Reduction of circulating soluble Flt-1 alleviates preeclampsia-like symptoms in a mouse model

Preeclampsia (PE) is characterized by widespread endothelial damage with hypertension, proteinuria, glomeruloendotheliosis and elevated soluble Flt-1 (sFlt-1), a natural occurring antagonist of vascular endothelial growth factor (VEGF). Cancer patients receiving anti-VEGF therapy exhibit similar symptoms. We suggested that a decrease in circulating sFlt-1 would alleviate the symptoms associated with PE. Adenoviral (Adv) overexpression of sFlt-1 induced proteinuria, caused glomerular damage and increase in blood pressure in female Balb/c mice. Circulating level of sFlt-1 above 50 ng/ml plasma induced severe vascular damage and glomerular endotheliosis. Albumin concentration in urine was elevated up to 30-fold, compared to control AdvGFP-treated animals. The threshold of kidney damage was in the range of 20-30 ng/ml sFlt-1 in plasma (8-15 ng/ml in urine). Co-administration of AdvsFlt-1 with AdvVEGF to neutralize circulating sFlt-1 resulted in more than a 70% reduction in free sFlt-1 in plasma, more than 80% reduction in urine and rescued the damaging effect of sFlt-1 on the kidneys. This demonstrates that below a critical threshold sFlt-1 fails to elicit damage to the fenestrated endothelium and that co-expression of VEGF is able to rescue effects mediated by sFlt-1 overexpression.

α7 nicotinic receptor-mediated astrocytic gliotransmitter release:Aβ effects in a preclinical Alzheimer's mouse model

It is now recognized that astrocytes participate in synaptic communication through intimate interactions with neurons. A principal mechanism is through the release of gliotransmitters (GTs) such as ATP, D-serine and most notably, glutamate, in response to astrocytic calcium elevations. We and others have shown that amyloid-β (Aβ), the toxic trigger for Alzheimer's disease (AD), interacts with hippocampal α7 nicotinic acetylcholine receptors (nAChRs). Since α7nAChRs are highly permeable to calcium and are expressed on hippocampal astrocytes, we investigated whether Aβ could activate astrocytic α7nAChRs in hippocampal slices and induce GT glutamate release. We found that biologically-relevant concentrations of Aβ1-42 elicited α7nAChR-dependent calcium elevations in hippocampal CA1 astrocytes and induced NMDAR-mediated slow inward currents (SICs) in CA1 neurons. In the Tg2576 AD mouse model for Aβ over-production and accumulation, we found that spontaneous astrocytic calcium elevations were of higher frequency compared to wildtype (WT). The frequency and kinetic parameters of AD mice SICs indicated enhanced gliotransmission, possibly due to increased endogenous Aβ observed in this model. Activation of α7nAChRs on WT astrocytes increased spontaneous inward currents on pyramidal neurons while α7nAChRs on astrocytes of AD mice were abrogated. These findings suggest that, at an age that far precedes the emergence of cognitive deficits and plaque deposition, this mouse model for AD-like amyloidosis exhibits augmented astrocytic activity and glutamate GT release suggesting possible repercussions for preclinical AD hippocampal neural networks that contribute to subsequent cognitive decline. © 2013 Pirttimaki et al.

Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition

Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest. © 2011 Elsevier Inc.

Aminoglutethimide-induced leucopenia in a mouse model:effects of metabolic and structural determinates

A model of human leucopenia has been developed further in the female mouse. Following daily administration to female mice of 50 mg/kg of the aromatase inhibitor aminoglutethimide, significant falls in platelet and white cell counts occurred after 2 and 3 weeks. At week 4, drug dosage was stopped and the cell counts recovered at the end of that week, although on rechallenge at the beginning of week 5, both platelet and white cell counts fell rapidly. Administration to the mice of structural analogues of aminoglutethimide, such as WSP-3, glutethimide and 4-nitroglutethimide, showed no reductions in platelet and white cell counts. The haemotoxicity of aminoglutethimide over 21 days was unaffected by the presence of either the P-450 inhibitor SKF-525A or the hepatic P-450 inducer phenobarbitone. However, the co-administration of cimetidine abolished the haemotoxicity of aminoglutethimide in terms of platelet and white cell levels. In in vitro studies, both aminoglutethimide and WSP-3 were oxidised to cytotoxic species, although aminoglutethimide was significantly more cytotoxic than WSP-3. The NADPH-dependent covalent binding of 14C aminoglutethimide to mouse microsomes in vitro was significantly reduced by the presence of cimetidine. The activation of the compound to reactive species in vitro, the inhibitory effects of cimetidine in vivo and in vitro, as well as the rapid fall in the in vivo white cell count on rechallenge with aminoglutethimide suggest that this model illustrates a form of leucopenia which may be related to hapten formation and subsequent immune-mediated platelet and white cell lysis. © 2003 Elsevier B.V. All rights reserved.

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

Effects of Endothelin 3 on the Tumor-Angiogenic Response in a Novel Melanoma Mouse Model

Skin cancer is the most common form of cancer in the United States. Melanoma is a particular type of skin cancer, which arises from the malignant transformation of melanocytes and generally exhibits a high propensity to metastasize. Melanoma progression is dependent on angiogenesis to deliver the oxygen and nutrients required to maintain the altered metabolism of rapidly proliferating tumorigenic cells. Recent studies have implicated the growth factor Endothelin 3 (Edn3) in melanoma progression and metastasis. The aim of this study was to examine the role that Edn3 plays in the angiogenesis of melanocytic lesions. For this purpose, Dct-Grm1 transgenic mice, which spontaneously acquire melanocytic lesions through the aberrant expression of the metabotropic glutamate receptor 1 (mGluR1), were crossed with K5-Edn3 transgenic mice that overexpress Edn3. Tumors in the Dct-Grm1/K5-Edn3 experimental population were examined and compared to the control Dct-Grm1 population using immuno-fluorescent staining targeted against the vascular endothelial cell marker CD31. Proteomic arrays were also used and identified changes in the expression of specific angiogenic factors. CD31 antibody staining results revealed an increased vascular density in Dct-Grm1/K5-Edn3 tumors compared with tumors from the Dct-Grm1 controls. Analysis of the relative expression of angiogenic proteins showed an upregulation of various vascular factors in tumors from the Dct-Grm1/K5-Edn3 population, including VEGF-B, MMP-8, MMP-9, and Angiogenin. These results suggest that endothelin signaling promotes angiogenesis in melanocytic lesions. Targeting the factors upregulated by Edn3 signaling may prove effective in hindering melanoma progression.

UV-induced melanoma mouse model dependent on endothelin 3 over-expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.^

Characterization of plasmacytoid dendritic cells in the CD4C/HIV transgenic mouse model

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The effects of aging on the BTBR mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex heterogeneous neurodevelopmental disorder characterized by alterations in social functioning, communicative abilities, and engagement in repetitive or restrictive behaviors. The process of aging in individuals with autism and related neurodevelopmental disorders is not well understood, despite the fact that the number of individuals with ASD aged 65 and older is projected to increase by over half a million individuals in the next 20 years. To elucidate the effects of aging in the context of a modified central nervous system, we investigated the effects of age on the BTBR T + tf/j mouse, a well characterized and widely used mouse model that displays an ASD-like phenotype. We found that a reduction in social behavior persists into old age in male BTBR T + tf/j mice. We employed quantitative proteomics to discover potential alterations in signaling systems that could regulate aging in the BTBR mice. Unbiased proteomic analysis of hippocampal and cortical tissue of BTBR mice compared to age-matched wild-type controls revealed a significant decrease in brain derived neurotrophic factor and significant increases in multiple synaptic markers (spinophilin, Synapsin I, PSD 95, NeuN), as well as distinct changes in functional pathways related to these proteins, including "Neural synaptic plasticity regulation" and "Neurotransmitter secretion regulation." Taken together, these results contribute to our understanding of the effects of aging on an ASD-like mouse model in regards to both behavior and protein alterations, though additional studies are needed to fully understand the complex interplay underlying aging in mouse models displaying an ASD-like phenotype.

Hippocampal Transcriptomic and Proteomic Alterations in the BTBR Mouse Model of Autism Spectrum Disorder.

Autism spectrum disorders (ASD) are complex heterogeneous neurodevelopmental disorders of an unclear etiology, and no cure currently exists. Prior studies have demonstrated that the black and tan, brachyury (BTBR) T+ Itpr3tf/J mouse strain displays a behavioral phenotype with ASD-like features. BTBR T+ Itpr3tf/J mice (referred to simply as BTBR) display deficits in social functioning, lack of communication ability, and engagement in stereotyped behavior. Despite extensive behavioral phenotypic characterization, little is known about the genes and proteins responsible for the presentation of the ASD-like phenotype in the BTBR mouse model. In this study, we employed bioinformatics techniques to gain a wide-scale understanding of the transcriptomic and proteomic changes associated with the ASD-like phenotype in BTBR mice. We found a number of genes and proteins to be significantly altered in BTBR mice compared to C57BL/6J (B6) control mice controls such as BDNF, Shank3, and ERK1, which are highly relevant to prior investigations of ASD. Furthermore, we identified distinct functional pathways altered in BTBR mice compared to B6 controls that have been previously shown to be altered in both mouse models of ASD, some human clinical populations, and have been suggested as a possible etiological mechanism of ASD, including "axon guidance" and "regulation of actin cytoskeleton." In addition, our wide-scale bioinformatics approach also discovered several previously unidentified genes and proteins associated with the ASD phenotype in BTBR mice, such as Caskin1, suggesting that bioinformatics could be an avenue by which novel therapeutic targets for ASD are uncovered. As a result, we believe that informed use of synergistic bioinformatics applications represents an invaluable tool for elucidating the etiology of complex disorders like ASD.

Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, glucose metabolism, and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff-cell glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells.

A Novel Mouse Model of Diffuse Intrinsic Pontine Glioma Initiated in Pax3-Expressing Cells.

Diffuse intrinsic pontine glioma (DIPG) is a rare and incurable brain tumor that arises predominately in children and involves the pons, a structure that along with the midbrain and medulla makes up the brainstem. We have previously developed genetically engineered mouse models of brainstem glioma using the RCAS/Tv-a system by targeting PDGF-B overexpression, p53 loss, and H3.3K27M mutation to Nestin-expressing brainstem progenitor cells of the neonatal mouse. Here we describe a novel mouse model targeting these same genetic alterations to Pax3-expressing cells, which in the neonatal mouse pons consist of a Pax3+/Nestin+/Sox2+ population lining the fourth ventricle and a Pax3+/NeuN+ parenchymal population. Injection of RCAS-PDGF-B into the brainstem of Pax3-Tv-a mice at postnatal day 3 results in 40% of mice developing asymptomatic low-grade glioma. A mixture of low- and high-grade glioma results from injection of Pax3-Tv-a;p53(fl/fl) mice with RCAS-PDGF-B and RCAS-Cre, with or without RCAS-H3.3K27M. These tumors are Ki67+, Nestin+, Olig2+, and largely GFAP- and can arise anywhere within the brainstem, including the classic DIPG location of the ventral pons. Expression of the H3.3K27M mutation reduces overall H3K27me3 as compared with tumors without the mutation, similar to what has been previously shown in human and mouse tumors. Thus, we have generated a novel genetically engineered mouse model of DIPG, which faithfully recapitulates the human disease and represents a novel platform with which to study the biology and treatment of this deadly disease.