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

Neuroprotective properties of queen bee acid by autophagy induction

Autophagy is a conserved intracellular catabolic pathway that removes cytoplasmic components to contribute to neuronal homeostasis. Accumulating evidence has increasingly shown that the induction of autophagy improves neuronal health and extends longevity in several animal models. Therefore, there is a great interest in the identification of effective autophagy enhancers with potential nutraceutical or pharmaceutical properties to ameliorate age-related diseases, such as neurodegenerative disorders, and/or promote longevity. Queen bee acid (QBA, 10-hydroxy-2-decenoic acid) is the major fatty acid component of, and is found exclusively in, royal jelly, which has beneficial properties for human health. It is reported that QBA has antitumor, anti-inflammatory, and antibacterial activities and promotes neurogenesis and neuronal health; however, the mechanism by which QBA exerts these effects has not been fully elucidated. The present study investigated the role of the autophagic process in the protective effect of QBA. We found that QBA is a novel autophagy inducer that triggers autophagy in various neuronal cell lines and mouse and fly models. The beclin-1 (BECN1) and mTOR pathways participate in the regulation of QBA-induced autophagy. Moreover, our results showed that QBA stimulates sirtuin 1 (SIRT1), which promotes autophagy by the deacetylation of critical ATG proteins. Finally, QBA-mediated autophagy promotes neuroprotection in Parkinson’s disease in vitro and in a mouse model and extends the lifespan of Drosophila melanogaster. This study provides detailed evidences showing that autophagy induction plays a critical role in the beneficial health effects of QBA.This research was supported by a grant (IB18048) from Junta de Extremadura, Spain, and a grant (RTI2018-099259-A-I00) from Ministerio de Ciencia e Innovación, Spain. This work was also partially supported by “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. Part of the equipment employed in this work has been funded by Generalitat Valeciana and co-financed with ERDF funds (OP EDRF of Comunitat Valenciana 2014-2020). G.M-C is supported by University of Extremadura (ONCE Foundation). M.P-B is a recipient of a fellowship from the “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (University of Extremadura).” S.M.S.Y-D is supported by CIBERNED. E.U-C was supported by an FPU predoctoral fellowship FPU16/00684 from Ministerio de Educación, Cultura y Deporte. A.B. was supported by a postdoctoral fellowship (APOSTD2017/077). M.S.A. was supported by a predoctoral fellowship (ACIF/2018/071) both from the Conselleria d’Educació, Investigació, Cultura i Esport (Generalitat Valenciana). E.A-C was supported by a grant (IB18048) from Junta de Extremadura, Spain. S.C-C was supported by an FPU predoctoral fellowship FPU19/04435 from Ministerio de Educación, Cultura y Deporte. J.M.B-S. P was funded by the “Ramón y Cajal” program (RYC-2018-025099). J.M.F. received research support from the Instituto de Salud Carlos III, CIBERNED (CB06/05/004). M.N-S was funded by the “Ramon y Cajal” Program (RYC-2016-20883) Spain

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

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Análisis Seahorse

Para medir el metabolismo celular a partir de análisis Seahorse, hay que resuspender las células antes de contarlas y sembrarlas, y determinar la concentración ideal de siembra. Si las células no son adherentes, se puede pre-tratar la placa con polilisina o colágeno. Preparar más volumen de medio + células para utilizar la pipeta multicanal y no utilizar volúmenes demasiado pequeños y así, evitar errores. Después de 2 horas, dejar con 80 μl de medio para evitar mover las células. Éstas no estarán pegadas y se pueden ir a los bordes. Si se hidrata con H2O miliQ hay que cambiarlo, al menos una hora antes de realizar el experimento. Si se hidrata directamente con el tampón de calibrado, no debe estar más de 24 horas. Si las células no estuvieran el día 2, se cambia el agua y se rehidrata con el tampón 1 hora antes del experimento. El volumen del tratamiento es de 100 μl/pocillo. Preparar el medio Seahorse adicionando glutamina 1X, glucosa 10 mM y sodio piruvato 1X. Antes de añadir ningún inhibidor, meter y sacar varias veces el cartucho del líquido de calibrado para eliminar cualquier burbuja que se haya podido generar. Añadir de izquierda a derecha apoyando la punta de la pipeta multicanal en la pared y de forma muy lenta para que resbale poco a poco. Luego, meter las placas en el aparato sin las tapas. Al finalizar el calibrado, el aparato pide la placa de las células y devuelve la placa con el líquido de calibrado. La placa de las células estará no menos de 45 minutos en la estufa. Comenzará con una etapa de equilibrado. Tras 13 minutos, ofrecerá las medidas basales y las inyecciones. Al terminar todo el proceso, cuya duración dependerá del kit utilizado y de las modificaciones hechas, pedirá que se finalice el programa y la máquina devolverá el cartucho y la placa de las células.To measure cell metabolism from Seahorse assays, resuspend the cells before counting and seeding, and determine the ideal seeding concentration. If the cells are not adherent, the plate can be pre-treated with poly-L-lysine or collagen. Prepare more volume of medium + cells to use the multichannel pipette and do not use too small volumes to avoid errors. After 2 hours, leave with 80 μl of medium to avoid moving the cells. The cells will not be stuck together and may go to the edges. If hydrated with H2O miliQ, change the medium at least one hour before the experiment. If hydrated directly with the calibration buffer, it should not be more than 24 hours. If the cells were not present on day 2, the water is changed and rehydrated with the buffer 1 hour before the experiment. The treatment volume is 100 μl/well. Prepare Seahorse medium by adding 1X glutamine, 10 mM glucose and 1X sodium pyruvate. Before adding any inhibitor, dip the cartridge in and out of the calibration liquid several times to remove any bubbles that may have been generated. Add from left to right with the tip of the multichannel pipette resting on the wall and very slowly so that it slides off little by little. Then place the plates in the apparatus without the lids. At the end of the calibration, the apparatus asks for the cell plate and returns the plate with the calibration liquid. The cell plate shall remain in the oven for at least 45 minutes. It will start with a balancing step. After 13 minutes, it will provide the basal measurements and injections. At the end of the whole process, the duration of which will depend on the kit used and the modifications made, it will ask for the programme to be terminated and the machine will return the cartridge and the cell plate

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

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

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