Cellular Werner Phenotypes in Mice Expressing a Putative Dominant-Negative Human WRN Gene

Abstract Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.

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MITOCHONDRIA AS REVERSIBLE REGULATORS OF AGING ASSOCIATED SKIN WRINKLES AND HAIR LOSS IN MICE

Innovation in Aging ◽

10.1093/geroni/igz038.1461 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S395-S395

Author(s):

Keshav K Singh

Keyword(s):

Mouse Model ◽

Hair Loss ◽

Transgene Expression ◽

Skin Aging ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dominant Negative Mutation ◽

Polymerase Domain ◽

Skin Wrinkles

Abstract To evaluate the consequences of the decline in mtDNA content associated with aging we have created an inducible mouse model expressing, in the polymerase domain of POLG1, a dominant-negative mutation that induces depletion of mtDNA. We utilized this inducible mouse model to modulate mitochondrial function by depleting and repleting the mtDNA content. We demonstrate that, in mice, ubiquitous expression of dominant-negative mutant POLG1 leads to 1) reduction of mtDNA content in skin, 2) skin wrinkles, and 3) hair loss. By turning off the mutant POLG1 transgene expression in the whole animal, the skin and hair phenotypes revert to normal after repletion of mtDNA. Thus, we have developed whole-animal mtDNA depleter-repleter mice. These mice present evidence that mtDNA homeostasis is involved in skin aging phenotype and loss of hair and provide an unprecedented opportunity to create tissue-specific mitochondrial modulation to determine the role of the mitochondria in a particular tissue.

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Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain.

The Journal of Cell Biology ◽

10.1083/jcb.118.1.203 ◽

1992 ◽

Vol 118 (1) ◽

pp. 203-212 ◽

Cited By ~ 80

Author(s):

M Metsäranta ◽

S Garofalo ◽

G Decker ◽

M Rintala ◽

B de Crombrugghe ◽

...

Keyword(s):

Transgenic Mice ◽

Endochondral Ossification ◽

Electron Microscopic Examination ◽

Type Ii Collagen ◽

Dominant Negative ◽

Collagen Molecule ◽

Type Ii ◽

Electron Microscopic ◽

Dominant Negative Mutation ◽

Collagen Chain

We have generated transgenic mice by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7. This mutation was expected to disturb the assembly and processing of the homotrimeric type II collagen molecule in cartilage. Expression of transgene mRNA at levels equivalent or higher than the endogenous mRNA in the offspring of two founder animals resulted in a severe chondrodysplastic phenotype with short limbs, hypoplastic thorax, abnormal craniofacial development, and other skeletal deformities. The affected pups died at birth due to respiratory distress. Light microscopy of epiphyseal growth plates of transgenic pups demonstrated a marked reduction in cartilaginous extracellular matrix and disruption of the normal organization of the growth plate. The zone of proliferating chondrocytes was greatly reduced whereas the zone of hypertrophic chondrocytes was markedly increased extending deep into the diaphysis suggestive of a defect in endochondral ossification. Electron microscopic examination revealed chondrocytes with extended RER, a very severe reduction in the amount of cartilage collagen fibrils, and abnormalities in their structure. We postulate that the deletion in the alpha 1(II) collagen acts as a dominant negative mutation disrupting the assembly and secretion of type II collagen molecules. The consequences of the mutation include interference with normal endochondral ossification. These mice constitute a valuable model to study the mechanisms underlying human chondrodysplasias and normal bone formation.

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Abstract T MP114: PPARγ in Endothelial Cells Plays an Essential Role in Protecting Against Vascular Aging: Role of Oxidative Stress and Rho Kinase

Stroke ◽

10.1161/str.45.suppl_1.tmp114 ◽

2014 ◽

Vol 45 (suppl_1) ◽

Author(s):

Dale Kinzenbaw ◽

T. Micheal De Silva ◽

Curt Sigmund ◽

Frank M Faraci

Keyword(s):

Oxidative Stress ◽

Endothelial Function ◽

Vascular Disease ◽

Vascular Dysfunction ◽

Rho Kinase ◽

Dominant Negative ◽

Vascular Aging ◽

Dominant Negative Mutation ◽

Age Related

Although aging is the greatest risk factor for vascular disease and stroke, relatively little is known regarding mechanisms that regulate vascular aging. Endothelial dysfunction - a key element of carotid artery and cerebrovascular disease - progresses with age, greatly increasing the risk for ischemic stroke and cognitive impairment. The nuclear receptor peroxisome proliferator activated receptor-γ (PPARγ) is a ligand-activated transcription factor that may exert diverse effects depending on the cell type. Because little is known regarding the role of PPARγ in vascular aging, we used transgenic mice expressing a dominant negative mutation in human PPARγ (V290M) under control of the endothelial-specific vascular cadherin promoter (designated E-V290M) to examine the hypothesis that cell-specific interference with PPARγ would promote age-induced vascular dysfunction. Responses of carotid arteries from adult (11-12 mo) and old (24±1 mo) E-V290M mice and non-transgenic littermates were examined in vitro. Acetylcholine (an endothelium-dependent agonist) produced similar relaxation of arteries from adult control and E-V290M mice as well as old control mice. In contrast, responses to acetylcholine in arteries from old E-V290M mice were reduced by more than 50% in old E-V290M mice (P<0.01). Endothelial function in old E-V290M mice was not altered by indomethacin but was restored to normal by tempol (a superoxide scavenger) or VAS-2870 (an inhibitor of NADPH oxidase). Reactive oxygen species can activate Rho kinase (a potential mediator of vascular disease) and inhibition of Rho kinase with Y-27632 restored endothelial function to normal in old E-V290M mice. Relaxation of arteries to nitroprusside, which acts directly on vascular muscle, was similar in all groups. These findings provide the first evidence that age-related vascular dysfunction is accelerated following cell-specific interference with endothelial PPARγ through mechanisms involving oxidative stress and Rho kinase. This novel role for endothelial PPARγ has implications for understanding vascular pathophysiology as well as therapeutic approaches for age-induced large and small vessel disease.

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Attenuation of Transient Focal Cerebral Ischemic Injury in Transgenic Mice Expressing a Mutant ICE Inhibitory Protein

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-199704000-00002 ◽

1997 ◽

Vol 17 (4) ◽

pp. 370-375 ◽

Cited By ~ 171

Author(s):

Hideaki Hara ◽

Klaus Fink ◽

Matthias Endres ◽

Robert M. Friedlander ◽

Valeria Gagliardini ◽

...

Keyword(s):

Cerebral Ischemia ◽

Transgenic Mice ◽

Artery Occlusion ◽

Dominant Negative ◽

Neurological Deficits ◽

Wild Type ◽

Brain Swelling ◽

Inhibitory Protein ◽

Endogenous Expression ◽

Dominant Negative Mutation

We used transgenic mice expressing a dominant negative mutation of interleukin-1β converting enzyme (ICE) (C285G) in a model of transient focal ischemia in order to investigate the role of ICE in ischemic brain damage. Transgenic mutant ICE mice (n = 11) and wild-type littermates (n = 9) were subjected to 3 h of middle cerebral artery occlusion followed by 24 h of reperfusion. Cerebral infarcts and brain swelling were reduced by 44% and 46%, respectively. Neurological deficits were also significantly reduced. Regional CBF, blood pressure, core temperature, and heart rate did not differ between groups when measured for up to 1 h after reperfusion. Increases in immunoreactive IL-1β levels, observed in ischemic wild-type brain at 30 min after reperfusion, were 77% lower in the mutant strain, indicating that proIL-1β cleavage is inhibited in the mutants. DNA fragmentation was reduced in the mutants 6 and 24 h after reperfusion. Hence, endogenous expression of an ICE inhibitor confers resistance to cerebral ischemia and brain swelling. Our results indicate that down-regulation of ICE expression might provide a useful therapeutic target in cerebral ischemia.

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Loss of Caveolin-1 Gene Expression Accelerates the Development of Dysplastic Mammary Lesions in Tumor-Prone Transgenic Mice

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0503 ◽

2003 ◽

Vol 14 (3) ◽

pp. 1027-1042 ◽

Cited By ~ 100

Author(s):

Terence M. Williams ◽

Michelle W.-C. Cheung ◽

David S. Park ◽

Babak Razani ◽

Alex W. Cohen ◽

...

Keyword(s):

Gene Expression ◽

Tumor Suppressor ◽

Transgenic Mice ◽

Histological Grade ◽

Dominant Negative ◽

Breast Cancers ◽

Suppressor Activity ◽

Caveolin 1 ◽

Dominant Negative Mutation

Caveolin-1 is the principal structural component of caveolae microdomains, which represent a subcompartment of the plasma membrane. Several independent lines of evidence support the notion that caveolin-1 functions as a suppressor of cell transformation. For example, the human CAV-1 gene maps to a suspected tumor suppressor locus (D7S522/7q31.1) that is frequently deleted in a number of carcinomas, including breast cancers. In addition, up to 16% of human breast cancers harbor a dominant-negative mutation, P132L, in the CAV-1 gene. Despite these genetic associations, the tumor suppressor role of caveolin-1 still remains controversial. To directly assess the in vivo transformation suppressor activity of the caveolin-1 gene, we interbred Cav-1 (−/−) null mice with tumor-prone transgenic mice (MMTV-PyMT) that normally develop multifocal dysplastic lesions throughout the entire mammary tree. Herein, we show that loss of caveolin-1 gene expression dramatically accelerates the development of these multifocal dysplastic mammary lesions. At 3 wk of age, loss of caveolin-1 resulted in an approximately twofold increase in the number of lesions (foci per gland; 3.3 ± 1.0 vs. 7.0 ± 1.2) and an approximately five- to sixfold increase in the total area occupied by these lesions. Similar results were obtained at 4 wk of age. However, complete loss of caveolin-1 was required to accelerate the appearance of these dysplastic mammary lesions, because Cav-1 (+/−) heterozygous mice did not show any increases in foci development. We also show that loss of caveolin-1 increases the extent and the histological grade of these mammary lesions and facilitates the development of papillary projections in the mammary ducts. Finally, we demonstrate that cyclin D1 expression levels are dramatically elevated in Cav-1 (−/−) null mammary lesions, consistent with the accelerated appearance and growth of these dysplastic foci. This is the first in vivo demonstration that caveolin-1 can function as a transformation suppressor gene.

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Depletion of cartilage collagen fibrils in mice carrying a dominant negative Col2a1 transgene affects chondrocyte differentiation

AJP Cell Physiology ◽

10.1152/ajpcell.00579.2002 ◽

2003 ◽

Vol 285 (6) ◽

pp. C1504-C1512 ◽

Cited By ~ 37

Author(s):

Ottavia Barbieri ◽

Simonetta Astigiano ◽

Monica Morini ◽

Sara Tavella ◽

Anna Schito ◽

...

Keyword(s):

Transgenic Mice ◽

Bone Growth ◽

Collagen Type ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

Collagen Fibrils ◽

Dominant Negative ◽

Differentiation Markers ◽

Altered Expression ◽

Dominant Negative Mutation

We have generated transgenic mice harboring the deletion of exon 48 in the mouse α1(II) procollagen gene ( Col2a1). This was the first dominant negative mutation identified in the human α1(II) procollagen gene ( COL2A1). Patients carrying a single allele with this mutation suffer from a severe skeletal disorder called spondyloepiphyseal dysplasia congenita (SED). Transgenic mice phenotype was neonatally lethal with severe respiratory failure, short bones, and cleft palate. Transgene mRNA was expressed at high levels. Growth plate cartilage of transgenic mice presented morphological abnormalities and reduced number of collagen type II fibrils. Chondrocytes carrying the mutation showed altered expression of several differentiation markers, like fibroblast growth factor receptor 3 (Fgfr3), Indian hedgehog (Ihh), runx2, cyclin-dependent kinase inhibitor P21CIP/WAF (Cdkn1a), and collagen type X (Col10a1), suggesting that a defective extracellular matrix (ECM) depleted of collagen fibrils affects chondrocytes differentiation and that this defect participates in the reduced endochondral bone growth observed in chondrodysplasias caused by mutations in COL2A1.

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Abstract 108: Cell-Specific Interference with Endothelial PPARγ Produces Oxidative Stress and Accelerates Vascular Aging

Stroke ◽

10.1161/str.43.suppl_1.a108 ◽

2012 ◽

Vol 43 (suppl_1) ◽

Author(s):

Mary L Modrick ◽

Dale A Kinzenbaw ◽

Pimonrat Ketsawatsomkron ◽

Curt D Sigmund ◽

Frank M Faraci

Keyword(s):

Oxidative Stress ◽

Carotid Artery ◽

Vascular Disease ◽

Carotid Arteries ◽

Vascular Dysfunction ◽

Dominant Negative ◽

Nitric Oxide Donor ◽

Vascular Aging ◽

Dominant Negative Mutation ◽

Age Related

Despite the fact that aging is the greatest risk factor for vascular disease and stroke, relatively little is known regarding mechanisms that promote or protect against vascular aging in experimental models or people. Endothelial dysfunction is a key contributor to both the initiation and progression of vascular disease. Atherosclerosis in carotid arteries (carotid artery disease) greatly increases the risk for ischemic stroke and may contribute to dementia (including Alzheimer’s disease). Peroxisome proliferator activated receptor-γ (PPARγ) is a ligand-activated transcription factor that exerts diverse effects depending on the cell type. Recent work suggests that PPARγ can have beneficial effects in the vasculature including protection against oxidative stress. Relatively little is known regarding the potential role of PPARγ in aging. We used transgenic mice expressing a dominant negative mutation in human PPARγ (V290M) under control of the endothelial-specific vascular cadherin promoter (designated E-V290M) to examine the hypothesis that interference with endothelial PPARγ will promote age-induced vascular dysfunction. Responses of carotid arteries from adult (6±1 mo) and old (23±1 mo) E-V290M mice and non-transgenic littermates (controls) were examined in vitro. Acetylcholine (an endothelium-dependent agonist) produced similar relaxation of arteries from adult control and E-V290M mice as well as old control mice. In contrast, responses to acetylcholine in arteries from old E-V290M mice were markedly impaired. For example, relaxation of the carotid artery to 10 µmol/L acetylcholine was 84±3 and 40±6% in old control versus old E-V290M mice, respectively (P<0.01). The impaired response to acetylcholine in old E-V290M mice could be restored to normal (81±4% relaxation) by tempol, a scavenger of superoxide. Relaxation of the carotid artery to nitroprusside (a nitric oxide donor that acts directly on vascular muscle) was similar in all groups. These findings provide the first evidence that age-related vascular dysfunction is accelerated following cell-specific interference with endothelial PPARγ function. The mechanism that accounts for this change appears to involve reactive oxygen species. Our findings suggest a major protective role for endothelial PPARγ in age-induced oxidative stress and vascular dysfunction.

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