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1.
J Cell Sci ; 132(9)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30926624

RESUMO

Mitochondria play an essential role in regulating insulin secretion from beta cells by providing the ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine whether the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in rodent beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria twofold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely to be important for the generation of Ca2+ microdomains required for efficient insulin granule release.


Assuntos
Cálcio/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina , Mitocôndrias/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Exocitose/fisiologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Camundongos , Optogenética , Ratos
2.
Elife ; 72018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30179155

RESUMO

Catching primal functional changes in early, 'very far from disease onset' (VFDO) stages of Huntington's disease is likely to be the key to a successful therapy. Focusing on VFDO stages, we assessed neuronal microcircuits in premanifest Hdh150 knock-in mice. Employing in vivo two-photon Ca2+ imaging, we revealed an early pattern of circuit dysregulation in the visual cortex - one of the first regions affected in premanifest Huntington's disease - characterized by an increase in activity, an enhanced synchronicity and hyperactive neurons. These findings are accompanied by aberrations in animal behavior. We furthermore show that the antidiabetic drug metformin diminishes aberrant Huntingtin protein load and fully restores both early network activity patterns and behavioral aberrations. This network-centered approach reveals a critical window of vulnerability far before clinical manifestation and establishes metformin as a promising candidate for a chronic therapy starting early in premanifest Huntington's disease pathogenesis long before the onset of clinical symptoms.


Assuntos
Comportamento Animal , Córtex Cerebral/fisiopatologia , Doença de Huntington/fisiopatologia , Metformina/farmacologia , Rede Nervosa/fisiopatologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Comportamento Animal/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Cálcio/metabolismo , Respiração Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Modelos Animais de Doenças , Proteína Huntingtina/metabolismo , Doença de Huntington/patologia , Cinética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mutantes/metabolismo , Rede Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fótons , Agregados Proteicos/efeitos dos fármacos , Biossíntese de Proteínas , Imagem com Lapso de Tempo
3.
Front Cell Neurosci ; 10: 226, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27774050

RESUMO

Expansion of CAG repeats, which code for the disease-causing polyglutamine protein, is a common feature in polyglutamine diseases. RNA-mediated mechanisms that contribute to neuropathology in polyglutamine diseases are important. RNA-toxicity describes a phenomenon by which the mutant CAG repeat RNA recruits RNA-binding proteins, thereby leading to aberrant function. For example the MID1 protein binds to mutant huntingtin (HTT) RNA, which is linked to Huntington's disease (HD), at its CAG repeat region and induces protein synthesis of mutant protein. But is this mechanism specific to HD or is it a common mechanism in CAG repeat expansion disorders? To answer this question, we have analyzed the interaction between MID1 and three other CAG repeat mRNAs, Ataxin2 (ATXN2), Ataxin3 (ATXN3), and Ataxin7 (ATXN7), that all differ in the sequence flanking the CAG repeat. We show that ATXN2, ATXN3, and ATXN7 bind to MID1 in a CAG repeat length-dependent manner. Furthermore, we show that functionally, in line with what we have previously observed for HTT, the binding of MID1 to ATXN2, ATXN3, and ATXN7 mRNA induces protein synthesis in a repeat length-dependent manner. Our data suggest that regulation of protein translation by the MID1 complex is a common mechanism for CAG repeat containing mRNAs.

4.
PLoS One ; 9(7): e102420, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25025689

RESUMO

Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by two neuropathological hallmarks: senile plaques, which are composed of Aß peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated TAU protein. Diabetic patients with dysregulated insulin signalling are at increased risk of developing AD. Further, several animal models of diabetes show increased Aß expression and hyperphosphorylated tau. As we have shown recently, the anti-diabetic drug metformin is capable of dephosphorylating tau at AD-relevant phospho-sites. Here, we investigated the effect of metformin on the main amyloidogenic enzyme BACE1 and, thus, on the production of Aß peptides, the second pathological hallmark of AD. We find similar results in cultures of primary neurons, a human cell line model of AD and in vivo in mice. We show that treatment with metformin decreases BACE1 protein expression by interfering with an mRNA-protein complex that contains the ubiquitin ligase MID1, thereby reducing BACE1 activity. Together with our previous findings these results indicate that metformin may target both pathological hallmarks of AD and may be of therapeutic value for treating and/or preventing AD.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Proteínas dos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas Quinases S6 Ribossômicas/metabolismo , Ubiquitina-Proteína Ligases
5.
Nat Commun ; 4: 1511, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23443539

RESUMO

Expansion of CAG repeats is a common feature of various neurodegenerative disorders, including Huntington's disease. Here we show that expanded CAG repeats bind to a translation regulatory protein complex containing MID1, protein phosphatase 2A and 40S ribosomal S6 kinase. Binding of the MID1-protein phosphatase 2A protein complex increases with CAG repeat size and stimulates translation of the CAG repeat expansion containing messenger RNA in a MID1-, protein phosphatase 2A- and mammalian target of rapamycin-dependent manner. Our data indicate that pathological CAG repeat expansions upregulate protein translation leading to an overproduction of aberrant protein and suggest that the MID1-complex may serve as a therapeutic target for the treatment of CAG repeat expansion disorders.


Assuntos
Proteínas dos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas/genética , Proteína Fosfatase 2/metabolismo , Fatores de Transcrição/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Animais , Western Blotting , Células HeLa , Humanos , Proteína Huntingtina , Luciferases/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína Ligases
6.
Cytotherapy ; 14(1): 61-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21954838

RESUMO

BACKGROUND AIMS: Adipose-derived stromal/stem cells (ASC) possess a multilineage differentiation potential, can be used from an autologous origin, and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues and organs. Beside their well-known differentiation into cells of mesodermal origin, ASC are able to differentiate into cells of ecto- and endodermal origin. METHODS: Previous studies have shown that all trans retinoic acid (ATRA) induces the expression of cytokeratin 18 (CK18), indicating the beginning of differentiation into the epithelial lineage. Nevertheless, ATRA does not induce the expression of other epithelial markers. Therefore, we tested the additional influence of two growth factors on the onset of epithelial differentiation of ASC. The cells were cultured with ATRA, Activin A (ActA) and bone morphogenetic protein-7 (BMP-7), either alone or in combination. Differentiation into the epithelial lineage was assessed by the expression of the characteristic epithelial markers CK18 and zonula occludens protein 1 (ZO-1) using Western blot, immunofluorescence staining and polymerase chain reaction (PCR) analysis. RESULTS: The mixture of all three factors induced epithelial differentiation of ASC without enhancing cell proliferation. Upon induction, the ASC showed phenotypic changes consistent with an epithelial phenotype. The addition of the growth factors ActA and BMP-7 enhanced the inductive effect of ATRA, as shown by the de novo expression of ZO-1 in addition to CK18 expression. CONCLUSIONS: Our study highlights the onset of the epithelial differentiation of ASC induced by culture with a combination of ATRA, ActA and BMP-7.


Assuntos
Tecido Adiposo/citologia , Células Epiteliais/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Transplante de Células-Tronco , Ativinas/farmacologia , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 7/farmacologia , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratina-18/genética , Queratina-18/metabolismo , Proteínas de Membrana/genética , Fosfoproteínas/genética , Medicina Regenerativa , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Tretinoína/farmacologia , Proteína da Zônula de Oclusão-1
7.
Cells Tissues Organs ; 192(2): 106-15, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20185896

RESUMO

High hopes are put into the use of mesenchymal stem cells (MSCs) in various approaches for tissue engineering and regenerative medicine. MSCs are derived from different tissues with only small differences in their phenotype or their differentiation potential, but higher differences in the cell yield. Since fat is easily accessible and contains a high amount of MSCs to be isolated, adipose-derived stem cells (ASCs) are very promising for clinical approaches. ASCs are not a completely homogeneous cell population. Our study was initiated to explore an easy and convenient method to reduce heterogeneity. We tested different isolation methods: (1) the standard isolation method for ASCs based on plastic attachment, (2) the standard method with an initial washing step after 60 min of adherence and (3) immunomagnetic isolation by 4 typical markers (CD49a, CD90, CD105 and CD271). Cells isolated by these methods were evaluated using quantitative PCR and flow cytometry as well as by their differentiation potential. Washing led to a significantly lower expression of desmin, smA and six2, and a higher expression of the stem cell markers nestin, oct-4 and sall-1, compared to standard isolated cells, while the immunomagnetically isolated cells showed no significant changes. All cells independent of the isolation method could be induced to differentiate into adipocytes and osteoblasts. Our study demonstrates that a simple washing step reduces heterogeneity of cultured ASCs according to PCR analysis, whereas the immunomagnetic isolation only showed minor advantages compared to the standard method, but the disadvantage of significantly lower cell yields in the primary isolates.


Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Adulto , Antígenos CD/metabolismo , Diferenciação Celular , Endoglina , Feminino , Humanos , Integrina alfa1/metabolismo , Magnetismo , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos , Receptores de Superfície Celular/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Antígenos Thy-1/metabolismo
8.
Cytotherapy ; 12(1): 96-106, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19929458

RESUMO

BACKGROUND AIMS: The potential of cultured adipose-derived stem cells (ASC) in regenerative medicine and new cell therapeutic concepts has been shown recently by many investigations. However, while the method of isolation of ASC from liposuction aspirates depending on plastic adhesion is well established, a standard expansion medium optimally maintaining the undifferentiated state has not been described. METHODS: We cultured ASC in five commonly used culture media (two laboratory-made media and three commercially available media) and compared them with a standard medium. We analyzed the effects on cell morphology, proliferation, hepatocyte growth factor (HGF) expression, stem cell marker profile and differentiation potential. Proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a fluorescent assay. Release of HGF was assessed by an immunoassay. Expression of characteristic stem cell-related transcription factors and markers was evaluated by quantitative polymerase chain reaction (qPCR) (Nanog, Sox-2, Rex-1, nestin and Oct-4) and flow cytometry (CD44, CD73, CD90, CD105 and CD166), and differentiation was shown by adipogenic medium. RESULTS: The morphology and expansion of ASC were significantly affected by the media used, whereas none of the media influenced the ASC potential to differentiate into adipocytes. Furthermore, two of the media induced an increase in expression of transcription factors, an increased secretion of HGF and a decrease in CD105 expression. CONCLUSIONS: Culture of ASC in one of these two media before using the cells in cell therapeutic approaches may have a benefit on their regenerative potential.


Assuntos
Adipócitos/fisiologia , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Regeneração/fisiologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Antígenos CD/análise , Antígenos CD/metabolismo , Antígenos de Superfície/análise , Antígenos de Superfície/metabolismo , Bioensaio , Biomarcadores/análise , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Meios de Cultura/química , Endoglina , Feminino , Fator de Crescimento de Hepatócito/análise , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/metabolismo , Recuperação de Função Fisiológica/fisiologia , Regeneração/efeitos dos fármacos , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia
9.
Virology ; 373(2): 400-10, 2008 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-18191169

RESUMO

Human adenoviruses (hAdV) have been recognized as a highly prevalent virus family causing severe disease in immunocompromised patients. In xenotransplantation the xenograft therefore will be exposed to these viruses, which in case of its infection might contribute to posttransplant complications. To evaluate the susceptibility of porcine cells for hAdV, we infected the porcine cell line POEK with seven serotypes representing all six hAdV species. Additionally, a second porcine cell line (ST) was infected with two serotypes. Viral replication of serotypes varied: porcine cells were fully permissive for serotypes 1, 4 and 17, semi-permissive for 11 and 21, and non-permissive for 31 and 40. Furthermore, we demonstrated the interaction of serotype 1 with the porcine homologue of the coxsackie-adenovirus receptor, the receptor used by many hAdV serotypes for cell attachment. Thus, various adenovirus types of different hAdV species may be capable of infecting different porcine tissue types.


Assuntos
Adenovírus Humanos/patogenicidade , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Animais , Sequência de Bases , Células CHO , Proteínas do Capsídeo/biossíntese , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Cricetinae , Cricetulus , DNA Viral/genética , Células HeLa , Humanos , Receptores Virais/fisiologia , Sorotipagem , Especificidade da Espécie , Suínos , Transplante Heterólogo , Virulência , Replicação Viral
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