigkeitsumsatz hatten und eine gesteigerte Elektrolytausscheidung aufwiesen. Für die Beurteilung der funktionellen renalen Reserve wurde die Hyperfiltration nach Aminosäureinfusion untersucht. Die älteren CD-1-Mäuse hatten eine niedrigere basale GFR und zeigten keine GFR-Steigerung infolge der AS-Infusion. Bei den jungen Tiere nahm die GFR durch AS-Infusion stark zu. Eine gesteigerte Natriumausscheidung und Diurese nach Aminosäuregabe war bei jungen und älteren Tieren zu beobachten, bei den älteren CD-1-Mäusen jedoch nur in abgeschwächter Form.
Der zweite Teil der Arbeit untersucht die Bedeutung des D3-Rezeptors für die renale Hämodynamik und Ausscheidungsfunktion bei D3 (-/-) Mäusen und Wildtypmäusen. Die Basalwerte der D3 (-/-) Mäusen in wachem Zustand im Stoffwechselkäfig liessen keine Unterschiede gegenüber den Wildtyptieren erkennen. Sie zeigten sogar die Tendenz, mehr Natrium auszuscheiden als die Wildtyptiere. Diese Beobachtung wurde durch weitere Versuche mit einer oralen NaCl-Beladung von 1% bzw. 4% bestätigt. Auch hier konnte kein Defizit in der Menge und der Geschwindigkeit der Natriumausscheidung bei D3 knock-out Mäusen gegenüber den Wildtypmäusen beobachtet werden. Im Vergleich zu den Wildtyptieren büssten die D3 (-/-) Mäuse durch den Verlust des D3-Rezeptors ihre Fähigkeit zur suffizienten Elektrolyt- und Volumenausscheidung nicht ein.
Um die Bedeutung des D3-Rezeptors für die renale Hämodynamik zu untersuchen...
The serotonin-1A [5-hydroxytryptamine 1A (5HT1A)] receptor is important for emotional and homeostatic processes in the central nervous system. In the hippocampus, the 5HT1A receptor couples to inhibitory Gi/o proteins to decrease pyramidal cell excitability. Here we investigate the 5HT1A receptor in a mouse deficient in the α-subunit of Gz protein (Gαz knock-out). Behavioural tests showed heightened anxiety and depression-like behaviour in the Gαz knock-out mice. Whole-cell recording in CA1 pyramidal neurons showed a significantly greater 5HT1A receptor-mediated potassium current in Gαz knock-out mice. The effect was independent of 5HT4 receptors as the slow after-hyperpolarization was unaffected and a slow depolarization was absent in the Gαz knock-out mice. Other receptors linked to Gi/o proteins [γ-aminobutyric acid type B receptor (GABAg), adenosine A1 and muscarinic acetylcholine receptors] were not affected in Gαz knock-out mice. These results suggest that the 5HT1A receptor may be linked to Gαz protein, as reported previously in cell culture but shown here in an intact neural network.