An mtDNA mutation accelerates liver aging by interfering with the ROS response and mitochondrial life cycle

Author’s Accepted Manuscript An mtDNA mutation accelerates liver aging by interfering with the ROS response and mitochondrial life cycle Jan Niemann, Cindy Johne, Susanne Schröder, Franziska Koch, Saleh M. Ibrahim, Julia Schultz, Markus Tiedge, Simone Baltrusch www.elsevier.com

PII: DOI: Reference:

S0891-5849(16)31070-X http://dx.doi.org/10.1016/j.freeradbiomed.2016.11.035 FRB13090

To appear in: Free Radical Biology and Medicine Received date: 18 March 2016 Revised date: 10 November 2016 Accepted date: 21 November 2016 Cite this article as: Jan Niemann, Cindy Johne, Susanne Schröder, Franziska Koch, Saleh M. Ibrahim, Julia Schultz, Markus Tiedge and Simone Baltrusch, An mtDNA mutation accelerates liver aging by interfering with the ROS response and mitochondrial life cycle, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2016.11.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An mtDNA mutation accelerates liver aging by interfering with the ROS response and mitochondrial life cycle Jan Niemanna, Cindy Johnea, Susanne Schrödera, Franziska Kocha,b, Saleh M. Ibrahimc, Julia Schultza, Markus Tiedgea, Simone Baltruscha* a

Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Rostock,

Germany b

Current address: Institute of Nutritional Physiology “Oskar Kellner”, Leibnitz Institute for

Farm Animal Biology, Dummerstorf, Germany c

Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany

*

Correspondence to: Institute of Medical Biochemistry and Molecular Biology, University of

Rostock, D-18057 Rostock, Germany. [email protected]

Abstract Mitochondrial dysfunction affects liver metabolism, but it remains unclear whether this interferes with normal liver aging. We investigated several mitochondrial pathways in hepatocytes and liver tissue from a conplastic mouse strain compared with the control C57BL/6NTac strain over 18 months of life. The C57BL/6NTac-mtNODLtJ mice differed from C57BL/6NTac mice by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase. Young C57BL/6NTac-mtNODLtJ mice showed reduced mitochondrial metabolism but similar reactive oxygen species (ROS) production to C57BL/6NTac mice. Whereas ROS increased almost equally up to 9 months in both strains, different mitochondrial adaptation strategies resulted in decreasing ROS in advanced age in C57BL/6NTac mice, but persistent ROS production in C57BL/6NTac-mtNODLtJ mice. Only the conplastic strain developed elongated mitochondrial networks with artificial loop structures, depressed autophagy, high mitochondrial respiration and up-regulated antioxidative response. Our results indicate that mtDNA mutations accelerate liver ballooning degeneration and carry a serious risk of premature organ aging.

1

Abbreviations: CAT, catalase; COX, cytochrome c oxidase; DJ7, Parkinson 7; DNM1L, dynamin-1-like protein; FIS1, mitochondrial fission protein 1; MFF, mitochondrial fission factor; MFN1, mitofusin-1; MFN2, mitofusin-2; mtDNA, mitochondrial DNA; mtNOD, C57BL/6NTac-mtNODLtJ; nDNA, nuclear DNA; OPA1, optic atrophy 1; OXPHOS, mitochondrial oxidative phosphorylation; PCX, pyruvate carboxylase; PINK1, PTEN-induced putative kinase 1; ROS, reactive oxygen species; SOD1/2, superoxide dismutase 1 and 2; TFAM, mitochondrial transcription factor A; TRAP1, TNF receptor associated protein 1; UCP2, uncoupling protein 2 Keywords: aging; conplastic mice; liver; mitochondria; reactive oxygen species

1. Introduction Mitochondria are essential for ATP generation and are thus vital for cellular function. Most mitochondrial proteins are encoded by nuclear DNA (nDNA). However, 13 polypeptides of the mitochondrial oxidative phosphorylation (OXPHOS) enzyme complexes, and 22 tRNAs for mitochondrial biosynthesis and for mitochondrial 16S and 18S rRNA [1] are encoded by mitochondrial DNA (mtDNA), a closed circular, double-stranded DNA. The rate of mutation in mtDNA is higher than in nDNA [2], most probably due to the absence of efficient repair systems. The mechanisms that determine the assembly of mtDNA- and nDNA-encoded subunits of OXPHOS are currently not well understood [3]. Recent studies suggest that mtDNA variations might have a disruptive influence on OXPHOS assembly and ultimately on mitochondrial quality and function [4]. Thus, in cellular processes such as stress and aging which require precise adaptation of mitochondrial function, mtDNA mutations can contribute to the manifestation of many human pathologies, including gastrointestinal, neurological, 2

respiratory, endocrine, ophthalmological, hematological, renal, and hepatic disorders [5]. Electron transport in the respiratory chain is a major generator of reactive oxygen species (ROS) [6]. ROS are pivotal for cellular viability, triggering stress response, the cell cycle, and energy metabolism. However, high cellular ROS levels are toxic and cause damage to lipids, proteins, and DNA [7]. Reflecting their location, mtDNA mutations can lead to increased ROS production at the affected respiratory chain complex in the inner mitochondrial membrane [8]. Thus, mtDNA mutations caused by oxidative stress can trigger a vicious cycle with increasing ROS production during aging [9]. Antioxidative enzymes, namely superoxide dismutase 1 and 2 (SOD1/2) [10], catalase (CAT) [11] and Parkinson 7 (DJ7) [12] detoxify ROS and confer cellular stress adaptation. In addition, the dynamics of the mitochondrial network through fission and fusion processes contribute to the antioxidative response. By mixing and sharing mitochondrial components, fusion is crucial in compensating for mtDNA variation [13]. However, severely damaged mitochondria become fragmented and are then selectively removed by an autophagic process that is termed mitophagy [14]. According to the most recent research, cytochrome c oxidase, also known as complex IV of OXPHOS, consists of 14 subunits which include COX 1-3 encoded by mtDNA [15]. COX 3 has been identified as having a central role in the proton translocation activity of cytochrome c oxidase [16]. Furthermore this subunit seems to be important for stabilization of respirasomes by associating with complex III and IV via cardiolipin [17]. Single mutations in COX 3 have been reported to cause encephalopathy [18], MELAS [19], Leigh-like syndrome [20], and exercise intolerance and rhabdomyolysis [21] in humans. However, causality relationships between a single COX 3 mutation and age-dependent ROS production, mitochondrial dynamics and metabolism at the cellular level have not been investigated. To address this issue we used the conplastic C57BL/6NTac-mtNODLtJ (mtNOD) mouse strain, carrying mtDNA from the NOD/LtJ strain with nt9821-10A and an nt9348A polymorphism in COX 3 but the same nDNA as the C57BL/6NTac strain [22]. We demonstrated that this mutation results in altered mitochondrial and cellular adaptation during aging compared with C57BL/6NTac mice. Liver tissue sections and isolated hepatocytes from 12-month-old mtNOD mice (an age that corresponds to middle age in humans) showed higher mtDNA content, mitochondrial ROS accumulation, increased mitochondrial fusion, and elongated mitochondria. Liver tissue sections from old mtNOD mice (> 18-months) revealed more severe signs of ballooning degeneration, infiltration and fibrosis.

2. Materials and methods 3

2.1 Experimental animals The conplastic C57BL/6NTac-mtNODLtJ (mtNOD) mouse strain was generated by crossing females from the mitochondrial donor strain (NOD/LtJ) to males with the preferred genomic background (C57BL/6NTac). The female offspring were subsequently backcrossed to males of the recipient strain. After 10 generations, the offspring were regarded as a pure conplastic strain [22]. All mouse strains were housed at the central animal care facility of the Medical Faculty, University of Rostock, receiving conventional rodent chow (SSNIFF) and water ad libitum. The colonies were regularly monitored for murine pathogens, in line with the recommendations of the Federation for Laboratory Animal Science Associations. This study was carried out in accordance with the German Animal Welfare Act 2006 (last amended 2014) and has been approved by the State Department of Agriculture, Food Safety and Fisheries, Mecklenburg-Vorpommern (LALLF M-V). Male and female mice were used in equal numbers in all experiments. 2.2 Fluorescence microscopy Fluorescence microscopy was performed with a Fluoview FV10i (Olympus, Hamburg, Germany) confocal laser scanning microscope. Images of tissue sections and hepatocytes were taken with an UPLSAPO 60×1.35 numerical aperture oil-immersion objective (Olympus).

2.3 Quantification of ROS Mice were injected intraperitoneally with 25 µg of the superoxide indicator MitoSOX™ (Molecular Probes, Invitrogen, Carlsbad, CA, USA) one hour before euthanization. MitoSOX™ is rapidly oxidized by superoxide anions in tissue. Upon binding to nucleic acid the oxidized product is detectable at a wavelength of 578 nm. The accumulation of ROS in 5 µm thin liver tissue sections was investigated by fluorescence microscopy. ROS were quantified as the amount of pixels with a higher intensity than a defined threshold value using AutoQuant X image deconvolution software (Media Cybernetics, Rockville, MD, USA). 2.4 Quantification of the mitochondrial network structure The mitochondrial network structure in 5 µm thin liver tissue sections was visualized by staining with MitoTracker® Deep Red (Molecular Probes) (1:20000 dilution) for 30 min at 37°C. The dye is detectable at a wavelength of 514 nm. The degree of homogeneity of the mitochondrial network structure was determined as the amount of pixels with a higher intensity than a defined threshold value using AutoQuant X image deconvolution software (Media Cybernetics). 4

2.5 Culture of primary hepatocytes Hepatocytes were isolated as described [23] and seeded on collagen-coated dishes and cultured in Williams Medium E supplemented with fetal calf serum (10%), penicillinstreptomycin (1%), glutamine (2.5%), insulin (0.5%) and dexamethasone (0.01%). 2.6 Mitochondrial membrane potential and morphology 48 h after seeding, hepatocytes were stained with MitoTracker® Green (Molecular Probes) (1:10000 dilution) for 30 min at 37°C and thereafter with TMRE (Molecular Probes) (1:10000 dilution). Cells were washed with PBS, incubated for 1 h at 37°C in PBS and finally were cultured in modified Williams Medium E. MitoTracker® Green is detectable at a wavelength of 519 nm. To determine mitochondrial morphology, the mean elongation of mitochondria in fluorescence images was determined using AutoQuant X image deconvolution software (Media Cybernetics). In addition, mitochondria with a loop-shaped structure were counted per cell area using FV1000 software (Olympus). TMRE fluorescence at a wavelength of 578 nm appeared in mitochondria according to their membrane potential. To determine the membrane potential of mitochondria in hepatocytes a plug-in of the ImageJ software (Wright Cell Imaging Facility, Toronto, ON, Canada) was used. For quantification, overlap coefficients were calculated by co-localization analyses of the TMRE and MitoTracker® Green fluorescence.

2.7 Mitochondrial dynamics Mitochondrial dynamics were investigated as described [24] using the green-to-red photoswitchable protein Dendra2. For this purpose, hepatocytes were transfected with Dendra2-Mito using PromoFectine Hepatocyte (PromoCell, Heidelberg, Germany) according to the manufacturer’s instructions. Time-lapse series were recorded with a FVMPE-RS multiphoton microscope (Olympus) additionally equipped with a UV laser for photoconversion of Dendra2. 2.8 ATP and ADP measurements ATP content in hepatocytes was measured with the ATPlite® luminescence ATP detection assay system (PerkinElmer, Hamburg, Germany) 48 h after seeding. Luminescence was determined at 562 nm with a VICTOR3™ Multilabel Counter model 1420 (PerkinElmer). For determining the ADP content, the sum of the ATP content and the ADP content was measured by conversion of ADP to ATP with pyruvate kinase from rabbit muscle (Sigma-Aldrich, Munich, Germany) and phospho(enol)pyruvic acid tri(cyclohexylammonium) salt (Sigma-

5

Aldrich). Quantification of protein content was performed using a QuantiProTM BCA assay kit (Sigma-Aldrich) and protein standard (Micro standard, liquid, Sigma-Aldrich). 2.9 Enzyme activity assay COX activity was measured with a complex IV rodent enzyme activity microplate assay kit (Abcam, Cambridge, UK). Immunocapturing of cytochrome c oxidase was performed according to the manufacturer’s instructions. Subsequently, activity was measured with a VICTOR3™ Multilabel Counter model 1420 (PerkinElmer). A kinetic program was used to measure absorbance of cytochrome c at 550 nm and 30°C for 153.5 min. The measurement interval was set to 94 sec. To determine the activity, the rate of the slope (OD/min) was calculated between 470 sec and 4135 sec by linear regression. 2.10 Mitochondrial respiration Oxygen consumption rates of hepatocytes were measured using the Seahorse XF Cell Mito Stress Test and the Seahorse XFe96 Analyzer (Seahorse Bioscience, Agilent Technologies, Waldbronn, Germany). By stepwise addition of oligomycin, carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) and finally antimycin A and rotenone the following parameters were calculated: basal respiration, maximal respiration and ATP production. 10000 cells per well were analyzed, as confirmed by counting DAPI-stained nuclei after the experiment.

2.11 Gene expression analyses Hepatocytes were homogenized in QIAshredder Mini Spin Columns (Qiagen, Hilden, Germany) by centrifugation (2 min, 12000 rpm). RNA was isolated and purified using an RNeasy® Mini Kit (Qiagen). cDNA was synthesized using the Maxima™ First Strand cDNA synthesis kit for RT-qPCR (Thermo Scientific, Darmstadt, Germany). RNA solutions containing the probes of the cDNA synthesis kit were placed in a thermocycler (Labcycler, SensoQuest, Göttingen, Germany) programmed at 25°C for 10 min, followed by 50°C for 15 min and by 85°C for 5 min. For real-time PCR, cDNA solutions containing TaqMan Universal Master Mix (Applied Biosystems, Darmstadt, Germany) and the corresponding TaqMan® Gene Expression Assay (Applied Biosystems) of primer and gene probe (Table 1) were amplified and detected using a 7900HT Fast Real-Time System (Life Technologies, Darmstadt, Germany). The PCR system was programmed at 50°C for 2 min, followed by 95°C for 10 min and by 40 repeats of the steps 95°C for 15 sec and 60°C for 1 min. GAPDH served as a housekeeping gene for nuclear-encoded genes and RNR2 for mitochondrial6

encoded genes. Gene expression values were calculated with the SDS RQ Manager 1.2 software (Life Technologies). 2.12 mtDNA copy number DNA from liver tissue was isolated with the QIAamp DNA Mini Kit (Qiagen). The mtDNA copy number in liver was determined using the NovaQUANTTM Mouse Mitochondrial to Nuclear DNA Ratio kit (Novagen, Madison, WI, USA). Specific mitochondrial and nuclear genes were quantified by SYBR Green based real-time PCR. The 7900HT Fast Real-Time System (Life Technologies) was programmed at 95°C for 10 min, followed by 40 repeats of the steps 95°C for 15 sec and 60°C for 1 min. 2.13 Western blot analyses 40 µg of cellular proteins were separated by SDS-PAGE and blotted onto Roti®-Fluoro PVDF membrane (Carl Roth, Karlsruhe, Germany). Membranes were probed for one hour at room temperature with anti-DNM1L (Abnova, Teipei City, Taiwan, PAB12447, 1:1500), anti-MFN2 (Proteintech, Rosemont, IL, USA, 12186-1-AP, 1:500), anti-SOD2 (Abcam, ab13533, 1:5000), anti-PCX (Abcam, ab126707, 1:1000) and anti-actin (New England Biolabs, Frankfurt, Germany, 4970, 1:1000) antibodies. Immunoreactive bands were visualized using IRDye 680, IRDye 800 CW fluorescence-labelled secondary antibodies and analyzed via the Odyssey imaging system. Densitometry measurements of bands were performed using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA) and normalized to actin expression. 2.14 Immunofluorescence staining Hepatocytes were fixed with 4% formaldehyde for 15 minutes and permeabilized with 0.2% Tween 20 for 5 minutes in phosphate-buffered saline. Cells were stained for one hour with anti-Parkin (Santa Cruz, CA, USA, sc-32282, 1:100), anti-TOMM20 (Abcam, ab186735, 1:100) and anti-LC3 (Sigma-Aldrich, L8918, 1:100) antibody. Positive signals were visualized using Alexa488 or Alexa647 (Abcam, 1:250). The samples were counterstained with DAPI and mounted using VECTASHIELD® mounting medium (Vector Laboratories, Peterborough, UK). Images were generated using FV1000 software (Olympus) and the degree of co-localization was investigated using AutoQuant X image deconvolution software (Media Cybernetics). 2.15 Quantification of Autophagy Autophagic activity was determined in live hepatocytes with and without rapamycin (500 nmol/l) and chloroquine (30 µmol/l) treatment for 18 h using the CYTO-ID® Autophagy Detection Kit (Enzo Life Sciences, Exeter, UK) in line with [25]. The cationic amphiphilic 7

tracer exhibits bright green fluorescence when incorporated into pre-autophagosomes, autophagosomes, and autolysosomes. Cell nuclei were counterstained with Hoechst 33342. The degree of autophagy was quantified in fluorescence images as the number of autophagosomes per cell using FV1000 software (Olympus). 2.16 Liver histology and measurement of collagen content Liver histology was assessed using hematoxylin and eosin and Goldner’s trichrome stains in paraffin-embedded sections using standard methods [26]. Images were taken with a cellR/Olympus IX81 (Olympus) inverted microscope system equipped with a CCD camera. A blinded assessment was made by two independent observers according to the criteria proposed by Kleiner et al. [27] and Hübscher [28]. In addition, the collagen content of 20 mg mouse liver was measured with a hydroxyproline assay kit according to the manufacturer’s instructions (MAK008, Sigma-Aldrich, St. Louis, MO, USA). 2.17 Statistical analysis Statistical analyses were performed using the Prism® 5.00 analysis program (GraphPad, Witzenhausen, Germany) and the results are presented as means ± SEM. Differences were examined using Student’s t test or ANOVA/Bonferroni’s correction as indicated, and p values <0.05 were considered to be statistically significant.

3. Results 3.1 Accumulation of ROS and development of ballooning degeneration, infiltration and fibrosis during aging in liver of mtNOD and control mice Controls and mtNOD mice showed an age-dependent increase in mitochondrial ROS generation in the liver from the age of 3 to 9 months (Fig. 1A-C). Beyond 9 months ROS generation decreased in controls but remained consistently high in mtNOD mice. ROS was detected by intraperitoneal injection of MitoSOX™ into living animals one hour before euthanization. Because the liver was frozen in liquid nitrogen immediately afterwards, this use of MitoSOX™ together with observer-independent quantification of the fluorescence images supplies reliable results regarding mitochondrial ROS. Because of the known limitations of hydroethidine [29], on which the probe MitoSOX™ is based, quantified values (Fig. 1B) did not show mitochondrial superoxide formation in absolute terms, but did allow comparison of ROS generation between mouse strains during aging. Our ROS measurements are not based simply on differences in mtDNA, as confirmed by co-injection of the LC50 of antimycin A, which significantly increased mitochondrial ROS in 3-month-old control mice (Fig. 1C). Control and mtNOD mice older than 18 months showed distinct liver pathologies 8

(Fig 1D, E). Whereas liver steatosis was not the main cause of the alterations, mtNOD mice developed a significantly higher degree of ballooning degeneration (Fig. 1F). Inflammation (Fig. 1G) and fibrosis (Fig. 1H) scored lower in absolute terms, but were also significantly higher in mtNOD than in control mice older than 18 months. Significant differences in fibrosis were confirmed by hydroxyproline measurements, indicating higher collagen content in the liver of mtNOD mice (Fig. 1I). Furthermore, accumulation of Mallory-Denk bodies was higher in mtNOD than in control mice (Fig. 1J). 3.2 Age-dependent mitochondrial morphologies in liver of mtNOD and control mice In mtNOD and control mice continuous loss of homogeneous mitochondrial network structure in the liver was observed with aging up to the age of 12 months. However, in mtNOD mice this process had a much earlier onset (Fig. 2A-D), with significantly more areas of accumulated mitochondria detected in liver tissue sections from 6-month-old mtNOD mice compared with controls. Levels of accumulated mitochondria were reduced in controls aged 18 months or older but remained high in old mtNOD mice. Significantly greater numbers of loop-shaped mitochondria were detected in 3- and 6-monthold mtNOD mice compared with controls (Fig. 2E-G). These mitochondrial structures also became visible at a later age in control mice. The dynamics of loop-shaped mitochondria were further investigated using photoconversion and time-lapse imaging techniques. Unlike fragmented and elongated mitochondria, the loop-shaped structures did not participate in fission-fusion processes (Fig. 2H). 3.3 mtDNA copy number and gene expression of COX subunits in hepatocytes of mtNOD and control mice At 3 and 6 months the gene expression pattern of COX subunits was comparable in hepatocytes of both strains; the exception was COX6b1 expression, which was significantly higher in mtNOD mice (Fig. 3A-E). At 9 months gene expression levels for all COX subunits fell in controls, whereas those of subunits COX1, COX3 and COX6a1 remained unchanged in mtNOD mice. At 12 months gene expression of COX3, COX6a1 and COX6b1 was significantly higher in mtNOD mice compared with controls. Mitochondrial transcription factor A (TFAM) is an activator of mitochondrial transcription and regulates mitochondrial genome copy number. In both strains there was an age-dependent increase in TFAM gene expression, but with minimum values at 9 months (Fig. 3G). A comparable age-dependent pattern for mtDNA copy number confirmed this result (Fig. 3H). However, the strong increases detected in 12-month-old mtNOD mice compared with controls were more pronounced for mtDNA copy number than for TFAM values. 9

3.4 Expression of proteins involved in mitochondrial metabolism, regulation, antioxidative response and autophagy in hepatocytes of mtNOD and control mice Pyruvate carboxylase (PCX) is an enzyme located in mitochondria that catalyzes the carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in the citric acid cycle. Accordingly, increased gene expression levels of PCX in hepatocytes can be used as an indication of enhanced mitochondrial metabolism. Gene expression levels of PCX decreased in controls and increased in mtNOD mice with age (Fig. 4E). Uncoupling protein 2 (UCP2) channels anions through the inner mitochondrial membrane, and thereby plays a role in the control of free radical production by reducing mitochondrial membrane potential [30]. UCP2 gene expression increased with age in controls but decreased slightly in mtNOD mice (Fig 4F). The gene expression pattern of UCP2 is inversely proportional to that of PCX. At 12 months PCX protein expression was significantly higher in mtNOD mice than in control mice (Fig. 4J,K). Superoxide dismutases (SOD) catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. Isoform 1 (SOD1) is located in the cytoplasm and isoform 2 (SOD2) in mitochondria [10]. Protein deglycase (DJ1), also known as Parkinson disease protein 7, stimulates SOD1 activity by delivering copper [31]. Catalase (CAT) catalyzes the decomposition of hydrogen peroxide to water and oxygen [11]. Age-dependent gene expression of SOD1, CAT and DJ1 showed similar patterns in both mtNOD and control mice (Fig. 4A-C). Gene expression in those cases decreased from 3 to 9 months and then increased strongly at 12 months. The gene expression pattern of SOD2 was different (Fig. 4D), with controls showing peak expression at 9 months and comparable expression levels at 3, 6 and 12 months. By contrast, mtNOD mice showed a continuous increase in expression from 3 to 12 months. Higher SOD2 protein expression compared with controls at 12 months was confirmed by western blot analysis (Fig. 4J,L). PTEN-induced putative kinase 1 (PINK1) and PARK2 encoding Parkin specifically initiate turnover of mitochondria with a depolarized membrane potential [32]. By phosphorylating TNF receptor associated protein 1 (TRAP1), PINK1 protects cells against apoptosis [33]. We observed an age-dependent decrease in gene expression of both PINK1 and TRAP1 in controls and, conversely, an increase in their expression in mtNOD mice (Fig. 4G-I). Parkin gene expression was highest at 9 months in both strains (Fig. 4H). At 12 months it remained consistently high in mtNOD mice but had decreased significantly in controls. Autophagy was comparable in hepatocytes of 3-month-old mice but significantly lower in 12-month-old mtNOD mice than in control mice (Fig. 4M). Whereas autophagic activity was inducible by rapamycin in hepatocytes of young mice, resulting in significantly higher levels in 3-month-old mtNOD mice than in controls, 10

hepatocytes of old mice remained unaffected (Fig. 4M). In addition, LC3-positive aggregates co-localized with mitochondria in control hepatocytes but not in mtNOD mice (Fig. 4N,O). Immunofluorescence analyses confirmed higher Parkin expression in 12-month-old mtNOD mice (Fig. 4P,Q), but co-localization with mitochondria was significantly lower compared with control cells (Fig. 4R). 3.5 Mitochondrial membrane potential, ATP generation, COX activity and O2 consumption rate in hepatocytes of mtNOD and control mice Mitochondria in isolated hepatocytes of mtNOD mice showed a significantly lower membrane potential compared with controls at 3 and 6 months (Fig. 5A-D). However, the number of mitochondria with a high membrane potential (overlap value, Fig. 5D) increased stepwise in mtNOD mice between 6 and 12 months, whilst there was a slight decrease between 6 and 12 months in controls. Finally, at 12 months, mitochondrial membrane potential in mtNOD mice was significantly higher than in controls. The age-dependent pattern of mitochondrial membrane potential was similar to that of respiratory chain activity, measured as activity of cytochrome c oxidase (COX) (Fig. 5E). The ATP/protein, ADP/protein, and pooled ATP + ADP/protein ratios in controls at 3 months were higher than in mtNOD mice (Fig. 5F-H). However, at 12 months this relationship was reversed and the ratios were significantly higher in mtNOD mice compared with controls. The ratios decreased with age in controls and increased with age in mtNOD mice. The ATP/ADP ratio was somewhat higher in mtNOD mice than in controls. At 12 months there was an intensive and significant peak increase in the ATP/ADP ratio in control mice exceeding the value in mtNOD mice, due to a fall in ADP (Fig. 5I). However, the total amount of ATP and that of nucleoside phosphates was higher in mtNOD mice compared with controls. All investigations were performed after 48 h of hepatocyte culture to bring the results into line with the transfection experiments. The values obtained were confirmed by a representative measurement after 24 h (Fig. 5J). Moreover, it was demonstrated that ATP production was abolished by co-treatment with FCCP for 1 h (Fig. 5J). In addition, oxygen consumption rates were determined in 12-month-old animals, showing higher values in mtNOD mice than in control mice (Fig. 5K-M). Altogether, mitochondrial activity increased in mtNOD mice over the period from 3 to 12 months, but it decreased in controls. 3.6 Expression of proteins regulating mitochondrial fission and fusion in hepatocytes of mtNOD and control mice The optic atrophy 1 (OPA1) protein regulates inner membrane fusion, whereas mitofusin-1 and mitofusin-2 (MFN1, MFN2) are important for fusion of the outer mitochondrial 11

membrane. Mitochondrial fission is regulated by dynamin-1-like protein (DNM1L), mitochondrial fission protein 1 (FIS1) and mitochondrial fission factor (MFF) [34, 35]. Gene expression levels of both MFN1 and MFN2 in control mice showed an age-dependent decrease by 12 months, whereas OPA1 expression recovered at 12 months after a minimum level at 9 months (Fig. 6A-C). In mtNOD mice all fusion proteins showed the same agedependent pattern with maximum values at 6 and 12 months. At 12 months MFN2 protein expression was significantly higher in mtNOD mice than in control mice (Fig. 6I, K). The pooled gene expression pattern of fusion regulators revealed an age-dependent decrease in control mice, but was more variable in mtNOD mice, with maximum values at 6 and 12 months (Fig. 6G). Gene expression levels of the fission proteins DNM1L and FIS1 in mtNOD mice both increased over time to 9 months; however, at 12 months DNM1L decreased whereas FIS1 increased further (Fig. 6D,E). Significantly lower DNM1L protein expression compared with controls at 12 months was confirmed by western blot analysis (Fig. 6J, L). In control mice gene expression of DNM1L was constant, whereas FIS1 showed an oscillating pattern with maxima at 3 and 9 months. Gene expression of MFF showed comparable increases in both strains from 3 to 9 months followed by a decrease at 12 months (Fig. 6F). The pooled gene expression pattern of the three fission proteins was comparable, with a maximum at 9 months in both strains (Fig. 6H). In light of the age-dependent gene expression patterns of fission and fusion proteins, we suggest that mitochondria in hepatocytes of control mice are more elongated in younger animals, with a trend toward fragmentation during aging, as illustrated in Fig. 6M. By contrast, in mtNOD mice an oscillating pattern can be expected, with highly elongated mitochondria present at 12 months. 3.7 Mitochondrial network structure in hepatocytes of mtNOD and control mice In line with the gene expression patterns, hepatocytes of control mice revealed a decrease in mitochondrial elongation with age (Fig. 7). By contrast, mitochondria in hepatocytes of mtNOD mice showed an opposite trend toward elongation with age (Fig. 7).

4. Discussion Intense interest is currently focused on how ROS influence organ aging and contribute to organ failure and metabolic diseases. In this study, we investigated age-dependent ROS production in the liver of two mouse strains. The C57BL/6NTac mouse strain served as control, whereas the conplastic C57BL/6NTac-mtNODLtJ strain (mtNOD) with a point mutation 12

in the mitochondrial-encoded COX 3 gene represented the situation of mitochondrial restriction. Our comprehensive analyses revealed age-dependent interplay between ROS generation, antioxidative defense, mitochondrial metabolism and mitochondrial morphology, with different patterns for mtNOD and control mice. According to the gradual ROS response hypothesis, increased ROS generation results in agedependent cellular damage. Initially, ROS are signaling molecules modulating stress response pathways. However, with aging, the level of ROS becomes maladaptive and ROS toxicity starts to contribute to irreversible cellular damage that cannot be combated by ROS-dependent stress pathways. This can induce a vicious cycle in which ROS-dependent damage leads to further increases in ROS generation [36]. In line with this hypothesis, ROS production increased in both strains up to the age of 9 months. This suggests that at 9 months ROS reached a limit, above which they would be more harmful than useful. Lowering of ROS generation in mice older than 9 months was observed only in controls whereas ROS levels were maintained in mtNOD mice. Thus, both strains appear to have different adaptive mechanisms in the liver which, however, ultimately prevent a toxic run-away process with severe ROS generation. There is a direct interaction between DJ1 and SOD1 in the antioxidative response [37]. Accordingly, age-dependent gene expression patterns of both enzymes were similar and showed the same trend in both strains in terms of long-term adaptation. A marked increase was noted at 12 months compared with 6 and 9 months and this might be a response to the critically increased ROS levels at 9 months in both strains. Only SOD2 gene expression differed from the other antioxidative enzymes in a strain-specific manner, with a continuous increase from 3 to 12 months in mtNOD mice and a maximum value at 9 months in controls. The SOD1 gene is often constitutively expressed and is not as easily inducible as SOD2 [38]. Because expression of superoxide dismutase was decreased at 12 months in controls but not in mtNOD mice, we were able to demonstrate a strain-specific antioxidative response according to ROS generation. In addition, our results are consistent with the “uncoupling to survive” theory [39]. ROS-induced UCP2 activation has been implicated in minimizing ROS production from the electron transport chain [40]. Increased gene expression of UCP2 correlates with reduced ROS production in our study. The decreased mitochondrial metabolism rate with aging might be a further important adaptation in controls to inhibit the run-away process in ROS generation after 9 months. The high ATP/ADP ratio at 12 months in controls is indicative of COX inhibition [41]. However, it has also been shown that a decreased rate of metabolism does not necessarily prevent 13

oxidative damage [42]. mtNOD mice showed an opposite trend in age-dependent adaptation, with higher mitochondrial respiration at 12 months. Notably, although metabolism rate was lower in young mtNOD mice, ROS production was in the same range compared with controls. Thus, ROS production does not correlate directly with metabolism rate and may also be a result of the COX3 mutation in mtNOD mice. Our in vivo MitoSOX™ ROS measurements are in agreement with those from a recent study using MitoB-based HPLC quantification, demonstrating that in vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice [43]. It has been shown that oxidative stress leads to mitochondrial fragmentation of human lung adenocarcinoma cells (ASTC-a-1) and African green monkey SV40-transformed kidney fibroblast cells (COS-7) [44] and of young human endothelial cells (HUVECs) in tandem with decreased mitochondrial membrane potential [45]. In hepatocytes of controls this effect was observed at 9 months when ROS generation was highest. Oxidative stress most probably initiates mitochondrial fragmentation to segregate damaged structures in the mitochondrial network. Finally, initiation of mitophagy clears the cellular pool of damaged mitochondria [13]. The significant increase in gene expression of Parkin at 9 months substantiates this hypothesis. The low membrane potential of damaged fragmented mitochondria should lead to an accumulation of PINK1 at the outer mitochondrial membrane. Accumulated PINK1 selectively recruits Parkin to the depolarized mitochondria which initiates mitophagy [32]. Gene expression of both PINK1 and Parkin is balanced, preventing unwanted mitophagy of healthy mitochondria. Gene expression analyses of mitochondrial fusion and fission genes as well as morphological analyses by fluorescence microscopy indicated an increase in mitochondrial elongation in mtNOD mice at 9 months, possibly as a response to increased oxidative stress. In senescent HUVECs it has been shown that mitochondrial hyperfusion takes place after stress [45]. Other studies have demonstrated that elongation of mitochondria makes cells less vulnerable to apoptotic stimuli [46-48]. Elongated and interconnected mitochondria allow rapid distribution and exchange of molecules. Furthermore, slightly increased levels in gene expression of PINK1 initiate an apoptosis-protective pathway [45]. In such circumstances, PINK1 protects mitochondria against oxidative stress-induced cell death by phosphorylating TRAP1 which in turn suppresses cytochrome c release from mitochondria and concomitant apoptosis [33]. Our age-dependent gene expression analyses revealed a slight increase in PINK1 and a significant increase in TRAP1 in mtNOD mice, thus confirming the postulated pathway. However, the marked increase in mitochondrial elongation did not appear until the age of 9 months although gene expression of mitochondrial 14

fusion proteins was highest at 6 months. Our results suggest a complex regulatory response triggered by both the loss of homogeneous mitochondrial network structure and increasing ROS generation. High gene expression levels of Parkin together with low mtDNA copy numbers in hepatocytes at 9 months are indicative of mitophagic activity in the mtNOD mouse strain. However, high mitochondrial membrane potential and pronounced mitochondrial elongation appear to confer protection against excessive mitophagy and apoptosis, as was confirmed at the age of 12 months. Autophagy and co-localization between LC3 and mitochondria was lower in mtNOD mice than in controls, even though Parkin expression was higher. Autophagic activity could not be induced by the starvation mimic rapamycin in old mice of either strain, indicating reduced capacity for adaptation in old age. In contrast, autophagic activity was highly inducible in young mice of both strains. Interestingly, basal autophagy was higher in aged liver than in young liver of controls. A similar finding has recently been reported in the kidney [49]. In mtNOD mice this increase with age was absent, suggesting reduced cellular clearing activity of damaged structures. Different protective mechanisms against increased oxidative stress have been described, depending on cell age [45]. This might imply that hepatocytes of controls and mtNOD mice differ in terms of physiological age and degeneration. In fact, more mitochondria with a loopshaped structure were detected in hepatocytes of young mtNOD mice compared with controls at the same age. We showed that these mitochondria were not participating in fission and fusion processes. Furthermore, age-dependent loss of the homogeneous mitochondrial network structure commenced earlier in mtNOD mice compared with controls. The mitochondrial network structure of 18-month-old controls was even more homogeneous than at 12 months. This suggests that hepatocytes from controls were at least in part able to regenerate their mitochondrial network structure. In contrast, in hepatocytes from mtNOD mice carrying the COX3 mutation, stronger signs of aging were detectable. Ballooning degeneration, fibrosis and inflammatory infiltration were also detectable in control mice, but these were less pronounced than in mtNOD mice. In particular, large numbers of MalloryDenk bodies were detectable in mtNOD mice; this finding might be directly related to mitochondrial aggregation and also to ROS-induced protein degradation [27, 28, 50]. COX in mammals consists of 14 subunits. The mitochondrial-encoded subunits COX1, COX2 and COX3 form the catalytic core of the enzyme, while the remaining nuclear-encoded subunits are important for the stability and dimerization of the enzyme complex [51]. Down-regulation of COX3 and COX1 gene expression in 9-month-old compared with 6month-old control animals was remarkable. The reduced expression of the catalytic core 15

subunits necessarily entails a reduced rate of oxidative phosphorylation in the cells. In contrast, down-regulation of COX1 and COX3 was not detected in the mtNOD strain at 9 months. These data suggest that oxidative phosphorylation in controls was lower than in mtNOD mice at 9 months, a finding that is in line with the observed lower membrane potential and COX activity. COX1 and COX3 gene expression was up-regulated again in 12-month-old control animals. At that age, compared with 9-month-old animals, mtDNA copy number and TFAM gene expression were also elevated. This suggests a further response mechanism to the high ROS generation at 9 months. TFAM gene expression and the resultant mtDNA copy number also affect the age-dependent gene expression of nuclear-encoded COX subunits. The low rate of TFAM expression and the low mtDNA copy number in 9-month-old animals are indicative of generally reduced mitochondrial mass. The gene expression pattern of the nuclear-encoded subunit COX6a1 in control animals was similar to that of COX3. Due to its close location to COX3, COX6a1 is important for the stability and activity of the catalytic core [52]. Accordingly, the expression rates of COX3 and COX6a1 are directly interdependent and a defect in COX6a1 reduces COX activity and leads to reduced ATP synthesis. By contrast with controls, high COX3 and COX6a1 gene expression despite low TFAM expression was observed in 9-month-old mtNOD mice, indicating specific adaptation in the conplastic mouse strain. COX6b1 gene expression was in line with TFAM gene expression and was comparable in both strains. It is postulated that TFAM is located in the nucleus and regulates gene expression [53]. COX6b1 is important in stabilizing the dimeric structure of COX [54]. COX5a is a regulatory subunit that prevents ATP inhibition of COX by binding thyroid hormones [41]. The age-dependent gene expression pattern of COX5a was also comparable in both strains. Thus, the mutation in COX3 seems to affect only subunits in close proximity, but not those located peripherally in the complex.

5. Conclusion In conclusion, there is concurrent increased ROS generation up to a critical value at 9 months in the liver of both C57BL/6NTac strains. However, in hepatocytes of mtNOD mice but not of controls, the mutation in COX3 imposes changes in mitochondrial morphology and metabolism during aging. Whereas oxidative stress in controls initiates mitochondrial fragmentation, mitophagy and a decreased metabolic rate, in mtNOD mice it triggers mitochondrial elongation and an increased metabolic rate. Finally, controls are able to reduce 16

ROS generation and to maintain the vitality of the mitochondrial network in older age. By contrast, mtNOD mice are only able to keep ROS levels constant, rendering them vulnerable to further stressors. Our study explains mitochondrial aging in the liver of control mice and suggests that ROS is a pivotal regulator of health control. The conplastic mtNOD mouse strain represents an interesting model, demonstrating that mutations in mtDNA contribute to more pronounced liver ballooning degeneration and possibly accelerate liver failure in the event of overnutrition and alcohol consumption in older age.

Acknowledgments This work was supported by the BMBF GERONTOSYS 2 project ROSAge, the DFG ExC306 (S.M. Ibrahim) and the DDG (S. Baltrusch). We are grateful to A. Kott, R. Waterstradt, S. Giers, I. Klamfuß and L. Wengler for technical assistance, to Dr. J. Brenmöhl (Leibniz Institute for Farm Animal Biology, Institute for Genome Biology, Dummerstorf, Germany) for help with the Seahorse XF Cell Mito Stress Test and to D. Beattie (freelance medical writer/UK) for editorial assistance.

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Fig. 1. Age-dependent mitochondrial ROS accumulation persists beyond 9 months in liver tissue sections of mtNOD mice but not in controls and resulted in more pronounced ballooning degeneration, infiltration and fibrosis. (A) Red spots indicate MitoSOX-stained ROS in liver tissue (examples marked by yellow arrows). Nuclei were stained with DAPI (blue). Scale bar: 30 µm. (B) For quantification, we calculated the number of pixels with a higher intensity than a defined threshold value in the fluorescent channel. Age-dependent increases in ROS production were detected in controls and mtNOD mice. In both strains ROS generation was highest at 9 months. ROS decreased in controls older than 9 months but not in mtNOD mice (n = 15 - 25 sections from 3 - 5 animals per age stage and mouse strain). Data for 18- and 24-month-old animals were pooled (18+). Values are expressed as mean ± SEM (***, $$$, ### p < 0.001; 1-way ANOVA/Bonferroni’s test). (C) Antimycin A significantly increased ROS in 3-month-old control mice (n = 20 sections). Values are expressed as mean ± SEM (*p < 0.05; Student’s t test). Hepatocyte ballooning (F) and inflammatory infiltration (G) and Mallory-Denk bodies (J) were calculated from liver sections stained with hematoxylin-eosin (D), and fibrosis (H) was calculated from sections stained with Goldner’s trichrome (E). Scores (F-H, J) represent mean ± SEM (*p < 0.05; Student’s t test) from 4 animals each older than 18 months. Magnification: 200x and zoomed section. Measurements of hydroxyproline (I), an additional marker of fibrosis, were performed from 20 mg liver from the same animals. Values are expressed as mean ± SEM (*p < 0.05; Student’s t test).

Fig. 2. Mitochondrial network inhomogeneity with immobile loop-shaped mitochondria appears at an earlier age stage in liver tissue sections of mtNOD mice compared with controls. (A, C) Liver tissue sections were stained with MitoTracker Deep Red (red) and DAPI (blue). Red spots reveal accumulated mitochondria in the network (examples marked by yellow arrows). Scale bar: 75 µm. (B) For quantification, we calculated the number of pixels with a higher intensity than a defined threshold value (green lines) in the MitoTracker Deep Red channel. (D) Age-dependent loss of homogeneous mitochondrial structure occurred in both strains, but changes in the mitochondrial network were detected earlier in mtNOD mice (n = 24

15 - 40 sections from 3 - 8 animals per age stage and mouse strain). Data for 18- and 24month-old animals were pooled (18+). Values are expressed as mean ± SEM (* p < 0.05, **, ## p < 0.01, $$$ p < 0.001; 1-way ANOVA/Bonferroni’s test). (E) The number of loopshaped mitochondria (red arrows) was detected in living hepatocytes stained with MitoTracker Green. Scale bar: 20 µm. (F, G) Analysis of the number of loops per area revealed a significant difference between controls and mtNOD mice aged 3 - 6 months (n = 20 - 50 images from 4 - 10 animals). Values are expressed as mean number of loops/µm2 ± SEM; * p < 0.05, Student’s t test). (H) The dynamics of differently shaped mitochondria were analyzed by means of selective mitochondrial staining with Dendra2, a green-to-red photoswitchable fluorescent protein. By radiation with UV light (blue arrows), green Dendra2 in a straight mitochondrion (red arrow) and a circular mitochondrion (yellow arrow) was photoconverted to the stable red fluorescent Dendra2. Morphological changes and network fusion of the straight (but not of the circular) mitochondrion was detected over 2-minute timelapse recording (1 - 9). Scale bar: 5 µm.

Fig. 3. Mitochondrial copy number and gene expression patterns of COX subunits and TFAM in hepatocytes differ between mtNOD and control mice. Age-dependent gene expression of the COX subunits COX1 (A), COX3 (B), COX5a (C), COX6a1 (D) and COX6b1 (E) and TFAM (G) was analyzed. In addition, mtDNA copy number (H) in hepatocytes was calculated (n = 3). Values are expressed as mean ± SEM (*, # p < 0.05; **, $$, ## p < 0.01; ***, $$$, ### p < 0.001; 1-way ANOVA/Bonferroni’s test). With the exception of COX5a gene expression, all COX subunits showed a different age-dependent expression pattern in mtNOD mice compared with controls. The mtDNA copy number was significantly increased at 12 months in mtNOD mice compared with controls. Gene expression of TFAM revealed a similar pattern, with TFAM expression and mtDNA copy number in both strains being lowest at 9 months. (F) The structure of the bovine heart illustrates localization of the subunits in the cytochrome c oxidase dimer. Data [55] were taken from the RSCB protein data bank and processed using Jmol (Jmol Development Team, Version 13.0.4). COX3 (III, green), COX6a1 (VIa, red) and COX6b1 (VIb, cyan) are located centrally, whereas COX1 (I, yellow) and COX 5a1 (Va, orange) are located peripherally.

Fig. 4. Expression analyses of proteins pivotal in mitochondrial metabolism, regulation, antioxidative response and mitophagy revealed specific signs of adaptation in hepatocytes of mtNOD mice. Gene expression of the antioxidative enzymes SOD1 (A), DJ1 (B) and CAT 25

(C) decreased from 3 to 9 months and increased from 9 to 12 months in both strains. (D) SOD2 gene expression peaked at 9 months in the controls, but increased continuously from 3 to 12 months in mtNOD mice. (E) Gene expression levels of PCX decreased in controls and increased in mtNOD mice as a function of age. (F) Gene expression of UCP2 increased significantly in controls at 12 months, whereas a decrease was noted at the same time point in mtNOD mice. Gene expression of PINK1 (G) and TRAP1 (I) generally decreased with age in controls but increased in mtNOD mice. (H) Gene expression of Parkin showed peak levels at 9 months in controls and in mtNOD mice. By 12 months Parkin gene expression had declined significantly in controls but remained high in mtNOD mice. Values (n = 3) are expressed as mean ± SEM (*, $, # p < 0.05; **, $$, ## p < 0.01; ***, $$$, ### p < 0.001; 1-way ANOVA/Bonferroni’s test). PCX (K) and SOD2 (L) protein expression was investigated in 12-month-old mice by western blotting (J). Expression was quantified relative to actin expression. Values (K, L) represent mean ± SEM (*p < 0.05; Student’s t test) from 4 independent experiments. Autophagy was determined in 3- and 12-month-old control and mtNOD mice treated with and without rapamycin/chloroquine for 18 h using the CYTO-ID® Green detection reagent. For quantification, the number of autophagosomes per cell was counted (M). Values (n = 3) represent mean ± SEM (# p < 0.05; ***, $$$, ### p < 0.001; 1way ANOVA/Bonferroni’s test). Co-localization of LC3 (N, O) and Parkin (P, Q) with mitochondria was investigated by immunofluorescence. LC3 (green) co-localized with TOMM20-labeled mitochondria (red) in cells from 12-month-old control mice (N), but not in mtNOD mice (O) (white arrows). (P, Q) Parkin (green) expression is shown together with the TMRE-labeled mitochondrial network (red). Scale bars: 10 µm. Co-localization between Parkin and mitochondria was quantified. Values (R) represent mean ± SEM (*p < 0.05; Student’s t test) from 3 independent experiments.

Fig. 5. Age-dependent increase of mitochondrial membrane potential, COX activity, ATP generation and oxygen consumption rates in hepatocytes of mtNOD mice. After culturing for 48 h hepatocytes were stained with MitoTracker Green (A) and TMRE (B). Red TMRE fluorescence appears as a function of mitochondrial membrane potential. Co-localization analyses of the two channels were performed using ImageJ software to quantify mitochondrial membrane potential (C). Scale bar: 20 µm. (D) A high overlap value correlates with high mitochondrial membrane potential. The number of mitochondria with high membrane potential increased with age in mtNOD mice, but decreased slightly in controls (n = 30 - 90 images from 3 - 9 animals per age stage and mouse strain). (E) Activity of cytochrome c 26

oxidase in hepatocytes increased with age in mtNOD mice but decreased in controls (n = 9 from 3 animals per age stage and mouse strain). (F) A luminescence assay was used for ATP and ADP measurements. The ATP/protein ratio in controls was highest at 3 months. This ratio decreased significantly at 6 months before recovering at 12 months. In mtNOD mice the ATP/protein ratios were lower at 3 and 6 months compared with controls, but then increased significantly at 9 and 12 months. At 12 months the ATP/protein ratio was significantly higher than in controls. The ADP/protein ratio (G), ATP + ADP/protein ratio (H) and ATP/ADP ratio (I) were also calculated. Values (n = 3 – 4 animals per age stage and mouse strain) are expressed as mean ± SEM (*, $, # p < 0.05; **, $$, p < 0.01; ***, $$$, ### p < 0.001; 1-way ANOVA/Bonferroni’s test). For illustrative purposes the stability and specificity of ATP measurements were confirmed as ATP/protein ratios in 12-month-old hepatocytes cultured for 24 h and treated for one hour with and without FCCP (J). Oxygen consumption rates of 12month-old hepatocytes were measured using the Seahorse XF Cell Mito Stress Test and basal respiration (K), maximal respiration (L) and ATP production (M) were calculated. Values (n = 3 animals per mouse strain) are expressed as mean ± SEM (*p < 0.05; Student’s t test).

Fig. 6. Expression patterns of key regulators of mitochondrial fission and fusion indicate agedependent mitochondrial elongation in hepatocytes of mtNOD mice. Gene expression levels of the mitochondrial fusion proteins OPA1, MFN1 and MFN2 (A, B, C) and the mitochondrial fission proteins DNM1L, FIS1 and MFF (D, E, F) were determined as a function of age in mtNOD and control mice (n = 3 per age stage and mouse strain). (G) The pooled gene expression pattern of mitochondrial fusion proteins revealed a decrease with age in control mice, whereas mtNOD mice showed an oscillating pattern with high gene expression rates at 6 and 12 months and low rates at 3 and 9 months. (H) The pooled gene expression pattern of mitochondrial fission proteins was comparable in both strains, generally showing an increase from 3 to 9 months. Values are expressed as mean ± SEM (*, $, # p < 0.05; **, $$, p < 0.01; ***, $$$, ### p < 0.001; 1-way ANOVA/ Bonferroni’s test). MFN2 (I, K) and DNM1L (J, L) protein expression was quantified relative to actin expression in 12-month-old mice. Values (I, J) represent mean ± SEM (*p < 0.05, **p < 0.01; Student’s t test) from 3 independent experiments. (M) There is evidence to suggest that mitochondria in hepatocytes of 9- and 12-month-old controls are highly fragmented whereas those in 6- and 12-month-old mtNOD mice are highly elongated.

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Fig. 7. Age-dependent mitochondrial elongation in hepatocytes of mtNOD mice. Mitochondrial network structure was investigated in MitoTracker Green-stained hepatocytes. The original images (A) were processed with ImageJ (B) to create stronger edges between individual mitochondria. The processed images were then analyzed using AutoQuant algorithms (C). By individually detecting all mitochondria (D), the mean value of mitochondrial elongation in a single hepatocyte was then finally calculated from the original images (E). Scale bar: 20 µm. (F) NB: The greater the elongation level, the less the fragmentation of mitochondria in the network. At 9 months a reversal occurred between mtNOD and control mice. In control mice the previously more elongated mitochondrial network appeared more fragmented whereas in mtNOD mice the elongation level significantly increased. Values (n = 30 - 50 images from 3 - 5 animals per age stage and mouse strain) are expressed as mean ± SEM ($ p < 0.05; $$ p < 0.01; ***, ### p < 0.001; 1way ANOVA/Bonferroni’s test).

Table 1 Quantitative real-time PCR probes (Applied Biosystems)

28

CAT

Mm01340247_m1

COX1

Mm04225243_g1

COX3

Mm04225261_g1

COX5A

Mm00432638_m1

COX6A1 Mm01612194_m1 COX6B1 Mm00824357_m1 DJ1

Mm00498538_m1

DNM1L

Mm01342903_m1

FIS1

Mm00481580_m1

GAPDH

Mm99999915_g1

MFF

Mm00512718_m1

MFN1

Mm01289369_m1

MFN2

Mm01255785_m1

OPA1

Mm01349716_m1

PARK2

Mm00450187_m1

PCX

Mm00500992_m1

PINK1

Mm00550827_m1

RNR2

Mm04260181_s1

SOD1

Mm01700393_g1

SOD2

Mm00690588_m1

TFAM

Mm00447485_m1

TRAP1

Mm00446003_m1

UCP2

Mm00627599_m1

29

Highlights 

A COX3 mtDNA mutation affects ROS production and antioxidative response in liver

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Old mutant mice show increased metabolism and mitochondrial elongation/clustering

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Old control mice show regular metabolism and tend to mitochondrial fragmentation

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Liver ballooning degeneration is accelerated in conplastic C57BL/6NTac-mtNODLtJ mice

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31

32

33

34

35