Mitochondrial DNA and disease


  • No conflicts of interest were declared.


Mitochondrial DNA (mtDNA) defects are a relatively common cause of inherited disease and have been implicated in both ageing and cancer. MtDNA encodes essential subunits of the mitochondrial respiratory chain and defects result in impaired oxidative phosphorylation (OXPHOS). Similar OXPHOS defects have been shown to be present in a number of neurodegenerative conditions, including Parkinson's disease, as well as in normal ageing human tissues. Additionally, a number of tumours have been shown to contain mtDNA mutations and an altered metabolic phenotype. In this review we outline the unique characteristics of mitochondrial genetics before detailing important pathological features of mtDNA diseases, focusing on adult neurological disease as well as the role of mtDNA mutations in neurodegenerative diseases, ageing and cancer. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Mitochondria are subcellular organelles responsible for producing the majority of cellular ATP through the process of oxidative phosphorylation (OXPHOS) 1. In addition to OXPHOS, mitochondria are the location of important biochemical pathways, including the tricarboxylic acid cycle (TCA) and parts of the urea cycle. Mitochondria are also important regulators of apoptosis 2 and cytosolic calcium concentration 3, and are central to iron–sulphur cluster biogenesis 1. Mitochondrial dysfunction is increasingly being recognized as a major contributing factor in a number of metabolic and degenerative diseases, ageing and cancer. Here we describe the histopathological changes observed in disorders associated with mitochondrial DNA (mtDNA) mutations and their contribution to the underlying disease mechanisms. Understanding the molecular mechanisms precipitating these common features has implications for ageing, neurodegenerative disease and cancer and may provide clues for the development of treatments for disorders of OXPHOS.

Mitochondrial genetics

Mitochondria contain the only extranuclear source of DNA in animal cells 4. MtDNA is a circular, double-stranded, 16 569 base-pair molecule of DNA which encodes 37 genes, including 13 essential polypeptides for the OXPHOS system, two ribosomal RNAs (12S and 16S) and 22 tRNAs 5 (Figure 1). The remaining proteins required for mitochondrial metabolism and maintenance are synthesized in the cytosol and are specifically targeted, sorted and imported to their correct mitochondrial location 6. The mitochondrial genome has unique characteristics that distinguish it from the nuclear genome; it is strictly maternally inherited and there are several hundred to several thousands of copies within a single cell. The number of copies present varies between different cell types, depending on the energy demand within the tissue 7. There are no introns and genes have either none or very few non-coding bases between them, and in most cases termination codons are not present but are created post-transcriptionally by polyadenylation 5. The displacement loop (D-loop) is the only major non-coding region of the molecule and is formed by the displacement of the two genomic strands by a third DNA strand. The D-loop is a 1.1 kb region containing the important control elements for mtDNA transcription and replication 8. MtDNA mutations are thought to arise due to close proximity of the genome to the OXPHOS system, which is located on the inner mitochondrial membrane, thus making it vulnerable to damage through leakage of reactive oxygen species (ROS) during the OXPHOS process. This increased susceptibility of mtDNA to damage leads to a mutation frequency which is thought to be much higher than that of the nuclear DNA 9. The multi-copy nature of mtDNA means that mtDNA mutations can co-exist with wild-type molecules in a situation known as heteroplasmy 10. In the majority of cases these mutations do not cause a biochemical phenotype until they reach a threshold level, which has been shown to vary for different types of mutation, in the range 50–60% 11–14 for deleted mtDNA molecules to > 90% for some tRNA point mutations 15, 16. However, there has been one recent report of a dominant mutation, m.5545C → T, in the MT-TW gene, which showed tissues to be clinically affected with a mutation level of < 25% 17. Certainly, for mtDNA deletions, it is the amount of wild-type mtDNA, which is lowered in the presence of a mutation, that is most important in terms of defining the biochemical profile of the cell. Once this threshold is exceeded, a cellular OXPHOS defect can be demonstrated.

Figure 1.

A map of the human mitochondrial genome. Schematic diagram of the 16.6 kb circular, double-stranded human mitochondrial genome. The outer circle represents the heavy (H) strand of the genome and the inner circle the light (L) strand. Genes encoding subunits of respiratory chain complex I are shown in green, the MT-CYB gene of complex III in purple, catalytic subunits of complex IV in yellow and those of complex V in blue. The two ribosomal RNAs are shown in red and the 22 transfer RNAs represented as black bars and denoted by their single-letter abbreviations

An important phenomenon to consider for all diseases in which mtDNA mutations are involved is clonal expansion. In patients with primary mtDNA disease the difference in severity between different cells within a tissue [eg the mosaic pattern of cytochrome c oxidase (COX)-deficient cells in muscle biopsy] is due to adjacent cells containing a varying proportion of mutated and wild-type mtDNA. There is random replication of mitochondrial genomes, which allows the variation between cells. This process is even more apparent in the presence of acquired mtDNA mutations occurring due to nuclear genetic mutations (see below) or during the ageing process. Under these circumstances, either mtDNA deletions or point mutations occur randomly throughout the genome. The majority of these mutations will be lost but some mutations accumulate to high levels in individual cells. This process of clonal expansion will take time; this explains why we only detect these changes later in life and almost certainly why they are more prominent in humans than laboratory animals. It is also important to appreciate that, because of relaxed replication 18 of the mitochondrial genome (replication of the mitochondrial genome independently of the cell cycle) in post-mitotic cells such as muscle and neurons, clonal expansion of mtDNA mutations is very common in these tissues in both ageing and disease.

Mitochondrial DNA disease

Disease-causing mtDNA mutations were first reported in 1988 19, 20 and since then more than 300 pathogenic mtDNA mutations have been described 21. MtDNA disease has an extremely variable phenotype and can present at any age 22. A chronic state of energy failure is thought to be the main driving force underlying most mitochondrial cytopathies at the cellular level. Cells that cannot produce enough ATP via OXPHOS may shunt pyruvate to lactate in attempt to produce more ATP, resulting in systemic lactic acidosis. The failure to meet cellular energy demands results in the multi-systemic disorders observed at a clinical level. The clinical features usually affect tissues in which there is a high metabolic demand, such as the central nervous system, skeletal muscle or heart (Figure 2). However, other tissues are frequently involved, such as the β cells of the pancreas, leading to diabetes, the inner hair cells of the cochlea, causing deafness, or the renal tubules, leading to kidney dysfunction. There are a number of well-defined clinical syndromes but the majority of patients do not fall into easily defined clinical groups and definition by genotype is often helpful.

Figure 2.

Tissues commonly affected by mitochondrial DNA related disease. (a–g) Sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) histochemistry on sections of human tissue including: (a) cerebellum; (b) extra-ocular muscle; (c) heart; (d) liver; (e) smooth muscle of colon; (f) kidney; (g) skeletal muscle (quadriceps) from patients with primary mtDNA abnormalities. Cells with normal COX activity are brown following COX histochemistry; those with no COX activity are blue upon sequential SDH histochemistry

The exact prevalence of mtDNA disease is difficult to quantify due to the clinical heterogeneity and number of disease-causing mutations, but it is estimated that as many as 1 in 10 000 people in the North East of England have clinically manifesting mtDNA disease, with a further 1 in 6000 at risk 23. However, this is likely to be a major underestimate, since the frequency of pathogenic mtDNA mutations in the population is high (approximately 1 in 200 people) 24, 25. The majority of these individuals will not be affected by disease because the mtDNA mutation is present at low levels of heteroplasmy or, for example, the mutation only causes deafness when the patients are given a specific antibiotic 26, 27.

Most primary rearrangements of mtDNA are single large-scale deletions and more than 120 different mtDNA deletions have been identified 21 in association with disease. A large proportion of mtDNA deletions occur in regions of the genome flanked by tandem repeat sequences 28, and deletions are probably formed by repair of damaged mtDNA 29. Most mtDNA deletions are sporadic and not transmitted to offspring 30. The pathology of mtDNA deletion disorders is dependent on the pattern of tissue segregation, and reported deletions are without exception heteroplasmic 21. The three main clinical phenotypes associated with these mutations are: Pearson's syndrome, a severe disorder presenting in infancy, characterized by sideroblastic anaemia with pancytopenia and exocrine pancreatic failure 31; Kearns–Sayre syndrome (KSS) 32, associated with multiple problems, including retinitis pigmentosa, progressive external ophthalmoplegia, cardiomyopathy, deafness, short stature, endocrinopathies and dysphagia, and neurological symptoms such as cerebellar ataxia, deafness and cognitive impairment; and chronic progressive external ophthalmoplegia (CPEO), which commonly presents in adulthood and is characterized by progressive paralysis of the eye muscles, leading to ptosis and impaired eye movement 33.

Point mutations in protein-encoding rRNA and tRNA genes, in contrast to large-scale mtDNA rearrangements, are usually maternally inherited. Pathogenic mtDNA mutations in RNA genes are thought to cause an impairment of overall mitochondrial protein synthesis, whereas mutations in the protein-encoding genes affect specific respiratory chain complexes 34. More than half of disease-related point mutations reported are located within mt-tRNA genes, despite the fact that these genes represent only about 5% of the mitochondrial genome.

One of the most common heteroplasmic disease-causing mtDNA point mutations is m.3243 A → G in the MT-TL1 gene. The most frequent manifestation of the mutation is maternally inherited diabetes and deafness (MIDD) 35, but a more severe syndrome, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) 36, is also observed. Patients often present with seizures and stroke-like episodes particularly affecting the occipital region of the brain, leading to visual field defects. Pathological features such as enlarged mitochondria and mitochondria with complicated cristae have been reported in pericytes of capillaries, endothelial cells and smooth muscle cells of the small arteries, including terminal arterioles and precapillary sphincters, which may be responsible for the occasional occurrence of transient cerebral ischaemia causing stroke-like episodes 37. Abnormalities of mitochondrial enzyme function in the endothelium and smooth muscle of the leptomeningeal and cortical blood vessels of the brain have also been observed 38. Gastrointestinal involvement has also been implicated with this mutation, with constipation and gastric discomfort being the most common manifestations 39. A second common mtDNA point mutation associated with disease is an m.8344 A → G substitution in the MT-TK gene, which causes myoclonic epilepsy with ragged-red fibres (MERRF) 40. Other features include intermittent encephalomyopathy, ataxia, migraine and cognitive impairment. MERRF is a progressive neurodegenerative disease, often presenting in childhood or early adulthood, with proximal muscle wasting and central neurological features such as epilepsy, cerebellar ataxia and optic atrophy. Non-neurological symptoms include re-entrant atrio-ventricular tachycardias and multiple symmetrical lipomas. Table 1 describes the most common disease-associated mtDNA point mutations and their clinical manifestations.

Table 1. Common mitochondrial disorders associated with mtDNA point mutations
Mitochondrial DNA disorderMutationGene affectedPhenotype
  1. AID, aminoglycoside-induced deafness; LHON, Leber hereditary optic neuropathy; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes; MERRF, myoclonic epilepsy and ragged-red fibres; MIDD, maternally-inherited diabetes and deafness; NARP, neurogenic weakness, ataxia and retinitis pigmentosa.

LHON 112, 113m.3460 G→AMT-ND1Subacute bilateral visual failure and optic atrophy
 m.11778 G→AMT-ND4 
 m.14484 T→CMT-ND6 
Leigh syndrome 114m.8993 T→CMT-ATP6Onset 4–5 months, developmental delay, psychomotor delay, pyramidal signs, dystonia, seizures, respiratory failure
NARP 115m.8993 T→GMT-ATP6Sensory neuropathy, cerebellar ataxia, retinitis pigmentosa, dementia, proximal weakness
MELAS 36m.3243 A→GMT-TL1Onset ca.10 years, stroke-like episodes before 40, seizures, dementia, lactic
 m.3271 T→CMT-TL1acidosis
MERRF 116m.8344 A→GMT-TKMyoclonus, seizures, cerebellar ataxia, myopathy
 m.8356 T→CMT-TK 
AID 117m.1555 A→GMT-RNR1Aminoglycoside-induced non-syndromic deafness
MIDD 35m.3243A→GMT-TL1Diabetes and deafness

Nuclear DNA mutations causing secondary mitochondrial DNA disease

It is estimated that ∼1500 proteins are present in the mitochondrial proteome 41; therefore, as the mitochondrial genome only encodes 13 of these proteins, it is not surprising that a large proportion of mitochondrial disorders are caused by nuclear DNA mutations 41. A number of these nuclear gene mutations exert their effect by disturbing the function of the mitochondrial genome, and these can be broadly divided into the following groups:

  • 1.MtDNA maintenance and expression; eg POLG encoding the catalytic subunit of mitochondrial polγ 42, POLG2 encoding the accessory β-subunit of polγ 43, and PEO1 encoding mitochondrial helicase Twinkle 44. This group also includes genes encoding ribosomal proteins and binding factors, amino acyl tRNA synthetases (comprehensively reviewed in 45) and tRNA-modifying enzymes such as TRMU 46, 47
  • 2.Nucleoside transport, salvage or synthesis; eg SLC25A4, encoding adenine nucleotide translocator 1 48, and RRM2B, encoding a subunit of the p53-inducible ribonucleotide reductase protein required for maintaining balanced mitochondrial dNTP pools 49
  • 3.Mitochondrial dynamics; eg OPA1, which encodes a dynamin-related GTPase required for the maintenance of the inner mitochondrial membrane architecture 50, 51.

Histopathological features of mtDNA disease

A multidisciplinary approach to the diagnosis of mitochondrial disease is essential to piece together information from clinical, histochemical and biochemical testing to target molecular genetic screening, as guided by rational diagnostic algorithms 22. Laboratory-based diagnosis often requires the analysis of a clinically relevant tissue (often skeletal muscle, but other tissues, eg cardiac or liver, may be appropriate), with many patients showing histological and histochemical changes indicative of OXPHOS dysfunction. A central finding in patients with mtDNA disease is the demonstration of respiratory-deficient muscle fibres in skeletal muscle, in particular those with a lack of histocytochemical cytochrome c oxidase (COX) activity. COX-deficient fibres were first identified in patients with chronic progressive external ophthalmoplegia (CPEO) 52, as demonstrated by a loss of the oxidized 3,3′-diaminobenzidine tetrahydrochloride (DAB) reaction product used in the histochemical assay (Figure 3c). The demonstration of COX-deficiency was subsequently enhanced by combining the individual COX reaction with the histochemical demonstration of succinate dehydrogenase (SDH) activity (Figure 3d), in which a blue, nitroblue tetrazolium (NBT) reaction product is formed 53. The latter enzyme is fully encoded by the nuclear genome, whereas COX has three catalytic subunits encoded by the mitochondrial genome (mtDNA). The presence of a mosaic pattern of COX- deficient, SDH-reactive muscle fibres (Figure 3e) is a pathological hallmark of a mtDNA-related defect, which may be either a primary (mt-tRNA mutation or single, large-scale mtDNA deletion) or secondary (multiple mtDNA deletions or mtDNA depletion) defect. In these patients, there is typically a mosaic pattern of deficiency, with both COX-positive and COX-deficient fibres present in the same biopsy (Figure 3e) 54. Studies have extensively used this technique to explore the molecular mechanisms involved in mtDNA disease, with COX-deficient fibres showing high levels of mutated mtDNA (in patients with heteroplasmic mtDNA defects) 55 associated with lower levels of wild-type mtDNA 56. One of the observations made several years ago documents the presence of so-called ‘ragged red’ fibres in the histological Gomori trichrome stain in patients with mtDNA disease (Figure 3b). These fibres show focal accumulation of mitochondria around the periphery of the fibre, and can also be identified using the sequential COX/SDH activity reaction as deeply-staining ‘ragged-blue’ fibres (Figure 3d, e). An example of the histological and histochemical staining commonly used in the diagnosis of mtDNA disease is shown in Figure 3a–e. Figure 3f–i highlights the variability in COX activity observed with different mtDNA disorders. Global loss of COX activity often is associated with mtDNA disorders affecting COX assembly (Figure 3f, g), whereas mosaic loss of COX activity is commonly seen with primary mtDNA defects, when the proportion of COX deficient fibres can vary from relatively few (Figure 3h) associated with a large scale mtDNA deletion to the very high levels often observed with mt-tRNA mutations (Figure 3i).

Figure 3.

Histopathological abnormalities commonly observed in primary mtDNA-related disease. Illustrated (a–e) are examples of the histological and histochemical assessment of sequential, transverse-orientated sections of vastus lateralis biopsy tissue from a patient with a single, large-scale mtDNA deletion, which provides useful clues in the laboratory diagnosis of mtDNA disease. The sections highlight two fibres stained or reacted for the following: (a) haematoxylin and eosin (H&E) staining to show general muscle morphology, in this case identifying abnormal, granular fibres showing basophilic rims; (b) modified Gomori trichrome stain highlighting these as classical ragged-red muscle fibres; (c) COX histochemistry showing that these fibres are COX-deficient to differing degrees; (d) the SDH reaction which reveals the subsarcolemmal accumulation of mitochondrial activity is exclusively found in mitochondria; (e) sequential COX/SDH histochemistry highlighting COX-deficient fibres, which retain SDH activity. Mutations in mtDNA and nuclear-encoded proteins involved in COX assembly can lead to a complete, global loss of COX activity, which is illustrated in (f), reacted for COX alone and (g), sequential COX-SDH activities; the typical ‘mosaic’ distribution of COX-deficiency can vary for different mutations, as demonstrated by sequential COX/SDH histochemistry in muscle from patients, with (h) a single, large-scale mtDNA deletion or (i) a mitochondrial tRNA mutation

Sequential COX/SDH histochemistry is an excellent marker for cells deficient in complex IV of the respiratory chain; however, it will not highlight cells which are deficient in other respiratory chain complex activities, which can occur in association with mtDNA abnormalities, causing generalized mitochondrial translation defects. Immunohistochemical techniques, employing monoclonal antibodies to individual subunits of other respiratory chain complexes (eg complex I, complex III), in combination with the histochemical techniques described above, are extremely useful for highlighting muscle fibres deficient in these complexes and gaining a comprehensive picture of the extent of respiratory chain deficiency in muscle associated with an mtDNA defect 54 (Figure 4). These techniques are often more helpful than in vitro activity measurements in muscle homogenates, since in mtDNA disease the defect is often mosaic rather than generalized and may not be detectable with such biochemical techniques. It has been shown that for primary mtDNA mutations, loss of COX activity correlates very well with loss of expression of the catalytic subunits of COX 57 (Figure 4d, h); however, this was not shown to be the case for mitochondrial defects secondary to nuclear DNA mutations 57. This technique may therefore be useful as an initial screen for the likelihood of the defect being a primary mtDNA mutation or a nuclear DNA mutation. Figure 4 shows a typical immunohistochemical staining panel for a patient with the m.3243 A → G MT-TL1 gene mutation, which given causes multiple respiratory chain deficiency on account of the disturbance of tRNALeu (UUR) function. It is interesting to note that, although levels of COX deficiency are shown to correlate perfectly with both COX histochemical activity (Figure 4b, d) and complex IV subunit immunohistochemistry (Figure 4h), expression of the complex I NDUFS8 subunit is reduced or absent in a much higher proportion of the fibres (Figure 4e), highlighting the significant complex I deficiency which is often associated with this particular mt-tRNA defect 58.

Figure 4.

Histochemical and immunohistochemical analysis of skeletal muscle from a patient with m.3243A → G. (a) Haematoxylin and eosin staining to show general muscle morphology; (b) COX histochemistry; (c) SDH histochemistry; (d) sequential COX/SDH histochemistry; (e–g) immunohistochemistry showing expression of OXPHOS complex subunits (brown staining); (e) complex I NDUFB8 subunit; (f) complex II 70 KDa flavoprotein subunit; (g) complex III, Rieske subunit; (h) complex IV subunit 1. The white arrows highlight a fibre which is deficient in COX activity, complex IV subunit 1 expression and complex I NDUFB8 expression. This fibre is also shown to be ‘ragged-blue’ on the SDH reaction, indicating enhanced mitochondrial accumulation. The red arrows identify two fibres which also show subsarcolemmal mitochondrial accumulation, based on the SDH activity and reduced complex I expression but normal COX activity and complex IV subunit 1 expression. *Fibres which have normal COX activity but reduced or absent complex I expression. Scale bars = 50 µm

Neuropathological findings in patients with primary mitochondrial DNA disorders

Neurodegeneration caused directly by mitochondrial dysfunction is prevalent in primary mitochondrial DNA disorders (Figure 5a, b). In these patients there are many different neuropathological findings, which depend in part upon the nature of the underlying genetic defect. Patients harbouring the m.3243A → G mutation who develop MELAS show multiple infarcts in the cortex. These are found in particular in the temporal, parietal and occipital lobes, and are not related to a particular vascular territory. They are associated with neuronal loss, neuronal eosinophilia and astrogliosis 59 and are thought to underlie the stroke-like episodes common in this disorder. Calcification of the basal ganglia, particularly in the vasculature, is commonly observed and in severe cases cerebral and cerebellar atrophy occurs 60. Patients with the m.8344A → G MERRF mutation often show degenerative features within the olivocerebellar pathway, with severe neuronal loss involving inferior olivary nucleus, Purkinje cells and dentate nucleus. Enlarged mitochondria containing inclusions have been described in the surviving neurons of the cerebellum 61; however, there was no evidence of correlation between mtDNA mutation levels in the surviving neurons and the extent of neurodegeneration 62. This suggests that factors in addition to the mtDNA mutation load play an important role in determining which neurons are particularly vulnerable in this disorder. Patients with large-scale deletions leading to the KSS phenotype show severe spongiform degeneration of the white matter regions of the brain, including the cerebellum, cerebrum, thalamus, brain stem and spinal cord, and the severity of these lesions can vary widely 63. Neurodegeneration also affects patients who harbour mtDNA deletions as a consequence of a primary nuclear DNA mutation. For example, patients with mutations in the POLG gene can show degeneration and neuron loss in the cerebellum and inferior medullary olives, and degeneration within the spinal cord as also been described 64. Changes within the substantia nigra have also been reported and these changes have also been associated with Parkinson's disease-like symptoms and pathology. For example, a loss of substantia nigra neurons, the development of α-synuclein positive Lewy bodies as well as the presence of increased levels of respiratory-deficient neurons have been reported in association with POLG mutations. This patient also showed cerebellar changes, including a loss of Purkinje cells and dentate nucleus neurons 65.

Figure 5.

Mitochondrial complex I deficiency in neurons. Loss of immunoreactivity against the NDUFB8 subunit, highlighting complex I deficiency, can be detected in a number of different clinical scenarios and brain regions. Red arrows highlight complex I-deficient neurons in (a) the substantia nigra and (b) the cerebellum of a patient with primary mtDNA disease due to the m.3243A → G mutation in MT-TL1. (c, d) Complex I-deficient neurons in the substantia nigra of (c) a patient with Parkinson's disease, and (d) a normal elderly subject. The brown staining in (a, c, d) is the chromogen DAB; the blue staining is a haematoxylin counterstain highlighting cell nuclei. The red staining in (b) is Vector VIP chromogen and the nuclei are counterstained with haematoxylin (blue). Scale bar = 100 µm

Mitochondrial DNA mutations in other common neurodegenerative disorders

Although the implications of mitochondrial dysfunction are beginning to be understood in a number of neurodegenerative diseases, they have perhaps been best characterized in Parkinson's disease (PD). Recent genetic studies have shown that a number of the genes important in familial, early-onset forms of PD encode proteins essential for mitochondrial function. These include Pink1 and parkin, which act to maintain mitochondrial structural integrity and may also target mitochondria for degradation 66, 67, and DJ-1, which acts as a potential ROS scavenger/sensor 68. Deletions of mtDNA have been shown to accumulate in the neurons of the substantia nigra (the brain region primarily affected in PD) with advancing age and in patients with PD 69, 70. These deletions are sufficient to cause mitochondrial dysfunction (Figure 5c, d). Unlike inherited mtDNA mutations, these deletions accumulate and clonally expand over decades. Changes in the activity and expression of subunits of complex I of the electron transport chain are also reported to occur within the tissues of patients with PD and the complex I inhibitor MPP+ also causes PD-like symptoms when taken as the drug MPTP 71, 72. Respiratory chain dysfunction caused by mtDNA mutations has also been reported in AD neurons and recently also in the neurons of multiple sclerosis patients 73, 74. Again, the changes seen in these diseases also extend to changes in mitochondrial morphology, activity and axonal distribution 75.

Mitochondria and ageing

Biochemical defects in OXPHOS in ageing tissues were first documented in 1989 by Trounce and colleagues in skeletal muscle 76, and then in the same year by Muller-Hocker, who showed that there was an increase in the number of COX-deficient cardiomyocytes with increasing age 77. Since then, COX-deficient cells have been demonstrated to occur during ageing in a number of different tissues, such as various neuronal cell types 78, 79 and colonic 80 (Figure 6a, b) and stomach 81 epithelium. The striking observation in these ageing tissues is the similarity of the changes within certain cells to the pathological features seen in mtDNA disease, the major difference being that, in mtDNA disease, a much higher proportion of cells are affected. Why these mtDNA mutations accumulate with age is still uncertain. The mitochondrial theory of ageing proposes that the progressive accumulation of somatic mutations in mtDNA during life leads to a decline in mitochondrial function 82. MtDNA mutations are thought to impair cellular OXPHOS, leading to enhanced ROS production, leading to further damage to mtDNA, and so a vicious cycle of exponentially increasing oxidative damage and dysfunction is produced 83. A more likely scenario, in our view, is that mtDNA mutations occur throughout life, due to either oxidative damage or errors of the mtDNA polymerase, and these mutations clonally expand to cause cellular dysfunction, rather than accumulation of new mutations as predicted by the vicious cycle.

Figure 6.

Mitochondrial dysfunction in ageing and cancer. Mitochondrial dysfunction has been documented in a number of ageing tissues as well as in tumours of various tissues. (a) H&E staining of normal human colon. (b) Sequential COX/SDH reaction performed in human colon from an elderly individual (82 years), showing that 10% of all crypts were COX-deficient in this patient. H&E staining (c) and COX/SDH histochemistry (d) on a rectal adenocarcinoma present in the same 82 year-old patient as in (a, b)

An interesting observation is that in post-mitotic tissues, such as brain and muscle, the majority of clonally expanded mtDNA mutations within individual cells are large-scale mtDNA deletions, with little evidence of clonally expanded point mutations 84. As highlighted above, substantia nigra neurons are very prone to this accumulation, with over 40% of all mtDNA being deleted in elderly controls and slightly more in patients with PD 69, 85. This is thought to be due to high levels of ROS associated with dopamine production in these cells, causing double-strand breaks which, if repaired incorrectly, may lead to mtDNA deletion formation 29. In other tissues the level of total deleted species is much lower, rarely exceeding 1% 86, even in very old individuals, questioning the contribution of mtDNA deletions to the ageing process. However, since the deletions are mostly present within individual cells at high levels due to clonal expansion, the associated respiratory deficiency may well contribute to the ageing process.

In mitotic tissues, the mtDNA defect is much more commonly clonal expansion of point mutations. In certain human tissues, these mitochondrial defects can, with age, lead to a significant number of respiratory-deficient cells, as shown by studies on human colon (Figure 6a, b). There is an exponential increase in cells showing respiratory deficiency with age 80. One of the interesting observations is that the structure of the colonic epithelium highlighted that the clonal expansion and respiratory deficiency must originate from the crypt stem cell. The presence of respiratory deficiency has been detected in other aged stem cell populations, such as stomach 81, liver 87, prostate 88, 89 and urothelial epithelium 90, in association with respiratory chain deficiency. These mutations have been extremely useful in stem cell lineage-tracing experiments 81, 87–93.

The studies described above are correlative, and the causality of mtDNA mutations in human ageing is unclear. A mitochondrial ‘mutator mouse’ was developed to test this directly. The mutator mouse has a homozygous knock-in mutation (p.Asp257Ala) that affects a highly conserved residue in the proofreading domain of mitochondrial polymerase-γ 94, 95. The mice have an mtDNA mutator phenotype which shows a three- to five-fold increase in the frequency of mtDNA point mutations and also an increased incidence of large-scale mtDNA rearrangements (a linear form of mtDNA) 95. The mice appear normal up to ∼25 weeks of age, whereafter they elicit a premature ageing phenotype. Symptoms include hair loss and greying, kyphosis, reduced subcutaneous fat, osteoporosis, reduced fertility, anaemia and significantly reduced life-span. Surprisingly, this was not accompanied by an increase in oxidative stress or ROS-induced damage. There was no evidence of an increase in H2O2 or superoxide production, neither was there evidence of increased oxidative damage to proteins 94, 96; however, there was evidence of increased cleaved caspase-3, a marker of apoptosis in a variety of tissues. This suggests that the increased mutation burden could be inducing apoptosis, and that it is this that leads to tissue dysfunction rather that ROS-associated damage, as predicted by the vicious cycle theory of ageing 83. These mouse models were the first evidence that excessive mtDNA mutagenesis alone can cause degenerative age-related signs.

Mitochondrial DNA mutations and cancer

One of the major distinguishing features between normal and tumour cells is their intermediary metabolism. In 1956, Otto Warburg observed a higher glucose consumption and higher lactic acid production in tumour cells, even in the presence of sufficient oxygen, suggesting that tumour cells preferentially utilize glycolysis for ATP production over OXPHOS 97, 98. This was termed ‘aerobic glycolysis’, or the ‘Warburg Effect’. Warburg suggested that a key event in carcinogenesis was ‘injury’ to the respiratory machinery. Subsequent analysis of mitochondrial function showed that OXPHOS was frequently down-regulated in many tumours 99 (Figure 6c, d); however, it is still unknown whether these metabolic changes are a cause or a consequence of neoplastic transformation. In addition to OXPHOS defects, cells with high membrane potential have been shown to have an enhanced capacity to respond to hypoxia by avoiding apoptosis and initiating angiogenesis; they escape anoikis and grow under anchorage-independent conditions, and invade the basement membrane 100.

MtDNA point mutations were first detected in human colorectal cancer 101 and since then in numerous other cancers, including bladder, head and neck and lung tumours 102. Following these initial studies there have been a number of reports showing mutations in a number of solid tumours and haematological malignancies 103–107. A comprehensive analysis of all reported mtDNA point mutations in human tumours revealed that 72% were previously reported polymorphic variants or rare haplogroup markers 108. The remaining 28% were grouped into two classes; severe mutations that inhibit OXPHOS, increase ROS production and promote tumour cell proliferation, and milder mutations that may permit tumours to adapt to new environments 108. Transmitochondrial cybrid technology has been used to investigate the functional effects of mtDNA mutations in tumour initiation, progression and metastasis. These cells are generated by fusing enucleated cells (cytoplasts) containing the mtDNA mutation of interest with ρ0 cells (cells devoid of mtDNA) 109. This technique allows the effect of the mtDNA mutation to be investigated in a different nuclear background to its cell of origin. Two cybrid cell lines containing mutations in MT-ATP6, m.8993T → G and m.9176T > C, were generated 110, and upon transplantation into nude mice the mutations conferred an advantage in the early stage of tumour growth and showed lower levels of apoptosis. When a mixture (1 : 1) of mutant and wild-type cybrids was injected into nude mice, the proportion of mutant mtDNA in the tumour increased progressively and eventually entirely replaced the wild-type mtDNA. A second study using cybrid technology transferred mtDNA from a non-metastatic cell line to a highly metastatic cell line and vice versa 111. The mtDNA from the highly metastatic cell line was found to contain two mutations in MT-ND6 and had reduced complex I activity and an over-production of ROS. The cybrids containing the mutant mtDNA conferred higher metastatic potential, regardless of the nuclear background, and when they were pretreated with ROS scavengers they suppressed their metastatic potential. This suggests that ROS-induced oxidative stress might lead to the expression and regulation of nuclear genes related to metastasis. The crosstalk between the mitochondria and the nucleus is an important area for further research.

Concluding remarks

Mitochondrial dysfunction plays a role in three major classes of disease, primary mitochondrial disease, neurodegeneration and cancer, as well as contributing to the decline in tissue integrity with age. Although significant progress has been made in our understanding of the role of mitochondria in these disorders over the last two decades, a number of fundamental questions remain. In the case of primary mtDNA disease, there is still no effective therapy for patients and the ever-increasing number of reported nuclear and mtDNA mutations and widening clinical spectrum make diagnosis difficult. In the case of mitochondrial defects in neurodegeneration, ageing and cancer, the biochemical and molecular pathways involved are yet to be established, and dissecting causality from consequence is a constant challenge. It is critical that we further our understanding of the molecular mechanisms involved and develop potential therapeutic strategies.


Our work on mitochondrial diseases is currently supported by the Newcastle University Centre for Brain Ageing and Vitality, supported by BBSRC, EPSRC, ESRC and MRC as part of the cross-council Lifelong Health and Wellbeing Initiative (Grant No. G0700718), the Wellcome Trust (Grant No. 074454/Z/04/Z), the UK NIHR Biomedical Research Centre for Ageing and Age-related Disease award to the Newcastle upon Tyne Hospitals NHS Foundation Trust and the UK NHS Specialist Commissioners, which funds the ‘Rare Mitochondrial Disorders of Adults and Children’ Diagnostic Service in Newcastle upon Tyne.

Author contributions

All authors contributed to the writing of this article.