The Mitochondrion and Its Disorders

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100 PRACTICAL NEUROLOGY Pract Neurol: first published as 10.1046/j.1474-7766.2003.09117.x on 1 April 2003. Downloaded from HOW TO UNDERSTAND IT The mitochondrion and its disorders

Patrick F. Chinnery nalling and apoptosis (programmed cell death), and they have a crucial role in metabolism. Department of Neurology, Regional Neurosciences Many metabolic enzyme systems are contained within mitochondria, including components of Centre, Newcastle General Hospital and the tricarboxylic acid (Krebs) cycle enzymes, and University of Newcastle upon Tyne, UK. Email: the fatty acid β-oxidation pathway. However, the term ‘mitochondrial disorder’ usually refers [email protected] to a primary abnormality of the mitochondrial Practical Neurology, 2003, 3, 100–105 respiratory chain. Secondary mitochondrial dysfunction is seen as part of normal ageing and also in neurode- Mitochondria are ubiquitous intracellular generative disorders such as Alzheimer’s disease, organelles that play a pivotal role in cellular but the signiﬁ cance of these changes is not clear. energy metabolism. It therefore should come as Mitochondrial dysfunction also plays an im- no surprise that mitochondrial dysfunction can portant part in the pathophysiology of a group cause neurological disease. These disorders are of inherited neurological diseases that includes

not rare; each UK neurologist will have at least Friedreich’s ataxia, Wilson’s disease and heredi- http://pn.bmj.com/ 20 patients with mitochondrial disease within tary spastic paraparesis. Although related, these their catchment area of about 200 000 people. are not thought of as ‘primary mitochondrial This short article will focus on the basic science disorders’ and will not be considered here. that underpins our current understanding of mitochondrial disease. It will stick to the bare WHAT DOES THE MITOCHONDRIAL RESPIRATORY on September 29, 2021 by guest. Protected copyright. essential facts that will help the busy neurolo- CHAIN DO? gist to identify, investigate and manage these The mitochondrial respiratory chain is a group fascinating and challenging patients. of ﬁ ve large enzyme complexes that sit within The tables should serve as a useful reference. the inner mitochondrial membrane (Fig. 1). Table 1 illustrates the clinical relevance of the Each enzyme complex contains multiple basic science described in this article, but the subunits, and the largest is complex I with over reader should be aware that it will soon be out 70 components. The metabolism of carbohy- of date. Table 2 lists the features associated with drates, fats and proteins generates intermediary well–recognized mitochondrial ‘syndromes’. metabolites that feed electrons in to the respiratory chain. These electrons are passed from MITOCHONDRIA AND MITOCHONDRIAL DISORDERS: complex to complex, and this energy is used to WHAT ARE THEY? pump protons out of the mitochondrial matrix. Rather than thinking of mitochondria as dis- This generates the mitochondrial membrane crete membrane-bound structures, they prob- potential, which is harnessed by complex V to ably form a budding and fusing reticulum that is synthesize adenosine triphosphate (ATP), the integrated into the cellular network. Mitochon- principal intracellular energy source. The de- dria have a number of interrelated functions. tailed biochemistry of the respiratory chain is They are involved in intracellular calcium sig- not important clinically, but it is worth remem-

Table 1 Mitochondrial disorders bering that complex II is also called succinate Nuclear genetic disorders Inheritance pattern dehydrogenase (SDH) and complex IV is usu- Disorders of mtDNA maintenance ally called cytochrome c oxidase (COX). Autosomal dominant external ophthalmoplegia (with 2° multiple mtDNA deletions) MITOCHONDRIAL BIOGENESIS: A TALE OF TWO Ant 1 mutations AD POLG mutations AD or AR GENOMES Twinkle (C10orf2) mutations AD The respiratory chain has a dual genetic basis. Mitochondrial neuro-gastrointestinal encephalomyopathy The vast majority of the respiratory chain (with 2° multiple mtDNA deletions) complex subunits are synthesized within the Thymidine phosphorylase gene AR Myopathy with mtDNA depletion cytoplasm from nuclear gene transcripts (mes- Thymidine kinase defi ciency AR senger RNA molecules transcribed from genes Encephalopathy with liver failure within the cell nucleus). These are delivered Deoxyguanosine kinase defi ciency AR into mitochondria by a targeting sequence that Disorders of mitochondrial protein import enters through the mitochondrial protein im- Dystonia-deafness port machinery. Thirteen of the complex subu- DDP1/TIMM8a mutations XLR nits are synthesized within the mitochondria Primary disorders of the respiratory chain themselves from small circles of DNA called the Leigh’s syndrome Complex I defi ciency – mutations in NDUFS2,4,7,8 mitochondrial genome (mtDNA; Fig. 2). In ad- and FV1 complex I subunits AR dition to the protein coding genes, mtDNA also Complex II defi ciency – mutations in Fp subunit of complex II AR encodes for 24 RNA molecules that are needed Leukodystrophy and myoclonic epilepsy for intramitochondrial protein synthesis. As Complex I defi ciency – mutations in NDUFV1 complex I subunit (Scheulke et al. 1999) AR a result, genetic mutations of nuclear DNA Cardioencephalomyopathy (nDNA) or mtDNA can affect respiratory chain Complex I defi ciency – mutations in NDUFS2 AR activity. MtDNA defects fall into two groups: Optic atrophy and ataxia rearrangements (large chunks of deleted or du- Complex II defi ciency – mutations in Fp subunit of complex II AD plicated mtDNA) and point mutations (single Disorders of assembly of the respiratory chain base changes). These mutations can affect the Leigh’s syndrome Complex IV defi ciency – mutations in SURF I AR RNA genes and lead to a general defect of pro- Complex IV defi ciency – mutations in COX 10 AR tein synthesis within the mitochondria, or they Cardioencephalomyopathy can affect the protein-encoding genes them- Complex IV defi ciency – mutations in SCO 2 AR selves. MtDNA duplications are often found in Hepatic failure and encephalopathy http://pn.bmj.com/ Complex IV defi ciency – mutations in SCO 1 AR patients harbouring mtDNA deletions, but the Tubulopathy, encephalopathy and liver failure duplications are not thought to be pathogenic. Complex III defi ciency – mutations in BCS1L AR Mitochondria cannot survive on their own, Mitochondrial genetic disorders and there are many nuclear-encoded factors (mtDNA nucleotide positions refer to the L-chain) Inheritance pattern that play a crucial role in maintaining a healthy Rearrangements (deletions and duplications) respiratory chain. Over recent years we have

Chronic progressive external ophthalmoplegia (CPEO) S on September 29, 2021 by guest. Protected copyright. Kearns–Sayre syndrome S learned that these factors are important clini- Diabetes and deafness S cally. The nucleus maintains healthy mtDNA Point mutations thoughout human life. Disruption of the Protein-encoding genes mtDNA polymerase-γ (POLG), or the balance LHON (G11778A, T14484C, G3460A) M NARP/Leigh syndrome (T8993G/C) M of nucleotides (DNA building blocks) within tRNA genes the mitochondrial matrix, leads to the forma- MELAS (A3243G, T3271C, A3251G) M tion of many different secondary mutations of MERRF (A8344G, T8356C) M mtDNA throughout life, or the loss (depletion) CPEO (A3243G, T4274C) M Myopathy (T14709C, A12320G) M of mtDNA (Table 1, disorders of mtDNA main- Cardiomyopathy (A3243G, A4269G) M tenance). Finally, speciﬁ c proteins are needed Diabetes and deafness (A3243G, C12258A) M to assemble the various components of the Encephalomyopathy (G1606A, T10010C) M respiratory chain into the complete complexes, rRNA genes Non-syndromic sensorineural deafness (A7445G) M and disruption of these processes can also lead Aminoglycoside induced nonsyndromic deafness (A1555G) M to severe respiratory chain deﬁ ciencies that usually present in childhood (Table 1, disorders of AD, autosomal dominant; AR, autosomal recessive; M, maternal; S, sporadic; assembly of the respiratory chain). XLR, X-linked recessive. Two other mitochondrial components also need mentioning. Co-enzyme Q10

Table 2 Clinical syndromes associated with mitochondrial disease

DISORDER PRIMARY FEATURES ADDITIONAL FEATURES Chronic progressive External ophthalmoplegia Mild proximal myopathy external ophthalmoplegia (CPEO) and bilateral ptosis Infantile myopathy and lactic acidosis Hypotonia in the ﬁ rst year of life Fatal form may be associated (fatal and nonfatal forms) Feeding and respiratory difﬁ culties with a cardiomyopathy and/or the Toni– Fanconi–Debre syndrome Kearns–Sayre syndrome (KSS) PEO onset before age 20 with Bilateral deafness pigmentary retinopathy plus one of Myopathy the following: CSF protein greater than Dysphagia 1 g/L, cerebellar ataxia, heart block Diabetes mellitus Hypoparathyroidism Dementia Leber’s hereditary optic neuropathy Subacute painless bilateral visual failure Dystonia (LHON) Males:females approx. 4 : 1 Cardiac pre–excitation syndromes Median age of onset 24 years Leigh’s syndrome (LS) Subacute relapsing encephalopathy Basal ganglia lucencies with cerebellar and brain-stem signs presenting during infancy

Mitochondrial encephalomyopathy Stroke-like episodes before age 40 years Diabetes mellitus with lactic acidosis and stroke-like Seizures and/or dementia Cardiomyopathy (hypertrophic leading episodes Ragged-red ﬁ bres and/or lactic acidosis to dilated) (MELAS) Bilateral deafness Pigmentary retinopathy Cerebellar ataxia Myoclonic epilepsy with ragged-red ﬁ bres Myoclonus Dementia (MERF) Seizures Optic atrophy Cerebellar ataxia Bilateral deafness Myopathy Peripheral neuropathy Spasticity Multiple lipomata Neurogenic weakness with ataxia and Late childhood or adult onset Basal ganglia lucencies retinitis pigmentosa (NARP) peripheral neuropathy with associated Abnormal electroretinogram

ataxia and pigmentary retinopathy Sensorimotor neuropathy http://pn.bmj.com/ Pearson’s Syndome Sideroblastic anaemia of childhood Renal tubular defects Pancytopenia Exocrine pancreatic failure on September 29, 2021 by guest. Protected copyright.

Figure 1 Nuclear – mitochondrial interactions and the respiratory chain. The mitochondrial respiratory mitochondrion chain consists of ﬁ ve enzyme complexes that use the products of intermediary metabolism (of ATP proteins, carbohydrates and fats) to synthesize Complex A N subunits T adenosine triphosphate (ATP) which is shuttled mtDNA ATP out of the mitochondria by adenine nucleotide V transferrase (ANT). MtDNA is maintained by a Q Q number of nuclear encoded factors. Nuclear factors Q IV II III e- also regulate the transcription of mtDNA (forming a e-I e- e- messenger RNA template) and the translation of the Carbohydrates Factors for mtDNA Proteins maintenance, transcripts into proteins within the mitochondrion. Fats transcription and Nuclear DNA also codes for most of the respiratory Complex subunits & translation assembly proteins chain subunits and the complex assembly factors. MtDNA codes for 13 essential respiratory chain subunits and part of the machinery needed for nucleus protein synthesis within the mitochondrial matrix. e–, electrons; Q, coenzyme Q10 (ubiquinone).

D-loop

F T 12S RNA O H CYT b V Figure 2 The human mitochondrial genome. The 16S RNA P human mitochondrial genome (mtDNA) is a small E ND5 16.5kb molecule of double stranded DNA. The D- L(UUR) loop is the 1.1kb noncoding region that is involved in ND6 the regulation of transcription and replication of the ND1 molecule, and is the only region not directly involved I in the synthesis of respiratory chain polypeptides. Q MtDNA encodes for 13 essential components of L(CUN) M the respiratory chain. ND1-ND6, and ND4L encode S(AGY) ND2 seven subunits of complex I. Cyt b is the only mtDNA A H N encoded complex III subunit. COX I to III encode W C ND4 for three of the complex IV (cytochrome c oxidase, Y or COX) subunits, and the ATPase 6 and ATPase 8 R S(UCN) genes encode for two subunits of complex V. Two O L G ND4L ribosomal RNA genes (12S and 16S rRNA), and ND3 22 transfer RNA genes are interspaced between CO I D K CO III the protein-encoding genes. These provide the necessary RNA components for intramitochondrial CO II ATPase 6 protein synthesis. OH and OL are the origins of heavy ATPase 8 and light strand mtDNA replication.

(ubiquinone) has an important role shuttling drial disorders can affect any organ system this electrons between different respiratory chain is not strictly true, and certain tissues seem to be complexes, and which adenine nucleotide preferentially involved. In simple terms, tissues http://pn.bmj.com/ transferrase (ANT) exchanges ATP and ADP and organs that are heavily dependent upon across the mitochondrial membrane. ATP appear to be preferentially involved in patients with mitochondrial diseases. Neurones MITOCHONDRIAL DNA, HETEROPLASMY AND THE appear to be particularly vulnerable (including THRESHOLD EFFECT the retina and optic nerve), followed by skeletal Mononuclear human cells contain only two and cardiac muscle, and endocrine organs (par- on September 29, 2021 by guest. Protected copyright. copies of each nuclear gene, but many thou- ticularly the endocrine pancreas). However, a sands of copies of mtDNA. At birth all the wide range of tissues may be involved including mtDNA molecules are identical (a situation the cochlea, the gastrointestinal tract, the skin called homoplasmy). Patients with pathogenic and haematological tissues. mtDNA defects usually harbour a mixture of Patients with mtDNA disease have the added mutant and wild-type (normal) mtDNA within complexity of mtDNA heteroplasmy. Different each cell (heteroplasmy). The proportion of levels of mutant mtDNA in different tissues, mutant mtDNA can vary between 1 and 99%, coupled with tissue speciﬁ c thresholds, partly ex- and the cell only expresses a biochemical defect plains the pattern of clinical involvement. How- when the proportion of mutant mtDNA exceeds ever, if it were that simple then all patients with a critical threshold (typically 50–80% mutant, mitochondrial disease would look the same in the depending on the exact genetic defect). clinic – something that is clearly not the case.

TISSUE SPECIFICITY…GETTING MORE COMPLICATED PHENOCOPIES AND PHENOTYPIC VARIATION Perhaps the most difﬁ cult thing to explain is To pick an example, the A3243G point mutation the relative selectivity of clinical involvement. of mtDNA characteristically causes mitochon- Although the traditional view is that mitochondrial encephalopathy with lactic acidosis and

stroke-like episodes (MELAS). This mutation neurological disorder along with multiple ad- affects a mitochondrial tRNA gene that im- ditional organ involvement. pairs the synthesis of respiratory chain proteins within mitochondria and it is invariably hetero- PROBLEMS WITH MAKING A MOLECULAR plasmic. The A8344G point mutation of mtDNA DIAGNOSIS causes myoclonic epilepsy with ragged-red ﬁ bres If a patient has a suspected nuclear mitochon- (MERRF) and also affects a mitochondrial tRNA drial disorder, then DNA analysis can be carried gene with a consequent reduction in intramito- out on a blood sample. In some patients with a chondrial protein synthesis, and it is also in- mtDNA mutation it may be possible to identify variably heteroplasmic. Although there is some the genetic defect in a blood sample (for exam- clinical overlap in the phenotypes associated ple, in patients with Leber’s hereditary optic with these two mutations, they often cause quite neuropathy where most patients have only different disorders (Table 2), despite the fact that mutant mtDNA – homoplasmic mutant). Un- the molecular defects are strikingly similar. fortunately a simple blood sample may not be Unfortunately there is a poor relationship sufﬁ cient when investigating every patient with between the clinical features of mitochondrial suspected mitochondrial disease. This is because disease and the underlying genetic and bio- the level of mutant mtDNA may be very low in chemical abnormalities. Different genetic de- blood (below the threshold of detection), or fects can cause a similar clinical phenotype (for not present at all. This is almost always the case example, a mutation in mtDNA or nDNA can in patients with sporadic chronic progressive cause clinically indistinguishable Leigh’s syn- external ophthalmoplegia or the Kearns–Sayre drome), and yet the same genetic defect can also syndrome, which is usually due to a mtDNA cause very different clinical features even within deletion, but it also applies to point mutations the same family (for example A3243G can cause such as the common A3243G mutation. Thus, MELAS, but more often causes a milder disorder whilst it is entirely reasonable to send off a blood with deafness, diabetes or external ophthalmo- sample for molecular analysis in patients with plegia with ptosis). It is becoming clear that suspected mitochondrial disease, if the blood additional mitochondrial and nuclear genetic mtDNA test is negative the patient should then factors modulate the expression of the primary have a muscle biopsy. mtDNA defects, and that these interact with the environment. We clearly do not have all the CLUES FROM THE HISTOCHEMISTRY

answers at present. Routine muscle histochemisty includes a re- http://pn.bmj.com/ Despite the complexities, there are, however, action for COX and SDH (Fig. 3). COX has a number of well recognised syndromes that are both nuclear and mtDNA encoded subunits. usually due to mitochondrial disease (Table 2). A generalized decrease in COX in all muscle However, mitochondrial dysfunction should be fi bres suggests that the genetic defect is in the Figure 3 Skeletal muscle considered in any patient with an unexplained nuclear genome, probably involving a COX on September 29, 2021 by guest. Protected copyright. histochemistry from a patient with a pathogenic mtDNA defect. COX (cytochrome c oxidase; complex IV) showing a mosaic COX muscle histochemistry SDH muscle histochemistry distribution of COX negative fi bres. The COX negative fi bres contain a high percentage of mutant mtDNA (above the critical threshold level, 80% for the mutation in this patient). SDH (succinate dehydrogenase; complex II) showing mitochondrial proliferation. The subsarcolemmal proliferation corresponds to the ‘ragged-red’ COX negative fibre “ragged-red” fibre appearance of muscle fi bres with 17% 38% 55% 85% 90% the Gomori trichrome stain. Scale Percentage bar = 70 µm. mutant

© 2003 Blackwell Publishing Ltd APRIL 2003 105 Pract Neurol: first published as 10.1046/j.1474-7766.2003.09117.x on 1 April 2003. Downloaded from assembly gene. Patients with mtDNA defects MITOCHONDRIAL DISORDERS – SOME FACTS TO REMEMBER often have a mosaic COX defi ciency (Fig. 3) • Mitochondrial disorders are primary disorders of the respiratory chain. because different muscle fi bres contain differ- • Mitochondrial disorders can be due to genetic defects in either the nuclear ent amounts of mutant mtDNA and only some genome or the mitochondrial genome. fi bres contain supra-threshold levels. SDH is • Nuclear genetic mitochondrial disorders are inherited as autosomal domi- the only respiratory chain complex that is en- nant, recessive or rarely as X-linked traits. tirely encoded by nuclear genes. Patients with • Most adults with mitochondrial disease have an underlying defect of mito- mtDNA mutations usually show up-regulation chondrial DNA (mtDNA). of SDH in affected fi bres (thought to be a com- • In many patients there is a mixture of mutant and wild-type (normal) pensatory mechanism) and proliferation of mtDNA (heteroplasmy). mitochondria (corresponding to ragged red-fi - • The level of mutant mtDNA heteroplasmy may be undetectable in blood, and bres, Fig. 3). Unfortunately some patients have patients with suspected mtDNA disease should have a muscle biopsy if blood a biochemical defect that does not involve SDH DNA tests are negative. or COX, and these patients have normal muscle • MtDNA disorders are transmitted down the maternal line – males cannot histochemistry. As luck would have it, the most transmit the genetic defect. common heteroplasmic mtDNA point muta- • The recurrence risks for most mtDNA disorders are not well established. tion (the A3243G ‘MELAS’ mutation) does Some are sporadic and some are transmitted. just this. So, normal muscle histochemistry • There is no effective treatment for mitochondrial disorders – management does not mean no mitochondrial disease. If a is supportive. mitochondrial disease is still high on the list of differential diagnoses, then the next step is ANY TREATMENTS? to measure individual respiratory chain com- There are currently no established disease mod- plexes in muscle. This is only done well in a few ifying treatments for mitochondrial disease. specialist centres. Patients are often given ubiquinone (coenzyme Q10) because it is innocuous and there have INHERITANCE been reports of improvement in some cases. The nuclear genetic mitochondrial disorders Various vitamins and cofactors have also been are inherited as autosomal dominant, auto- used, and there is a clinical trial of dichloracetate somal recessive or rarely as X-linked recessive currently in progress. Management is largely traits (Table 1). By contrast, mtDNA is inherited supportive, with particular attention being paid down the maternal line. This means that a male to genetic counselling. cannot transmit the defect to their offspring http://pn.bmj.com/ – information that is always received by families CONCLUSION with great relief. The offspring of women with a Although mitochondrial disease is confusing, it mtDNA defect are at risk of inheriting the dis- is relatively straightforward to avoid the clinical order. Approximately one-third of pathogenic pitfalls if a few basic facts are kept in mind (see mtDNA defects are deletions, and the risk of text in box). We may have no treatments, but women transmitting mtDNA deletions is low a precise diagnosis has important implications on September 29, 2021 by guest. Protected copyright. (< 1%, the reasons for this are not known). Ap- for the management of these patients who are proximately one-third of pathogenic mtDNA being increasingly recognized. defects are homoplasmic (i.e. all of the mtDNA is mutant). In this situation, a woman will pass ACKNOWLEDGEMENTS on the defect to all her offspring who will also be PFC is funded by the Wellcome Trust homoplasmic (this is usually the case for Leber’s hereditary optic neuropathy, where about 40% FURTHER READING of male offspring and 10% of female offspring Genetests: a medical genetics information resource. become affected). For the remaining one-third, http://www.geneclinics.org/ Mitomap: a human mitochondrial genome database. the mutation is heteroplasmic and a varying http://www.mitomap.org/ proportion of mutant mtDNA may be passed DiMauro, S and Schon, E.A. (2001) Mitochondrial DNA on to the offspring. The inherited mutation mutations in human disease. Am. J. Medical Genet, load roughly correlates with the likelihood of 106, 18–26. becoming clinically affected. There are no ro- Suomalainen, A and Kaukonen, J. (2001) Diseases caused by nuclear genes affecting mtDNA stability bust counselling guidelines available for women Am. J. Medical Genet, 106, 53–61. with heteroplasmic mtDNA mutations. This is Servidei, S. (2002) Mitochondrial encephalomyopathies: an area of current study. gene mutation Neuromuscul Disord 12, 101–110.