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Interactions between environmental and genetic factors may contribute to neurodegenerative disease. In this issue of Neuron, Calon et al. report that a diet low in an essential omega-3 polyunsaturated fatty acid (docosahexaenoic acid) depletes postsynaptic proteins and exacerbates behavioral alterations in a transgenic mouse model of Alzheimer's disease.
PURPOSE: To determine if vascular occlusion and nonperfusion is associated with the outer retinal atrophy, retinopathy, and choroidopathy (chorioretinopathy) that occurs in the alpha H beta S[beta MDD] and alpha H beta S [alpha MD beta MDD] transgenic mouse models of sickle cell disease. METHODS: Mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] transgenic mouse lines that express high levels of human beta S globin were anesthetized and administered horseradish peroxidase (HRP) intracardially. After 1 min, the animals were sacrificed, and the retina from one eye was excised, fixed, and developed in diaminobenzidine (DAB). The contralateral eye was fixed, embedded whole in glycol methacrylate, and HRP developed in 2.5 microns sections. RESULTS: HRP reaction product (HRP-RP) and stained erythrocytes (RBCs) (due to endogenous peroxidase) were diffusely distributed within all vascular lumens in flatmount retinas from control animals (littermates homozygous for the mouse Beta Major deletion not expressing the beta S transgene). In 42.5% of the transgenic mice expressing beta S without any proliferative retinopathy, many blood vessels contained RBC plugs and lacked lumenal HRP-RP. In addition to packed RBCs, fibrin was sometimes present at sites of occlusion. In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion (lumen that lacked HRP-PR). In areas with normal photoreceptors, the choriocapillaris appeared perfused (HRP-RP was present). In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-RP, suggesting autoinfarction. CONCLUSIONS: Nonperfused retinal and choroidal vessels were observed in mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] lines without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Furthermore, small foci of PR loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell-mediated vaso-occlusions are an initial event in the chorioretinopathy and outer retinal atrophy that occurs in these models.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A(2A) adenosine receptor (A(2A)-R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-microMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. (1)H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5'AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.
OBJECTIVE: To determine the capacity of chondrocytes in aging and degenerating articular cartilage to produce major components of the extracellular matrix and maintain the normal structure of articular cartilage in a transgenic mouse model of osteoarthritis. METHODS: Transcription factor Sox9 was used as an indicator of the activation and maintenance of the articular chondrocyte phenotype. Knee joints of Del1 mice carrying 6 copies of the pro alpha1(II) collagen transgene with a short deletion mutation were analyzed at the age of 10 days and at 2, 3, 4, 6, 9, and 15 months by Northern hybridization, RNase protection assay, quantitative reverse transcription-polymerase chain reaction, and immunohistochemistry. Nontransgenic littermates were used as controls. RESULTS: We demonstrated the presence of Sox9 in articular chondrocytes during development, growth, and aging, with the highest messenger RNA levels during the period of rapid growth. With the appearance of degenerative lesions in articular cartilage, 2 repair processes were observed. Local proliferation and activation of chondrocytes rich in Sox9, surrounded by type IIA procollagen and proteoglycans, was seen in articular cartilage. In contrast, metabolically inactive chondrocytes were observed at the margins of the defects. They were devoid of Sox9 and were surrounded by a proteoglycan-poor matrix. Sometimes, the lesions were filled with repair tissue that contained type III collagen but little proteoglycan or type II collagen. CONCLUSION: The results indicate that chondrocytes in mature articular cartilage are capable of inducing the production of Sox9 and type IIA procollagen, which is typical of early chondrogenesis. Degenerative defects in the knee joints of transgenic Del1 mice are associated with local activation of chondrocytes, which probably contributes to the repair process. In other areas, the repair process produces a noncartilaginous matrix, which is insufficient to maintain the integrity of articular cartilage and which allows degeneration to proceed.
Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine disease caused by the expansion of a CAG repeat in the androgen receptor (AR) gene. We generated a transgenic mouse model carrying a full-length AR containing 97 CAGs. Three of the five lines showed progressive muscular atrophy and weakness as well as diffuse nuclear staining and nuclear inclusions consisting of the mutant AR. These phenotypes were markedly pronounced in male transgenic mice, and dramatically rescued by castration. Female transgenic mice showed only a few manifestations that markedly deteriorated with testosterone administration. Nuclear translocation of the mutant AR by testosterone contributed to the phenotypic difference with gender and the effects of hormonal interventions. These results suggest the therapeutic potential of hormonal intervention for SBMA.
Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.
Human immunodeficiency virus (HIV)-1 causes lung disease by increasing the host's susceptibility to pathogens. HIV-1 also causes an increase in systemic oxidative/nitrosative stress, perhaps enhancing the deleterious effects of secondary infections. Here we examined the ability of HIV-1 proteins to increase lung oxidative/nitrosative stress after lipopolysaccharide (LPS) (endotoxin) administration in an HIV-1 transgenic mouse model. Lung oxidative/nitrosative stress biomarkers studied 3 and 6 h after LPS administration were as follows: lung edema, tissue superoxide, NO metabolites, nitrotyrosine, hydrogen peroxide, and bronchoalveolar lavage fluid (BALF) glutathione (GSH). Blood serum cytokine levels were quantified to verify immune function of our nonimmunocompromised animal model. Results indicate that 3 h after LPS administration, HIV-1 transgenic mouse lung tissue has significantly greater edema and superoxide. Furthermore, NO metabolites are significantly elevated in HIV-1 transgenic mouse BALF, lung tissue, and blood plasma compared with those of wild-type mice. HIV-1 transgenic mice also produce significantly greater lung nitrotyrosine and hydrogen peroxide than wild-type mice. In addition, HIV-1 transgenic mice produce significantly less BALF GSH than wild-type mice 3 h after LPS treatment. Without treatment, serum cytokine levels are similar for HIV-1 transgenic and wild-type mice. After treatment, serum cytokine levels are significantly elevated in both HIV-1 transgenic and wild-type mice. Therefore, HIV-1 transgenic mice have significantly greater lung oxidative/nitrosative stress after endotoxin administration than wild-type mice, independent of immune function. These results indicate that HIV-1 proteins may increase pulmonary complications subsequent to a secondary infection by altering the lung redox potential.
The value of the chronic rodent carcinogenicity assay in adequately predicting cancer risk in humans has become a matter of debate over the past few years. Therefore, more rapid and accurate alternative tests are urgently needed. Transgenic mouse models, those harboring genetic changes that are relevant to the multistage cancer process, may provide such alternative tests. Transgenic Emu-pim-1 mice, developed by Berns and coworkers in 1989, contain the pimn-1 oncogene, which is expressed at elevated levels in their lymphoid compartments. As a result, these mice are predisposed to the development of T-cell lymphomas. Because of the low incidence of spontaneous tumors and the increased sensitivity to N-ethyl-N-nitrosourea-induced carcinogenesis, Emu-pim-1 mice were suggested to be one of the first potential and attractive candidates to be used in short-term carcinogenicity testing. In the present article, the results from 2 recent short-term assays (with mitomycin C and x-rays) are briefly presented, together with a review of all 11 performed bioassays and their corresponding histopathologic and molecular data. The overall results allow the first evaluation of the Emu-pim-1 mouse model with regard to its usefulness in short-term carcinogenicity testing. It has been shown that the model is primarily suitable as a sensitive short-term assay for genotoxic carcinogens that not only induce (at least) gene mutations and/or large deletions and rearrangements but that also sufficiently target the lymphoid system. However, the Emu-pim-1 mice lack sufficient sensitivity to justify their routine use in short-term carcinogenicity testing in general.
The immune response to hepatitis viruses is believed to be involved in the development of chronic hepatitis; however, its pathogenetic potential has not been clearly defined. The current study, using a transgenic mouse model of chronic hepatitis B, was designed to determine the relative contributions of the immune cell subsets to the progression of liver disease that induces hepatocellular carcinogenesis. Hepatitis B virus transgenic mice were adoptively transferred with CD4+ and CD8+ T cell-enriched or -depleted and B cell-depleted splenocytes obtained from hepatitis B surface antigen-primed, syngeneic nontransgenic donors. The resultant liver disease, hepatocyte apoptosis, regeneration, and tumor development were assessed and compared with the manifestations in mice that had received unfractionated spleen cells. Transfer of CD8(+)-enriched splenocytes caused prolonged disease kinetics, and a marked increase in the extent of hepatocyte apoptosis and regeneration. In 12 of 14 mice the transfer resulted in multiple hepatocellular carcinomas (HCCs) comparable with the manifestations seen in the mice transferred with total splenocytes. In contrast, mice that had received CD4(+)-enriched cells demonstrated lower levels of liver disease and developed fewer incidences of HCC (4 of 17). The experiment also revealed that all of the groups of mice complicated with HCC developed comparable mean numbers and sizes of tumors. B-cell depletion had no effect on disease kinetics in this model. Taken together, these results demonstrate that the pathogenetic events induced by CD8+ T-cell subset are primarily responsible for the induction of chronic liver disease that increases tumor incidence, suggesting their potential in triggering the process of hepatocarcinogenesis.
Current theories of breast cancer progression have been greatly influenced by the development and refinement of mouse transgenic and gene targeting technologies. Early transgenic mouse models confirmed the involvement of oncogenes, previously implicated in human breast cancer, by establishing a causal relationship between overexpression or activation of these genes and mammary tumorigenesis. More recently, the importance of genes located at sites of loss of heterozygosity in human breast cancer have been examined in mice by their targeted disruption via homologous recombination. The union of these two approaches allows the generation of complex animal models that more accurately reflect the multistep nature of human breast cancer. This review will examine how the study of transgenic mice has increased our understanding of the molecular events responsible for oncogenic transformation of the mammary gland. BioEssays 22:554-563, 2000.
Cytochrome p450 3A4 (CYP3A4) plays an important role in drug metabolism, and the enzymatic activity of CYP3A4 contributes to many adverse drug-drug interactions. Here we describe a transgenic mouse model that is useful in monitoring the in vivo transcriptional regulation of the human CYP3A4 gene. A reporter construct consisting of 13 kilobases of the human CYP3A4 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line [FVB/N-Tg(CYP3A4-luc)Xen]. Reporter gene expression was assessed using an in vivo imaging system (IVIS) in anesthetized mice. Basal expression of the reporter was highest in liver and kidney, and moderate in the duodenum in male transgenic mice, whereas the basal luciferase activity was highest in the duodenum and lower in kidney and liver in females. Injections of pregnenolone, phenobarbital, rifampicin, nifedipine, dexamethasone, 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), and clotrimazole resulted in a time-dependent induction of luciferase expression, primarily in liver, that peaked at 6 h post injection. The greatest induction was found with clotrimazole, dexamethasone, and PCN, whereas the lowest induction followed pregnenolone, phenobarbital, and rifampicin injection. In general, male mice responded to these drugs more strongly than did females. Our results suggest that the human CYP3A4 promoter functions in transgenic mice and that this in vivo model can be used to study transcriptional regulation of the CYP3A4 gene.
There is substantial evidence that excitotoxicity and oxidative damage may contribute to Huntington's disease (HD) pathogenesis. We examined whether the novel anti-oxidant compound BN82451 exerts neuroprotective effects in the R6/2 transgenic mouse model of HD. Oral administration of BN82451 significantly improved motor performance and improved survival by 15%. Oral administration of BN82451 significantly reduced gross brain atrophy, neuronal atrophy and the number of neuronal intranuclear inclusions at 90 days of age. These findings provide evidence that novel anti-oxidants such as BN82451 may be useful for treating HD.
Transgenic mice carrying human Amyloid Precursor Protein mutations present amyloid plaque deposition in the brain upon aging. In this study, we characterized the changes of cortex proteome and endogenous Apolipoprotein E in these mice. Differential analysis of two-dimensional electrophoresis images revealed spots altered upon aging, transgene addition and plaque deposition. Alpha-synuclein and cytochrome oxidase polypeptide Va were up-regulated in transgenic mice. Upon aging, expression of ATP synthase alpha, alpha enolase, UMP-CMP kinase, and dihydropyrimidinase like-2 protein was modified. These proteins and their modification probably play a role in the amyloid aggregate formation in these mice.
Transgenic mice carrying amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. c-Jun is a transcription factor frequently induced in injured neurons. In this study we have examined the distribution of c-Jun-immunoreactivity in the brainstem and spinal cord of transgenic SOD1 mice with a glycine 93 alanine (G93A) mutation. In non-transgenic littermates c-Jun immunostaining was predominantly situated in motoneurons. The number of c-Jun immunoreactive motoneuron was reduced in SOD1(G93A) mice due to pronounced loss of motoneurons. In SOD1(G93A) mice, however, c-Jun-immunoreactivity was strongly induced in neurons in the intermediate zone (Rexed's laminae V-VIII and X) of the spinal cord and throughout the brainstem reticular formation. These findings are of interest since increased levels of c-jun also have been found in the intermediate zone of the spinal cord of ALS patients. This c-Jun may be involved in the neurodegenerative processes both in ALS and in motoneuron disease in SOD1(G93A) mice.
Transgenic mice expressing the superoxide dismutase G93A mutation (SOD1(G93A)) were used to investigate the role of glial inwardly rectifying K(+) (Kir)4.1 channels, which buffer extracellular K(+) increases in response to neuronal excitation. A progressive decrease in Kir4.1 immunoreactivity was observed predominantly in the ventral horn of SOD1(G93A) mutants. Immunoblotting of spinal cord extracts mirrored these changes by showing a loss of Kir4.1 channels from presymptomatic stages onwards. Kir4.1 channels were found to be expressed in the spinal cord grey matter, targetting astrocytes and clustering around capillaries, supporting their role in clearance of extracellular K(+). To understand the functional implications of extracellular K(+) increases, we challenged the NSC34 motor neurone cell line with increasing extracellular K(+) concentrations. Exposure to high extracellular K(+) induced progressive motor neurone cell death. We suggest that loss of Kir4.1 impairs perineural K(+) homeostasis and may contribute to motor neurone degeneration in SOD1(G93A) mutants by K(+) excitotoxic mechanisms.
Previous experiments have shown that a vascular endothelial growth factor (VEGF)-DT385 toxin conjugate inhibits endothelial cell proliferation, angiogenesis and solid tumor growth in a xenotransplant model system. Here, we report that VEGF-DT385 toxin conjugate effectively inhibits spontaneous tumorigenesis. The C3(1)/SV40 TAg transgenic mouse model of mammary gland carcinogenesis was used to determine the effectiveness of VEGF-DT385 toxin conjugate in delaying the onset of disease and the development of solid tumors. Animals were treated daily with conjugate for a period of 7 days. Therapy was initiated at week 14 of development before any visible adenocarcinomas were evident. Treatment of mice with VEGF-DT385 toxin conjugate significantly delayed the onset of tumorigenesis and inhibited solid tumor growth by more than 92%. Furthermore, conjugate treated animals showed less than twice the number of tumor nodules when compared to control mice. Finally, this vascular targeting agent significantly increased survival time of animals by 5 weeks. VEGF-DT385 toxin conjugate resulted in temporary weight loss and no long-lasting toxicity was seen. More importantly, using this established tumor model, VEGF-DT385 toxin conjugate appeared to be as effective as a similar treatment schedule with recombinant human endostatin. Our results suggest that VEGF-DT385 toxin conjugate is a potent inhibitor of mammary adenocarcinoma growth and might be useful in breast cancer therapy.
Brain-derived neurotrophic factor (BDNF) is an attractive component for the treatment of various neurodegenerative diseases such as Alzheimer's or Parkinson's disease. Innovative non-invasive therapeutic approaches involve appropriate pharmacological induction of endogenous BDNF synthesis in brain. A transgenic mouse model has been established to study human BDNF gene expression and permit the screening of compounds capable of stimulating its activity. A 145-kb yeast artificial chromosome carrying the human BDNF gene has been engineered to produce the transgene which contains the extended BDNF promoter and 3' flanking regions and has integrated the enhanced green fluorescent protein (E-GFP) coding sequence in place of the BDNF coding exon. Five transgenic lines have been obtained through microinjection of the YAC into fertilized mouse oocytes. From the three lines expressing the transgene, one displays the specific pattern of BDNF expression. Faithful tissue-restricted transcription of BDNF 5' exons and localization of the fluorescent reporter gene product in the expected brain subregions are reported. This line constitutes an exploitable system for investigating human BDNF gene regulation in vivo.
Alzheimer's disease (AD) is characterized by cognitive decline and deposition of beta-amyloid (Abeta) plaques in cortex and hippocampus. A transgenic mouse AD model (Tg2576) that overexpresses a mutant form of human Abeta precursor protein exhibits age-related cognitive deficits, Abeta plaque deposition, and oxidative damage in the brain. We tested the ability of Ginkgo biloba, a flavonoid-rich antioxidant, to antagonize the age-related behavioral impairment and neuropathology exhibited by Tg2576 mice. At 8 months of age, 16 female Tg2576 and 15 female wild-type (wt) littermate mice were given ad lib access to tap water or Ginkgo biloba (70 mg/kg/day in water). After 6 months of treatment, all mice received Morris water maze training (4 trials/day for 10 days) to assess hippocampal dependent spatial learning. All mice received a 60-s probe test of spatial memory retention 24 h after the 40th trial. Untreated Tg2576 mice exhibited a spatial learning impairment, relative to wt mice, while Ginkgo biloba-treated Tg2576 mice exhibited spatial memory retention comparable to wt during the probe test. Spatial learning was not different between Ginkgo biloba-treated and untreated wt mice. There were no group differences in learning to swim to a visible platform. Soluble Abeta and hippocampal Abeta plaque burden did not differ between the Tg2576 groups. Brain levels of protein carbonyls were paradoxically elevated in Ginkgo biloba-treated mice. These data indicate that chronic Ginkgo biloba treatment can block an age-dependent decline in spatial cognition without altering Abeta levels and without suppressing protein oxidation in a transgenic mouse model of AD.