Loss of KEAP1 causes an accumulation of nondegradative organelles

KEAP1 is a cytoplasmic protein that functions as an adaptor for the Cullin-3-based ubiquitin E3 ligase system, which regulates the degradation of many proteins, including NFE2L2/NRF2 and p62/SQSTM1. Loss of KEAP1 leads to an accumulation of protein ubiquitin aggregates and defective autophagy. To better understand the role of KEAP1 in the degradation machinery, we investigated whether Keap1 deficiency affects the endosome-lysosomal pathway. We used KEAP1-deficient mouse embryonic fibroblasts (MEFs) and combined Western blot analysis and fluorescence microscopy with fluorometric and pulse chase assays to analyze the levels of lysosomal-endosomal proteins, lysosomal function, and autophagy activity. We found that the loss of keap1 downregulated the protein levels and activity of the cathepsin D enzyme. Moreover, KEAP1 deficiency caused lysosomal alterations accompanied by an accumulation of autophagosomes. Our study demonstrates that KEAP1 deficiency increases nondegradative lysosomes and identifies a new role for KEAP1 in lysosomal function that may have therapeutic implications

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

Estudio del uso de metabolitos derivados de la microbiota intestinal (y sus precursores) como estrategia terapéutica en modelos de enfermedad de Parkinson

Programa de Doctorado de Salud Pública y AnimalLa enfermedad de Parkinson es la segunda enfermedad neurodegenerativa de mayor prevalencia a nivel mundial. En los últimos años, ha surgido un creciente interés en el uso de diferentes compuestos de origen natural con capacidad antiinflamatoria y antioxidante y que son capaces de restablecer estos procesos degradativos que están alterados, pudiendo ejercer un papel importante en el tratamiento de este tipo de patologías. En base a esto nos planteamos analizar el posible efecto protector que pueden tener diferentes compuestos bioactivos procedentes del cascabullo (un subproducto) de la bellota en modelos de la enfermedad de Parkinson, debido a las propiedades beneficiosas descritas para este fruto, por su elevado contenido en polifenoles. En este trabajo se ha comprobado la efectividad de estos extractos de inducir autofagia y mitofagia. así como aumentar la calidad mitocondrial, protegiendo frente al estrés de este organelo asociado a la exposición de pesticidas. Además se ha comprobado su eficacia frente a la protección de la muerte celular en presencia de mutaciones asociadas a la enfermedad de Parkinson así como a un aumento de la vida media y la mejora de la actividad locomotora en Un modelo de Drosophila melanogaster portador de estas mutaciones. Por tanto, aunque son necesarios estudios más exhaustivos para esclarecer los mecanismos a través de los cuales los extractos de este subproducto de la bellota ejerce su acción, podemos pensar en la posibilidad de su uso como diana terapéutica de este tipo de desórdenes.Parkinson's disease is the second most prevalent neurodegenerative disease worldwide. In recent years, there has been a growing interest in the use of different compounds of natural origin with anti-inflammatory and antioxidant capacity that are capable of restoring these degradation processes that are altered, which could play an important role in the treatment of this type of pathology. Based on this, we proposed to analyze the possible protective effect that different bioactive compounds from the acorn (a by-product) of the acorn may have in models of Parkinson's disease, due to the beneficial properties described for this fruit, due to its high polyphenol content. In this work, the effectiveness of these extracts to induce autophagy and mitophagy, as well as to increase the mitochondrial quality, protecting against the stress of this organelle associated to pesticide exposure, has been proved. In addition, its efficacy in protecting against cell death in the presence of mutations associated with Parkinson's disease as well as increasing half-life and improving locomotor activity in a Drosophila melanogaster model carrying these mutations has also been demonstrated. Therefore, although more exhaustive studies are needed to clarify the mechanisms through which the extracts of this acorn by-product exert their action, we can think of the possibility of its use as a therapeutic target for this type of disorders..-Proyecto PID2021-122576OB-I00 financiado por MCIN/ AEI /10.13039/501100011033. .- Proyecto financiado por la Consejería de Economía e Infraestructuras– Comunidad Autónoma de Extremadura. .- Programa para el Fomento de la Investigación Científica y Desarrollo Tecnológico del VI Plan Regional de I+D+i (2018-2021). Expediente: GR18063. .- Programa para el Fomento de la Investigación Científica y Desarrollo Tecnológico del VI Plan Regional de I+D+i (2021-2022). Expediente: GR21063.- Ayuda financiada por MCIN/AEI /10.13039/501100011033 y por FSE invierte en tu futuro

Citometría de flujo

La siembra celular se hace en placas de 6 pocillos (130184, Biolite) o de 24 pocillos (3524,Corning). A continuación, se aplican los tratamientos según corresponda en tiempo y concentración. El marcaje y análisis permiten recuperar las células con tripsina y se añaden junto a las que se encuentran en suspensión a tubos de citometría (1 por pocillo). Después, se centrifugan a 1.230 xg durante 5 min y se elimina el sobrenadante. Según el análisis a realizar, resuspender el pellet en diferentes tampones.Cell seeding is done in 6-well plates (130184, Biolite) or 24-well plates (3524, Corning). Treatments are then applied according to time and concentration. Cells are trypsin labelled and analysed and recovered and added together with the cells in suspension to cytometry tubes (1 per well). They are then centrifuged at 1230 xg for 5 min and the supernatant is removed. Depending on the analysis to be performed, resuspend the pellet in different buffers

Microscopia electrónica

Protocolo de procesamiento de muestras de cultivos celulares en microscopia electrónica. Se aplica a la investigación de enfermedades neurodegenerativas en un laboratorio de neurociencias.Protocol for processing cell culture samples in electron microscopy. Applied to the investigation of neurodegenerative diseases in a neuroscience laboratory

Toxicity of Necrostatin-1 in Parkinson’s Disease Models

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. This neuronal loss, inherent to age, is related to exposure to environmental toxins and/or a genetic predisposition. PD-induced cell death has been studied thoroughly, but its characterization remains elusive. To date, several types of cell death, including apoptosis, autophagy-induced cell death, and necrosis, have been implicated in PD progression. In this study, we evaluated necroptosis, which is a programmed type of necrosis, in primary fibroblasts from PD patients with and without the G2019S leucine-rich repeat kinase 2 (LRRK2) mutation and in rotenone-treated cells (SH-SY5Y and fibroblasts). The results showed that programmed necrosis was not activated in the cells of PD patients, but it was activated in cells exposed to rotenone. Necrostatin-1 (Nec-1), an inhibitor of the necroptosis pathway, prevented rotenone-induced necroptosis in PD models. However, Nec-1 affected mitochondrial morphology and failed to protect mitochondria against rotenone toxicity. Therefore, despite the inhibition of rotenone-mediated necroptosis, PD models were susceptible to the effects of both Nec-1 and rotenone

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

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

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

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