Changes of Mitochondrial Ultrastructure and Function During Ageing in Mice and 2 Drosophila
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1 Changes of mitochondrial ultrastructure and function during ageing in mice and 2 Drosophila 3 Tobias Brandt1†, Arnaud Mourier2,3,4†, Luke Tain5, Linda Partridge5,6, Nils-Göran Larsson2,7, 4 Werner Kühlbrandt1* 5 6 1Max-Planck-Institute of Biophysics, Department of Structural Biology, Max-von-Laue-Str. 7 3, 60438 Frankfurt am Main, Germany 8 2Max Planck Institute for Biology of Ageing, Department of Mitochondrial Biology, Joseph- 9 Stelzmann-Str. 9b, 50931 Cologne, Germany 10 3Université de Bordeaux, Institut de Biochimie et Génétique Cellulaires UMR 5095, Saint- 11 Saëns, F-33077 Bordeaux, France. 12 4CNRS, Institut de Biochimie et Génétique Cellulaires UMR 5095, Saint-Saëns, F-33077 13 Bordeaux, France. 14 5Max Planck Institute for Biology of Ageing, Department of Biological Mechanisms of 15 Ageing, Joseph-Stelzmann-Str. 9b, 50931 Cologne, Germany 16 6Institute of Healthy Ageing, Department of Genetics, Evolution, and Environment, 17 University College London, Darwin Building, Gower Street, London WC1E 6BT, UK 18 7Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 19 Stockholm, Sweden 20 21 * Corresponding author 22 23 Author contributions. WK and NGL initiated and supervised the project. TB and AM 24 performed and analyzed experiments. NGL, LT and LP provided materials. WK and TB 25 interpreted the data and wrote the paper. 26 1 27 † These authors contributed equally to this work 28 2 29 Abstract 30 Ageing is a progressive decline of intrinsic physiological functions. We examined the impact 31 of ageing on the ultrastructure and function of mitochondria in mouse and fruit flies 32 (Drosophila melanogaster) by electron cryo-tomography and respirometry and discovered 33 distinct age-related changes in both model organisms. Mitochondrial function and 34 ultrastructure are maintained in mouse heart, whereas subpopulations of mitochondria from 35 mouse liver show age-related changes in membrane morphology. Subpopulations of 36 mitochondria from young and old mouse kidney resemble those described for apoptosis. In 37 aged flies, respiratory activity is compromised and the production of peroxide radicals is 38 increased. In about 50% of mitochondria from old flies, the inner membrane organization 39 breaks down. This establishes a clear link between inner membrane architecture and 40 functional decline. Mitochondria were affected by ageing to very different extents, depending 41 on the organism and possibly on the degree to which tissues within the same organism are 42 protected against mitochondrial damage. 43 3 44 Introduction 45 Mitochondria produce most of the ATP in non-photosynthetic eukaryotes, providing the 46 energy to drive a multitude of cellular processes. Mitochondria have an inner membrane, 47 which surrounds the matrix, and an outer membrane, which surrounds the inner membrane 48 and separates the mitochondrial compartments from the cytoplasm. In addition to a multitude 49 of soluble enzymes and ribosomes, the matrix houses the mitochondrial genome (mtDNA), 50 which in humans and flies encodes 13 hydrophobic subunits of the oxidative phosphorylation 51 system. Thus nearly all mitochondrial proteins are nuclear-encoded and imported into the 52 different mitochondrial compartments by a set of protein translocases (1). The oxidative 53 phosphorylation (OXPHOS) system consists of the respiratory chain complexes I to IV and 54 the mitochondrial F1Fo-ATP synthase, sometimes referred to as complex V. Complexes I to 55 IV transfer electrons from soluble electron donors to molecular oxygen. In this process, 56 complexes I, III and IV generate an electrochemical proton potential across the mitochondrial 57 inner membrane that is used by the ATP synthase to produce ATP by rotary catalysis (2). 58 Oxidative phosphorylation occurs mostly, if not entirely, in the deeply invaginated cristae of 59 the inner mitochondrial membrane (3,4). The ATP synthase forms long rows of dimers at the 60 tightly curved cristae ridges, while the respiratory chain complexes are confined to the 61 remaining membrane regions (5-7). 62 Ageing has long been linked to mitochondrial dysfunction. In mammals, the age-related 63 weakening of physiological functions frequently goes along with a decline in health. While 64 there has been considerable progress in the study of age-associated diseases, the biological 65 mechanisms of ageing remain elusive. Ageing is attributed to either ‘programmed’ or 66 ‘damage-based’ processes (8). More than fifty years ago, Harman postulated that ageing 67 results from the accumulation of molecular damage caused by oxygen radicals, often called 68 reactive oxygen species (ROS) (9). Oxygen radicals are side products of electron transfer 69 reactions in respiratory chain complexes I and III (10). Later refinements of Harman’s free 70 radical theory of ageing postulated that an impairment of respiratory chain function increases 71 ROS production, resulting in higher levels of mtDNA damage, which in turn would 72 compromise the turnover of damaged respiratory chain complexes, resulting in a vicious 73 circle of damage and decline (11). The mitochondrial DNA mutator mouse model has 74 provided experimental evidence of a causative link between mtDNA mutations and an ageing 75 phenotype in mammals (12), although mitochondrial defects in this model were found not to 76 be associated with an increase in ROS production (13). Several other key predictions of the 4 77 free-radical theory (14) have also not been substantiated. For example, in some model 78 organisms, such as the nematode Caenorhabditis elegans, moderately elevated levels of ROS, 79 induced either chemically or through genetically engineered defects in the respiratory chain, 80 actually increase lifespan (15-17). In the same organism, removal of the ROS-scavenging 81 superoxide dismutase (SOD) does not increase oxidative damage to mtDNA and has no 82 apparent effect on life span (18-20). In the fly, SOD knockouts do have a decreased lifespan, 83 but they do not accumulate mtDNA mutations more quickly than wildtype (21). 84 In addition to energy conversion and ATP production, the oxidative phosphorylation system 85 also has a key role in shaping the inner membrane cristae (5-7). Accumulating oxidative 86 damage and OXPHOS dysfunction might, therefore, be expected to affect mitochondrial 87 ultrastructure, which should be visible by electron microscopy. Recent analyses of yeast and 88 mouse mitochondria show that a defect in supra-molecular organisation of the ATP synthase 89 results in aberrant cristae morphology (5,22). In aged cultures of the short-lived filamentous 90 fungus Podospora anserina, the inner membrane strikingly vesiculates and ATP synthase 91 dimers break down (23,24). However, little is known about the impact of ageing on 92 mitochondrial morphology and membrane structure in metazoans. Previous studies of the 93 ultrastructure of mitochondria in mouse liver by electron microscopy of thin plastic sections 94 revealed anomalous cristae in a subpopulation of the organelles (25). A study of mouse 95 skeletal muscle mitochondria found that occasionally mitochondria from trained animals lost 96 their cristae, whereas no differences were observed in non-trained animals (26). Mitochondria 97 from aged D. melanogaster flight muscle were found to have cristae ‘swirls’ that were 98 attributed to oxidative damage (27), and mitochondria in aged Drosophila repleta heart 99 muscle were enlarged (28). 100 We chose D. melanogaster (average life span >50 days depending on environmental 101 conditions (29)) and mouse (average life span 107 weeks for females and 114 weeks for males 102 (30)) as two well-established metazoan ageing models (31) for a systematic study of 103 mitochondrial ultrastructure, respiration and ROS production in young and old animals. Our 104 results reveal clear tissue- and organism-related age-specific differences, establishing an 105 apparent link between mitochondrial ultrastructure and function. 5 106 Results 107 Effects of ageing on function and ultrastructure of mouse mitochondria 108 (a) Mitochondrial OXPHOS activity and ROS homeostasis 109 To find out how ageing impacts mitochondrial function and ultrastructure, mitochondria from 110 heart, liver and kidney of young (20 weeks) and old (80-96 weeks) mice were isolated for 111 electron cryo-tomography (cryoET) and high-resolution respirometry. Isolated mitochondria 112 were incubated with respiratory substrates that deliver electrons at the level of complex I 113 (pyruvate, glutamate and malate) or complex II (succinate and rotenone). The mitochondrial 114 oxygen consumption was recorded under phosphorylating (addition of ADP and Pi; state 3), 115 non-phosphorylating (addition of oligomycin to inhibit ATP synthase; state 4) and uncoupled 116 conditions (addition of CCCP). Mitochondrial respiratory rates normalised to protein content 117 were found to be highest in heart mitochondria, ~2-3-fold lower in kidney and ~5-fold lower 118 in liver (Figure 1A). In line with previous reports (32) and liver (33,34), our investigation 119 showed no significant differences in respiratory rates between mitochondria isolated from 20 120 week-old and 80 week-old heart tissue (Figure 1A), and no age-related change in the rate of 121 ATP production was observed (Figure 1C). Moreover, maximal activities of key 122 mitochondrial enzymes remained unchanged (Figure 1E). These results prompted us to 123 investigate how ageing impacts mitochondrial ROS homeostasis. We first assessed