Mitochondrial encephalopathies

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Mitochondrial encephalopathies

The mitochondrial genome is a circular molecule of DNA (mtDNA) comprising 16 569 base pairs. Each mitochondrion contains 2–10 copies of the genome, which encodes 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and 13 subunits of the respiratory chain (Fig. 24.1). Since spermatozoa contribute no or minimal mitochondria during fertilization, mitochondria and their genomes are essentially all maternally inherited (Figs 24.2, 24.3).

24.1 Diagram of human mtDNA.
The outer and inner circles represent the heavy and light strands respectively. The genes encoding the mitochondrial rRNAs are shown as 12 s and 16 s rRNA, those encoding the reduced form of nicotinamide adenine dinucleotide dehydrogenase (NADH) subunits as ND1–6, the cytochrome oxidase subunits as COI–III, and cytochrome b as Cyt b. Also shown are the genes encoding subunits 6 and 8 of adenosine triphosphatase (ATPase). Conventional single letter amino acid abbreviations are used to indicate the tRNA genes. The broad gray arc shows the region involved to a greater or lesser extent in most deletions of mtDNA. The blue arrows indicate the main sites of point mutations or deletions in mitochondrial disorders that affect the CNS.

24.2 Maternal inheritance of mitochondria and mitochondrial DNA.

24.3 Maternal inheritance and offspring mitochondrial genotype.

A wide range of disorders affecting the central nervous system (CNS) is attributable to defective mitochondrial function. Most mtDNA defects are heteroplasmic (i.e. cells contain a mixture of mutant and wild-type mtDNA) (Fig. 24.4). As mitochondria are randomly segregated during mitosis, the proportion of mitochondria containing mutant genomes varies from cell to cell. However, the levels of mutated mtDNA tend to be highest in non-dividing cells such as neurons, and skeletal and cardiac muscle. The frequency of mutations and deletions increases with age to a much greater extent in mitochondrial than in nuclear DNA. Several factors probably contribute: much of the damage is mediated by reactive oxygen species that are generated within the mitochondrion itself; there is no ‘redundant’ non-coding mtDNA; mtDNA undergoes progressively more frequent replication (exceeding that of nuclear DNA); the responsible polymerase lacks the proof-reading and repair activities of nuclear DNA polymerases.

24.4 Homoplasmy and heteroplasmy.

Tissues with a high energy demand, such as neural tissue and muscle, are particularly susceptible to impairment of mitochondrial function (Fig. 24.5). Probably because relatively few mitochondria (and therefore mitochondrial genomes) are transmitted by a mother to her progeny during embryogenesis, pronounced shifts in the degree of mitochondrial heteroplasmy can occur from generation to generation (Figs 24.6, 24.7).

24.5 The threshold effect.

24.6 Mitochondrial genetic bottleneck effect.
Rapid and marked shifts in degree of mitochondrial heteroplasmy can occur in one generation. A mitochondrial genetic bottleneck that permits only a small subset of maternal mtDNA genomes to be effectively transmitted to progeny could account for these incidences of sudden genetic drift. In this figure, the genetic bottleneck effect has resulted in a drastic shift to a state of wild-type homoplasmy for progeny 2. Given that the maternal mitochondria are approximately 50% mutant in this example, progeny homoplasmy would be unlikely if there was simply a random distribution of mtDNA genomes alone. Without the bottleneck effect, accumulated maternal mitochondrial mutations would always be transmitted in the germline. Over generations, additional accumulated mutations would result inexorably in non-functional mitochondria, non-viable oocytes or progeny, and species extinction. With random mitotic segregation, which may have a greater role in somatic cells, genetic drift is likely to be less pronounced. In contrast, the bottleneck genetic effect has the theoretical evolutionary advantage of potentially producing some progeny that have no mutant mtDNA. In the short term, deleterious mutations may accumulate in some oocytes or progeny, e.g. progeny 1, but these would be selected against.

24.7 Possible mechanisms for the mitochondrial genetic bottleneck.
Whether one or all of these mechanisms contribute to the putative genetic bottleneck remains under investigation.

In disorders with an increased level of oxidative stress, the age-related accumulation of mutations in mtDNA tends to be exacerbated. Some studies have suggested that this may impair oxidative phosphorylation and contribute to cell death in neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. (These neurodegenerative diseases are covered in Chapters 28 and 31). Several other diseases are either due to mutations in nuclear genes that encode mitochondrial proteins (e.g. Alpers syndrome, Menkes syndrome, and a range of defects of fatty acid, amino acid, and pyruvate metabolism), or cause secondary mitochondrial dysfunction (e.g. Batten’s disease). These too are considered separately, in Chapters 6 and 7.

This chapter covers only those mitochondrial diseases that involve the CNS and are due to mutations of mtDNA. In most of the diseases that are included in this chapter, the mutation in mtDNA is the primary genetic defect, but the chapter also covers a few disorders in which abnormalities in mtDNA are secondary to mutations of nuclear DNA (Table 24.1). Although Leigh syndrome can be caused by point mutations or deletions of mtDNA, it more often results from mutations in nuclear DNA, and the etiology, clinical and pathologic findings are therefore described in Chapter 6. An algorithm for genetic analysis of mitochondrial disorders is shown in Figure 24.8. Mitochondrial disorders that do not involve the CNS are not considered in this book.

Table 24.1

Mitochondrial syndromes

24.8 Algorithm for genetic analysis of mitochondrial disorders. (Algorithm adapted from the 2009 European Federation of Neurological Sciences Guidelines.)

MITOCHONDRIAL MYOPATHY, ENCEPHALOPATHY, LACTIC ACIDOSIS, AND STROKE-LIKE EPISODES (MELAS)

ETIOLOGY OF MELAS

In over 80% of cases, MELAS is caused by an A-to-G transition at nucleotide 3243 in the mitochondrial tRNA^Leu(UUR) gene. This transition is within the region of termination of transcription of the mitochondrial rRNAs, but whether this is relevant to the genesis of MELAS lesions is uncertain.

• This same mutation can also cause Kearns–Sayre syndrome (KSS), at least some features of which are often present (see below).

• When only a relatively low proportion of mtDNA is mutated at nucleotide 3243, patients may develop adult-onset, non-insulin-dependent diabetes mellitus (type I diabetes) with or without deafness (DMDF).

MELAS can also be caused by a T-to-C transition at nucleotide 3271 in the tRNA^Leu(UUR) gene.

Occasionally, MELAS is due to other point mutations in mtDNA, usually within the genes that encode mitochondrial tRNAs.

Pathogenesis

The lesions of MELAS probably result from defective mitochondrial metabolism, in which case strictly-speaking they are not infarcts in that the necrosis is not due to ischemia/oligemia or hypoxia. Some researchers have suggested that the stroke-like lesions are due to impaired cerebral blood flow resulting from an accumulation of mitochondria within the cerebrovascular endothelium and smooth muscle cells. However, the stroke-like lesions have been shown to be associated with hyperemia rather than reduced perfusion.

The likelihood of developing symptomatic MELAS is related to the proportion of mtDNA that contains the mutation. However, there is wide variation in the degree of heteroplasmy in different tissues and in most cases the correlation between the distribution of lesions and that of the mutant mtDNA is imprecise.

In vitro studies have shown that the mutant mtDNA replicates more efficiently than wild-type mtDNA and therefore tends to increase as a proportion of total mtDNA with time.

MACROSCOPIC APPEARANCES

Scattered foci of necrosis and cavitation are usually evident in the cerebral cortex (Fig. 24.9). These infarct-like lesions may be of varying ages. Although they can occur in any part of the cerebral cortex, the occipital lobe is often most severely affected. The basal ganglia, thalamus, and cerebellum are much less commonly involved.

24.9 Macroscopic appearance of brain in MELAS.
Infarct-like lesions of varying ages. (a) Recent lesions (arrows) in the frontal cortex. The distribution of necrosis in these lesions differs from that associated with hypoxia/ischemia in that the crests of the gyri are more extensively affected than the depths of sulci, and the deep laminae are largely spared. The occipital lobes (b) are often severely affected in this case by lesions of differing ages. The older lesions have produced shrinkage and brown discoloration of the cortical ribbon.

MELAS

MELAS is usually a disorder of children and young adults.

The syndrome has a variable presentation, which may include sudden onset of headache, vomiting, seizures, hemiplegia, hemianopia, and occasionally, loss of consciousness. Milder attacks may simulate migraine.

There is often some neurologic recovery between attacks, but the natural history is of recurrent stroke-like episodes leading to increasing impairment.

The degree of lactic acidosis is variable, and the myopathy may be mild.

MELAS due to the nucleotide 3243 mutation, may be associated with sensorineural hearing loss, pigmentary retinal degeneration, migraine, hypothalamic hypogonadism, hypertrophic cardiomyopathy, and non-insulin-dependent diabetes mellitus.

MICROSCOPIC APPEARANCES

The necrotic foci are histologically indistinguishable from infarcts of various ages (Fig. 24.10), although their distribution does not correspond to that of any particular arterial perfusion territory (Fig. 24.10, see also Fig. 24.9) and the crests of gyri are often more extensively affected than the depths of sulci. As with infarcts, there is capillary proliferation around and within the lesions. In many cases there is also mineralization in and around the walls of blood vessels in the globus pallidus, and occasionally in the cerebral white matter, thalamus, and dentate nucleus. This may be associated with fibrous thickening of the affected blood vessels and severe narrowing of the lumen. There may be spongy vacuolation of cerebral and cerebellar white matter, but this is unusual. Histology, histochemistry, and electron microscopy of skeletal muscle reveal ‘ragged-red’ fibers containing accumulations of morphologically abnormal mitochondria, some with paracrystalline inclusions. There are also accumulations of mitochondria in the endothelium and smooth muscle of intramuscular blood vessels (Fig. 24.10).

24.10 Histology of MELAS.
(a) Infarct-like acute lesion in MELAS. This section is through the edge of the lesion. (b) The boundary between the acutely affected superficial cortex and the intact deeper cortex has a serpiginous shape. (c) Chronic lesion, which is partly cavitated. (d) ‘Punched-out’ chronic lesions within cortex that is gliotic and depleted of neurons. (e) Skeletal muscle in MELAS. Demonstration of abnormal accumulations of mitochondria in paraffin sections of muscle from a patient with MELAS by in situ hybridization with probes to mitochondrial rRNA. An abnormally strong signal is present not only in several muscle fibers but also in some of the blood vessels (arrows).

MYOCLONIC EPILEPSY WITH RAGGED-RED FIBERS (MERRF)

ETIOLOGY OF MERFF

An A-to-G transition at nucleotide 8344 in the mitochondrial tRNA^Lys gene is demonstrable in 80–90% of cases.

This mutation consistently results in neurosensory hearing loss, and mitochondrial myopathy with ragged-red fibers. The likelihood of also developing myoclonic epilepsy is related to the proportion of mtDNA that contains the mutation.

Less common mutations producing MERRF include a C-to-T transition at nucleotide 3256 in the tRNA^Leu(UUR) gene, and MERRF/MELAS overlap syndromes have been associated with T-to-C transitions at nucleotide 8356 in the tRNA^Lys gene or at nucleotide 7512 in the tRNA^Ser gene.

MACROSCOPIC AND MICROSCOPIC APPEARANCES

The brain usually appears unremarkable macroscopically, but there may be obvious brown discoloration and shrinkage of the dentate nuclei (Fig. 24.11) and inferior olives. Microscopy reveals neuronal loss and gliosis, which is most severe in the dentate nuclei (Fig. 24.11), inferior olivary nuclei, substantia nigra, red nuclei, and basal ganglia (Fig. 24.11). The gracile and cuneate nuclei and Clarke’s column in the spinal cord may also be affected. Blood vessels in the basal ganglia and cerebral white matter may show mineralization. Skeletal muscle reveals histologic and histochemical changes of mitochondrial myopathy, with ‘ragged-red’ fibers containing accumulations of morphologically abnormal mitochondria, some with paracrystalline inclusions.

24.11 MERRF.
(a) On macroscopic examination, the dentate nucleus (arrow) appears shrunken and shows slight brown discoloration. (b) Histology reveals loss of neurons from the ribbon of the nucleus and of myelinated fibers from the hilum. (c) Higher magnification of the ribbon of the dentate nucleus shown in (b). (d) Marked pallor of myelin staining in the putamen in a case of MERRF. (Courtesy of Professor F Scaravilli, Institute of Neurology, London, UK.) (e) The putamen is gliotic, as demonstrated using immunohistochemistry for glial fibrillary acidic protein.

The findings in Leigh syndrome, which is also occasionally caused by an A-to-G transition at nucleotide 8344 in the mitochondrial tRNA^Lys gene, are considered in Chapter 6. Other point mutations in mtDNA that can cause Leigh syndrome, include T8993G, T8993C and T9176C mutations in the gene for ATPase 6.

LEBER’S HEREDITARY OPTIC NEUROPATHY (LHON), BILATERAL STRIATAL NECROSIS, AND MULTIPLE SCLEROSIS (MS)-LIKE MITOCHONDRIAL DISEASE

MACROSCOPIC AND MICROSCOPIC APPEARANCES

While photoreceptors and the retinal pigment epithelium are spared, the retinal ganglion cell layer of the eye is degenerate. Atrophy and demyelination of the optic nerve and tracts have been described. LHON and multiple sclerosis lesions involving the brain and spinal cord coexist at an estimated 50-fold increased incidence over chance alone. The lesions include both active and inactive demyelinating plaques.

BILATERAL STRIATAL NECROSIS

This rare disorder is often preceded by a febrile illness, and manifests with choreoathetosis, abnormal eye movements, seizures and mental retardation. Some cases are due to the nucleotide 14 459 mutation that can also cause severe LHON. CT scans reveal bilateral striatal lucencies. Patients with this form of bilateral striatal necrosis usually also show features of LHON.

ETIOLOGY OF LEBER’S HEREDITARY OPTIC NEUROPATHY

Over 90% of families with LHON have missense G-to-A mutations at nucleotides 11 778, 3460, or 14 484.

An A-to-G mutation at nucleotide 14 459 is responsible for a severe form of LHON and has been reported in some cases of LHON with dystonia and bilateral striatal necrosis.

A further 18 mutations have been reported in association with LHON, many of which influence the severity of the disease caused by the ‘primary’ LHON mutations noted above.

Some of the mutations are heteroplasmic, affecting only a proportion of the mitochondrial genomes; others are homoplasmic.

Retinal ganglion cells are known to be selectively vulnerable in LHON. As photoreceptors also have high oxidative requirements but are preserved, energy needs alone cannot explain the selective vulnerability. One hypothesis suggests that an actively maintained mitochondrial gradient at the lamina cribrosa, which demarcates the unmyelinated optic nerve from myelinated nerve, may be disrupted. In turn, axonal transport is perturbed leading to further lamina cribrosa mitochondrial imbalances. Retinal ganglion cell apoptosis is the end result.

All the mutations affect mitochondrial genes encoding subunits in complexes I, III, IV, or V of the respiratory chain, and defective functioning of this chain is thought to cause the manifestations of this disease. However, several aspects of the pathophysiology remain unclear, including:

• the factors determining the timing and close concordance of onset between the two eyes

• the considerably greater risk of visual loss in males than in females, which may lead to mistaken diagnosis of an X-linked recessive disorder. A recessive X-linked susceptibility gene that works in concert with the LHON mutation has been postulated.

LHON

LHON usually presents in the third or fourth decade with acute or subacute loss of central vision.

Both eyes are affected simultaneously, or sequentially over a period of up to 2 years.

Variable recovery of vision occurs depending on the site of the mutation, with the mutation at nucleotide 11 778 having the worst prognosis.

LHON is associated with non-ophthalmologic manifestations, including cardiac conduction defects and lesions involving the brain and spinal cord.

If due to the 11 778 mutation or, rarely, the 3460 mutation, LHON may be associated with a multiple sclerosis-like disease (see Chapter 19). The MRI characteristics of this condition are strongly suggestive of foci of demyelination in the cerebral white matter.

If due to the 14 459 mutation LHON may be associated with neuropathy, ataxia, dystonia, and the development of lesions in the basal ganglia.

MACROSCOPIC AND MICROSCOPIC APPEARANCES

The putamen and, less consistently, the caudate nucleus and intervening white matter, are symmetrically cavitated (Fig. 24.12). Histology reveals accumulation of lipid laden macrophages, and surrounding gliosis (Fig. 24.12).

24.12 Infantile bilateral striatal necrosis.
(a) Brain slice showing bilateral necrosis of the striatum (arrows) and intervening strip of internal capsule. (b) Accumulation of lipid-laden macrophages in the cavitated putamen.

NEUROPATHY, ATAXIA, AND RETINITIS PIGMENTOSA (NARP)

T-to-C transition or T-to-G transversion at nucleotide 8993 in the mitochondrial ATPase 6 gene can produce a syndrome of sensory neuropathy, cerebellar ataxia, retinitis pigmentosa (NARP), and mental retardation. These mutations are invariably heteroplasmic and the precise clinical manifestations and severity depend on the percentage of mutant mtDNAs. Scant information is available concerning the histologic changes in the CNS. Cerebellar atrophy with neuronal loss and gliosis has been described. Optic, auditory, and olfactory pathways may show neuronal loss as well. Biopsies of skeletal muscle from patients with NARP do not show ‘ragged-red’ fibers, but contain accumulations of lipid and glycogen within vacuoles. Hypocitrullinemia is a frequent finding. Patients in whom a high proportion of mtDNA is mutated may develop Leigh syndrome, which is considered in Chapter 6. Some authors consider NARP and maternally inherited Leigh syndrome (MILS) to be a continuum.

KEARNS–SAYRE SYNDROME (KSS), AND CHRONIC PROGRESSIVE EXTERNAL OPHTHALMOPLEGIA (CPEO)

MACROSCOPIC AND MICROSCOPIC APPEARANCES

The commonest neuropathologic abnormality in KSS is vacuolation of the white matter in the brain stem, cerebellum, and cerebrum (in decreasing order of frequency). Myelinated fibers in the deep gray nuclei and spinal cord may also be involved. This change is usually not discernible on macroscopic examination, but affected white matter may appear slightly gray (Fig. 24.13). On microscopy the affected white matter has a spongy appearance (Fig. 24.13) due to separation of myelin lamellae by clear vacuoles (Fig. 24.13), which may be elongated in the long axis of the nerve fibers. Occasionally, severe spongiform vacuolation in the brain stem or cerebellum leads to oligodendrocyte degeneration and rather poorly demarcated foci of demyelination.

24.13 Kearns–Sayre syndrome: brain.
(a) In this case, the cerebral white matter shows ill-defined gray discoloration (e.g. between arrows). On histology, spongy vacuolation of the white matter (b) and (c), is typical. (d) At higher magnification, some of the vacuoles can be seen to be within myelin sheaths.

ETIOLOGY OF KSS AND CPEO

CPEO is a component of KSS but often occurs without other features of that syndrome.

KSS is usually associated with mtDNA deletions, which vary in size in different patients.

In most cases the deletions are bracketed by direct repeats of 13–18 nucleotides, suggesting that the deletions occur as a result of a recombinational event during replication of mtDNA.

An identical 4977 base pair deletion is present in approximately one-third of patients with KSS and most patients with ‘pure’ CPEO due to mitochondrial disease. This same deletion also occurs in patients with Pearson pancreas–marrow syndrome, some of whom eventually develop KSS.

Differential tissue distribution of the deleted and wild-type mtDNAs in these disorders may partly account for the differences in phenotype.

Many patients with KSS have tandem duplications as well as deletions of mtDNA whereas those with ‘pure’ CPEO do not usually have duplications.

Other, less common causes of CPEO include MNGIE (see below), and point mutations of mtDNA.

CPEO may also be a manifestation of several neuromuscular disorders that are not due to a defect in mitochondrial DNA.

KEARNS–SAYRE SYNDROME

KSS presents in childhood or adolescence.

KSS is characterized by:

• progressive external ophthalmoplegia

• pigmentary degeneration of the retina

• cardiomyopathy with conduction defects

• various combinations of short stature, proximal myopathy, high levels of protein in the cerebrospinal fluid (CSF), deafness, and electroencephalographic abnormalities.

Mineral deposits are found in the walls of blood vessels in the globus pallidus, thalamus, dentate nucleus, midbrain, and medulla in approximately one-third of cases. There may be associated fibrous thickening of the vessel walls and severe narrowing of the lumina.

Histologic, histochemical, and electron microscopic examination of skeletal muscle usually reveals ‘ragged-red’ fibers (Fig. 24.14) containing accumulations of morphologically abnormal mitochondria, some with paracrystalline inclusions (Fig. 24.14).

24.14 Kearns–Sayre syndrome: muscle.
(a) ‘Ragged-red’ muscle fibers (asterisks) in KSS. (b) Many of the muscle fibers are deficient in cytochrome oxidase activity (asterisks). (c) Electron micrograph of part of a muscle fiber with enlarged mitochondria containing paracrystalline inclusions.

MYONEUROGASTROINTESTINAL ENCEPHALOPATHY (MNGIE) SYNDROME

MNGIE is also known as polyneuropathy, ophthalmoplegia, leukoencephalopathy and intestinal pseudo-obstruction (POLIP). It is an uncommon syndrome and its main clinical features are encapsulated in the two acronyms by which it is usually known. This rare, autosomally inherited disease is due to mutations in the gene for thymidine phosphorylase (chromosome 22q13.32-qter). This enzyme is also known as platelet-derived endothelial cell growth factor or as gliostatin. Thymidine phosphorylase is critical for phosphorylating thymidine and for forming thymine and deoxyribose-phosphate. Impaired thymidine phosphorylase activity leads to a severe deoxynucleotide pool imbalance, culminating in depletion and multiple deletions of mtDNA. Patients have diffusely abnormal white matter on MRI, but the histologic substrate of the leukoencephalopathy is poorly documented. Sural nerve biopsies have been reported to show axonal atrophy and demyelination. Muscle biopsies show ragged-red fibers, scattered fibers devoid of cytochrome c oxidase activity, and features of denervation.

The differential diagnosis includes familial visceral myopathy with external ophthalmoplegia (also known as oculogastrointestinal muscular dystrophy), and intestinal dysplasia with neuronal intranuclear inclusions (see Table 24.2). Intestinal pseudo-obstruction has also been reported in association with MELAS. Allogeneic hematopoietic stem cell transplantation has shown promise as a therapy.

Table 24.2

The diagnosis of mitochondrial disorders

Important investigations

In many cases coming to autopsy, the precise genetic abnormality will not have been determined.

If a mitochondrial disorder it is advisable to:

• Sample skeletal muscles for histology and, if possible, enzyme histochemistry and electron microscopy.

• Examine the heart, eyes, and peripheral nerves.

Findings suggestive of mitochondrial disorder

The morphologic findings on examination of the CNS may suggest or indicate a mitochondrial disorder although without genetic analysis precise classification is often difficult. There is extensive overlap between the neuropathologic findings in different mitochondrial disorders. Features suggestive of a mitochondrial disorder include:

• Intramyelinic edema or spongy vacuolation of white matter (but see also differential diagnosis on p. 525).

• Extensive calcification in the basal ganglia or cerebral white matter, thalamus, dentate nucleus, and brain stem (but see Chapter 1 and Fahr’s disease, on p. 469).

• Neuronal loss and gliosis involving the dentate nucleus, inferior olives, striatum, and gracile and cuneate nuclei. Differential diagnoses include Friedreich’s ataxia and other system degenerations (see Chapters 27, 29, and 30).

• Rarefaction and coarse vacuolation of gray matter with preservation of neurons, particularly in the striatum, brain stem, and dentate nucleus (see also Wernicke’s encephalopathy on p. 453 and Leigh’s disease on p. 150).