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Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction [1]

['Dusanka Milenkovic', 'Max Planck Institute For Biology Of Ageing', 'Cologne', 'Adrián Sanz-Moreno', 'Institute Of Experimental Genetics', 'German Mouse Clinic', 'Helmholtz Zentrum München', 'German Research Center For Environmental Health Gmbh', 'Neuherberg', 'Julia Calzada-Wack']

Date: 2022-07

Abstract Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.

Author summary We have addressed the controversy of the role of the mitochondrial genome and maintenance exonuclease 1 (MGME1) in mtDNA metabolism by characterization of knockout mice. Our findings show that loss of MGME1 leads to increased de novo formation of linear deleted mtDNA, thus contradicting previous report that MGME1 degrades long linear mtDNA molecules. In addition, we report that loss of MGME1 leads to age-associated pathology manifested as progressive weight loss, cataract and retinopathy. Aged knockout mice also develop kidney inflammation leading to glomerular changes, fibrosis and nephrotic syndrome. Defective mtDNA replication causing the formation of linear deleted mtDNA can thus trigger an immune response that leads to the development of progressive kidney disease in ageing animals.

Citation: Milenkovic D, Sanz-Moreno A, Calzada-Wack J, Rathkolb B, Veronica Amarie O, Gerlini R, et al. (2022) Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction. PLoS Genet 18(5): e1010190. https://doi.org/10.1371/journal.pgen.1010190 Editor: Carlos T. Moraes, University of Miami, UNITED STATES Received: December 15, 2021; Accepted: April 5, 2022; Published: May 9, 2022 Copyright: © 2022 Milenkovic et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by grants to NGL from the Swedish Research Council (2015-00418), Swedish Cancer Foundation (2021.1409), the Knut and Alice Wallenberg foundation, European Research Council (ERC Advanced Grant 2016-741366), Novo Nordisk Foundation (NNF20OC0063616), Diabetesfonden (DIA2020-516) and grants from the Swedish state under the agreement between the Swedish government and the county councils (SLL2018.0471). GMC is supported by grants from the German Federal Ministry of Education and Research (Infrafrontier grant 01KX1012 to MHdA) and the German Center for Diabetes Research (DZD) (MHdA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests. NGL is a scientific founder and holds stock in Pretzel Therapeutics, Inc. The other authors have no competing interests.

Introduction Impaired replication or maintenance of mitochondrial DNA (mtDNA) lead to mitochondrial diseases, a clinically and genetically heterogeneous group of multisystemic disorders affecting various organs [1,2]. Defects in mtDNA can be either quantitative, causing mtDNA depletion, or qualitative, causing accumulation of deletions and/or point mutations of mtDNA. The expression of mtDNA is completely dependent on nuclear genes that encode proteins that are synthesized in the cytosol and imported into the mitochondrial matrix [3]. At least two hundred nucleus-encoded proteins are needed for maintenance, replication and transcription of mtDNA, as well as biogenesis of mitochondrial ribosomes [4]. The basic components of the mtDNA replication machinery are known and mutations in the catalytic and accessory subunits of mitochondrial DNA polymerase (POLγA and POLγB) [5, 6] the replicative DNA helicase (TWNK) [7], the mitochondrial single-stranded DNA binding protein (SSBP1) [8] and the mitochondrial genome and maintenance exonuclease 1 (MGME1) [9,10], cause mutations and/or depletion of mtDNA, which, in turn, impair mitochondrial function and cause mitochondrial disease syndromes. According to one model supported by biochemical data, MGME1 is a mitochondrial nuclease that processes newly replicated 5’ DNA ends to facilitate ligation when mtDNA synthesis is completed [9,11–13]. Based on studies in cell lines, an additional function for MGME1 in degradation of long linear mtDNA fragments was proposed [14]. Both models predict that the absence of MGME1 will lead to the formation of long linear deleted mtDNA molecules, but due to different mechanisms, i.e. increased formation or decreased degradation, respectively. Loss of function mutations in the MGME1 gene cause human disease syndromes with mtDNA depletion and accumulation of mtDNA rearrangements (MIM#615084) [9,10]. Affected patients develop a range of symptoms in various organs, including brain, skeletal muscle, heart and gastrointestinal organs [9,10]. The majority of the patients develop adult onset disease, but onset in childhood has also been described [10]. We recently generated Mgme1 knockout mice and found that they had prominent mtDNA replication aberrations manifested as replication stalling, mtDNA depletion, formation of long linear deleted molecules and an increase of short single stranded DNA products caused by prematurely aborted replication. In our initial characterization, we established that Mgme1 knockout mice were born at Mendelian proportions and had no obvious changes in gross appearance up until one year of age [13]. Consistent with our results, the international mouse phenotyping consortium project (https://www.mousephenotype.org/data/genes/MGI:1921778#section-associations) revealed only minor changes in an independent Mgme1 knockout mouse strain analyzed at the age of 10 weeks. Because humans with MGME1 mutations typically develop late-onset-mitochondrial disease [9,12], we decided to characterize ageing cohorts of Mgme1 knockout mice by an extensive phenotypic analysis, including clinical chemistry (haematology, metabolism and organ function), energy metabolism (indirect calorimetry, body composition), evaluation of different organ systems, immune system characterization and pathological assessment of tissue changes. We report here that Mgme1 knockout mice show reduced weight gain during aging and even weight loss later in life. In addition, the Mgme1 knockout mice develop cataracts and retinopathy at 65–70 weeks of age. Strikingly, aged mice also develop kidney inflammation, glomerular changes and chronic progressive nephropathy with albuminuria, and die prematurely at ~70 weeks of age. Our findings provide a direct link between defective mtDNA replication and ageing-associated inflammation.

Discussion In this study we report a detailed phenotypic characterization of mice lacking the mitochondrial exonuclease MGME1. Despite having clear aberrations in mtDNA, young Mgme1 knockout animals are healthy and fertile without apparent morphological alterations [13]. However, weight loss and functional changes of multiple organs become apparent as the animals age. Lack of the MGME1 protein alters body composition and causes a reduction in fat mass. There are also pathological changes of the lens and the retina. Most importantly, mutant animals develop a severe kidney pathology with glomerular changes, tubulointerstitial fibrosis and inflammation ultimately leading to a nephrotic syndrome coupled with reduced life span. The mtDNA maintenance diseases are genetically and clinically heterogenous with severities ranging from infantile fatal forms to adult onset of mild disease. Whereas organs with high energy demands, such as heart, skeletal muscle and the central nervous system, often are affected, manifestations can occur in many different organs [19]. Kidneys contain a high density of mitochondria, particularly in the cortical tubules, but all sections of the nephron may be affected by mitochondrial dysfunction. Proximal tubulopathy (Fanconi syndrome) is a common renal phenotype frequently seen in early-onset mitochondrial disease patients, whereas glomerular disease is more frequent in adults [20]. Human patients with MGME1 deficiency were reported to have an adult onset, multisystem mitochondrial disorder including progressive external ophthalmoplegia (PEO), muscle wasting, muscle weakness, exercise intolerance, cerebellar atrophy, cerebellar ataxia and cardiomyopathy [9]. With regard to kidney-related phenotypes, those patients were reported to develop nephrolithiasis [9]. Also, mitochondrial dysfunction has been linked to chronic kidney disease in humans [21]. Interestingly, some end-stage alterations in human chronic kidney disease are similar to those in rodents, e.g. proteinuria, tubular atrophy, scarring, as well as increases of blood urea nitrogen (BUN) and serum creatinine levels [22]. MGME1 deficiency in patients and mice leads to high levels of a linear deleted mtDNA fragment in many different tissues, but the involved mechanisms are much debated. To address this question, we used Mgme1-/- knockout mice [13] to study linear deletion formation. Our data argue against a proposed model where MGME1 functions as a key enzyme that degrades linear mtDNA fragments [14]. Firstly, our data show that deleted linear mtDNA fragments do not accumulate with age in MGME1 knockout mice. Secondly, in organello mtDNA replication assays show that there is a substantial de novo formation of linear deleted mtDNA in the absence of MGME1, whereas the degradation of this linear fragment is not affected. Two additional replication factors, POLγA and the replicative mtDNA helicase TWINKLE have been suggested to act together with MGME1 to degrade linear mtDNA fragments [14,16,17]. However, as POLγA possesses a 3´-5´exonuclease activity and MGME1 preferentially degrades single-stranded DNA in the 5′-3′ direction [9], it seems unlikely that any POLγA can compensate for loss of MGME1. Instead, the results in our study support a previously proposed model where MGME1 removes flaps generated when mtDNA replication is reaching completion [23]. In the absence of MGME1, these flaps will persist and prevent the ligation step necessary to finalize mtDNA replication, which will result in the formation of linear deleted mtDNA [23]. Unexpectedly, the Mgme1-/- mouse model also links defective mtDNA replication to inflammatory disease manifestations. Ageing Mgme1-/- mice develop kidney inflammation, tubulointerstitial fibrosis and glomerular changes leading to nephrotic syndrome. It is unclear at this point how defective mtDNA replication triggers age-associated inflammation. An increasing literature indicates that mitochondria are key participants in innate immune pathways, representing signaling platforms and participating in effector responses [24]. Under certain pathological conditions various mitochondrial ligands or damage-associated molecular patterns (DAMPs), including mtDNA, can be released from mitochondria and recognized by different pattern recognition receptors (PRRs). It is now appreciated that mtDNA can stimulate different PRRs, including cytosolic cGAS, endosomal localised TLR9 and inflamasomes to activate various pro-inflammatory signaling pathways [25–27]. Future studies should aim to investigate if the inflammation associated with MGME1 defficiency is triggered by mtDNA release.

Materials and methods Ethics statement This study was performed in strict accordance with the recommendations and guidelinesof the Federation of European Laboratory Animal Science Associations (FELASA). The protocol was approved by the “Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein- Westfalen” (reference numbers 81.02.04.2020.A082, 84–02.04.2015.A103 and 84–02.50.15.004) and by the Stockholm ethical committee (Stockholms djurförsöksetiska nämnd) under the ethical permit 1206–2019. The mice were maintained according to the GMC housing conditions (www.mouseclinic.de) in strict accordance with directive 2010/63/EU, the local government and German laws. The GMC holds a general license to run phenotype assessments in mice and all tests are approved by the responsible authority of the district government of Upper Bavaria. Animals and housing This study was performed in strict accordance with the recommendations and guidelinesof the Federation of European Laboratory Animal Science Associations (FELASA). The protocol was approved by the “Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein- Westfalen” (reference numbers 81.02.04.2020.A082, 84–02.04.2015.A103 and 84–02.50.15.004). The Mgme1 knockout and wild-type mice on a C57BL/6N background were housed in standard individually ventilated cages (45 x 29 x 12 cm) under a 12h light/ dark schedule in controlled environmental conditions of 22 ± 2°C and 50 + 10% relative humidity and fed a normal chow diet and water ad libitum. Generation of the Mgme1 knockout mice was described before [13]. Characterization on 14 female Mgme1-/- and 15 Mgme1+/+ littermate controls and 15 male Mgme1-/- and 15 Mgme1+/+ littermate controls was performed at the German Mouse Clinic. Body weight measurements started at the age of 8 weeks, the phenotyping examination of 3 female Mgme1-/- and 4 females controls, 5 male Mgme1-/- and 6 male controls was performed at 68 weeks of age, comprising tests examining Neurology, Dysmorphology, Metabolism, Cardiology, Clinical Chemistry and Pathology. The mice were maintained according to the GMC housing conditions (www.mouseclinic.de) in strict accordance with directive 2010/63/EU, the local government and German laws. The GMC holds a general license to run phenotype assessments in mice and all tests are approved by the responsible authority of the district government of Upper Bavaria. The phenotypic tests were performed as outlined in the standard operating procedures (SOP) linked to the EMPReSS website http://empress.har.mrc.ac.uk. Experimental groups were assigned according to the genotype of the animals. The selection of the mice for testing was balanced, control and mutants were measured alternately. Most of the tests were not conducted in blinded conditions because the results were recorded directly by the machines and, therefore, not influenceable by the examiner. The experiment was conducted in blinded conditions whenever there could have been an influence from the investigator. All the procedures are described in SOPs. Metadata for each data point was recorded throughout the measurements. Body composition assessment (GMC) Body composition was analysed with time domain-nuclear magnetic resonance (Bruker Minispec LF 50) in live mice without the administration of anesthesia at 68 weeks of age of 4 wild-type and 3 mutant females and 6 wild-type and 5 mutant males. Body weight measured at the same time of the analysis was used to determine the body fat and lean percent. Pathological analyses and immunohistochemistry (GMC) Microscopy and histopathological analyses using hematoxylin and eosin (H&E) staining on formalin-fixed paraffin-embedded sections (3 μm) were performed as described in www.mouseclinic.de/screens/pathology. A Leica Bond III (Leica Biosystems) automatic stainer was used for immunohistochemistry. Heat-induced antigen retrieval was performed with citrate buffer (pH 6) for 30 minutes (AR9961; Bond Epitope Retrieval Solution; Leica Biosystems). Antibodies against CD3 (Clone SP7; ZYT-RBG024; Zytomed systems) and CD45R/B220 (Clone RA3-6B2; 550286; BD Pharmingen) were employed and the staining was detected with DAB chromogen. PAS (Periodic acid-Schiff) staining was performed to study glomerular changes in the kidneys (in particular mesangial expansion). Tubulointerstitial fibrosis was assessed with Sirius Red staining using standard protocols. The slides were scanned using a Hamamatsu NanoZoomer 2.0HT digital scanner and analyzed by two independent pathologists using NDP.view2 software (Hamamatsu Photonics). Eye histology: At sacrifice the eyes were enucleated and after 24 hour Davidson fixation were embedded in Technovit 8100 (Heraeus Kulzer, Wehrheim, Germany) and kept for polymerization for 6–10 hours at 4°C. Samples, cut in 2 μm sagitally through the middle of the eye ball, were stained with basic fuchsin and methylene blue. Slides were scanned (NanoZoomer 2.0HT Digital slide scanner, Hamamatsu, Japan) and taken images were processed with an image-processing program (Adobe Inc., 2019. Adobe Illustrator). Blood collection Blood samples were collected under isoflurane anaesthesia by retrobulbar puncture as a final blood withdrawal without prior fasting of the animals from 5 homozygous mutant and 6 wild-type males as well as 3 homozygous and 4 wild-type females. An aliquot of 50μl whole blood was collected in EDTA-coated end-to-end capillaries and diluted 1:5 with buffer provided from Sysmex (Cell-Pack buffer) for subsequent analysis of basic haematological parameters. Blood samples for clinical chemistry analyses were collected in Li-heparin-coated tubes and stored at room temperature until centrifugation (4500xg, 10 min) and separation of plasma aliquots for further analyses. Plasma samples were frozen at -80°C until analysis within one week after collection. Clinical chemistry and cytokines For the clinical chemistry analyses plasma samples were thawed at room temperature, diluted 1:2 with deionised water, mixed thoroughly, and centrifuged again (5000xg, 10 min) to remove clots from the sample. Measurement of circulating biochemical parameters was performed using a clinical chemistry analyser (Beckman Coulter AU 480 autoanalyzer, Krefeld, Germany). A broad set of parameters was measured using the respective kits provided by Beckman Coulter, in order to determine various enzyme activities as well as plasma concentrations of specific substrates and electrolytes in ad libitum fed mice [28]. Multiplex measurement of the proinflammatory cytokines IL-6, TNF-α, KC/GRO, IL-2, IL-10, IFN-γ, IL-5, IL-4 and IL-1b was performed in plasma in plasma of a separate cohort of mice at x weeks of age (wt n = 11 ; Mgme-/- n = 20). Nitrotetrazolium blue exclusion (NBTx) staining assay Tissue Preparation—kidneys were quickly frozen in 2-methylbutane, in a glass beaker cooled by immersion in liquid nitrogen. Frozen tissues were stored at -80°C until ready to use. Thin sections of 10 μm were cut with a cryostat at -20°C (OFT 5000, Bright Instruments, Luton, UK) and mounted on Superfrost Plus microscope slides (Menzel, Thermo Scientific, Waltham, MA, USA) and air-dried for 5 to 10 min. Slides were kept at -80°C for maximum a few months to avoid the loss of enzyme activity. Staining Protocol—slides were taken out of the -80°C freezer and thawed briefly at room temperature on a slide holder without lid. Sections (3 sections per slide) were then covered with 1ml PBS for 10 min in an incubator set at 21°C (1 ml per slide). PBS was discarded and replaced with 1 ml NBTx solution. Sections of kidneys were left 30 min at 21°C in the incubator and washed briefly in purified water followed by dehydration in ethanol (2 min in 50%, 75%, 96%, 100% followed by an extra 5 min in 100% ethanol). Finally, slides were immersed for 5 min in two changes of xylene before mounted on coverslips with Cytoseal (Thermo Scientific, Darmstadt, Germany). Isolation of mitochondria from mouse tissues Mitochondria were isolated from mouse tissues using differential centrifugation as previously described [29]. Briefly, freshly obtained tissues were cut, washed with ice cold PBS and homogenized in mitochondrial isolation buffer containing (320 mM sucrose, 10 mM Tris/HCl pH 7.4, and 1 mM EDTA) supplemented with 1× Complete protease inhibitor cocktail (Roche) by using a Teflon pestle (Schuett Biotec). After 10 min centrifugation at 1000×g using swing-out rotor at 4°C the supernatants were subsequently spun at 10000×g for 10 min at 4°C to isolate the mitochondria. mtDNA extraction and Southern blot analysis Total DNA or mtDNA was isolated from pulverized tissue or purified mitochondria respectively, using Gentra Puregene Tissue Kit (Qiagen) according to kit instructions. DNA quantification was performed with the Qubit 1.0 fluorometer (Thermofisher). 300–1000 μg of DNA were digested with SacI restriction nuclease and DNA fragments were separated by agarose gel electrophoresis, transferred to nitrocellulose membranes (Hybond-N+ membranes, GE Healthcare) and hybridized with αP32-dCTP-labeled probes. For 7S DNA, Southern blot samples were heated for 3 min at 93°C prior to loading. Southern blot signals were quantified using MultiGauge or ImageJ softwares. In organello replication 1 mg of freshly isolated heart mitochondria were resuspended in 0.5 ml of incubation buffer (25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 10 mM K 2 HPO 4 , 0.05 mM EDTA, 5 mM MgCl 2 , 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM Tris–HCl, pH 7.4, 1 mg/ml fatty acid-free bovine serum albumin, 50 μM each of dTTP, dCTP and dGTP and 20 μCi α-32P-dATP (3000 Ci/mmol). Incubation was carried out at 37°C for 2h on a rotating wheel. For the chase reisolated mitochondria were incubated in 0.5 ml of incubation buffer supplemented with all four non-radiolabeled dNTPs (50 μM) for indicated time. After incubation, mitochondria were pelleted at 9000 rpm for 4 min and washed twice with washing buffer (10% glycerol, 10 mM Tris–HCl, pH 6.8, 0.15 mM MgCl 2 ). In the following step DNA isolation and Southern blot analysis were performed as described above. Western blot analysis and BN-PAGE 20 μg of isolated mitochondria were resuspended in 4X Lämmli-Buffer (4% SDS, 20% Glycerol, 120mM Tris, 0,02% Bromophenol Blue), proteins were separated on 4–12% NuPAGE gels (Invitrogen) and transferred on Hybond-P membrane (GE Helthcare). MitoProfile total OXPHOS antibody cocktail (MitoSciences) antibody was used for the western blotting. Western blot signals were quantified using the ImageJ processing program. BN-PAGE and subsequent in gel activity were performed as previously described [30]. For BN-PAGE, 75 μg of isolated kidney mitochondria were lysed in 50 μl solubilization buffer (20 mM Tris pH 7.4; 0.1 mM EDTA; 50 mM NaCl; 10% [v/v] glycerol) containing 1% (w/v) digitonin (Calbiochem) and mixed with loading dye (5% [w/v] Coomassie Brilliant Blue G-250, 150 mM Bis-Tris, and 500 mM ε-amino-n-caproic acid [pH 7.0]). BN-PAGE samples were resolved on self-made 3%– 13% gels. Protein complexes were visualized using in gel activity staining for complexes I, II and IV. For CI in gel activity the BN-PAGE gel was incubated in 2 mM Tris/HCl pH 7.4, 0.1 mg/ml NADH (Roche) and 2.5 mg ml-1 iodonitrozolium (Sigma) for about 10 minutes. In gel CIV activity was determined by incubating the BN-PAGE gels in 10 ml of 0.05 mM phosphate buffer pH 7.4, 25 mg 3.3´-diamidobenzidine tetrahydrochloride (DAB), 50 mg Cyt c, 3.75 g Sucrose and 1 mg Catalase for approximately 1h. For the CII assay, the buffer contained 200 μl of sodium succinate (1 M), 8 μl of phenazine methosulfate (250 mM dissolved in DMSO), and 25 mg of NTB in 10 ml of 5 mM Tris/HCl, pH 7.4. Incubation of 10–30 min was required. All in gel staining reactions were carried out at room temperature and stopped using solution containing 50% methanol, 10% acetic acid for 30 min. Statistics Tests for genotype effects of the phenotyping data were made by Wilcoxon rank sum test for parametric data, if not indicated otherwise. A P < 0.05 has been used as a level of significance; a correction for multiple testing has not been performed. Figures were prepared using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, California, USA).

Acknowledgments We are grateful to Petra Kirschner for expert technical assistance.We thank the FACS & Imaging core facility of Max Planck Institute for biology of Ageing in Cologne.

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