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1.
Hum Mol Genet ; 22(4): 633-45, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23118351

RESUMO

Gaucher's disease (GD) is caused by mutations in the GBA1 gene, which encodes acid-ß-glucosidase, an enzyme involved in the degradation of complex sphingolipids. While the non-neuronopathic aspects of the disease can be treated with enzyme replacement therapy (ERT), the early-onset neuronopathic form currently lacks therapeutic options and is lethal. We have developed an induced pluripotent stem cell (iPSc) model of neuronopathic GD. Dermal fibroblasts of a patient with a P.[LEU444PRO];[GLY202ARG] genotype were transfected with a loxP-flanked polycistronic reprogramming cassette consisting of Oct4, Sox2, Klf4 and c-Myc and iPSc lines derived. A non-integrative lentiviral vector expressing Cre recombinase was used to eliminate the reprogramming cassette from the reprogrammed cells. Our GD iPSc express pluripotent markers, differentiate into the three germ layers, form teratomas, have a normal karyotype and show the same mutations and low acid-ß-glucosidase activity as the original fibroblasts they were derived from. We have differentiated them efficiently into neurons and also into macrophages without observing deleterious effects of the mutations on the differentiation process. Using our system as a platform to test chemical compounds capable of increasing acid-ß-glucosidase activity, we confirm that two nojirimycin analogues can rescue protein levels and enzyme activity in the cells affected by the disease.


Assuntos
1-Desoxinojirimicina/análogos & derivados , Adamantano/análogos & derivados , Doença de Gaucher/tratamento farmacológico , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , 1-Desoxinojirimicina/farmacologia , Adamantano/farmacologia , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Análise Mutacional de DNA , Neurônios Dopaminérgicos/enzimologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática/efeitos dos fármacos , Doença de Gaucher/patologia , Expressão Gênica , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fator 4 Semelhante a Kruppel , Lisossomos/enzimologia , Macrófagos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transporte Proteico , Bibliotecas de Moléculas Pequenas , Transcriptoma
2.
Int J Mol Sci ; 14(1): 2119-34, 2013 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-23340651

RESUMO

Optimization of mesenchymal stem cells (MSC) culture conditions is of great importance for their more successful application in regenerative medicine. O(2) regulates various aspects of cellular biology and, in vivo, MSC are exposed to different O(2) concentrations spanning from very low tension in the bone marrow niche, to higher amounts in wounds. In our present work, we isolated mouse bone marrow stromal cells (BMSC) and showed that they contained a population meeting requirements for MSC definition. In order to establish the effect of low O(2) on cellular properties, we examined BSMC cultured under hypoxic (3% O(2)) conditions. Our results demonstrate that 3% O(2) augmented proliferation of BMSC, as well as the formation of colonies in the colony-forming unit assay (CFU-A), the percentage of quiescent cells, and the expression of stemness markers Rex-1 and Oct-4, thereby suggesting an increase in the stemness of culture when exposed to hypoxia. In contrast, intrinsic differentiation processes were inhibited by 3% O(2). Overall yield of differentiation was dependent on the adjustment of O(2) tension to the specific stage of BMSC culture. Thus, we established a strategy for efficient BMSC in vitro differentiation using an initial phase of cell propagation at 3% O(2), followed by differentiation stage at 21% O(2). We also demonstrated that 3% O(2) affected BMSC differentiation in p53 and reactive oxygen species (ROS) independent pathways. Our findings can significantly contribute to the obtaining of high-quality MSC for effective cell therapy.


Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Oxigênio/farmacologia , Animais , Células da Medula Óssea/citologia , Hipóxia Celular , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição/metabolismo
3.
Biochim Biophys Acta ; 1757(5-6): 624-30, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16829231

RESUMO

The abundance of mitochondria is regulated by biogenesis and division. These processes are controlled by cellular factors, given that, for example, mitochondria have to replicate their DNA prior to cell division. However, the mechanisms that allow a synchronization of cell proliferation with mitochondrial genome replication are still obscure. We report here our investigations on the role of proliferation and the contribution of Ras and p66Shc in the regulation of mitochondrial DNA copy number. Ras proteins mediate a variety of receptor-transduced mitogenic signals and appear to play an essential role in the cellular response to growth factors. P66Shc is a genetic determinant of life span in mammals and has been implicated in the regulation of receptor signaling and various mitochondrial functions. First, we confirmed previous reports showing that mitochondrial DNA is replicated during a specific phase of the cell cycle (the pre-S phase) and provided novel evidences that this process is regulated by mitogenic growth factors. Second, we showed that mitochondrial DNA replication is activated following Ras-induced cellular hyper-proliferation. Finally, we showed that p66Shc expression induces mitochondrial DNA replication, both in vitro and in vivo. We suggest that mitochondria are target of intracellular signaling pathways leading to proliferation, involving Ras and p66Shc, which might function to integrate cellular bio-energetic requirements and the inheritance of mitochondrial DNA in a cell cycle-dependent manner.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proliferação de Células , DNA Mitocondrial/genética , Dosagem de Genes , Proteínas ras/fisiologia , Adipócitos , Animais , Ciclo Celular/fisiologia , Núcleo Celular/fisiologia , Células Cultivadas , Replicação do DNA , Genes ras , Substâncias de Crescimento/fisiologia , Camundongos , Camundongos Knockout , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteínas ras/genética
4.
Int J Dev Biol ; 56(9): 729-35, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23319348

RESUMO

Induced pluripotent stem cells (iPSCs) have the ability to differentiate towards various cell types of the adult organism and are a potential source of transplantable material in regenerative medicine. The entire process of conversion of iPSCs into terminally differentiated cells takes place in vitro and requires long periods of time. During in vitro culture, cells are exposed to environmental factors, which are capable of decreasing cellular performance and viability. Oxidative stress is the major underlying mechanism of such negative impact of in vitro environmental factors. We aimed to study the alteration of cellular properties during in vitro hematopoietic differentiation of human iPSCs and the ability of N-acetylcysteine (NAC), a potent free radical scavenger, to prevent such alterations. IPSCs were differentiated towards hematopoietic cells in the presence of 1 mM NAC. Intracellular reactive oxygen species (ROS), nitric oxide (NO), senescence, apoptosis and mitochondrial membrane potential (MMP) were evaluated at 1 and 3 weeks of differentiation. In the course of hematopoietic differentiation of iPSCs, cells progressively accumulated intracellular ROS and NO, increased the levels of apoptosis and senescence, and showed a decrease in mitochondrial functionality. NAC supplementation reversed all these phenomena. NAC administration also improved hematopoietic differentiation of iPSCs in terms of production of CD34, CD45 and CD43 positive cells. In conclusion, when supplemented during hematopoietic differentiation of iPSCs, NAC decreased oxidative stress, rescued the decline in cellular properties induced by long-term in vitro culture and promoted hematopoietic differentiation of iPSCs.


Assuntos
Acetilcisteína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Aging Cell ; 11(1): 162-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22081964

RESUMO

Deletion of the p66(Shc) gene results in lean and healthy mice, retards aging, and protects from aging-associated diseases, raising the question of why p66(Shc) has been selected, and what is its physiological role. We have investigated survival and reproduction of p66(Shc)-/- mice in a population living in a large outdoor enclosure for a year, subjected to food competition and exposed to winter temperatures. Under these conditions, deletion of p66(Shc) was strongly counterselected. Laboratory studies revealed that p66(Shc)-/- mice have defects in fat accumulation, thermoregulation, and reproduction, suggesting that p66(Shc) has been evolutionarily selected because of its role in energy metabolism. These findings imply that the health impact of targeting aging genes might depend on the specific energetic niche and caution should be exercised against premature conclusions regarding gene functions that have only been observed in protected laboratory conditions.


Assuntos
Envelhecimento/genética , Longevidade/genética , Proteínas Adaptadoras da Sinalização Shc/genética , Envelhecimento/metabolismo , Animais , Evolução Biológica , Regulação da Temperatura Corporal/genética , Metabolismo Energético/genética , Feminino , Aptidão Genética/genética , Heterozigoto , Homozigoto , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Knockout , Estações do Ano , Proteínas Adaptadoras da Sinalização Shc/deficiência , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
6.
Aging Cell ; 10(1): 55-65, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21040401

RESUMO

Longevity of a p66Shc knockout strain (ShcP) was previously attributed to increased stress resistance and altered mitochondria. Microarrays of ShcP tissues indicated alterations in insulin signaling. Consistent with this observation, ShcP mice were more insulin sensitive and glucose tolerant at organismal and tissue levels, as was a novel p66Shc knockout (ShcL). Increasing and decreasing Shc expression in cell lines decreased and increased insulin sensitivity, respectively - consistent with p66Shc's function as a repressor of insulin signaling. However, differences between the two p66Shc knockout strains were also observed. ShcL mice were fatter and susceptible to fatty diets, and their fat was more insulin sensitive than controls. On the other hand, ShcP mice were leaner and resisted fatty diets, and their adipose was less insulin sensitive than controls. ShcL and ShcP strains are both highly inbred on the C57Bl/6 background, so we investigated gene expression at the Shc locus, which encodes three isoforms, p66, p52, and p46. Isoform p66 is absent in both strains; thus, the remaining difference to which to attribute the 'lean' phenotype is expression of the other two isoforms. ShcL mice have a precise deletion of p66Shc and normal expression of p52 and p46Shc isoforms in all tissues; thus, a simple deletion of p66Shc results in a 'fat' phenotype. However, ShcP mice in addition to p66Shc deletion have a fourfold increase in p46Shc expression in white fat. Thus, p46Shc overexpression in fat, rather than p66Shc deletion, is the likely cause of decreased adiposity and reduced insulin sensitivity in the fat of ShcP mice, which has implications for the longevity of the strain.


Assuntos
Adiposidade/genética , Insulina/metabolismo , Isoformas de Proteínas/genética , Proteínas Adaptadoras da Sinalização Shc/genética , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Gorduras na Dieta/metabolismo , Feminino , Loci Gênicos/fisiologia , Glucose/metabolismo , Resistência à Insulina/genética , Longevidade/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/genética , Isoformas de Proteínas/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Magreza/genética , Magreza/metabolismo
7.
Aging (Albany NY) ; 1(6): 503-10, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-20157533

RESUMO

Oxygen metabolism is thought to impact on aging through the formation of reactive oxygen species (ROS) that are supposed to damage biological molecules. The study of p66(Shc), a crucial regulator of ROS level involved in aging dysfunction, suggests that the incidence of degenerative disease and longevity are determined by a specific signaling function of ROS other than their unspecific damaging property.


Assuntos
Envelhecimento/metabolismo , Regulação da Expressão Gênica/fisiologia , Longevidade/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Envelhecimento/genética , Animais , Humanos , Longevidade/fisiologia , Mutação , Oxigênio/metabolismo , Isoformas de Proteínas , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
8.
J Biol Chem ; 283(49): 34283-93, 2008 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18838380

RESUMO

Reactive oxygen species (ROS) and insulin signaling in the adipose tissue are critical determinants of aging and age-associated diseases. It is not clear, however, if they represent independent factors or they are mechanistically linked. We investigated the effects of ROS on insulin signaling using as model system the p66(Shc)-null mice. p66(Shc) is a redox enzyme that generates mitochondrial ROS and promotes aging in mammals. We report that insulin activates the redox enzyme activity of p66(Shc) specifically in adipocytes and that p66(Shc)-generated ROS regulate insulin signaling through multiple mechanisms, including AKT phosphorylation, Foxo localization, and regulation of selected insulin target genes. Deletion of p66(Shc) resulted in increased mitochondrial uncoupling and reduced triglyceride accumulation in adipocytes and in vivo increased metabolic rate and decreased fat mass and resistance to diet-induced obesity. In addition, p66(Shc-/-) mice showed impaired thermo-insulation. These findings demonstrate that p66(Shc)-generated ROS regulate the effect of insulin on the energetic metabolism in mice and suggest that intracellular oxidative stress might accelerate aging by favoring fat deposition and fat-related disorders.


Assuntos
Estresse Oxidativo , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Oxirredução , Oxigênio/metabolismo , Fosforilação , Espécies Reativas de Oxigênio , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
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