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1.
Artigo em Inglês | MEDLINE | ID: mdl-38576540

RESUMO

Aging is the most significant risk factor for age-related diseases in general, which is true for age-related diseases in the eye including age-related macular degeneration (AMD). Therefore, in order to identify potential therapeutic targets for these diseases, it is crucial to understand the normal aging process and how its mis-regulation could cause age-related diseases at the molecular level. Recently, abnormal lipid metabolism has emerged as one major aspect of age-related symptoms in the retina. Animal models provide excellent means to identify and study factors that regulate lipid metabolism in relation to age-related symptoms. Central to this review is the role of transmembrane protein 135 (TMEM135) in the retina. TMEM135 was identified through the characterization of a mutant mouse strain exhibiting accelerated retinal aging and positional cloning of the responsible mutation within the gene, indicating the crucial role of TMEM135 in regulating the normal aging process in the retina. Over the past decade, the molecular functions of TMEM135 have been explored in various models and tissues, providing insights into the regulation of metabolism, particularly lipid metabolism, through its action in multiple organelles. Studies indicated that TMEM135 is a significant regulator of peroxisomes, mitochondria, and their interaction. Here, we provide an overview of the molecular functions of TMEM135 which is crucial for regulating mitochondria, peroxisomes, and lipids. The review also discusses the age-dependent phenotypes in mice with TMEM135 perturbations, emphasizing the importance of a balanced TMEM135 function for the health of the retina and other tissues including the heart, liver, and adipose tissue. Finally, we explore the potential roles of TMEM135 in human age-related retinal diseases, connecting its functions to the pathobiology of AMD.

2.
J Vis Exp ; (193)2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36971449

RESUMO

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.


Assuntos
Degeneração Macular , Camundongos , Animais , Camundongos Endogâmicos C57BL , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/metabolismo , Retina/metabolismo , Modelos Animais de Doenças , Epitélio/metabolismo
3.
Commun Biol ; 6(1): 8, 2023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36599953

RESUMO

Transmembrane protein 135 (TMEM135) is thought to participate in the cellular response to increased intracellular lipids yet no defined molecular function for TMEM135 in lipid metabolism has been identified. In this study, we performed a lipid analysis of tissues from Tmem135 mutant mice and found striking reductions of docosahexaenoic acid (DHA) across all Tmem135 mutant tissues, indicating a role of TMEM135 in the production of DHA. Since all enzymes required for DHA synthesis remain intact in Tmem135 mutant mice, we hypothesized that TMEM135 is involved in the export of DHA from peroxisomes. The Tmem135 mutation likely leads to the retention of DHA in peroxisomes, causing DHA to be degraded within peroxisomes by their beta-oxidation machinery. This may lead to generation or alteration of ligands required for the activation of peroxisome proliferator-activated receptor a (PPARa) signaling, which in turn could result in increased peroxisomal number and beta-oxidation enzymes observed in Tmem135 mutant mice. We confirmed this effect of PPARa signaling by detecting decreased peroxisomes and their proteins upon genetic ablation of Ppara in Tmem135 mutant mice. Using Tmem135 mutant mice, we also validated the protective effect of increased peroxisomes and peroxisomal beta-oxidation on the metabolic disease phenotypes of leptin mutant mice which has been observed in previous studies. Thus, we conclude that TMEM135 has a role in lipid homeostasis through its function in peroxisomes.


Assuntos
Ácidos Docosa-Hexaenoicos , Metabolismo dos Lipídeos , Proteínas de Membrana , Peroxissomos , Animais , Camundongos , Ácidos Docosa-Hexaenoicos/metabolismo , Homeostase , Oxirredução , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Peroxissomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo
4.
Sci Rep ; 12(1): 756, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35031662

RESUMO

Aging is a significant factor in the development of age-related diseases but how aging disrupts cellular homeostasis to cause age-related retinal disease is unknown. Here, we further our studies on transmembrane protein 135 (Tmem135), a gene involved in retinal aging, by examining the transcriptomic profiles of wild-type, heterozygous and homozygous Tmem135 mutant posterior eyecup samples through RNA sequencing (RNA-Seq). We found significant gene expression changes in both heterozygous and homozygous Tmem135 mutant mouse eyecups that correlate with visual function deficits. Further analysis revealed that expression of many genes involved in lipid metabolism are changed due to the Tmem135 mutation. Consistent with these changes, we found increased lipid accumulation in mutant Tmem135 eyecup samples. Since mutant Tmem135 mice have similar ocular pathologies as human age-related macular degeneration (AMD) eyes, we compared our homozygous Tmem135 mutant eyecup RNA-Seq dataset with transcriptomic datasets of human AMD donor eyes. We found similar changes in genes involved in lipid metabolism between the homozygous Tmem135 mutant eyecups and AMD donor eyes. Our study suggests that the Tmem135 mutation affects lipid metabolism as similarly observed in human AMD eyes, thus Tmem135 mutant mice can serve as a good model for the role of dysregulated lipid metabolism in AMD.


Assuntos
Olho/metabolismo , Metabolismo dos Lipídeos/genética , Degeneração Macular/etiologia , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Mutação , Animais , Modelos Animais de Doenças , Humanos , Degeneração Macular/genética , Camundongos Mutantes
5.
Invest Ophthalmol Vis Sci ; 61(12): 16, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33064130

RESUMO

Purpose: Aging is a critical risk factor for the development of retinal diseases, but how aging perturbs ocular homeostasis and contributes to disease is unknown. We identified transmembrane protein 135 (Tmem135) as a gene important for regulating retinal aging and mitochondrial dynamics in mice. Overexpression of Tmem135 causes mitochondrial fragmentation and pathologies in the hearts of mice. In this study, we examine the eyes of mice overexpressing wild-type Tmem135 (Tmem135 TG) and compare their phenotype to Tmem135 mutant mice. Methods: Eyes were collected for histology, immunohistochemistry, electron microscopy, quantitative PCR, and Western blot analysis. Before tissue collection, electroretinography (ERG) was performed to assess visual function. Mouse retinal pigmented epithelium (RPE) cultures were established to visualize mitochondria. Results: Pathologies were observed only in the RPE of Tmem135 TG mice, including degeneration, migratory cells, vacuolization, dysmorphogenesis, cell enlargement, and basal laminar deposit formation despite similar augmented levels of Tmem135 in the eyecup (RPE/choroid/sclera) and neural retina. We observed reduced mitochondria number and size in the Tmem135 TG RPE. ERG amplitudes were decreased in 365-day-old mice overexpressing Tmem135 that correlated with reduced expression of RPE cell markers. In Tmem135 mutant mice, RPE cells are thicker, smaller, and denser than their littermate controls without any signs of degeneration. Conclusions: Overexpression and mutation of Tmem135 cause contrasting RPE abnormalities in mice that correlate with changes in mitochondrial shape and size (overfragmented in TG vs. overfused in mutant). We conclude proper regulation of mitochondrial homeostasis by TMEM135 is critical for RPE health.


Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Contagem de Células , Células Cultivadas , Modelos Animais de Doenças , Eletrorretinografia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/fisiopatologia , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/patologia
6.
Exp Biol Med (Maywood) ; 245(17): 1571-1583, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32515224

RESUMO

IMPACT STATEMENT: Mitochondria are dynamic organelles undergoing fission and fusion. Proper regulation of this process is important for healthy aging process, as aberrant mitochondrial dynamics are associated with several age-related diseases/pathologies. However, it is not well understood how imbalanced mitochondrial dynamics may lead to those diseases and pathologies. Here, we aimed to determine metabolic alterations in tissues and cells from mouse models with over-fused (fusion > fission) and over-fragmented (fusion < fission) mitochondria that display age-related disease pathologies. Our results indicated tissue-dependent sensitivity to these mitochondrial changes, and metabolic pathways likely affected by aberrant mitochondrial dynamics. This study provides new insights into how dysregulated mitochondrial dynamics could lead to functional abnormalities of tissues and cells.


Assuntos
Proteínas de Membrana/genética , Metaboloma/genética , Proteínas Mitocondriais/genética , Mutação/genética , Animais , Células Cultivadas , Cerebelo/metabolismo , Hipocampo/metabolismo , Redes e Vias Metabólicas , Metabolômica , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Análise de Componente Principal , Epitélio Pigmentado da Retina/metabolismo
7.
Genetics ; 214(1): 121-134, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31754016

RESUMO

One major aspect of the aging process is the onset of chronic, low-grade inflammation that is highly associated with age-related diseases. The molecular mechanisms that regulate these processes have not been fully elucidated. We have identified a spontaneous mutant mouse line, small with kinky tail (skt), that exhibits accelerated aging and age-related disease phenotypes including increased inflammation in the brain and retina, enhanced age-dependent retinal abnormalities including photoreceptor cell degeneration, neurodegeneration in the hippocampus, and reduced lifespan. By positional cloning, we identified a deletion in chondroitin sulfate synthase 1 (Chsy1) that is responsible for these phenotypes in skt mice. CHSY1 is a member of the chondroitin N-acetylgalactosaminyltransferase family that plays critical roles in the biosynthesis of chondroitin sulfate, a glycosaminoglycan (GAG) that is attached to the core protein to form the chondroitin sulfate proteoglycan (CSPG). Consistent with this function, the Chsy1 mutation dramatically decreases chondroitin sulfate GAGs in the retina and hippocampus. In addition, macrophage and neutrophil populations appear significantly altered in the bone marrow and spleen of skt mice, suggesting an important role for CHSY1 in the functioning of these immune cell types. Thus, our study reveals a previously unidentified impact of CHSY1 in the retina and hippocampus. Specifically, chondroitin sulfate (CS) modification of proteins by CHSY1 appears critical for proper regulation of immune cells of the myeloid lineage and for maintaining the integrity of neuronal tissues, since a defect in this gene results in increased inflammation and abnormal phenotypes associated with age-related diseases.


Assuntos
Sulfatos de Condroitina/metabolismo , Glucuronosiltransferase/metabolismo , Inflamação/metabolismo , Enzimas Multifuncionais/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Doenças Neurodegenerativas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/genética , Degeneração Retiniana/metabolismo , Fatores Etários , Animais , Apoptose/fisiologia , Feminino , Glucuronosiltransferase/genética , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enzimas Multifuncionais/genética , Mutação , N-Acetilgalactosaminiltransferases/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia
8.
PLoS One ; 13(8): e0201986, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30102730

RESUMO

Tissues with high-energy demand including the heart are rich in the energy-producing organelles, mitochondria, and sensitive to mitochondrial dysfunction. While alterations in mitochondrial function are increasingly recognized in cardiovascular diseases, the molecular mechanisms through which changes in mitochondria lead to heart abnormalities have not been fully elucidated. Here, we report that transgenic mice overexpressing a novel regulator of mitochondrial dynamics, transmembrane protein 135 (Tmem135), exhibit increased fragmentation of mitochondria and disease phenotypes in the heart including collagen accumulation and hypertrophy. The gene expression analysis showed that genes associated with ER stress and unfolded protein response, and especially the pathway involving activating transcription factor 4, are upregulated in the heart of Tmem135 transgenic mice. It also showed that gene expression changes in the heart of Tmem135 transgenic mice significantly overlap with those of aged mice in addition to the similarity in cardiac phenotypes, suggesting that changes in mitochondrial dynamics may be involved in the development of heart abnormalities associated with aging. Our study revealed the pathological consequence of overexpression of Tmem135, and suggested downstream molecular changes that may underlie those disease pathologies.


Assuntos
Expressão Gênica , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Miocárdio/metabolismo , Animais , Biomarcadores , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/mortalidade , Cardiopatias/patologia , Imuno-Histoquímica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Análise de Sequência de DNA
9.
Elife ; 52016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27863209

RESUMO

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Envelhecimento , Dinâmica Mitocondrial , Proteínas Mutantes/genética , Proteínas Nucleares/genética , Doenças Retinianas/patologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Animais , Camundongos , Mitocôndrias/química , Proteínas Mutantes/análise , Proteínas Nucleares/análise
10.
Invest Ophthalmol Vis Sci ; 56(6): 3776-87, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26066746

RESUMO

PURPOSE: The purpose of this study was to identify the molecular basis and characterize the pathological consequences of a spontaneous mutation named cone photoreceptor function loss 8 (cpfl8) in a mouse model with a significantly reduced cone electroretinography (ERG) response. METHODS: The chromosomal position for the recessive cpfl8 mutation was determined by DNA pooling and by subsequent genotyping with simple sequence length polymorphic markers in an F2 intercross phenotyped by ERG. Genes within the candidate region of both mutants and controls were directly sequenced and compared. The effects of the mutation were examined in longitudinal studies by light microscopy, marker analysis, transmission electron microscopy, and ERG. RESULTS: The cpfl8 mutation was mapped to Chromosome 12, and a premature stop codon was identified in the spectrin repeat containing nuclear envelope 2 (Syne2) gene. The reduced cone ERG response was due to a significant reduction in cone photoreceptors. Longitudinal studies of the early postnatal retina indicated that the cone photoreceptors fail to develop properly, rod photoreceptors mislocalize to the inner nuclear layer, and both rods and cones undergo apoptosis prematurely. Moreover, we observed migration defects of secondary neurons and ectopic Müller cell bodies in the outer nuclear layer in early postnatal development. CONCLUSIONS: SYNE2 is important for normal retinal development. We have determined that not only is photoreceptor nuclear migration affected, but also the positions of Müller glia and secondary neurons are disturbed early in retinal development. The cpfl8 mouse model will serve as an important resource for further examining the role of nuclear scaffolding and migration in the developing retina.


Assuntos
Mutação , Proteínas do Tecido Nervoso/genética , Neuroglia/patologia , Neurônios/patologia , Proteínas Nucleares/genética , Células Fotorreceptoras/patologia , Retina/patologia , Animais , Camundongos , Camundongos Endogâmicos C57BL
11.
Nat Commun ; 6: 6855, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25897778

RESUMO

Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of crosslinked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed ß-actin-GFP in adult mouse hair cells in vivo and by directly measuring ß-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of stereocilia, which have mechanically gated ion channels, show more variable actin turnover than the tallest stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to stereocilia length maintenance. Altogether, the data support a model whereby stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Mecanotransdução Celular/fisiologia , Órgão Espiral/fisiologia , Animais , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Camundongos , Órgão Espiral/ultraestrutura
12.
Mamm Genome ; 26(1-2): 21-32, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25273269

RESUMO

Understanding the normal aging process will help us determine the mechanisms of how age-related diseases are caused and progress. A/J inbred mice have been shown to exhibit accelerated aging phenotypes in the retina including increased inflammation and photoreceptor cell degeneration, which resemble human aging symptoms. C57BL/6J (B6) inbred mice are less susceptible for these abnormalities, indicating the existence of genetic factor(s) that affect their severity. In this study, we determined that another age-dependent phenotype, ectopic synapse formation, is also accelerated in the A/J retina compared to the B6 retina. Through genetic mapping utilizing recombinant inbred strains, we identified quantitative trait loci (QTLs) on chromosome 7 and 19, which contribute to abnormal retinal synapses as well as other age-dependent phenotypes. Using consomic single chromosome substitution lines where a single chromosome is from A/J and the rest of the genome is B6, we investigated the individual effect of each QTL on retinal aging phenotypes. We observed that both QTLs independently contribute to abnormal retinal synapses, reduction in the number of cone cells, and an up-regulation of retinal stress marker, glial fibrillary acidic protein (GFAP). Mice with a single chromosome substitution on chromosome 19 also exhibited an increase in inflammatory cells, which is characteristic of aging and age-related macular degeneration. Thus, we identified QTLs that are independently capable of affecting the severity and progression of age-dependent retinal abnormalities in mice.


Assuntos
Envelhecimento/fisiologia , Regulação da Expressão Gênica/genética , Fenótipo , Locos de Características Quantitativas/genética , Retina/anormalidades , Sinapses/genética , Envelhecimento/genética , Análise de Variância , Animais , Mapeamento Cromossômico , Fluorescência , Proteína Glial Fibrilar Ácida , Técnicas Histológicas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Especificidade da Espécie , Sinapses/patologia
13.
Physiol Genomics ; 46(8): 277-89, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24550211

RESUMO

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstn(corn1) mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstn(corn1) mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstn(corn1) cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstn(corn1) cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstn(corn1) mice. Comparison of gene expression values from WT, Dstn(corn1) mutant, and Dstn(corn1) rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstn(corn1) mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstn(corn1) mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstn(corn1) mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.


Assuntos
Córnea/metabolismo , Destrina/genética , Células Epiteliais/metabolismo , Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Fator de Resposta Sérica/genética , Actinas/genética , Actinas/metabolismo , Animais , Destrina/metabolismo , Regulação para Baixo/genética , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Camundongos , Mutação/genética , Fenótipo , Fator de Resposta Sérica/metabolismo , Transcrição Gênica/genética
14.
Mamm Genome ; 24(9-10): 349-57, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23929036

RESUMO

Mutations in the gene for destrin (Dstn), an actin depolymerizing factor, lead to corneal abnormalities in mice. A null mutation in Dstn, termed Dstn (corn1) , isolated and maintained in the A.BY background (A.BY Dstn (corn1) ), results in corneal epithelial hyperproliferation, inflammation, and neovascularization. We previously reported that neovascularization in the cornea of Dstn (corn1) mice on the C57BL/6 background (B6.A.BY-Dstn (corn1) ) is significantly reduced when compared to A.BY Dstn (corn1) mice, suggesting the existence of genetic modifier(s). The purpose of this study is to identify the genetic basis of the difference in corneal neovascularization between A.BY Dstn (corn1) and B6.A.BY-Dstn (corn1) mice. We generated N2 mice for a whole-genome scan by backcrossing F1 progeny (A.BY Dstn (corn1) × B6.A.BY-Dstn (corn1) ) to B6.A.BY-Dstn (corn1) mice. N2 progeny were quantitatively phenotyped for the extent of corneal neovascularization and genotyped for markers across the mouse genome. We identified significant association of variability in corneal neovascularization with a locus on chromosome 3 (Chr3). The validity of the identified quantitative trait locus (QTL) was tested using B6 consomic mice carrying Chr3 from A/J mice. Dstn (corn1) mice from F1 and F2 intercrosses (B6.A.BY-Dstn (corn1)  × C57BL/6J-Chr3(A/J)/NaJ) were phenotyped for the extent of corneal neovascularization. This analysis showed that mice carrying the A/J allele at the QTL show significantly increased neovascularization. Our results indicate the existence of a modifier that genetically interacts with the Dstn gene. This modifier demonstrates allelic differences between C57BL6 and A.BY or A/J. The modifier is sufficient to increase neovascularization in Dstn (corn1) mice.


Assuntos
Neovascularização da Córnea/genética , Destrina/genética , Animais , Destrina/metabolismo , Epistasia Genética , Estudos de Associação Genética , Escore Lod , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Locos de Características Quantitativas
15.
Mol Vis ; 18: 606-16, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22419854

RESUMO

PURPOSE: Mutations in destrin (Dstn) cause corneal abnormalities in mice. A null mutation, Dstn(corn1), results in corneal epithelial hyperproliferation, inflammation, and neovascularization in the A.BY background (A.BY Dstn(corn1)). Homozygosity for a point mutation, Dstn(corn1-2J), results in mild thickening of the corneal epithelium but no corneal neovascularization in a C57BL/6 (B6) background (B6 Dstn(corn1-2J)). The goal of this study was to determine whether phenotypic differences are due to allelic differences between Dstn(corn1) and Dstn(corn1-2J), or are the result of genetic background effects. METHODS: We generated two congenic (Cg) mouse lines, B6.Cg-Dstn(corn1) and A.BY.Cg-Dstn(corn1-2J), to compare to the original A.BY Dstn(corn1) and B6 Dstn(corn1-2J) lines. We performed immunohistochemistry to assay F-actin accumulation, neovascularization, proliferation, and inflammation. By western blot analysis we tested the expression of serum response factor (SRF), a known regulator of the Dstn(corn1) phenotype. RESULTS: The Dstn(corn1) mutation leads to neovascularization, hyperproliferation, and inflammation in the cornea of A.BY Dstn(corn1) as well as B6.Cg-Dstn(corn1) mice. We did not observe significant corneal neovascularization or hyperproliferation in either A.BY.Cg-Dstn(corn1-2J) or B6 Dstn(corn1-2J) mice. Actin accumulation, neovascularization, epithelial proliferation and inflammation in B6.Cg-Dstn(corn1) cornea are significantly reduced when compared to A.BY Dstn(corn1)cornea. SRF changes are consistent in Dstn(corn1) mutants, regardless of genetic background. CONCLUSIONS: Differences in the abnormal phenotypes of Dstn mutants result from allelic differences between Dstn(corn1) and Dstn(corn1-2J) . Moreover, phenotypes of Dstn(corn1) mice are modified by genetic background, suggesting the existence of genetic modifiers. Protein analysis suggests that a genetic modifier affects phenotypic severity functionally downstream from or in a pathway independent from SRF. These data demonstrate that natural genetic variation affects phenotypic severity in Dstn(corn1) mice.


Assuntos
Alelos , Córnea/metabolismo , Neovascularização da Córnea/genética , Destrina/genética , Mutação , Actinas/genética , Actinas/metabolismo , Animais , Córnea/irrigação sanguínea , Córnea/patologia , Neovascularização da Córnea/metabolismo , Destrina/metabolismo , Efeito Fundador , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Camundongos , Camundongos Transgênicos , Fenótipo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Índice de Gravidade de Doença
16.
Invest Ophthalmol Vis Sci ; 53(3): 1663-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22323461

RESUMO

PURPOSE: To identify genes that modify photoreceptor cell loss in the retinas of homozygous Tulp1(tm1Pjn) and Tub(tub) mice, which exhibit juvenile retinitis pigmentosa. METHODS: Modifier loci were identified by genetic quantitative trait locus analysis. F2 Tulp1(tm1Pjn/tm1Pjn) mutant mice from a B6-Tulp1(tm1Pjn/tm1Pjn) × AKR/J intercross were genotyped with a panel of single nucleotide polymorphism markers and phenotyped by histology for photoreceptor nuclei remaining at 9 weeks of age. Genotype and phenotype data were correlated and examined with Pseudomarker 2.02 using 128 imputations to map modifier loci. Thresholds for the 63%, 10%, 5%, and 1% significance levels were obtained from 100 permutations. A significant, protective candidate modifier was identified by bioinformatic analysis and confirmed by crossing transgenic mice bearing a protective allele of this gene with Tulp1- and Tub-deficient mice. RESULTS: A significant, protective modifier locus on chromosome 2 and a suggestive locus on chromosome 13 that increases photoreceptor loss were identified in a B6-Tulp1(tm1Pjn/tm1Pjn) × AKR/J intercross. The chromosome 2 locus mapped near Mtap1a, which encodes a protein associated with microtubule-based intracellular transport and synapse function. The protective Mtap1a(129P2/OlaHsd) allele was shown to reduce photoreceptor loss in both Tulp1(tm1Pjn/tm1Pjn) and Tub(tub/tub) mice. CONCLUSIONS: It was demonstrated that the gene Mtap1a, which modifies hearing loss in Tub(tub/tub) mice, also modifies retinal degeneration in Tub(tub/tub) and Tulp1(tm1Pjn/tm1Pjn) mice. These results suggest that functionally nonredundant members of the TULP family (TUB and TULP1) share a common functional interaction with MTAP1A.


Assuntos
Proteínas do Olho/genética , Proteínas Associadas aos Microtúbulos/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas/genética , Degeneração Retiniana/metabolismo , Retinose Pigmentar/genética , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos AKR , Células Fotorreceptoras de Vertebrados/patologia , Polimorfismo de Nucleotídeo Único , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia
17.
Methods Mol Biol ; 770: 293-312, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21805269

RESUMO

Establishment of methods to inactivate genes by homologous recombination in embryonic stem (ES) cells has provided great advantages to the field of mouse genetics. Using this technology, a number of null mutant mice, so-called knock-out mice, have been generated. The gene-targeting technology offers a strong tool that allows us to understand the function of a particular gene of interest in the whole animal and has contributed to studies in a wide variety of biological research areas. More recently, the original knock-out technology has been further refined to develop advanced strategies to generate conditional knock-out and knock-in mice. In this chapter, an overview of gene-targeting strategies is presented and procedures to generate these genetically engineered mice are discussed.


Assuntos
Marcação de Genes/métodos , Camundongos Knockout/genética , Animais , Técnicas de Cultura de Células , Linhagem Celular , Células Clonais/citologia , Células Clonais/metabolismo , Criopreservação , DNA/genética , Eletroporação , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Técnicas de Introdução de Genes , Genótipo , Camundongos
18.
Genetics ; 186(4): 1337-44, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20876567

RESUMO

X-linked retinoschisis (XLRS) is a form of macular degeneration with a juvenile onset. This disease is caused by mutations in the retinoschisin (RS1) gene. The major clinical pathologies of this disease include splitting of the retina (schisis) and a loss in synaptic transmission. Human XLRS patients display a broad range in phenotypic severity, even among family members with the same mutation. This variation suggests the existence of genetic modifiers that may contribute to disease severity. Previously, we reported the identification of a modifier locus, named Mor1, which affects severity of schisis in a mouse model of XLRS (the Rs1tmgc1 mouse). Homozygosity for the protective AKR allele of Mor1 restores cell adhesion in Rs1tmgc1 mice. Here, we report our study to identify the Mor1 gene. Through collecting recombinant mice followed by progeny testing, we have localized Mor1 to a 4.4-Mb region on chromosome 7. In this genetic region, the AKR strain is known to carry a mutation in the tyrosinase (Tyr) gene. We observed that the schisis phenotype caused by the Rs1 mutation is rescued by a Tyr mutation in the C57BL/6J genetic background, strongly suggesting that Tyr is the Mor1 gene.


Assuntos
Monofenol Mono-Oxigenase/fisiologia , Retinosquise/enzimologia , Animais , Adesão Celular , Mapeamento Cromossômico , Cromossomos , Proteínas do Olho/genética , Homozigoto , Camundongos , Monofenol Mono-Oxigenase/genética , Mutação , Retinosquise/genética
19.
Genetics ; 186(1): 147-57, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20610412

RESUMO

Cell hyperproliferation, inflammation, and angiogenesis are biological processes central to the pathogenesis of corneal disease, as well as other conditions including tumorigenesis and chronic inflammatory disorders. Due to the number of disease conditions that arise as a result of these abnormalities, identifying the molecular mechanisms underlying these processes is critical. The avascular and transparent cornea serves as a good in vivo model to study the pathogenesis of cell hyperproliferation, inflammation, and angiogenesis. Corneal disease 1 (Dstn(corn1)) mice are homozygous for a spontaneous null allele of the destrin (Dstn) gene, which is also known as actin depolymerizing factor (ADF). These mice exhibit abnormalities in the cornea including epithelial cell hyperproliferation, stromal inflammation, and neovascularization. We previously identified that the transcription factor, serum response factor (SRF) and a number of its target genes are upregulated in the cornea of these mice. In this study, we show that conditional ablation of Srf in the corneal epithelium of a diseased Dstn(corn1) cornea results in the rescue of the epithelial cell hyperproliferation, inflammation, and neovascularization phenotypes, delineating an epithelial cell-specific role for SRF in the development of all of these abnormalities. Our study also demonstrates that Dstn is genetically upstream of Srf and defines a new functional role for SRF as the master regulator of a hyperproliferative, inflammatory phenotype accompanied by neovascularization.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Fator de Resposta Sérica/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Destrina/genética , Destrina/metabolismo , Doxiciclina/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/citologia , Epitélio Corneano/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fenótipo , Fator de Resposta Sérica/deficiência , Fator de Resposta Sérica/genética
20.
PLoS Genet ; 6(5): e1000956, 2010 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-20502676

RESUMO

Intracellular trafficking is critical for delivering molecules and organelles to their proper destinations to carry out normal cellular functions. Disruption of intracellular trafficking has been implicated in the pathogenesis of various neurodegenerative disorders. In addition, a number of genes involved in vesicle/organelle trafficking are also essential for pigmentation, and loss of those genes is often associated with mouse coat-color dilution and human hypopigmentary disorders. Hence, we postulated that screening for mouse mutants with both neurological defects and coat-color dilution will help identify additional factors associated with intracellular trafficking in neuronal cells. In this study, we characterized a mouse mutant with a unique N-ethyl-N-nitrosourea (ENU)-induced mutation, named nur17. nur17 mutant mice exhibit both coat-color dilution and ataxia due to Purkinje cell degeneration in the cerebellum. By positional cloning, we identified that the nur17 mouse carries a T-to-C missense mutation in archain 1 (Arcn1) gene which encodes the delta subunit of the coat protein I (COPI) complex required for intracellular trafficking. Consistent with this function, we found that intracellular trafficking is disrupted in nur17 melanocytes. Moreover, the nur17 mutation leads to common characteristics of neurodegenerative disorders such as abnormal protein accumulation, ER stress, and neurofibrillary tangles. Our study documents for the first time the physiological consequences of the impairment of the ARCN1 function in the whole animal and demonstrates a direct association between ARCN1 and neurodegeneration.


Assuntos
Proteína Coatomer/genética , Cor de Cabelo , Células de Purkinje/patologia , Frações Subcelulares/metabolismo , Animais , Animais Geneticamente Modificados , Clonagem Molecular , Proteína Coatomer/fisiologia , Etilnitrosoureia/farmacologia , Camundongos , Mutação de Sentido Incorreto
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