Not all mitochondrial DNAs are made equal and the nucleus knows it.

The oxidative phosphorylation (OXPHOS) system is the only structure in animal cells with components encoded by two genomes, maternally transmitted mitochondrial DNA (mtDNA), and biparentally transmitted nuclear DNA (nDNA). MtDNA-encoded genes have to physically assemble with their counterparts encoded in the nucleus to build together the functional respiratory complexes. Therefore, structural and functional matching requirements between the protein subunits of these molecular complexes are rigorous. The crosstalk between nDNA and mtDNA needs to overcome some challenges, as the nuclear-encoded factors have to be imported into the mitochondria in a correct quantity and match the high number of organelles and genomes per mitochondria that encode and synthesize their own components locally. The cell is able to sense the mito-nuclear match through changes in the activity of the OXPHOS system, modulation of the mitochondrial biogenesis, or reactive oxygen species production. This implies that a complex signaling cascade should optimize OXPHOS performance to the cellular-specific requirements, which will depend on cell type, environmental conditions, and life stage. Therefore, the mitochondria would function as a cellular metabolic information hub integrating critical information that would feedback the nucleus for it to respond accordingly. Here, we review the current understanding of the complex interaction between mtDNA and nDNA.Fundacion Alfonso Martin Escudero (Spain), Grant/Award Number: Postdoctoral Fellowship; Human Frontier Science Program, Grant/Award Number: RGP0016/2018; Ministerio de Ciencia e Innovacion, Grant/Award Numbers: RTI2018-099357-B-I00, SAF2015-65633-R; Ministerio de Economia y Competitividad, Grant/Award Number: RGP0016S

The portrait of liver cancer is shaped by mitochondrial genetics.

Cancer heterogeneity and evolution are not fully understood. Here, we show that mitochondrial DNA of the normal liver shapes tumor progression, histology, and immune environment prior to the acquisition of oncogenic mutation. Using conplastic mice, we show that mtDNA dictates the expression of the mitochondrial unfolded protein response (UPRmt) in the normal liver. Activation of oncogenic mutations in UPRmt-positive liver increases tumor incidence and histological heterogeneity. Further, in a subset of UPRmt-positive mice, invasive liver cancers develop. RNA sequencing (RNA-seq) analysis of the normal liver reveals that, in this subset, the PAPP-A/DDR2/SNAIL axis of invasion pre-exists along with elevated collagen. Since PAPP-A promotes immune evasion, we analyzed the immune signature and found that their livers are immunosuppressed. Further, the PAPP-A signature identifies the immune exhausted subset of hepatocellular carcinoma (HCC) in humans. Our data suggest that mtDNA of normal liver shapes the entire liver cancer portrait upon acquisition of oncogenic mutations.This work was supported by an RO1 AG059635 award from the NIH to D.G.S

Delayed alveolar clearance of nanoparticles through control of coating composition and interaction with lung surfactant protein A

The coating composition of nanomedicines is one of the main features in determining the medicines' fate, clearance, and immunoresponse in the body. To highlight the coatings' impact in pulmonary administration, two micellar superparamagnetic iron oxide nanoparticles (SPION) were compared. These nanoparticles are similar in size and charge but have different coatings: either phosphatidylcholine (PC-SPION) or bovine serum albumin (BSA-SPION). The aim of the study was to increase the understanding of the nano-bio interaction with the cellular and non-cellular components of the lung and underline valuable coatings either for local lung-targeted drug delivery in theranostic application or patient-friendly route systemic administration. PC-SPION and BSA-SPION were deposited in the alveoli by in vivo instillation and, despite the complexity of imaging the lung, SPION were macroscopically visualized by MRI. Impressively, PC-SPION were retained within the lungs for at least a week, while BSA-SPION were cleared more rapidly. The different lung residence times were confirmed by histological analysis and supported by a flow cytometry analysis of the SPION interactions with different myeloid cell populations. To further comprehend the way in which these nanoformulations interact with lung components at the molecular level, we used fluorescence spectroscopy, turbidity measurements, and dynamic light scattering to evaluate the interactions of the two SPION with surfactant protein A (SP-A), a key protein in setting up the nanoparticle behavior in the alveolar fluid. We found that SP-A induced aggregation of PC-SPION, but not BSA-SPION, which likely caused PC-SPION retention in the lung without inducing inflammation. In conclusion, the two SPION show different outcomes from interaction with SP-A leading to distinctive fate in the lung. PC-SPION hold great promise as imaging and theranostic agents when prolonged pulmonary drug delivery is required

mtDNA variability determines spontaneous joint aging damage in a conplastic mouse model.

Mitochondria and mtDNA variations contribute to specific aspects of the aging process. Here, we aimed to investigate the influence of mtDNA variation on joint damage in a model of aging using conplastic mice. A conplastic (BL/6NZB) mouse strain was developed with the C57BL/6JOlaHsd nuclear genome and NZB/OlaHsd mtDNA, for comparison with the original C57BL/6JOlaHsd strain (BL/6C57). Conplastic (BL/6NZB) and BL/6C57 mice were sacrificed at 25, 75, and 90 weeks of age. Hind knee joints were processed for histological analysis and joint pathology graded using the Mankin scoring system. By immunohistochemistry, cartilage expression of markers of autophagy (LC3, Beclin-1, and P62) and markers of senescence (MMP13, beta-Galactosidase, and p16) and proliferation (Ki67) were analyzed. We also measured the expression of 8-oxo-dG and cleaved caspase-3. Conplastic (BL/6NZB) mice presented lower Mankin scores at 25, 75, and 90 weeks of age, higher expression of LC3 and Beclin-1 and lower of P62 in cartilage than the original strain. Moreover, the downregulation of MMP13, beta-Galactosidase, and p16 was detected in cartilage from conplastic (BL/6NZB) mice, whereas higher Ki67 levels were detected in these mice. Finally, control BL/6C57 mice showed higher cartilage expression of 8-oxo-dG and cleaved caspase-3 than conplastic (BL/6NZB) mice. This study demonstrates that mtDNA genetic manipulation ameliorates joint aging damage in a conplastic mouse model, suggesting that mtDNA variability is a prognostic factor for aging-related osteoarthritis (OA) and that modulation of mitochondrial oxidative phosphorylation (OXPHOS) could be a novel therapeutic target for treating OA associated with aging.This work was supported by grants from Fondo de Investigación Sanitaria (PI16/02124, PI19/01206 and RETIC-RIER-RD16/0012/0002) integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016, and funded by the ISCIII-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe”, by Grant IN607A2021/07 from GAIN, Xunta de Galicia (F.J.B.) and by CIBERFES-ISCIII, MINECO: SAF2015-65633-R, RTI2018-099357-BI00, and HFSP (RGP0016/2018) to J.A.E.S

mtDNA variability determines spontaneous joint aging damage in a conplastic mouse model

[Abstract] Mitochondria and mtDNA variations contribute to specific aspects of the aging process. Here, we aimed to investigate the influence of mtDNA variation on joint damage in a model of aging using conplastic mice. A conplastic (BL/6NZB) mouse strain was developed with the C57BL/6JOlaHsd nuclear genome and NZB/OlaHsd mtDNA, for comparison with the original C57BL/6JOlaHsd strain (BL/6C57). Conplastic (BL/6NZB) and BL/6C57 mice were sacrificed at 25, 75, and 90 weeks of age. Hind knee joints were processed for histological analysis and joint pathology graded using the Mankin scoring system. By immunohistochemistry, cartilage expression of markers of autophagy (LC3, Beclin-1, and P62) and markers of senescence (MMP13, beta-Galactosidase, and p16) and proliferation (Ki67) were analyzed. We also measured the expression of 8-oxo-dG and cleaved caspase-3. Conplastic (BL/6NZB) mice presented lower Mankin scores at 25, 75, and 90 weeks of age, higher expression of LC3 and Beclin-1 and lower of P62 in cartilage than the original strain. Moreover, the downregulation of MMP13, beta-Galactosidase, and p16 was detected in cartilage from conplastic (BL/6NZB) mice, whereas higher Ki67 levels were detected in these mice. Finally, control BL/6C57 mice showed higher cartilage expression of 8-oxo-dG and cleaved caspase-3 than conplastic (BL/6NZB) mice. This study demonstrates that mtDNA genetic manipulation ameliorates joint aging damage in a conplastic mouse model, suggesting that mtDNA variability is a prognostic factor for aging-related osteoarthritis (OA) and that modulation of mitochondrial oxidative phosphorylation (OXPHOS) could be a novel therapeutic target for treating OA associated with aging.Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI19/01206Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/000

Regulation of Mother-to-Offspring Transmission of mtDNA Heteroplasmy

mtDNA is present in multiple copies in each cell derived from the expansions of those in the oocyte. Heteroplasmy, more than one mtDNA variant, may be generated by mutagenesis, paternal mtDNA leakage, and novel medical technologies aiming to prevent inheritance of mtDNA-linked diseases. Heteroplasmy phenotypic impact remains poorly understood. Mouse studies led to contradictory models of random drift or haplotype selection for mother-tooffspring transmission of mtDNA heteroplasmy. Here, we show that mtDNA heteroplasmy affects embryo metabolism, cell fitness, and induced pluripotent stem cell (iPSC) generation. Thus, genetic and pharmacological interventions affecting oxidative phosphorylation (OXPHOS) modify competition among mtDNA haplotypes during oocyte development and/or at early embryonic stages. We show that heteroplasmy behavior can fall on a spectrum from random drift to strong selection, depending on mito-nuclear interactions and metabolic factors. Understanding heteroplasmy dynamics and its mechanisms provide novel knowledge of a fundamental biological process and enhance our ability to mitigate risks in clinical applications affecting mtDNA transmission.Peer reviewe

Enhanced Immunogenicity of Mitochondrial-Localized Proteins in Cancer Cells.

Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8+ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.S

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

Mitochondrial Na+ controls oxidative phosphorylation and hypoxic redox signalling

All metazoans depend on O2 delivery and consumption by the mitochondrial oxidative phosphorylation (OXPHOS) system to produce energy. A decrease in O2 availability (hypoxia) leads to profound metabolic rewiring. In addition, OXPHOS uses O2 to produce reactive oxygen species (ROS) that can drive cell adaptations through redox signalling, but also trigger cell damage1–4, and both phenomena occur in hypoxia4–8. However, the precise mechanism by which acute hypoxia triggers mitochondrial ROS production is still unknown. Ca2+ is one of the best known examples of an ion acting as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential and collaborating in ion transport10. Here we show that Na+ acts as a second messenger regulating OXPHOS function and ROS production by modulating fluidity of the inner mitochondrial membrane (IMM). We found that a conformational shift in mitochondrial complex I during acute hypoxia11 drives the acidification of the matrix and solubilization of calcium phosphate precipitates. The concomitant increase in matrix free-Ca2+ activates the mitochondrial Na+/Ca2+ exchanger (NCLX), which imports Na+ into the matrix. Na+ interacts with phospholipids reducing IMM fluidity and mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III, generating a redox signal. Inhibition of mitochondrial Na+ import through NCLX is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ import into the mitochondrial matrix controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences in cellular metabolism

Mitochondrial OxPhos : the integrator of the cellular metabolic status

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-11-2018Esta tesis tiene embargado el acceso al texto completo hasta el 29-05-2020El sistema de fosforilación oxidativa (OXPHOS) es el único sistema en la célula animal cuyos componentes están codificados en dos genomas, el ADN mitocondrial (mtDNA) y el ADN nuclear (nDNA). El mtDNA es poliploide, de herencia materna y carece de un sistema de recombinación durante su replicación, cuyas consecuencias son una tasa de mutación un orden de magnitud mayor que el nDNA y el origen de diversos haplotipos mitocondriales. Las proteínas del sistema OXPHOS codificadas en el nDNA presentan opciones alternativas debido a las variantes alélicas y las variantes específicas de tejido. Es por ello que se pueden dar diversas combinaciones de nDNA y mtDNA en el mismo individuo. Esta asimetría podría provocar un desajuste entre componentes del sistema: las proteínas codificadas en el mtDNA tienen que interaccionar físicamente con las proteínas codificadas en el nDNA para formar los complejos repiratorios. Todas las copias de mtDNA de una misma célula son idénticas, situación llamada homoplasmia. La heteroplasmia es el término utilizado para referirnos a la coexistencia de más de un tipo de mtDNA en el mismo citoplasma. En la actualidad, aún quedan cuestiones por resolver: (i) ¿son intercambiables los distintos haplotipos de mtDNA sin provocar ningún impacto funcional o fenotípico en el individuo?; (ii) ¿presenta la generación artificial de heteroplasmia un conflicto genómico capaz de provocar riesgos para la salud de los individuos?. Durante el desarrollo de esta tesis doctoral se han estudiado (i) cómo las modificaciones en genes nucleares relacionados con la función mitocondrial impactan en la fisiología del individuo bajo la presencia de genomas mitocondriales alternativos, (ii) cómo la coexistencia de dos variantes de mtDNA en el mismo citoplasma celular afecta al metabolismo y (iii) la identificación de posibles mecanismos relacionados con esta regulación. Para responder estas preguntas se han utilizado modelos de ratones conplásticos (idéntico nDNA y dos variantes de mtDNA no patológicas intercambiables, C57 y NZB) y ratones heteteroplásmicos (coexistencia de ambas variantes de mtDNA en la misma célula). La caracterización de los modelos animales conplásticos se ha llevado a cabo desde el desarrollo embrionario hasta la muerte natural de los mismos a través de diversas metodologías ómicas, análisis bioquímicos y fisiológicos así como diversos estudios fenotípicos. Se ha observado que los distintos haplotipos de mtDNA influyen en la producción de especies reactivas de oxígeno y en la homeostasis celular. Nuestros recientes análisis confirman que: (1) las variantes de mtDNA inducen diferencias en el sistema OXPHOS bajo un mismo contexto nuclear; (2) hay un proceso de adaptación celular que modula el rendimiento OXPHOS para mantener la salud del individuo a pesar del desajuste entre nDNA/mtDNA; (3) la sensibilidad frente a ese desajuste entre genomas es dependiente del tipo celular y (4), las diferencias en el ajuste nDNA/mtDNA dan lugar a alteraciones metabólicas, manifestadas en el animal adulto y que impactan dramáticamente en el proceso de envejecimiento. En la naturaleza, la heteroplasmia es activamente combatida por diversos mecanismos, como por ejemplo la degradación del mtDNA paterno tras la fertilización del ovocito o la existencia de un cuello de botella genético en el desarrollo del mismo. La condición de heteroplasmia puede generarse de forma natural durante la replicación del mtDNA por mutagénesis, pero también puede ser originada por nuevas tecnologías médicas. Entre ellas, cabe destacar aquellas empleadas para (i) la prevención de la transmisión de enfermedades mitocondriales, (ii) la mejora de la fertilidad a través de un rejuvenecimiento de ovocitos humanos y (iii) la recuperación de la función mitocondrial en células dañadas mediante la transferencia de mitocondrias exógenas. Durante esta tesis doctoral se ha demostrado que la heteroplasmia puede modular la viabilidad y el metabolismo de las células embrionarias y del individuo adulto. Durante el período postnatal, la mayoría de los tejidos combaten la heteroplasmia eliminando y/o seleccionado una de las variantes de mtDNA, preservando de esta forma la función tisular. A pesar de ello, existen tejidos incapaces de eliminar la heteroplasmia durante la vida del individuo, como es el caso del corazón, pulmón o músculo esquelético. Estos tejidos están sometidos a un estrés metabólico progresivo seguido de un fenotipo patológico en el animal adulto que incluye el daño cardiopulmonar, la pérdida de músculo esquelético, fragilidad y una muerte temprana en el individu