Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Excitability properties of mouse motor axons in the mutant SOD1(G93A) model of amyotrophic lateral sclerosis

Non-invasive excitability studies of motor axons in patients with amyotrophic lateral sclerosis (ALS) have revealed a changing pattern of abnormal membrane properties with disease progression, but the heterogeneity of the changes has made it difficult to relate them to pathophysiology. The SOD1(G93A) mouse model of ALS displays more synchronous motoneuron pathology. Multiple excitability measures of caudal and sciatic nerves in mutant and wild-type mice were compared before onset of signs and during disease progression (4-19 weeks), and they were related to changes in muscle fiber histochemistry. Excitability differences indicated a modest membrane depolarization in SOD1(G93A) axons at about the time of symptom onset (8 weeks), possibly due to deficient energy supply. Previously described excitability changes in ALS patients, suggesting altered sodium and potassium conductances, were not seen in the mice. This suggests that those changes relate to features of the human disease that are not well represented in the animal model.

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2-3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.

von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models

Deep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, the mechanism of DVT initiation remains elusive. Here we show that 48-hour flow restriction in the inferior vena cava (IVC) results in the development of thrombi structurally similar to human deep vein thrombi. von Willebrand factor (VWF)-deficient mice were protected from thrombosis induced by complete (stasis) or partial (stenosis) flow restriction in the IVC. Mice with half normal VWF levels were also protected in the stenosis model. Besides promoting platelet adhesion, VWF carries Factor VIII. Repeated infusions of recombinant Factor VIII did not rescue thrombosis in VWF(-/-) mice, indicating that impaired coagulation was not the primary reason for the absence of DVT in VWF(-/-) mice. Infusion of GPG-290, a mutant glycoprotein Ib?-immunoglobulin chimera that specifically inhibits interaction of the VWF A1 domain with platelets, prevented thrombosis in wild-type mice. Intravital microscopy showed that platelet and leukocyte recruitment in the early stages of DVT was dramatically higher in wild-type than in VWF(-/-) IVC. Our results demonstrate a pathogenetic role for VWF-platelet interaction in flow disturbance-induced venous thrombosis.

Preponderance of cells with stem cell characteristics in metastasising mouse mammary tumours induced by deregulated EphB4 and ephrin-B2 expression

We have previously shown that EphB4 and ephrin-B2 are differentially expressed in the mammary gland and that their deregulated expression in the mammary epithelium of transgenic mice leads to perturbations of the mammary parenchyma and vasculature. In addition, overexpression of EphB4 and expression of a truncated ephrin-B2 mutant, capable of receptor stimulation but incapable of reverse signalling, confers a metastasising phenotype on NeuT initiated mouse mammary tumours. We have taken advantage of this transgenic tumour model to compare stem cell characteristics between the non-metastasising and metastasising mammary tumours. We analysed the expression of the proliferation attenuating p21(waf) gene, which was significantly increased in the metastasising tumours. Moreover, we compared the expression of CK-19, Sca-1, CD24 and CD49f as markers for progenitor cells exhibiting a decreasing differentiation grade. Sca-1 expressing cells were the earliest progenitors detected in the non-metastasising NeuT induced tumours. The metastasising NeuT/EphB4 tumours were enriched in CD24 expressing cells, whereas the metastasising NeuT/truncated ephrin-B2 tumours contained in addition significant amounts of CD49f expressing cells. The same cell populations were also enriched in mammary glands of single transgenic MMTV-EphB4 and MMTV-truncated ephrin-B2 females indicating that deregulated EphB4-ephrin-B2 signalling interferes with the homeostasis of the stem/progenitor cell pool before tumour formation is initiated. Since the same cell populations are enriched in the normal tissue, primary mammary tumours and metastases we conclude that these progenitor cells were the origin of tumour formation and that this change in the tumour origin has led to the acquisition of the metastatic tumour phenotype.

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.

Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.

An adaptive ERG technique to measure normal and altered dark adaptation in the mouse

The time-course of dark adaptation provides valuable insights into the function and interactions between the rod and cone pathways in the retina. Here we describe a technique that uses the flash electroretinogram (ERG) response to probe the functional integrity of the cone and rod pathways during the dynamic process of dark adaptation in the mouse. Retinal sensitivity was estimated from the stimulus intensity required to maintain a 30 microV criterion b-wave response during a 40 min period of dark adaptation. When tracked in this manner, dark adaptation functions in WT mice depended upon the bleaching effects of initial background adaptation conditions. Altered dark adaptation functions, commensurate with the functional deficit were recorded in pigmented mice that lacked cone function (Gnat2 ( cplf3 )) and in WT mice injected with a toxin, sodium iodate (NaIO(3)), which targets the retinal pigment epithelium and also has downstream effects on photoreceptors. These data demonstrate that this adaptive tracking procedure measures retinal sensitivity and the contributions of the rod and/or cone pathways during dark adaptation in both WT control and mutant mice.

Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.

Presence of the Gpr179(nob5) allele in a C3H-derived transgenic mouse

PURPOSE To identify the mutation responsible for an abnormal electroretinogram (ERG) in a transgenic mouse line (tg21) overexpressing erythropoietin (Epo). The tg21 line was generated on a mixed (C3H; C57BL/6) background and lacked the b-wave component of the ERG. This no-b-wave (nob) ERG is seen in other mouse models with depolarizing bipolar cell (DBC) dysfunction and in patients with the complete form of congenital stationary night blindness (cCSNB). We determined the basis for the nob ERG phenotype and screened C3H mice for the mutation to evaluate whether this finding is important for the vision research community. METHODS ERGs were used to examine retinal function. The retinal structure of the transgenic mice was investigated using histology and immunohistochemistry. Inverse PCR was performed to identify the insertion site of the Epo transgene in the mouse genome. Affected mice were backcrossed to follow the inheritance pattern of the nob ERG phenotype. Quantitative real-time PCR (qRT PCR), Sanger sequencing, and immunohistochemistry were used to identify the mutation causing the defect. Additional C3H sublines were screened for the detected mutation. RESULTS Retinal histology and blood vessel structure were not disturbed, and no loss of DBCs was observed in the tg21 nob mice. The mutation causing the nob ERG phenotype is inherited independently of the tg21 transgene. The qRT PCR experiments revealed that the nob ERG phenotype reflected a mutation in Gpr179, a gene involved in DBC signal transduction. PCR analysis confirmed the presence of the Gpr179(nob5) insertional mutation in intron 1 of Gpr179. Screening for mutations in other C3H-derived lines revealed that C3H.Pde6b(+) mice carry the Gpr179 (nob5) allele whereas C3H/HeH mice do not. CONCLUSIONS We identified the presence of the Gpr179(nob5) mutation causing DBC dysfunction in a C3H-derived transgenic mouse line. The nob phenotype is not related to the presence of the transgene. The Gpr179(nob5) allele can be added to the list of background alleles that impact retinal function in commonly used mouse lines. By providing primers to distinguish between Gpr179 mutant and wild-type alleles, this study allows investigators to monitor for the presence of the Gpr179(nob5) mutation in other mouse lines derived from C3H.

Sleep-Wake Cycle Dysfunction in the TgCRND8 Mouse Model of Alzheimer's Disease: From Early to Advanced Pathological Stages.

In addition to cognitive decline, individuals affected by Alzheimer's disease (AD) can experience important neuropsychiatric symptoms including sleep disturbances. We characterized the sleep-wake cycle in the TgCRND8 mouse model of AD, which overexpresses a mutant human form of amyloid precursor protein resulting in high levels of β-amyloid and plaque formation by 3 months of age. Polysomnographic recordings in freely-moving mice were conducted to study sleep-wake cycle architecture at 3, 7 and 11 months of age and corresponding levels of β-amyloid in brain regions regulating sleep-wake states were measured. At all ages, TgCRND8 mice showed increased wakefulness and reduced non-rapid eye movement (NREM) sleep during the resting and active phases. Increased wakefulness in TgCRND8 mice was accompanied by a shift in the waking power spectrum towards fast frequency oscillations in the beta (14-20 Hz) and low gamma range (20-50 Hz). Given the phenotype of hyperarousal observed in TgCRND8 mice, the role of noradrenergic transmission in the promotion of arousal, and previous work reporting an early disruption of the noradrenergic system in TgCRND8, we tested the effects of the alpha-1-adrenoreceptor antagonist, prazosin, on sleep-wake patterns in TgCRND8 and non-transgenic (NTg) mice. We found that a lower dose (2 mg/kg) of prazosin increased NREM sleep in NTg but not in TgCRND8 mice, whereas a higher dose (5 mg/kg) increased NREM sleep in both genotypes, suggesting altered sensitivity to noradrenergic blockade in TgCRND8 mice. Collectively our results demonstrate that amyloidosis in TgCRND8 mice is associated with sleep-wake cycle dysfunction, characterized by hyperarousal, validating this model as a tool towards understanding the relationship between β-amyloid overproduction and disrupted sleep-wake patterns in AD.