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1.
Biochem Biophys Res Commun ; 511(3): 658-664, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30826061

RESUMO

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation.


Assuntos
Ciclo do Ácido Cítrico , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/metabolismo , Diferenciação Celular , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Células-Tronco Neurais/citologia
2.
J Biol Chem ; 288(4): 2521-31, 2013 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-23209290

RESUMO

γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-ß production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-ß production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-ß levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-ß generation. Likewise, purified recombinant GSAP had no effect on amyloid-ß generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-ß levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-ß levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-ß generation are uncertain.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Regulação da Expressão Gênica , Piperazinas/farmacologia , Proteínas/química , Pirimidinas/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Animais , Benzamidas , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Mesilato de Imatinib , Masculino , Camundongos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo
3.
Nat Commun ; 12(1): 6393, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34737297

RESUMO

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.


Assuntos
Doença de Depósito de Glicogênio Tipo II/enzimologia , alfa-Glucosidases/metabolismo , Animais , Autofagia , Terapia de Reposição de Enzimas , Feminino , Doença de Depósito de Glicogênio Tipo II/terapia , Fígado/enzimologia , Masculino , Camundongos , alfa-Glucosidases/genética
4.
Acta Neuropathol Commun ; 7(1): 167, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31661040

RESUMO

Glycogen storage disorder type III (GSDIII), or debranching enzyme (GDE) deficiency, is a rare metabolic disorder characterized by variable liver, cardiac, and skeletal muscle involvement. GSDIII manifests with liver symptoms in infancy and muscle involvement during early adulthood. Muscle biopsy is mainly performed in patients diagnosed in adulthood, as routine diagnosis relies on blood or liver GDE analysis, followed by AGL gene sequencing. The GSDIII mouse model recapitulate the clinical phenotype in humans, and a nearly full rescue of muscle function was observed in mice treated with the dual AAV vector expressing the GDE transgene.In order to characterize GSDIII muscle morphological spectrum and identify novel disease markers and pathways, we performed a large international multicentric morphological study on 30 muscle biopsies from GSDIII patients. Autophagy flux studies were performed in human muscle biopsies and muscles from GSDIII mice. The human muscle biopsies revealed a typical and constant vacuolar myopathy, characterized by multiple and variably sized vacuoles filled with PAS-positive material. Using electron microscopy, we confirmed the presence of large non-membrane bound sarcoplasmic deposits of normally structured glycogen as well as smaller rounded sac structures lined by a continuous double membrane containing only glycogen, corresponding to autophagosomes. A consistent SQSTM1/p62 decrease and beclin-1 increase in human muscle biopsies suggested an enhanced autophagy. Consistent with this, an increase in the lipidated form of LC3, LC3II was found in patients compared to controls. A decrease in SQSTM1/p62 was also found in the GSDIII mouse model.In conclusion, we characterized the morphological phenotype in GSDIII muscle and demonstrated dysfunctional autophagy in GSDIII human samples.These findings suggest that autophagic modulation combined with gene therapy might be considered as a novel treatment for GSDIII.


Assuntos
Autofagia , Doença de Depósito de Glicogênio Tipo III/patologia , Músculo Esquelético/patologia , Vacúolos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Músculo Esquelético/ultraestrutura
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