Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies

Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation.Fundacion Poco Frecuente (Almeria)Asociacion Espanola de Enfermos de Glucogenosis (AEEG)Asociacion Espanola de Enfermos de Pompe (AEEP

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38β, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.We thank S Bartlett and F Chanut for English editing. We are grateful to RJ Davis, A Padmanabhan, M Costa and C López-Otín for critical reading of the manuscript. We thank Dr. RJ Davis for the MKK3 and MKK6 KO animals and Dr. Erwin F Wagner for the p38α flox mice. We thank AC Silva (ana@anasilva illustrations.com) for help with figure editing and design. This work was funded by a CNIC Intramural Project Severo Ochoa (Expediente 12–2016 IGP) to GS and JJ and PID2019-104399RB-I00 funded by MCIN/AEI/10.13039/501100011033 to GS. BGT was a fellow of FPI Severo Ochoa CNIC Program (SVP-2013-067639) and is an American Heart Association Postdoctoral Fellow (18POST34080175). RRB is a fellow of the FPU Program (FPU17/03847). The following grants provided additional funding: GS is granted by funds from European Regional Development Fund (ERDF): EFSD/Lilly European Diabetes Research Programme Dr Sabio, Fundación AECC PROYE19047SABI and Comunidad de Madrid IMMUNOTHERCAN-CM B2017/BMD-3733; US National Heart, Lung, and Blood Institute (R01 Grant HL122352), Fondos FEDER, Madrid, Spain, and Fundación Bancaria “La Caixa (project HR19/52160013); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); and Instituto de Salud Carlos III to JJ. IN was funded by EFSD/Lilly grants (2017 and 2019), the CNIC IPP FP7 Marie Curie Programme (PCOFUND-2012–600396), EFSD Rising Star award (2019), JDC-2018-Incorporación (MIN/JDC1802). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC FoundationS

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.G.S. is a YIP EMBO member. B.G.T. was a fellow of the FPI Severo Ochoa CNIC program (SVP-2013-067639) and currently is funded by the AHA-CHF (AHA award number: 818798). V.M.R. is a FPI fellow (BES-2014-069332) and A.M.S. is a fellow of the FPI Severo Ochoa CNIC program (BES-2016-077635). This work was funded by the following grants: to G.S.: funding from the EFSD/Lilly European Diabetes Research Programme Dr Sabio, from Spanish Ministry of Science, Innovation and Universities (MINECO-FEDER SAF2016-79126-R and PID2019-104399RB-I00), Comunidad de Madrid (IMMUNOTHERCAN-CM S2010/BMD-2326 and B2017/BMD-3733) and Fundación Jesús Serra; to P.A.: Ayudas para apoyar grupos de investigación del sistema Universitario Vasco (IT971-16 to P.A.), MCIU/AEI/FEDER, funding from Spanish Ministry of Science, Innovation and Universities (RTI2018-095134-B-100); Excellence Network Grant from MICIU/AEI (SAF2016-81975-REDT and 2018-PN188) to PA and GS; to J.V.: funding from Spanish Ministry of Science, Innovation and Universities (PGC2018-097019-B-I00), the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria grant PRB3 (PT17/0019/0003- ISCIII-SGEFI / ERDF, ProteoRed), and “la Caixa” Banking Foundation (project code HR17-00247); to J.P.B.: funding from Spanish Ministry of Science, Innovation and Universities (PID2019-105699RB-I00, RED2018‐102576‐T) and Escalera de Excelencia (CLU-2017-03); to J.A.E.: funding from Spanish Ministry of Science, Innovation and Universities MINECO (RED2018-102576-T, RTI2018-099357-B-I00), CIBERFES (CB16/10/00282), and HFSP (RGP0016/2018). RAP (XPC/BBV1602 and MIN/RYC1102). The CNIC is supported by the Ministry of Science, Innovation and Universities and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

MKK6 controls T3-mediated browning of white adipose tissue

El aumento de la capacidad termogénica del tejido adiposo para mejorar el gasto de energía del organismo se considera una estrategia terapéutica prometedora para combatir la obesidad. Aquí nosotros informe que la expresión del activador MAPK p38 MKK6 está elevada en el tejido adiposo blanco de individuos obesos. Usando animales knockout y shRNA, mostramos que la eliminación de Mkk6 aumenta el gasto de energía y la capacidad termogénica del tejido adiposo blanco, protegiendo a los ratones contra la obesidad inducida por la dieta y el desarrollo de la diabetes. La eliminación de Mkk6 aumenta la expresión de UCP1 estimulada por T3 en los adipocitos, lo que aumenta su capacidad termogénica. De manera mecánica, demostramos que, en el tejido adiposo blanco, p38 se activa mediante una ruta alternativa que involucra AMPK, TAK y TAB. Nuestros resultados identifican MKK6 en los adipocitos como un posible objetivo terapéutico para reducir la obesidad.Increasing the thermogenic capacity of adipose tissue to enhance organismal energy expenditure is considered a promising therapeutic strategy to combat obesity. Here, we report that expression of the p38 MAPK activator MKK6 is elevated in white adipose tissue of obese individuals. Using knockout animals and shRNA, we show that Mkk6 deletion increases energy expenditure and thermogenic capacity of white adipose tissue, protecting mice against diet-induced obesity and the development of diabetes. Deletion of Mkk6 increases T3-stimulated UCP1 expression in adipocytes, thereby increasing their thermogenic capacity. Mechanistically, we demonstrate that, in white adipose tissue, p38 is activated by an alternative pathway involving AMPK, TAK, and TAB. Our results identify MKK6 in adipocytes as a potential therapeutic target to reduce obesity.• Guadalupe Sabio Buzo y Rebeca Acin Pérez pertenecen a Programa Ramón y Cajal • Elisa Manieri pertenece a Caixa • Ministerio de Economía y Competitividad. Proyecto FPI BES-2014-069332, para Valle Montalvo Romeral • Ministerio de Economía y Competitividad. Proyecto FPI BES-2011-043428, para Edgar Bernardo • Ministerio de Economía y Competitividad y FEDER SAF2016-79126-R y Comunidad de Madrid S2010 / BMD-2326, para Guadalupe Sabio Buzo • ISCIII y FEDER, PI10 / 01692 e I3SNS-INT12 / 049, para Miguel Marcos Martín • Junta de Castilla y León GRS 681 / A / 11, para Lourdes Hernández Cosido • Ministerio de Economía y Competitividad. BFU2015-70664-R, Xunta de Galicia 2015-CP080 y PIE13 / 00024, y ERC281408, para Rubén Nogueiras Pozo • Unión Europea. Becas europeas UE0 / MCA1108 y UE0 / MCA1201; y la Comunidad de Madrid CAM / API1009, para Rubén Nogueiras Pozo • Junta de Extremadura y FEDER BR15164, para Francisco Centeno Velázquez • Ministerio de Economía y Competitividad. . BFU2013-46109-R, para Clara V. Álvarez Villamarín • European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. ERC 260464peerReviewe

p38γ is essential for cell cycle progression and liver tumorigenesis

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)–cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease

Methionine adenosyltransferase 1a antisense oligonucleotides activate the liver-brown adipose tissue axis preventing obesity and associated hepatosteatosis

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis. High methionine and S-adenosylmethionine serum levels are related with obesity. Here the authors show that knockdown of methionine adenosyltransferase by using antisense oligonucleotides provides beneficial effects in obesity and comorbidities.This work was supported by Ayudas para apoyar grupos de investigacion del sistema Universitario Vasco (IT971-16) and MCIU/AEI/FEDER, UE (RTI2018-095134-B-100) (to P.A.), (RTI2018-099413-B-I00 and RED2018-102379-T) (to R.N.), PID2020119486RB-100 (to M.V.R.) and (RTI2018-096759-A-100) (to T.C.D). EFSD/Lilly European Diabetes Research Program, MICIU (PID2019-104399RB-I00), Fundacion AECC PROYE19047SABI, and Comunidad de Madrid IMMUNOTHERCAN-CM B2017/BMD-3733 (to G.S.). La CAIXA Foundation LCF/PR/HP17/52190004, MINECO-FEDER SAF2017-87301-R, AYUDAS FUNDACION BBVA A EQUIPOS DE INVESTIGACION CIENTIFICA UMBRELLA 2018 and AECC Scientific Foundation, grant name: Rare Cancers 2017 (to M.L.M.-C.). AECC Scientific Foundation (to T.C.D.). Xunta de Galicia 2020-PG015 (to R.N.) Gilead Sciences International Research Scholars Program in Liver Disease (to M.V.R.). Personal fellows: E.P.F. was awarded with Juan de la Cierva-Formacion, FJC2018-035449-I. C.F. was awarded with Sara Borrell (CD19/00078). CIC bioGUNE thanks MCIU for the Severo Ochoa Excellence Accreditation (SEV-2016-0644). The authors thank Dr. Manuel Lafitas laboratory (Getxo, Bizkaia, Spain) for his valuable help in the analysis of biochemical parameters

Role of hepatic p38δ MAPK in liver metabolism

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-06-2019Esta tesis tiene embargado el acceso al texto completo hasta el 14-12-2020Hepatic metabolism is a complex regulatory network which controls whole body homeostasis. Therefore, it is crucial to deeply understand its function, to better comprehend human physiology and to find novel targets and effective clinical approaches to treat metabolic diseases that are reaching pandemic proportions such as type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Contrary to the role of c-Jun NH2-terminal kinases (JNK) in hepatocytes, the role of the p38 mitogen-activated protein kinases (MAPK), other stress kinases, and among them, the role of the p38δ MAPK isoform in hepatocytes is unknown. Interestingly, since the expression of this kinase is increased in livers from obese patients with NAFLD, p38δ might play a main function in liver metabolism. Therefore, the main purpose of this thesis was to clarify the function of the hepatic p38δ MAPK in liver metabolism, its repercussion in whole body homeostasis and its function in obesity, to open new avenues for this kinase as a putative target in obesity-related T2D and NAFLD. We demonstrated that p38δ MAPK is a key regulator of glucose metabolism and it is essential to keep normoglycemia. Particularly, p38δ MAPK controls glycogen metabolism and glycolysis, through phosphorylation of glycogen synthase 2 (GYS2) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) respectively. We showed that mice lacking p38δ in hepatocytes presented decreased glycogenesis, hepatic glycogen storage and consequently, reduced blood glucose levels. This protected against hyperglycemia in high fat diet (HFD)-fed mice and promoted mild hypoglycemia in chow diet (CD)-fed mice. Moreover, HFD-fed mice lacking p38δ in hepatocytes showed reduced glycolysis, which may impair the hepatic de novo lipogenesis (DNL), the subsequent intrahepatic lipid accumulation and therefore, protect against NAFLD and insulin resistance development. Additionally, p38δ MAPK may also control lipid metabolism, regulating PPARα pathway and FGF21 plasma levels. Moreover, it might have an important role controlling different lipid oxidation pathways. Lastly, p38δ might also have a main function in the hepatic metabolism of long-chain acyl CoA (LCCoA) and diacylglycerides (DAG). Summarizing, this Thesis work firstly defines the function of the hepatic p38δ MAPK, identifying substrates for this kinase; giving therefore new insights into the complex stress kinases network. Secondly, this thesis reveals that p38δ MAPK is a central metabolic regulator, mainly controlling glucose but also lipid metabolism. Lastly, p38δ MAPK might be a target to treat T2D and NAFLD, because not only the lack of p38δ MAPK in hepatocytes, but also the downregulation of the hepatic p38δ in obese mice; protect against hyperglycemia, insulin resistance and NAFLD. SummaryEl metabolismo hepático es una compleja red reguladora que controla la homeostasis de todo el organismo. Es por tanto crucial entender completamente su función, para comprender mejor los mecanismos que gobiernan la fisiología humana y así encontrar nuevas dianas y tratamientos efectivos para complejas enfermedades metabólicas, como la diabetes tipo 2 y la enfermedad del hígado graso no alcohólico (EHGNA), que están alcanzando proporciones pandémicas en la actualidad. Mientras que el papel de las quinasas de estrés c-Jun NH2-terminal (JNK) en hepatocitos ha sido más estudiado, poco se sabe de la función de las quinasas de estrés p38 activadas por mitógenos (MAPK), particularmente el papel de la p38δ MAPK en hepatocitos es desconocido. Sin embargo, ya que la expresión de esta quinasa está aumentada en los hígados de pacientes obesos con EHGNA; p38δ MAPK debe desempeñar una función principal en el metabolismo hepático. Por lo tanto, el objetivo principal de esta tesis fue entender la función de p38δ MAPK en hepatocitos y en el metabolismo hepático; así como su repercusión en la homeostasis global del organismo. También se estudió su función en obesidad, abriendo así la posibilidad de poder ser estudiada como una diana para tratar la diabetes tipo 2 y el EHGNA asociados a la obesidad. En esta tesis demostramos que p38δ MAPK es un regulador clave del metabolismo de la glucosa, siendo esencial para mantener la normoglucemia. Particularmente, p38δ MAPK controla el metabolismo del glucógeno y la glucólisis hepática, mediante la regulación por fosforilación de la proteína glucógeno sintasa 2 (GYS2) y de la 6-fosfofructo-2-quinasa/fructosa-2,6-bifosfatasa 3 (PFKFB3). Hemos visto que ratones que carecen de p38δ MAPK en hepatocitos, tienen reducida la glucogénesis, el contenido de glucógeno hepático y consecuentemente los niveles de glucosa en sangre. Esto protege frente a hiperglucemia cuando los ratones son alimentados con una dieta alta en grasa y colesterol, pero produce hipoglucemia suave cuando tienen una dieta normal. Además, ratones sin p38δ MAPK en hepatocitos alimentados con dieta alta en grasa y colesterol tienen reducida la glucólisis, reduciendo así la síntesis de novo y la acumulación hepática de lípidos, estando por tanto protegidos frente al desarrollo de EHGNA y de resistencia a insulina. Adicionalmente, p38δ MAPK podría tener un papel importante en el metabolismo hepático lipídico, regulando la vía de PPARα y los niveles de FGF21 en plasma. Además, p38δ MAPK debe controlar el uso de diferentes vías de oxidación lipídica. Por último, nuestros datos indican que p38δ MAPK parece tener un papel en el metabolismo hepático de los acil-coenzima A (acil-CoA) de cadena larga y de los diacilglicéridos. Resumiendo, esta Tesis en primer lugar define la función de p38δ MAPK en hepatocitos, identificando nuevos substratos para esta quinasa; ampliando así el conocimiento sobre la compleja red de las quinasas de estrés. En segundo lugar, esta Tesis revela que p38δ MAPK es un regulador metabólico central, controlando principalmente el metabolismo glucídico, pero teniendo también una función en el metabolismo lipídico. Por último, hemos visto que p38δ MAPK puede ser una diana para tratar la diabetes tipo 2 y el EHGNA asociados a la obesidad

Role of Hepatic p38delta MAPK in Liver Metabolism

Hepatic metabolism is a complex regulatory network which controls whole body homeostasis. Therefore, it is crucial to deeply understand its function, to better comprehend human physiology and to uncover novel targets and effective clinical approaches to treat metabolic diseases that are reaching pandemic proportions such as type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Contrary to the role of c-Jun NH2-terminal kinases (JNK) in hepatocytes, the role of the p38 mitogen-activated protein kinases (MAPK), other stress kinases, and particularly, the role of the p38δ MAPK isoform in hepatocytes is unknown. Interestingly, the expression of p38δ MAPK is increased in livers from obese patients with NAFLD, suggesting that this kinase might play an important function in liver metabolism. Therefore, the main purpose of this thesis was to clarify the function of the hepatic p38δ MAPK in liver metabolism, its repercussion in whole body homeostasis and its function in obesity, to open new avenues for this kinase as a putative target in obesityrelated T2D and NAFLD. We demonstrated that p38δ is a key regulator of glucose metabolism and it is essential to keep normoglycemia. Particularly, we found that p38δ controls glycogen metabolism and glycolysis, through phosphorylation of glycogen synthase 2 (GYS2) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) respectively. We showed that mice lacking p38δ in hepatocytes (Albp38δKO) presented decreased glycogenesis, hepatic glycogen storage and consequently, reduced blood glucose levels. This protected against high fat diet (HFD)induced hyperglycemia and promoted mild hypoglycemia in chow diet (CD)-fed mice. Moreover, HFD-fed Albp38δKO mice showed reduced glycolysis, which may impair the hepatic de novo lipogenesis (DNL), the subsequent intrahepatic lipid accumulation and therefore, protect against NAFLD and insulin resistance development. Additionally, p38δ might control lipid metabolism, regulating peroxisome-proliferator-activated receptor α (PPARα) pathway and hepatokine fibroblast growth factor 21 (FGF21) plasma levels. Moreover, p38δ might regulate different lipid oxidation pathways. Lastly, p38δ might also have a main function in the hepatic metabolism of long-chain acyl CoAs (LCCoA) and diacylglycerides (DAG). Summarizing, this Thesis work firstly defines the function of the hepatic p38δ MAPK, identifying substrates for this kinase; giving therefore new insights into the complex stress kinases network. Secondly, this thesis reveals that p38δ MAPK is a central metabolic regulator, mainly controlling glucose but also lipid metabolism. Lastly, p38δ MAPK might be a target to treat T2D and NAFLD, because the lack of p38δ MAPK in hepatocytes protects against hyperglycemia, insulin resistance and NAFLD.The support received from the following Grants and Fellowships has permitted to develop this PhD work: -Programa de ayudas predoctorales FPI, del Ministerio de Economía, Industria y Competitividad del 2014 (BES-2014-069332). -Quinasas del estrés en el cáncer y las enfermedades metabólicas. Principal Investigador: Guadalupe Sabio. SAF2013-43506-R. Ministerio de Economía, Industria y Competitividad 2014-2016 -Programa de ayudas a la movilidad predoctoral para la realización de estancias breves en centros de I+D 2017 (EEBB-I-18-12998). -The company of Biologist. Travelling fellowship grant (JCSTF-170505

A functional mini-GDE transgene corrects impairment in models of glycogen storage disease type III

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