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[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; PI16/02124 Instituto de Salud Carlos III; PI19/01206 Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002

Supplementary File S1

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.

Supplementary Figures 1-8 and Tables 1-2

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1,2,3,4, a phenomenon that occurs in hypoxia4,5,6,7,8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism., This research has been financed by Spanish Government grants (ISCIII and AEI agencies, partially funded by the European Union FEDER/ERDF) CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 programme to A.M.-R. and J.A.E.); CP07/00143, PS09/00101, PI12/00875, PI15/00107 and RTI2018-094203-B-I00 (to A.M.-R.); CP12/03304 and PI15/01100 (to L.M.); CP14/00008, CPII19/00005 and PI16/00735 (to J.E.); SAF2016-77222-R (to A. Cogolludo); PI17/01286 (to P.N.); SAF2015-65633-R, RTI2018-099357-B-I00 and CB16/10/00282 (to J.A.E.); RTI2018-095793-B-I00 (to M.G.L.); and SAF2017-84494-2-R (to J.R.-C.), by the European Union (ITN GA317433 to J.A.E. and MC-CIG GA304217 to R.A.-P.), by grants from the Comunidad de Madrid B2017/BMD-3727 (to A. Cogolludo) and B2017/BMD-3827 (to M.G.L.), by a grant from the Fundación Domingo Martínez (to M.G.L. and A.M.-R.), by the Human Frontier Science Program grant HFSP-RGP0016/2018 (to J.A.E.), by grants from the Fundación BBVA (to R.A.-P. and J.R.-C.), by the UCM-Banco Santander grant PR75/18-21561 (to A.M.-d.-P.), by the Programa Red Guipuzcoana de Ciencia, Tecnología e Información 2018-CIEN-000058-01 (to J.R.-C.) and from the Basque Government under the ELKARTEK Program (grant no. KK-2019/bmG19 to J.R.-C.), by the Swiss National Science Foundation (SNF) grant 310030_124970/1 (to A.B.), by a travel grant from the IIS-IP (to P.H.-A.) and by the COST actions TD0901 (HypoxiaNet) and BM1203 (EU-ROS). The CNIC is supported by the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (Spanish Government award SEV-2015-0505). CIC biomaGUNE is supported by the María de Maeztu Units of Excellence Program from the Spanish Government (MDM-2017-0720). P.H.-A. was a recipient of a predoctoral FPU fellowship from the Spanish Government. E.N. is a recipient of a predoctoral FPI fellowship from the Universidad Autónoma de Madrid (UAM). A.M.-R., L.M. and J.E. are supported by the I3SNS or ‘Miguel Servet’ programmes (ISCIII, Spanish Government; partially funded by the FEDER/ERDF).

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.

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 NP 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.

[Abstract] It has been suggested that mitochondrial dysfunction and mtDNA variations may contribute to osteoarthritis (OA) pathogenesis. However, the causative link to support this claim is lacking. Here, we surgically-induced OA in conplastic mice in order to evaluate the functional consequences of mtDNA haplotypes in their joint degeneration. BL/6NZB strain was developed with C57BL/6JOlaHsd nuclear genome and NZB/OlaHsdmtDNA while BL/6C57, which is the original, was developed with C57BL/6JOlaHsd nuclear genome and C57/OlaHsdmtDNA for comparison. The surgical DMM OA model was induced in both strains. Their knees were processed and examined for histopathological changes. Cartilage expression of markers of autophagy, apoptosis, oxidative stress and senescence were also analyzed by immunohistochemistry. The joints of BL/6NZB mice that were operated presented more cellularity together with a reduced OARSI histopathology score, subchondral bone, menisci score and synovitis compared to those of BL/6C57 mice. This was accompanied with higher autophagy and a lower apoptosis in the cartilage of BL/6NZB mice that were operated. Therefore, the study demonstrates the functional impact of non-pathological variants of mtDNA on OA process using a surgically-induced OA model. Conplastic (BL/6NZB ) mice develop less severe OA compared to the BL/6C57original strain. These findings demonstrate that mitochondria and mtDNA are critical targets for potential novel therapeutic approaches to treat osteoarthritis. This work is supported by grants from Fondo de Investigación Sanitaria (CIBERCB06/01/0040-Spain, RETIC-RIER-RD16/0012/0002, PI16/02124, PI19/01206) integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Sub direction of Assessment and Promotion of Research-European Regional Development Fund (FEDER)“A way of making Europe”. MS is “Sara Borrell” researcher funded by ISCIII and FEDER (CD16/00099). IRP is funded by the Instituto de Salud Carlos III through a Miguel Servet-II programme (CPII17/00026). CVG is supported by Contrato Posdoctoral Xunta de Galicia (ED481D2017/023).

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: RGP0016 Sí

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. Sí

Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. Video Abstract: [Figure presented] A system of macrophages in the heart supports cardiomyocyte health by phagocytosing exopher particles ejected from cardiomyocytes that contain defective mitochondria, among other cellular contents., This study was supported by Intramural grants from the Severo Ochoa program (IGP-SO); grants SAF2015-71878-REDT and SAF2014-56819-R from the Ministerio de Ciencia e Innovacion (MICINN) to A.C.; European Research Council grant EU-rhythmy (ERC-ADG-2014-ID:669387) to S.G.P., and MATRIX (ERC-COG-2018-ID: 819775) to B.I.; L.G.N. is supported by SIgN core funding from A∗STAR; grant BFU2016-75144-R from the Ministry of Science and Innovation to J.A.B,; grants PGC2018-096486-B-I00 and RD16/0011/0019 (ISCIII) from MICINN, TNE-17CVD04 from the Leducq Foundation, and S2017/BMD-3875 from the Comunidad de Madrid to M.T; intramural grant TPC/O-SO and grants SAF2015-65633-R, RTI2018-099357-B-I00, and HFSP (RGP0016/2018) to J.A.E.; intramural grant IGP-SO to J.A.-C. and A.H.; BIO2017-83640-P and RYC-2014-16604 to J.A-C; grants PRB3 (IPT17/0019-ISCIII-SGEFI/ERDF, ProteoRed) from the Carlos III Institute of Health and Fondo de Investigaciones Sanitarias, BIO2015-67580-P and PGC2018-097019-B-I00 from MICINN to J.V.; RTI2018-096068 from MICINN, AFM, MDA, LaCaixa-HR17-00040, UPGRADE-H2020-825825, and European Research Council (ERC-741538) to P.M.C.; S2017/BMD-3867 RENIM-CM from the Comunidad de Madrid and cofunded with European structural and investment funds to M.D.; 120/C/2015-20153032 from Fundació la Marató de TV3, SAF2015-65607-R and RTI2018-095497-B-I00 from MICINN, HR17_00527 from La Caixa Foundation, and TNE-18CVD04 from the Leducq Foundation to A.H.; C.V.R. is a Howard Hughes Medical Institute Faculty Scholar; J.A.N-A is supported by fellowship SVP-2014-068595, A.V.L.-V. by SVP-2013-068089, L.E.-M. by FJCI-2016-29384, and A.R.-P. by BES-2016-076635, all from MICINN; and the CNIC International Postdoctoral Program (EU grant agreement 600396 to D.J.S.). The CNIC is supported by the MICINN and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MICINN award SEV-2015-0505).

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-to-offspring 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. A.V.L.-V. was supported by fellowship SVP-2013-068089 from MCIU. I.G.J. thanks ERC StG EvoConBiO, and a Turing fellowship from the Alan Turing Institute. N.J. thanks EP/N014529/1. SAF2017-84494-C2-R and Programa Red Guipuzcoana de Ciencia, Tecnologıa e Informacion 2018-CIEN-000058-01, and the Basque Government under its ELKARTEK research program (ref: KK-2019/00015) to J.R.-C. The work at CIC biomaGUNE was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720. This study was supported by grants from the MCNU (SAF2015-65633-R), the EU (UE0/MCA317433), the Biomedical ResearchNetworking Center on Frailty and Healthy Ageing (CIBERFES-ISCiii), and the HFSP agency (RGP0016/2018) to J.A.E. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacion y Universidades (MCNU), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). AGRADECIENTOS: ProCNIC; Severo Ochoa (SEV-2015-0505) Sí

Iron oxide nanomaterial is a typical example of a magnetic resonance imaging probe for negative contrast. It has also been shown how this nanomaterial can be synthesized for positive contrast by modification of the composition and size of the core. However, the role of the organic coating in the relaxometric properties is largely unexplored. Here, maghemite nanoparticles with either excellent positive or very good negative contrast performance are obtained by modifying coating thickness while the core is kept unchanged. Different nanoparticles with tailored features as contrast agent according to the coating layer thickness have been obtained in a single-step microwave-driven synthesis by heating at different temperatures. A comprehensive analysis is conducted of how the composition and structure of the coating affects the final magnetic, relaxometric, and imaging performance. These results show how the organic coating plays a fundamental role in the intrinsic relaxometric parameters of iron oxide-based contrast media., This study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (grant nos. MAT2013-47303-P, MAT2014-52069-R, SAF2016-79593-P). L.G. received financial support from the Ramón y Cajal subprogram (RYC-2014-15512). The CNIC is supported by the MEyC and the Pro-CNIC Foundation and is a SO-Center of Excellence (SEV-2015-0505).

Background Several studies indicated that osteoarthritis has a strong genetic component with a prevalent role of mitochondria and mtDNA variations (1). Objectives In the present study, we aimed to investigated the influence of the mtDNA variation in the joint deterioration using mice with the same nuclear genome but different mtDNA variants (named conplastic mice) (2) during aging and forced exercise. Methods Conplastic mice (BL/6NZB) strain was developed with the C57BL/6JOlaHsd nuclear genome and the NZB/OlaHsd mtDNA to compare with the original C57BL/6JOlaHsd strain (BL/6C57). Knee joints from BL/6NZB mice as well as from BL/6C57 mice were processed and cut into coronal sections. The mice were sacrificed at 25, 75 and 90 weeks of age and knee joints were collected for histological analysis. All sections were stained with Hematoxylin-Eosine and Safranin O-fast green and graded using a Mankin scoring system. Another group of mice from both BL/6NZB and BL/6C57 strains were subjected to exercise by running in a treadmill 400m/day three times a week. After 75 and 90 weeks of age, mice were sacrificed and knee joints were processed for histological analysis. Cartilage expression of markers of autophagy like LC3 and metalloproteinases like MMP-13 were also analysed by immunohistochemistry in both strains. The results are given as mean ± SEM and statistical analysis was performed using non parametric unpaired t-test (Graph Pad Prism v 6.0). Results In response to aging, conplastic mice BL/6NZB presented reduced cartilage Mankin score at 25 (p=0.0079), 75 (p=0.0087) and 90 (p=0.064) weeks when compared with mice of the original strain BL/6C57 at the same age. Specifically, we showed a reduced score in both femoral condyle (FC) and tibial plateau (TP) of BL/6NZB mice that reached the statistical significance at 25 (FC: p=0.0317; TP: p=0.0079), 75 (FC: p=0.0411; TP: p=0.0238) and borderline the statistical significance at 90 (FC: p=0.0649; TP: p=0.0628) weeks of age. These results were accompanied with more expression of LC3 in cartilage from BL/6NZB mice at 75 weeks when compared with cartilage from BL/6C57 at the same age (p=0.0152). We also reported a significant decrease of LC3 expression in cartilage from mice at 75 weeks when compared with mice at 25 weeks in both strains confirming the decrease of autophagy with aging. Difference in MMP13 cartilage expression between the two mice strains were also found. In the mice subjected to exercise, BL/6C57 presented an increased cartilage score in the medial compartment (p=0.0286) and lateral compartment (p=0.057) of the joint at 90 weeks when compared with BL/6NZB mice at the same age. Conclusion This study demonstrated that aging and forced exercise in conplastic mice BL/6NZB are associated with a reduced joint deterioration compared with the original strain BL/6C57. Moreover, we showed that mtDNA variants can improve the aging process at joint level through the modulation of autophagy. These results support the hypothesis that mtDNA background has a role in the process of joint damage, suggesting that mtDNA has potential as novel therapeutic target in OA associated to aging. References [1] Mitochondrial DNA variation and the pathogenesis of osteoarthritis phenotypes. Nat Rev Rheumatol. 2018Jun;14(6):327-340 [2] Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing; Latorre-Pellicer, et al. Nature2016;535(7613):561–5 Disclosure of Interests Morena Scotece: None declared, Ignacio Rego-Perez: None declared, Ana Victoria Lechuga Vieco: None declared, Purificacion Filgueira-Fernandez : None declared, Jose Antonio Enriquez: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grunenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD+ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan., This study was supported by a grant from the Andalusian regional government (Grupo de Investigacion Junta de Andalucia CTS113), Consejería de Salud de la Junta de Andalucia: PI‐0036‐2014 and Ministerio de economía y competitividad: SAF2017‐84494‐C2‐1‐R. FMA has the benefit of a FPU Fellowship (FPU 13/03173) from The Ministry of Education, Science and Sport. IF laboratory was funded by grants from Ministerio de Ciencia, Innovación y Universidades (SAF2016‐80406‐R) and Comunidad de Madrid (S2017/BMD‐3875). The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV‐2015‐0505)

All metazoans depend on O2delivery and consumption by the mitochondrial oxidative phosphorylation (OXPHOS) system to produce energy. A decrease in O2availability (hypoxia) leads to profound metabolic rewiring. In addition, OXPHOS uses O2to 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 hypoxia11drives 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.

Background Several studies showed interesting associations between mtDNA haplogorups and different OA-related features, including prevalence, incidence or progression of the disease1–3. The use of conplastic animals-individuals with the same nuclear genome but different mtDNA variants-provides an accurate tool to study the influence of the mitochondrial background in the ageing process4. Objectives To study the influence of mtDNA variation in the degree of joint deterioration of the knees of aged animals using a conplastic mouse model of ageing Methods mtDNAs from C57BL/6 and NZB/OlaHsd mice were used. These mtDNAs differ by 12 missense mutations, 4 tRNAs mutations, 8rRNAs mutations and 10 non-coding region mutations. Then, a conplastic mice strain was developed with the C57BL/6 nuclear genome and the NZB/OlaHsd mtDNA (BL/6NZB) to compare with the original C57BL/6 strain (BL/6C57) in animals of 25 and 75 weeks. A total of 38 limbs from 19 mice were processed to perform histologic analyses: 8 BL/6NZB25 w, 10 BL/6NZB75 w, 10 BL/6C5725 w and 10 BL/6C5775 w. Results Mankin score data showed significantly increased values in all knees from both strains at 75 w compared with 25 w (p The width of the epiphyseal plate was analysed in both tibia and femur bones. The results showed significantly decreased values in BL/6C57 at 75 w compared with the same strain at 25 w in tibial plateau (p Conclusions This study demonstrates the functional impact of mtDNA variation in the process of joint deterioration associated to ageing, leading to consider the mtDNA as a potential therapeutic target in osteoarthritis associated to ageing References [1] Shen, et al. PLoS One2014;9(10):e108896. [2] Fernandez-Moreno, et al. Ann Rheum Dis2017;76(6):1114–1122. [3] Fernandez-Moreno, et al. Rheumatology2017;56(2):263–270. [4] Latorre-Pellicer, et al. Nature2016;535(7613):561–5. Disclosure of Interest None declared

ApoB-100 and Phosphatidylcholine-specific phospholipase C (PC-PLC) are important contributors to atherosclerosis development. ApoB-100 is the main structural protein of LDL, being directly associated with atherosclerosis plaque generation. PC-PLC is highly expressed in atherosclerosis lesions and contributes to their progression. We show how phosphatidylcholine-coated nanomicelles can be used for specific characterisation of atherosclerosis plaque. Results show that ApoB-100 in the protein corona of the nanomicelle targets the particles to atherosclerotic areas in apolipoprotein E-/- mice. Furthermore, PC-PLC selectively removes the polar heads from the phospholipid coating of the nanomicelles leading to their accumulation. To fully characterise the behaviour of the nanomicelles, we developed multimodal probes using a nanoemulsion step. Hybrid imaging revealed plaque accumulation of the nanomicelles and colocalisation with PC-PLC expression and ApoB-100 in the plaque. This study shows how protein corona composition and enzyme-driven nanomaterial accumulation can be used for detection of atherosclerosis.

Post-translational modifications hugely increase the functional diversity of proteomes. Recent algorithms based on ultratolerant database searching are forging a path to unbiased analysis of peptide modifications by shotgun mass spectrometry. However, these approaches identify only one-half of the modified forms potentially detectable and do not map the modified residue. Moreover, tools for the quantitative analysis of peptide modifications are currently lacking. Here, we present a suite of algorithms that allows comprehensive identification of detectable modifications, pinpoints the modified residues, and enables their quantitative analysis through an integrated statistical model. These developments were used to characterize the impact of mitochondrial heteroplasmy on the proteome and on the modified peptidome in several tissues from 12-week-old mice. Our results reveal that heteroplasmy mainly affects cardiac tissue, inducing oxidative damage to proteins of the oxidative phosphorylation system, and provide a molecular mechanism explaining the structural and functional alterations produced in heart mitochondria. We thank Simon Bartlett (CNIC) for English editing. This study was supported by competitive grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (BIO2015-67580-P) through the Carlos III Institute of Health-Fondo de Investigacion Sanitaria (PRB2, IPT13/0001-ISCIII-SGEFI/FEDER; ProteoRed), by Fundacion La Marato TV3, and by FP7-PEOPLE-2013-ITN ``Next-Generation Training in Cardiovascular Research and Innovation-Cardionext.'' N.B. is a FP7-PEOPLE-2013-ITN-Cardionext Fellow. The CNIC is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO Award SEV-2015-0505). Sí

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health., This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).

The use of click chemistry reactions for the functionalization of nanoparticles is particularly useful to modify the surface in a well-defined manner and to enhance the targeting properties, thus facilitating clinical translation. Here it is demonstrated that olefin metathesis can be used for the chemoselective functionalization of iron oxide nanoparticles with three different examples. This approach enables, in one step, the synthesis and functionalization of different water-stable magnetite-based particles from oleic acid-coated counterparts. The surface of the nanoparticles was completely characterized showing how the metathesis approach introduces a large number of hydrophilic molecules on their coating layer. As an example of the possible applications of these new nanocomposites, a focus was taken on atherosclerosis plaques. It is also demonstrated how the in vitro properties of one of the probes, particularly its Ca(2+) -binding properties, mediate their final in vivo use; that is, the selective accumulation in atherosclerotic plaques. This opens promising new applications to detect possible microcalcifications associated with plaque vulnerability. The accumulation of the new imaging tracers is demonstrated by in vivo magnetic resonance imaging of carotids and aorta in the ApoE(-/-) mouse model and the results were confirmed by histology.

Multifunctional nanoparticles are usually produced by sequential synthesis, with long multistep protocols. Our study reports a generic modular strategy for the parallel one-step multifunctionalization of different hydrophobic nanoparticles. The method was designed and developed by taking advantage of the natural noncovalent interactions between the fatty acid binding sites of the bovine serum albumin (BSA) and the aliphatic surfactants on different inorganic nanomaterials. As a general example of the approach, three different nanoparticles-iron oxide, upconverting nanophosphors, and gold nanospheres-were nanoemulsified in water with BSA. To support specific applications, multifunctional capability was incorporated with a variety of previously modified BSA modules. These modules include different conjugated groups, such as chelating agents for (68)Ga or (89)Zr and ligand molecules for enhanced in vivo targeting. A large library of 13 multimodal contrast agents was developed with this convergent strategy. This platform allows a highly versatile and easy tailoring option for efficient incorporation of functional groups. Finally, as demonstration of this versatility, a bimodal (PET/MRI) probe including a maleimide-conjugated BSA was selectively synthesized with an RGD peptide for in vivo imaging detection of tumor angiogenesis.

In vivo detection and quantification of inflammation is a major goal in molecular imaging. Furthermore, cell-specific detection of inflammation would be a tremendous advantage in the characterization of many diseases. Here, we show how this goal can be achieved through the synergistic combination of nanotechnology and nuclear imaging. One of the most remarkable features of this hybrid approach is the possibility to tailor the pharmacokinetics of the nanomaterial-incorporated biomolecule and radionuclide. A good example of this approach is the covalent binding of a large amount of a neutrophil-specific, hydrophobic peptide on the surface of 68Ga core-doped nanoparticles. This new nano-radiotracer has been used for non-invasive in vivo detection of acute inflammation with very high in vivo labelling efficiency, i.e. a large percentage of labelled neutrophils. Furthermore, we demonstrate that the tracer is neutrophil-specific and yields images of neutrophil recruitment of unprecedented quality. Finally, the nano-radiotracer was successfully detected in chronic inflammation in atherosclerosis-prone ApoE-/- mice after several weeks on a high-fat diet. This study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (grant number: SAF2016-79593-P) and from Carlos III Health Research Institute (grant number: DTS16/00059). We thank Simon Bartlett for editorial assistance and manuscript preparation. The CNIC is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (MEIC award SEV-2015-0505). Sí

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 UC P1 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., Brown and beige adipose tissues dissipate heat via uncoupling protein 1 (UCP1). Here the authors show that the stress activated kinase MKK6 acts as a repressor of UCP1 expression, suggesting that its inhibition promotes adipose tissue browning and increases organismal energy expenditure.

Heart failure (HF) is a major health and economic burden in developed countries. It has been proposed that the pathogenesis of HF may involve the action of mitochondria. We evaluate three different mouse models of HF: tachycardiomyopathy, HF with preserved left ventricular (LV) ejection fraction (LVEF), and LV myocardial ischemia and hypertrophy. Regardless of whether LVEF is preserved, our results indicate that the three models share common features: an increase in mitochondrial reactive oxygen species followed by ultrastructural alterations in the mitochondrial cristae and loss of mitochondrial integrity that lead to cardiomyocyte death. We show that the ablation of the mitochondrial protease OMA1 averts cardiomyocyte death in all three murine HF models, and thus loss of OMA1 plays a direct role in cardiomyocyte protection. This finding identifies OMA1 as a potential target for preventing the progression of myocardial damage in HF associated with a variety of etiologies.

et al., Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development., This work was funded by the Spanish Ministerio de Economia y Competitividad (grants BFU2011-23083 and BFU2014-54608-P to MM, BFU2013-41322-P to JLGS and BFU2014-58449-JIN to JJT), the Comunidad Autónoma de Madrid (grant CELLDD-CM to MM), the Andalusian regional government (grant BIO-396 to JLGS), the Universidad Pablo de Olavide (postdoctoral grant to JJT). PDO is funded by the Heart and Stroke Foundation of Canada (G-17-0018613), Operational Funds from the Hospital for Sick Children, the Natural Sciences and Engineering Research Council of Canada (NSERC) (500865 to PDO), and the Canadian Institutes of Health Research (CIHR) (PJT-149046). The CNIC is supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

The production of magnetic nanoparticles of utmost quality for biomedical imaging requires several steps, from the synthesis of highly crystalline magnetic cores to the attachment of the different molecules on the surface. This last step probably plays the key role in the production of clinically useful nanomaterials. The attachment of the different biomolecules should be performed in a defined and controlled fashion, avoiding the random adsorption of the components that could lead to undesirable byproducts and ill-characterized surface composition. In this work, we review the process of creating new magnetic nanomaterials for imaging, particularly for the detection of atherosclerotic plaque, in vivo. Our focus will be in the different biofunctionalization techniques that we and several other groups have recently developed. Magnetic nanomaterial functionalization should be performed by chemoselective techniques. This approach will facilitate the application of these nanomaterials in the clinic, not as an exception, but as any other pharmacological compound. The authors thank the Spanish Ministry of Science (SAF2011-25445), the Comunidad de Madrid (S2010/BMD-2326, Inmunothercan-CM, NANOCOPD-CIBERES-CIBERBBN-SEPAR), and the EU 7th Framework Program (FP7-PEOPLE-ITN-264864 Pinet and FP7-PEOPLE-2013-ITN CardioNext). Sí

BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment.

Human mitochondrial DNA (mtDNA) shows extensive within-population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic, metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation, insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

A fast and reproducible microwave-driven process has allowed us to synthesise neridronate-functionalised nanoparticles. Contrary to tradition, the phosphate groups decorate the outside layer of the particles providing Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. In vivo and ex vivo detection by T2-weighted MRI is demonstrated and validated by histology. The accumulation in the plaque takes place in less than one hour following the intravenous injection, which is particularly suitable for clinical applications.

We report the synthesis of micellar phosphatidylcholine-coated superparamagnetic iron oxide nanoparticles as a new long circulation contrast agents for magnetic resonance imaging. Oleic acid-coated Fe3 O4 nanoparticles were first prepared through thermal degradation and then encapsulated into small clusters with a phosphatidylcholine coating to obtain hydrophilic nanomicelles. A thorough characterization confirmed the chemical nature of the coating and the excellent colloidal stability of these nanomicelles in aqueous media. Magnetization and relaxivity properties proved their suitability as magnetic resonance imaging (MRI) contrast agent and in vitro cell viability data showed low toxicity. Vascular lifetime and elimination kinetics in the liver were assessed by blood relaxometry and by in vivo MRI in rats and compared with "control" particles prepared with a polyethylene glycol derivative. These micellar particles had a lifetime in blood of more than 10 h, much longer than the control nanoparticles (≈2 h), which is remarkable considering that the coating molecule is a small biocompatible zwitterionic phospholipid. The protein corona was characterized after incubation with rat serum at different times by high-throughput proteomics, showing a higher proportion of bound apolipoproteins and other dysopsonins for the phosphatidylcholine particles. The antibiofouling properties of this corona and its resistance to the adsorption of proteins corroborate the observed enhanced stability and prolonged systemic circulation. This study is supported by a grant from FP7 Marie Curie, Pulmonary imaging network (PINET), by Fundacio La Maratode TV3 (70/C/2012) and by a grant from the Comunidad de Madrid (S2010/BMD-2326, Inmunothercan-CM) and by Spanish Economy Ministry (MAT2013-47303-P). We thank E. Urones (Centro Nacional de Microscopia de la Universidad Complutense de Madrid) for the transmission electronic microscopy imaging; P. Morales (Instituto de Ciencia de Materiales de la Universidad Autonoma de Madrid) for the thermogravimetric and magnetization analysis and B. Salinas (Fundacion Centro Nacional de Investigaciones Cardiovasculares and CIBER de Enfermedades Respiratorias) for the TEM picture of oleic acid coated Fe3 O4. The authors declare no competing financial interests. Sí

Nature 535, 561–565 (2016); doi:10.1038/nature18618 In this Letter, the number of mice monitored in Fig. 1a should have stated: ‘n = 31 BL/6C57 and n = 29 BL/6NZB’, rather than: ‘n = 31 per genotype (19 males, 12 females)’. In addition, in Extended Data Table 1, the non-synonymous mutation at nucleotide position 9985 (mt-Nd4l) was inadvertently omitted.