Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1)

Funding Information: This research was supported by the Isabel Gemio Foundation (P18–13) and was also partially supported by the “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. E.A.-C. was supported by a pre-doctoral fellowship of Valhondo Calaff Foundation. S.C.-C. and E.U.-C. were supported by FPU fellowships (FPU19/04435 and FPU16/00684, respectively) from the Ministerio de Ciencia, Innovación y Universidades, Spain. M.P.-B. and A.G.-B. received fellowships from the “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (Universidad de Extremadura). M.N.-S. was supported by the “Ramon y Cajal” Program (RYC-2016–20883), and P.G.-S., was funded by “Juan de la Cierva Incorporación” Program (IJC2019–039229-I), Spain. S.M.S.Y.-D. was supported by the Isabel Gemio Foundation and CIBERNED (CB06/05/0041). J.M.F received research support from the Isabel Gemio Foundation and the “Instituto de Salud Carlos” III, CIBERNED (CB06/05/0041). Publisher Copyright: © 2022 by the authors.Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation.publishersversionpublishe

Mitochondrial Dysfunction in Repeat Expansion Diseases

Repeat expansion diseases are a group of neuromuscular and neurodegenerative disorders characterized by expansions of several successive repeated DNA sequences. Currently, more than 50 repeat expansion diseases have been described. These disorders involve diverse pathogenic mechanisms, including loss-of-function mechanisms, toxicity associated with repeat RNA, or repeat-associated non-ATG (RAN) products, resulting in impairments of cellular processes and damaged organelles. Mitochondria, double membrane organelles, play a crucial role in cell energy production, metabolic processes, calcium regulation, redox balance, and apoptosis regulation. Its dysfunction has been implicated in the pathogenesis of repeat expansion diseases. In this review, we provide an overview of the signaling pathways or proteins involved in mitochondrial functioning described in these disorders. The focus of this review will be on the analysis of published data related to three representative repeat expansion diseases: Huntington’s disease, C9orf72-frontotemporal dementia/amyotrophic lateral sclerosis, and myotonic dystrophy type 1. We will discuss the common effects observed in all three repeat expansion disorders and their differences. Additionally, we will address the current gaps in knowledge and propose possible new lines of research. Importantly, this group of disorders exhibit alterations in mitochondrial dynamics and biogenesis, with specific proteins involved in these processes having been identified. Understanding the underlying mechanisms of mitochondrial alterations in these disorders can potentially lead to the development of neuroprotective strategies

LA DISTROFIA MIOTÓNICA TIPO 1 Y EL RECICLAJE CELULAR

Resumen de la publicación de una participación en formato vídeo corto en las segundas jornadas del congreso Divulga NextGen que se celebrará online, de manera gratuita y en las redes sociales los días 28, 29 y 30 de noviembre de 2023.Fundación Valhondo, CIBERNED, la Fundación ISABEL GEMIO y FUNDESALU

Cultivos celulares

Se utilizan diferentes modelos celulares para validar los resultados en todas las líneas según la procedencia de la línea celular. Los siguientes protocolos se realizan en células de neuroblastoma humano (SH-SY5Y), fibroblastos embrionarios de ratón (MEF), células de astrocitoma (U251), células de neuroglioma (H4), fibroblastos humanos (FH) y células de osteosarcoma (U2OS). El modelo celular basado SH-SY5Y nos permitirá realizar una buena aproximación a los mecanismos que medían la muerte celular en células nerviosas. Estas células son ampliamente utilizadas en estudios de neurodegeneración, incluyendo estudios de modelos in vitro de la Enfermedad de Parkinson. Así también el modelo de las células U251 nos permitirán realizar el estudio en unas condicionas más fisiológicas de las células de la glía y así aproximarnos a la interacción de estas células con las células nerviosasDifferent cell models are used to validate the results in all lines depending on the origin of the cell line. The following protocols are performed on human neuroblastoma cells (SH-SY5Y), mouse embryonic fibroblasts (MEF), astrocytoma cells (U251), neuroglioma cells (H4), human fibroblasts (FH) and osteosarcoma cells (U2OS). The SH-SY5Y-based cell model will allow us to make a good approximation of the mechanisms that mediate cell death in nerve cells. These cells are widely used in neurodegeneration studies, including studies of in vitro models of Parkinson's disease. The U251 cell model will also allow us to study glial cells under more physiological conditions and thus approach the interaction of these cells with nerve cells

Análisis de extracto proteico por Western blotting

Se estudia cómo hacer el extracto proteico a partir de Western blotting. Para ello, se prepara para la transferencia de cada gel, dos papeles Whatman (Extra Thick Blod PAPER Bio-Rad), el uso de dos esponjillas y una membrana de PVDF. Todo deberá estar equilibrado en su tampón de transferencia antes de proceder a realizar el proceso de transferencia. Estas piezas deben estar embebidas durante al menos 10 minutos en tampón de trasferencia. Se aplicará una tensión de 75 V y migrar durante 45 minutos en agitación continua y refrigeración en el caso de los geles de 18 pocillos (Criterion TGX). El tampón utilizado en la transferencia de geles de 18 pocillos es el tampón Tris Glicina Metanol. El anticuerpo secundario se diluye de 1:5.000 a 1:10.000 en 10 % de leche desnatada en una solución de TTBS. La elección del anticuerpo secundario entre monoclonal o policlonal depende siempre del anticuerpo primario. Finalmente, la membrana estará lista para ser reutilizada (al menos en dos o tres ocasiones más). Hay que tener en cuenta que antes del primer borrado, hay que incubar la membrana con los anticuerpos fosforilados de interés. Después se puede proceder al borrado de la membrana e incubar con los anticuerpos totales, específicos de los anticuerpos fosforilados.We study how to make the protein extract from Western blotting. To do this, two Whatman papers (Extra Thick Blod PAPER Bio-Rad), the use of two sponges and a PVDF membrane are prepared for the transfer of each gel. Everything must be balanced in its transfer buffer before proceeding with the transfer process. These parts shall be soaked for at least 10 minutes in transfer buffer. A voltage of 75 V shall be applied and migrate for 45 minutes under continuous agitation and cooling in the case of 18-well gels (Criterion TGX). The buffer used in the transfer of 18-well gels is Tris Glycine Methanol buffer. The secondary antibody is diluted 1:5,000 to 1:10,000 in 10 % skimmed milk in TTBS solution. The choice of monoclonal or polyclonal secondary antibody always depends on the primary antibody. Finally, the membrane is ready to be reused (at least two or three more times). Before the first blotting, the membrane must be incubated with the phosphorylated antibodies of interest. The membrane can then be blotted and incubated with the total antibodies specific to the phosphorylated antibodies

Extracción de proteínas

El estudio sobre la extracción de proteínas consiste en que, antes de usar, el reactivo A y reactivo B se complementaron con cóctel inhibidor de proteasa 10X (Sigma-Aldrich, P2714), ortovanadato de sodio al 0,5 M al 20 % (S6508, Sigma) y fluoruro de sodio al 0,1 M al 1 % (131675, Panreac). Las mitocondrias aisladas se lisaron en CHAPS al 2 % (C3023, Sigma). Las extracciones citosólicas y mitocondriales se analizaron mediante transferencia Western blotting. El protocolo B debe ser rápido, intentando no sobrepasar 30 segundos. Por otro lado, la adición de 1 μg/ml Leupeptina. 1 μg/ml Pepstatina. 1 μg/ml Aprotinina. 1 μg/ml Benzamidina. El PMSF se diluye en 1 ml de etanol absoluto filtrado. Si hay problemas con el método, probar modificando la concentración de digitonina.The protein extraction study consists of reagent A and reagent B supplemented with 10X protease inhibitor cocktail (Sigma-Aldrich, P2714), 0,5 M 20 % sodium orthovanadate (S6508, Sigma) and 0,1 M 1 % sodium fluoride (131675, Panreac) before use. Isolated mitochondria were lysed in 2 % CHAPS (C3023, Sigma). Cytosolic and mitochondrial extractions were analysed by Western blotting. Protocol B should be fast, trying not to exceed 30 seconds. On the other hand, the addition of 1 μg/ml Leupeptin. 1 μg/ml Pepstatin. 1 μg/ml Aprotinin. 1 μg/ml Benzamidine. PMSF is diluted in 1 ml of filtered absolute ethanol. If there are problems with the method, try modifying the digitonin concentration

Técnica de silenciamiento génico

Para realizar silenciamiento génico en placas de 6 pocillos (130184, Biolite) se emplean el reactivo HiPerfect Transfection Reagent (509301704, Qiagen); el reactivo Lipofectamine RNAiMAX Transfection (13778150, Invitrogen) y el reactivo Lipofectamine 2000 Reagent (11668-019, Invitrogen). En los tres procesos se elabora la misma mezcla con siRNA para el control negativo (scrambled) en paralelo. A las 24 horas de incubación con cualquiera de los reactivos se cambia el medio a la placa y se añade medio normal con antibiótico. Con el primer reactivo, pasadas otras 24 horas se añade el tratamiento de interés y se procede al lisado para realizar la técnica de Western blotting. Con el segundo reactivo, tras las 24 horas y el tratamiento adecuado, se procede a fijar la placa con PFA. Con el tercer reactivo, se dejan pasar 24 horas, se aplica el tratamiento previsto y se pasa a la recogida de células y posterior lisado.For gene silencing in 6-well plates (130184, Biolite), HiPerfect Transfection Reagent (509301704, Qiagen); Lipofectamine RNAiMAX Transfection Reagent (13778150, Invitrogen) and Lipofectamine 2000 Reagent (11668-019, Invitrogen) are used. In all three processes, the same mixture with siRNA for the negative control (scrambled) is prepared in parallel. After 24 hours of incubation with any of the reagents, the medium is changed to the plate and normal medium with antibiotic is added. With the first reagent, after another 24 hours, the treatment of interest is added and the lysate is lysed for Western blotting. With the second reagent, after 24 hours and the appropriate treatment, the plate is fixed with PFA. With the third reagent, 24 hours are allowed to elapse, the expected treatment is applied and the cells are collected and subsequently lysed

Técnica de sobreexpresión génica

Para la introducción de proteínas exógenas marcadas (plásmidos bacterianos en ocasiones marcados) se realiza una transfección, que consiste en la introducción de ADN o ARN de un virus o bacteriófago procariota en el interior celular. Después, se realiza una transfección química. Finalmente, se observan las células al microscopio de fluorescencia y si la transfección presenta una buena eficiencia. 24 horas más tarde, se realiza el tratamiento en medio completo, se fijan (placa de 24 o 96 pocillos) y se montan las lamelas (placa 24 pocillos) para su observación si se va a realizar la técnica de inmunofluorescencia o se lisan (placa 6 pocillos) si se va a realizar análisis de la sobreexpresión por Western blotting.For the introduction of tagged exogenous proteins (sometimes tagged bacterial plasmids), a transfection is performed, which involves the introduction of DNA or RNA from a prokaryotic virus or bacteriophage into the cell interior. This is followed by chemical transfection. Finally, the cells are observed under a fluorescence microscope to determine whether the transfection is efficient. 24 hours later, the cells are treated in complete medium, fixed (24- or 96-well plate) and lamellae are mounted (24-well plate) for observation if immunofluorescence is to be performed or lysed (6-well plate) if overexpression analysis by Western blotting is to be performed