Oxidative phosphorylation selectively orchestrates tissue macrophage homeostasis

We are grateful to N.-G. Larsson, F. Sa´ nchez-Madrid, G. Sabio, R.D. Palmiter, E. Gottlieb, C.T.Moraes, and M.A. del Pozofor sharing essential reagents.We thank S. Iborra, his team, M. Sa´ nchez-A´ lvarez, I. Nikolic, and members of the D.S. laboratory for discussions and critical reading of the manuscript. We thank the staff at the CNIC technical units; foremost the animal, cellomics, histology, metabolomics, genomics,microscopy, and bioinformaticsfacilities; and the SIdI of the Universidad Auto´ noma de Madrid for technical support. This project was supported by the ‘‘la Caixa’’ Foundation (ID 100010434) Postdoctoral Junior Leader Fellowship code LCF/BQ/PR20/11770008 (S.K.W.); ‘‘la Caixa’’ Foundation (ID 100010434) INPhINIT Fellowship code LCF/BQ/IN17/11620074 (I.H.-M.); Spanish Ministry of Education FPU fellowship code FPU20/01418 (M.G.); Ministerio de Ciencia e Innovacio´ n (MCIN) PID2019-104233RB-100/AEI/10.13039/ 501100011033 (S.L.); and NIH grants P01AG049665-08, RO1A148190, and P01HL154998 (N.S.C.). The J.A.E. laboratory is supported by the CNIC and a grant by Ministerio de Ciencia, Innovacio´ n y Universidades (MCNU); Agencia Estatal de Investigacio´ n (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (RTI2018-099357-B-I00); the Biomedical Research Networking Center on Frailty and Healthy Ageing (CIBERFES-ISCiii-CB16/10/00289); and the HFSP agency (RGP0016/2018). Work in the D.S. laboratory is funded by the CNIC; by the European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator grant 725091; by Spanish Ministerio de Ciencia e Innovacio´ n PID2019-108157RB/AEI/ and CPP2021-008310/AEI/10.13039/ 501100011033; by Comunidad de Madrid (P2022/BMD-7333 INMUNOVARCM); and by ‘‘la Caixa’’ Foundation (LCF/PR/HR20/00075 and LCF/PR/HR22/ 00253). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011033).S

CD69 expression on regulatory T cells protects from immune damage after myocardial infarction.

Increasing evidences advocate for an important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the underlying molecular mechanisms remain elusive. In this study, a broad analysis of immune markers in 283 patients revealed a significant CD69 overexpression on Treg cells after MI. Our results in mice showed that CD69 expression on Treg cells increased survival after left-anterior-descending coronary artery (LAD)-ligation. Cd69-/- mice developed strong IL-17+ γδT cell responses after ischemia that increased myocardial inflammation and, consequently, worsened cardiac function. CD69+ Treg cells, by induction of AhR-dependent CD39 ectonucleotidase activity, induced apoptosis and decreased IL-17A production in γδT cells. Adoptive transfer of CD69+ Treg cells to Cd69-/- mice after LAD-ligation reduced IL-17+ γδT cell recruitment, thus increasing survival. Consistently, clinical data from two independent cohorts of patients indicated that increased CD69 expression in peripheral blood cells after acute MI was associated with a lower risk of re-hospitalization for heart failure (HF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex and traditional cardiac damage biomarkers. Our data highlight CD69 expression on Treg cells as a potential prognostic factor and a therapeutic option to prevent HF after MI.This study was supported by competitive grants from the Ministerio de Ciencia e Innovación (MCIN), through the Carlos III Institute of Health (ISCIII)-Fondo de Investigación Sanitaria (PI22/01759) to P.M.; RTI2018-094727-B-100 to J. M-G; Comunidad de Madrid grants S2017/BMD-3671-INFLAMUNE-CM to P.M. and FSM.; Fundació La Marató TV3 (20152330 31) to J.M-G and F.S-M.; Ministerio de Ciencia e Innovación (MCIN) RTI2018-099357-B-I00, and CIBERFES (CB16/10/00282), Human Frontier Science Program (grant RGP0016/2018), and Leducq Transatlantic Networks (17CVD04) to JAE. AC is supported by Marie Skłodowska- Curie grant (agreement No. 713673). R.B-D. is supported by Formación de Profesorado Universitario (FPU16/02780) program from the Spanish Ministry of Education, Culture and Sports. The CNIC is supported by the ISCIII, the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Nuevos conocimientos sobre las manifestaciones heterogéneas de la disfunción mitocondrial

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 19-04-2023Esta tesis tiene embargado el acceso al texto completo hasta el 19-10-2024Las mitocondrias son orgánulos que pueblan las células eucariotas. Los defectos genéticos en los componentes mitocondriales son la causa de un grupo de trastornos heterogéneos conocidos colectivamente como enfermedades mitocondriales. El estudio de estos pacientes ha ayudado a comprender cómo responden las células a la disfunción mitocondrial y ha contribuido en gran medida a nuestros conocimientos sobre este orgánulo. Varias líneas de evidencia indican que diferentes defectos mitocondriales activan una respuesta similar a nivel celular, sin embargo, sus consecuencias son todavía difíciles de predecir y no sabemos por qué se manifiestan de forma tan heterogénea. En consecuencia, las opciones terapéuticas son escasas para la mayoría de los pacientes. Definir las bases moleculares de la patogénesis de estos trastornos es, por tanto, un reto actual para la investigación biomédica. Aquí estudiamos los efectos de la disfunción mitocondrial mediante dos enfoques complementarios. En primer lugar, comparamos los efectos sobre el crecimiento celular y los complejos respiratorios de dos conocidos inhibidores del complejo I, la rotenona y el diphenyleniodonium (DPI). Encontramos que, a pesar de que ambos bloquean la respiración dependiente del complejo I y suprimen el crecimiento celular en galactosa, el DPI provoca una rápida degradación del complejo I y de los supercomplejos que lo contienen. Demostramos que el DPI reacciona irreversiblemente con los cofactores de flavina de la célula, FMN y FAD, desestabilizando el módulo N del complejo I y bloqueando su ensamblaje en su último paso. En consecuencia, las células cultivadas en medios deficientes en riboflavina o con knockout para el transportador de FAD de la mitocondria SLC25A32 muestran una disminución de los niveles de ensamblaje del complejo I y de la respiración dependiente del complejo I. En segundo lugar, estudiamos las consecuencias fenotípicas de las alteraciones en la oligomerización de la ATP sintasa in vivo eliminando dos subunidades del dominio FO del complejo V, Atp5mk y Atp5mj. Los ratones Atp5mk -/- presentan alteraciones en los niveles de glucosa en ayunas y disminución de la capacidad de ejercicio. Los ratones Atp5mj -/- desarrollan una neuromiopatía progresiva cuyas manifestaciones (ataxia, oftalmopatías y atrofia del cerebelo) se asemejan al síndrome clínico observado en algunos pacientes con mutaciones en los genes MT-ATP6 y MTATP8. En las mitocondrias Atp5mj -/- observamos marcadas anomalías en la ultraestructura y un menor consumo de oxígeno. A pesar de la pérdida del complejo V oligomérico, la actividad ATPasa no se ve afectada, lo que sugiere que el complejo V tiene funciones importantes más allá de la fosforilación del ADP. Además, aunque la actividad de Oma1 es elevada en los ratones Atp5mj - /-, la supresión de Oma1 en los ratones Atp5mj -/- no produce cambios fenotípicos relevantes. En conjunto, nuestros resultados revelan una compleja interacción entre las reservas de cofactores de flavina mitocondriales, el ensamblaje del complejo I, la oligomerización de la ATP sintasa y la morfología de las mitocondriasMitochondria are organelles that populate eukaryotic cells. Genetic defects in mitochondrial components are cause of a group of heterogeneous disorders known collectively as mitochondrial diseases. The study of these patients has helped our understanding on how cell respond to mitochondrial dysfunction and contributed greatly to our knowledge about this organelle. Several lines of evidence indicate that different mitochondrial defects activate a similar response at the cell level, however, its consequences are still hard to predict and we don’t know why it manifest in such an heterogenous way. As a result, therapeutic options are scarce for most patients. Defining the molecular basis of the pathogenesis of these disorders is therefore a current challenge for biomedical research. Here, we studied the effects of mitochondrial dysfunction by two complementary approaches. Firstly, we compare the effects on cell growth and respiratory complexes of two known complex I inhibitors, rotenone and diphenyleneiodonium (DPI). We find that, despite both block complex I-dependent respiration and suppressed cell growth in galactose, DPI causes a rapid degradation of complex I and complex Icontaining supercomplexes. We show that DPI reacts irreversibly with cell flavin cofactors FMN and FAD, destabilizing complex I N-module and halting complex I assembly at its very last step. Accordingly, cells grown in riboflavin deficient media or knockout for the mitochondria FAD carrier SLC25A32 show decrease levels of assembled complex I and complex I-dependent respiration. Secondly, we study the phenotypic consequences of alterations in ATP synthase oligomerization in vivo by knocking out two subunits of the FO domain of complex V, Atp5mk and Atp5mj. Atp5mk -/- mice present alteration in fasting glucose levels and decreased exercise capacity. Atp5mj -/- mice develop a progressive neuromyopathy whose manifestation (ataxia, ophthalmopathies and cerebellar atrophy) resemble the clinical syndrome observed in some patient with mutations in MT-ATP6 and MT-ATP8 genes. In Atp5mj -/- mitochondria we observe marked ultrastructure abnormalities and reduced oxygen consumption. Despite the loss of oligomeric complex V, ATPase activity is unaffected, suggesting that complex V has important functions beyond ADP phosphorylation. Moreover, even though Oma1 activity is elevated in Atp5mj -/- mice, deleting Oma1 in Atp5mj -/- mice does not result in relevant phenotypic changes. Collectively, our results reveal a complex interplay between mitochondrial flavin cofactor pools, complex I assembly, ATP synthase oligomerization and mitochondria morpholog

Regulation of respiratory complex I assembly by FMN cofactor targeting

Respiratory complex I plays a crucial role in the mitochondrial electron transport chain and shows promise as a therapeutic target for various human diseases. While most studies focus on inhibiting complex I at the Q-site, little is known about inhibitors targeting other sites within the complex. In this study, we demonstrate that diphenyleneiodonium (DPI), a N-site inhibitor, uniquely affects the stability of complex I by reacting with its flavin cofactor FMN. Treatment with DPI blocks the final stage of complex I assembly, leading to the complete and reversible degradation of complex I in different cellular models. Growing cells in medium lacking the FMN precursor riboflavin or knocking out the mitochondrial flavin carrier gene SLC25A32 results in a similar complex I degradation. Overall, our findings establish a direct connection between mitochondrial flavin homeostasis and complex I stability and assembly, paving the way for novel pharmacological strategies to regulate respiratory complex I

Mitochondrial dynamics maintain muscle stem cell regenerative competence throughout adult life by regulating metabolism and mitophagy

Skeletal muscle regeneration depends on the correct expansion of resident quiescent stem cells (satellite cells), a process that becomes less efficient with aging. Here, we show that mitochondrial dynamics are essential for the successful regenerative capacity of satellite cells. The loss of mitochondrial fission in satellite cells-due to aging or genetic impairment-deregulates the mitochondrial electron transport chain (ETC), leading to inefficient oxidative phosphorylation (OXPHOS) metabolism and mitophagy and increased oxidative stress. This state results in muscle regenerative failure, which is caused by the reduced proliferation and functional loss of satellite cells. Regenerative functions can be restored in fission-impaired or aged satellite cells by the re-establishment of mitochondrial dynamics (by activating fission or preventing fusion), OXPHOS, or mitophagy. Thus, mitochondrial shape and physical networking controls stem cell regenerative functions by regulating metabolism and proteostasis. As mitochondrial fission occurs less frequently in the satellite cells in older humans, our findings have implications for regeneration therapies in sarcopenia.Work in the PMC laboratory was supported by Spanish Ministerio de Ciencia e Innovación ( RTI2018-096068 to P.M.-C. and E.P), ERC - 2016-AdG-741966 , LaCaixa - HEALTH-HR17-00040 , MDA , UPGRADE-H2020-825825 , AFM-Telethon , DPP-Spain , Fundació La Marató TV3-80/19-202021 to P.M.-C; Fundació La Marató TV3-137/38-202033 to A.L.S.; partly supported by Milky Way Research Foundation (MWRF) to P.M.-C; Severo Ochoa Program for Centers of Excellence to CNIC ( SEV-2015-0505 ) and Maria de Maeztu Program for Units of Excellence to UPF ( MDM-2014-0370 ). Work in the JAE laboratory was supported by Ministerio de Ciencia e Innovacion ( RTI2018-099357-B-I00 , RED2018-102576-T ), Human Frontier Science Program HFSP ( RGP0016/2018 ), Centro de Investigación Biomédica en Red en Fragilidad y Envejecimento Saludable ( CIBERFES16/10/00282 ), and Leduq Foundation award ( REDOX-17CVD04 ). Work in JMV laboratory was supported by the Spanish Ministerio de Ciencia e Innovación ( RTI2018-100695-B-I00 ), Spanish Junta de Andalucía ( P18-RT-4264 , 1263735-R and BIO-276 ), the FEDER Funding Program from the European Union , and Universidad de Córdoba . The authors are indebted to the personnel from the Servicio Centralizado de Apoyo a la Investigación (SCAI; University of Córdoba) for technical support with the transmission electron microscope. Work in MS laboratory was funded by the Italian Assoc. for Cancer Research ( AIRC IG-D17388 and ID23257 ) and ASI (MARS-PRE, project DC-VUM-2017-006). X.H., S.C., I.R.-P, and A.C were supported by Severo Ochoa PFI , PI , FPI , and H2020 Marie Skłodowska-Curie Actions predoctoral fellowships, respectively. P.H.-A was supported by Juan de la Cierva-Incorporación fellowship