Neurodegeneration in Primary Mitochondrial Disorders 2 · 2.2.2 Chronic Progressive External Ophthalmoplegia Chronic progressive external ophthalmoplegia (CPEO) is deÞ ned by a slowly

21A.K. Reeve et al. (eds.), Mitochondrial Dysfunction in Neurodegenerative Disorders, DOI 10.1007/978-0-85729-701-3_2, © Springer-Verlag London Limited 2012

Abstract Mitochondria are critically responsible for the generation of energy in the form of ATP through the electron transport chain (ETC). The central nervous system (CNS) performs highly energy-intensive tasks and is therefore particularly dependent on ATP. Defects residing within the complexes comprising the ETC can affect the synthesis of ATP and consequently severely compromise neuronal function. It is unsurprising then that primary mitochondrial DNA mutations are an important cause of neurological disease. The clinical presentation is often heterogeneous in terms of age of onset and different neurological signs and symptoms which might include ataxia, seizures, cognitive decline, blindness, and stroke. The clinical course can vary considerably but in many patients there is progressive neurological decline and marked neurodegeneration. Our under-standing of the mechanisms underpinning neurodegenerative changes due to mitochondrial DNA mutations is limited due to the availability of appropriate animal models of disease. However, studies on human postmortem CNS tissues have provided an invaluable insight into the distribution and severity of neuronal degeneration in patients harboring mitochondrial DNA defects. In this chapter, we describe the neuropathological changes occurring in the CNS associated with

N. Lax (*) Mitochondrial Research Group , Institute for Ageing and Health, Newcastle University , Newcastle upon Tyne, Tyne and Wear , UK

Institute for Ageing and Health/UK NTHR Biomedical Research Centre for Ageing and Age-Related diseases, Newcastle University , Newcastle upon Tyne, Tyne and Wear , UK e-mail: [email protected]

E. Jaros Neuropathology/Cellular Pathology , Newcastle upon Tyne Hospitals, NHS Foundation Trust, Royal Victoria Infi rmary , Newcastle upon Tyne, Tyne and Wear , UK

Institute for Ageing and Health/UK NTHR Biomedical Research Centre for Ageing and Age-Related diseases, Newcastle University , Newcastle upon Tyne, Tyne and Wear , UK e-mail: [email protected]

Neurodegeneration in Primary Mitochondrial Disorders

Nichola Lax and Evelyn Jaros

2

22 N. Lax and E. Jaros

different mutations of the mitochondrial genome and discuss the mechanisms which might contribute to neural dysfunction and cell death.

Keywords Mitochondria • Mitochondrial DNA • Neurodegeneration

2.1 Introduction

Mitochondrial disease was fi rst described in 1962, when Luft and colleagues described a patient with non-thyroidal hypermetabolism [ 1 ] . Since this discovery, major advances in our understanding of mitochondrial biology and genetics have permitted the recognition of a number of mitochondrial disorders. Mitochondrial diseases are a heterogeneous group of disorders that arise from defects in either the mitochondrial or nuclear genome. As has been discussed elsewhere in this book ( Chap. 1 ), mitochondria contain their own circular double-stranded DNA encoding for 13 polypeptides of the electron transport chain (ETC), 22 transfer RNAs (tRNA), and 2 ribosomal RNAs (rRNA) [ 2 ] . This genome is under dual genetic control, with many mitochondrial maintenance genes encoded by the nuclear genome.

Mitochondrial diseases present an important social and economic burden with the estimated prevalence of 1 in 10,000 of people clinically manifesting the disease and a further 1 in 6,000 who are at risk of developing mitochondrial disease [ 3 ] .

In mitochondrial disease, the central nervous system (CNS) can be particularly vulnerable to neuronal cell loss. Firstly, the brain is highly metabolically active and therefore particularly susceptible to bioenergetic failure. Secondly, it has consider-ably fewer antioxidant defenses than other tissues, so is less able to protect against excessive reactive oxygen species (ROS) production. Thirdly, with the exception of subventricular zone, olfactory epithelium, and the hippocampus, most neurons within the brain are postmitotic, and therefore irreplaceable. Consequently, any par-ticular neuronal insult will prove fatal if not alleviated in some way. In addition, there is increasing evidence linking mitochondrial dysfunction to neuronal cell loss in age-related neurodegenerative disorders, including Alzheimer’s disease and Parkinson’s disease.

2.2 Neurological Features and Genetics of Mitochondrial Diseases

Patients with mitochondrial diseases present with a wide variety of neurological manifestations, in particular optic atrophy, ataxia, seizures, dementia, stroke-like episodes, extrapyramidal features, and neuropathy. Whilst it is recognized that some of the symptoms are associated with particular patterns of CNS pathology and with certain mitochondrial DNA (mtDNA) mutations, little is understood about the mechanisms that underlie these associations.

232 Neurodegeneration in Primary Mitochondrial Disorders

2.2.1 Kearns–Sayre Syndrome

Kearns–Sayre syndrome (KSS) presents with a characteristic triad of symptoms including development of retinitis pigmentosa, progressive external ophthalmople-gia, and an onset before 20 years of age [ 4 ] . Additional neurological features include cerebellar ataxia, raised cerebrospinal fl uid (CSF) protein levels, subclinical neu-ropathy, cognitive impairment (defi ned by defects in visuospatial attention and executive function), and deafness. As this is a multisystem disorder, other clinical manifestations such as proximal myopathy, complete heart block, cardiomyopathy, endocrinopathies, short stature, and dysphagia are particularly prominent [ 5 ] .

This is predominantly a sporadic condition caused by a single large-scale dele-tion or complex rearrangements of mtDNA [ 6 ] . There is no specifi c deletion respon-sible for the disease as all deletions will remove multiple genes encoding ETC proteins and tRNAs. However, the majority of mtDNA deletions are located within the major arc between the two proposed origins of replication (O

H and O

L ). Often

mtDNA deletions may be fl anked by short direct repeats. Approximately, one-third of these patients harbor a 4,977 bp deletion, known as the common deletion which has a 13 bp repeat sequence [ 7 ] .

2.2.2 Chronic Progressive External Ophthalmoplegia

Chronic progressive external ophthalmoplegia (CPEO) is defi ned by a slowly pro-gressive ophthalmoplegia and ptosis. The age of onset is variable, with some patients fi rst noticing ptosis in the third or fourth decade of life. As the disease progresses, associated proximal muscle weakness and fatigue commonly occur but are rarely debilitating.

The genetic defect in CPEO can vary and can be caused by mtDNA rearrange-ments [ 8 ] , single nucleotide substitutions in specifi c tRNA genes [ 9– 11 ] , or muta-tions in nuclear-encoded mitochondrial maintenance genes which cause multiple mtDNA deletions [ 12 ] .

2.2.3 Mitochondrial Encephalopathy Lactic Acidosis and Stroke-Like Episodes

Mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS) presents with a triad of lactic acidosis, seizures, and stroke-like episodes which remain central to diagnosis and pathology. Although the m.3243A>G mutation was the fi rst and most frequently described mutation in association with MELAS, a number of other mutations including m.3253A>G [ 13 ] , m.3271T>C [ 14 ] , and m.3291T>C [ 15 ] have subsequently been identifi ed. Furthermore, m.1642G>A in mt-tRNA Val [ 16 ] , mutations in complex IV of the ETC, cytochrome c oxidase (COX) subunit III (such as m.9957T>C) [ 17 ] , and mutations within the mtDNA-encoded complex I (MTND) subunit genes have been described [ 18– 22 ] . It is also important to note that the m.3243A>G mutation can lead to other distinct clinical phenotypes, such as maternally inherited diabetes and deafness [ 23 ] and CPEO [ 9 ] .


2.2.4 Myoclonic Epilepsy Ragged Red Fibers

Myoclonic epilepsy ragged red fi bers (MERRF) is a severe neurodegenerative dis-order which presents during childhood or early adulthood. Clinically, myoclonus is often the fi rst presenting feature which becomes progressive. Other neurological symptoms comprise focal and generalized epilepsy, cerebellar ataxia, dementia, pyramidal signs, neuropathy, sensorineural hearing loss, and optic atrophy. Non-neurological symptoms include muscle weakness and wasting, hypertrophic cardio-myopathy, multiple lipomas, and Wolff–Parkinson–White syndrome [ 24 ] . Initially, MERRF was described in association with m.8344A>G point mutation in the MT-TK gene encoding for tRNA Lys . Whilst other mutations in the same tRNA have been reported, the m.8344A>G change remains the most common [ 25 ] .

2.2.5 Leigh Syndrome

Leigh syndrome is a progressive neurodegenerative disorder which has an onset in infancy and childhood. This disease is characterized by symmetric necrotic lesions distributed along the brainstem, diencephalon, and basal ganglia which have been documented in both neuroimaging and postmortem studies. Common neurological features include ataxia, dystonia, and developmental delay. Other clinical features include respiratory abnormalities, nystagmus, and hypotonia [ 26 ] . A severe failure of oxidative metabolism within the brain of infants with Leigh syndrome has been described in association with a variety of nuclear and mtDNA mutations.

2.2.6 Leber’s Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) was fi rst described by Theodore Leber in 1871 [ 27 ] . The salient clinical feature of LHON is the almost exclusive involve-ment of the optic nerve. Patients typically present with a subacute or acute, painless, loss of central vision usually between the ages of 20 and 40 years with a strong predilection for males. The fi rst mtDNA point mutation identifi ed was m.11778G>A [ 28 ] , however additional point mutations m.3460G>A and m.14484T>C have sub-sequently been identifi ed. It has been shown that over 95% of LHON pedigrees harbor one of these mtDNA point mutations, all involving genes encoding complex I subunits of the ETC [ 29 ] .

2.2.7 Mitochondrial Diseases Associated with Primary Mutations in POLG Gene

Mitochondrial DNA polymerase g is the only polymerase responsible for the syn-thesis and repair of mtDNA in mammalian cells. The human mtDNA polymerase is a 195 kDa heterotrimer consisting of a 140 kDa catalytic subunit (POLG1) and two identical 55 kDa accessory units (POLG2). The C-terminus of the catalytic


subunit of POLG1 is responsible for the polymerase function whilst the N-terminus is responsible for exonuclease activity and proofreading of mtDNA. The linker mediates a focal contact with the dimeric accessory subunit. POLG1 is encoded by the nuclear POLG gene. Primary mutations in the POLG gene represent one of the mechanisms that cause secondary mutations in the mtDNA, including depletion of mtDNA, multiple mtDNA deletions, or multiple point mutations in mtDNA [ 30 ] .

Recently, numerous mutations have been described in POLG in association with a spectrum of clinical/neurodegenerative phenotypes, including autosomal domi-nant and autosomal recessive progressive external ophthalmoplegia (PEO) [ 31– 33 ] , autosomal recessive sensory ataxic neuropathy with dysarthria and ophthalmople-gia (SANDO) [ 34 ] , mitochondrial inherited recessive ataxia syndrome without oph-thalmoplegia (MIRAS) [ 35 ] , Parkinsonism [ 36 ] , and Alpers syndrome [ 37 ] . In a large cohort of patients with POLG mutations, childhood presentation has been associated with the Alpers-Huttenlocher syndrome, characterized by fatal hepatoen-cephalopathy with refractory epilepsy, stroke-like episodes, cortical blindness, and the development of acute cerebral lesions with a propensity for the striate cortex. Presentation in teenage years or adulthood has been associated with ptosis, PEO, and unexplained multisystem neurological disorder or isolated neurological disor-der with family history [ 38 ] . The reasons for the large variability in the clinical/neurodegenerative phenotype in relation to the POLG genotype, and in turn to the secondary mtDNA mutations, are poorly understood. Involvement of some, as yet unidentifi ed, epigenetic factors has been suggested by a report on two siblings with identical compound heterozygous POLG mutations, one of whom presented with Alpers syndrome and the other with SANDO [ 39 ] .

2.3 Neurodegeneration in Mitochondrial Diseases

2.3.1 Brain Atrophy

Postmortem brain atrophy is a common observation in patients with mitochondrial diseases, though quantitative data are sparse. In a Newcastle series of 15 brains dis-sected at postmortem from patients with genetically diverse mitochondrial disease, evidence of atrophy was present in 12 (Professor Robert Perry, personal communi-cation). The average fresh weight was reduced by about 19% (range: 6–37%; Table 2.1 ) compared to brains from normal individuals matched for age and gender [ 40 ] . Of the 12 patients with atrophic brains, six were aged between 20 and 60 years, received MELAS diagnosis and harbored m.3243A>G, three patients were aged between 18 and 34 years and harbored mutations in POLG , one patient was aged 42 years with MERRF diagnosis and m.8344A>G, one patient was aged 40 years with KSS diagnosis and a single, large-scale mtDNA deletion, and the fi nal patient was aged 55 years and harbored homoplasmic m.14709T>C. Three of the 15 brains had the fresh weight within normal limits. This included a brain from a patient, aged 34 years, with neuropathological features of Leigh/MELAS and m.13094T>C, and two brains from patients aged 59 and 71 years, who had mutations in POLG .


2.3.2 Neuroradiological Imaging

Neuroradiological imaging in patients with mitochondrial diseases often demon-strates focal lesions in cerebral cortex, white matter, basal ganglia and brainstem and, particularly in patients with longer disease duration, generalized cerebral and cerebellar atrophy, all suggestive of CNS neuron loss. Enlargement of the fourth ventricles is often profound in younger patients and could be a predictor of cerebel-lar atrophy [ 41, 42 ] .

Most of the imaging fi ndings can be diverse and nonspecifi c with little correla-tion with genotype or biochemical phenotype but in some of the mitochondrial dis-eases the lesions show predilection for particular anatomical areas of the brain. For instance, in MELAS patients there is often prominent dilation of occipital horns with focal stroke-like cortical lesions (SLCL) in a nonvascular distribution gener-ally in the posterior of the brain consistent with stroke-like episodes (SLEs). In early stages of the disease, the SLCL show features of vasogenic edema, rather than isch-emic-like lesions [ 43 ] and often exhibit rapid resolution associated with clinical improvement. However, presence of cerebral atrophy in long-standing disease sug-gests association with gradual attrition of neuronal cells [ 41, 42 ] . In MERRF patients harboring the m.8344A>G mutation, neuroradiological imaging most often shows atrophy in cerebral cortex, cerebellum, superior cerebellar peduncle and brainstem [ 44, 45 ] . More recently, MRI showed evidence of thalamic demyelination in a young male harboring m.8344A>G but without the typical clinical presentation [ 46 ] . In patients with Leigh syndrome necrotic-like lesions distributed along the brainstem,

Table 2.1 Postmortem brain weights Mitochondrial cases Normal cases (Dekaban [ 40 ] )

Age Sex Genetic defect Wt (g) Wt (% normal)

Wt (% reduction)

Age range Sex

Wt (g) mean ± SD

18 F POLG 1,098 81.9 18.1 16–18 F 1,340 ± 40 20 F m.3243A>G 829 63.3 36.7 19–21 F 1,310 ± 50 24 F POLG 1,081 83.2 16.8 22–30 F 1,300 ± 10 30 M m.3243A>G 1,174 81.5 18.5 22–30 M 1,440 ± 20 34 F m.13094T>C 1,260 97.7 0 31–40 F 1,290 ± 30 34 F POLG 973 75.4 24.6 31–40 F 1,290 ± 30 36 F m.3243A>G 930 72.1 27.9 31–40 F 1,290 ± 30 40 F Single mtDNA

deletion 1,040 80.6 19.4 31–40 F 1,290 ± 30

42 F m.3243A>G 1,070 82.9 17.1 41–50 F 1,290 ± 20 42 F m.8344A>G 1,075 83.3 16.7 41–50 F 1,290 ± 20 55 M m.14709T>C 1,325 94 6 51–55 M 1,410 +/− 10 57 F m.3243A>G 1,150 92 8 56–60 F 1,250 ± 20 59 M POLG 1,480 108 0 56–60 M 1,370 ± 20 60 F m.3243A>G 984 78.7 21.3 56–60 F 1,250 ± 20 71 M POLG 1,340 99.3 0 71–75 M 1,350 ± 20


thalamus, and basal ganglia are considered characteristic [ 41 ] . Patients harboring POLG mutations demonstrate a combination of thalamic and cortical lesions with-out basal ganglia involvement but with lesions in deep cerebellar nuclei and inferior olivary nuclei which can be indicative of mitochondrial spinocerebellar ataxia and epilepsy. In contrast, the main fi nding in patients with POLG mutations and Alpers disease with pediatric onset are stroke-like lesions in occipital cortex [ 47 ] , although thalamic changes have also been described, particularly in patients presenting as young adults [ 48, 49 ] .

In some mitochondrial diseases imaging shows predominantly white matter lesions. In KSS patients symmetrical lesions in the white matter of the cerebrum, cerebellum, globus pallidus, dorsal midbrain, and thalamus are consistently reported [ 41 ] . In mitochondrial neurogastrointestinal encephalopathy (MNGIE) patients there are diffuse white matter lesions in cerebral hemispheres, brainstem, and cere-bellar peduncle in addition to demyelinating peripheral neuropathy [ 50 ] .

Intracerebral calcifi cation has been commonly reported in MELAS, KSS, and Leigh syndrome. A neuroimaging study from 1998 revealed that basal ganglia (BG) calcifi cation, involving the caudate, putamen, thalamus, and global pallidus, was the most common fi nding in 54% of MELAS patients harboring the m.3243A>G muta-tion [ 42 ] . A similar distribution pattern is seen in ageing suggesting that BG calcifi -cation in mitochondrial disease, and particularly in MELAS, may occur due to accelerated ageing [ 51 ] . This particular feature is also seen in a range of insults to the brain including anoxia [ 52 ] , exposure to radiation [ 53 ] , and parathyroid [ 54 ] , and hypothyroid disorders [ 55 ] suggesting that this brain region responds to damage by laying down minerals. Although BG calcifi cation in mitochondrial diseases is often severe, it has not been reported in association with BG atrophy. It is also unclear if there are any functional symptoms related to this pathology in the patients with mitochondrial diseases, and whilst there is evidence of BG calcifi cation in other diseases, there does appear to be a lack of correlation between the degree of pathology and emergence of clinical symptoms [ 56, 57 ] . It is intriguing that in the very old and also in younger patients with psychiatric illnesses psychotic symptoms are strongly associated with basal ganglia calcifi cation [ 51 ] .

2.4 Neuropathology of Mitochondrial Diseases

When postmortem brains from patients with mitochondrial diseases are examined microscopically there is profound neuron loss, often with microcystic change and astrogliosis and/or axon and glial loss from the anatomical areas of focal lesions seen by neuroradiological imaging. There is also more subtle microscopic pathol-ogy, not detectable by neuroradiology. These include variable reductions in neu-ronal density and in levels of mitochondrial ETC proteins detectable within neurons and cerebral vasculature using histochemistry and immunohistochemistry. Such microscopic changes are often reported in the cerebellar cortex, cerebellar dentate nucleus, medullary inferior olives, and other brain areas of many mitochondrial diseases but quantitative data are sparse (Fig. 2.1 ).


2.4.1 MELAS

MELAS is neuropathologically characterized by the presence of multiple “infarct-like” lesions in the occipital, parietal, and temporal lobes of the brain and less in the cerebellar cortex (Fig. 2.1a ). The lesions may be asymmetric, and when severe may be observed externally on the surface of the brain. Although microscopically they resemble true infarcts, commonly involving considerable neuronal cell loss, neuronal eosinophilia, astrogliosis, and microvacuolation, unlike infarcts, they

a b

ed

g h i j

c

f

Fig. 2.1 Cerebellar pathology in patients harboring primary mtDNA mutations. ( a ) Infarct-like lesion involving the molecular layer, Purkinje cells, granular cell layer, and the white matter of the cerebellar cortex in a patient harboring the m.3243A>G mutation (Cresyl fast violet stain, scale bar = 100 m m). ( b ) Gross atrophy of the cerebellar cortex in a patient harboring a m.3243A>G muta-tion (Cresyl fast violet stain, scale bar = 100 m m). ( c ) Foci of mild spongiform degeneration and moderate myelin loss in the cerebellar deep white matter from a patient with KSS harboring a single large-scale mtDNA deletion (Loyez myelin stain). ( d ) Formation of an axonal torpedo ( arrow ) in the granular cell layer in a patient with KSS harboring a single large-scale mtDNA deletion (Anti-phosphorylated neurofi lament, scale bar = 100 m m). ( e ) Multiple axonal torpedos ( arrows ) evident in the granular cell layer of a patient harboring m.14709T>C mutation (Bielschowsky’s silver stain, scale bar = 100 m m). ( f ) Trapped mitochondria within swollen Purkinje cell dendrites in the molecu-lar layer in a patient with KSS due to a single, large-scale deletion of mtDNA (Anti-porin, scale bar = 100 m m). ( g ) Porin immunohistochemistry in the dentate nucleus of a patient harboring the m.3243A>G mutation reveals abundant neuronal mitochondria (scale bar = 100 m m). ( h ) Complex I 30 kDa defi cient neurons ( arrowhead ) are evident in the dentate nucleus of a patient harboring the m.3243A>G mutation (scale bar = 100 m m). ( i ) Intact complex II 70 kDa proteins levels in the den-tate nucleus of a patient harboring the m.3243A>G mutation (scale bar = 100 m m). ( j ) Marked reduction in complex IV subunit I protein expression in the neuronal population ( arrowhead ) of the dentate nucleus of a patient harboring m.3243A>G (scale bar = 100 m m)


have cortical laminar distribution and are not be restricted to particular vascular territories of specifi c cerebral arteries or watershed zones [ 58 ] . These “infarct-like” lesions represent the chronic stage of the disease. They are seen as SLCL by neu-roradiological imaging and may be responsible for the SLE in MELAS. The patho-genic mechanism underlying the preferentially posterior distribution of the neuron loss in MELAS is not fully understood. There appears to be no correlation between distribution of the lesions, COX (cytochrome c oxidase)-defi cient neurons and intraneuronal levels of m.3243A>G. Based on fi ndings of profound defi ciency of COX in the walls of leptomeningeal and cortical vessels, it has been proposed that coupling of vascular mitochondrial dysfunction with cortical spreading depression originating in the occipital lobe are involved [ 59 ] .

Microscopic examination of areas of basal ganglia calcifi cation commonly seen on neuroimaging of MELAS patients show, often prominent, mineralization of the vessel walls within the basal ganglia. However, within the basal ganglia, there are no intraneuronal calcium deposits, the neuronal populations are usually spared, and there is no evidence of infarct-like lesions [ 42 ] .

2.4.2 MERRF

Microscopically, MERRF is characterized by severe neuronal cell loss and astro-gliosis within the cerebellar dentate nucleus, cerebellar cortex, including Purkinje cells, in the inferior olivary nuclei, red nucleus and substantia nigra [ 60 ] . Within these regions, abnormally enlarged mitochondria containing inclusion bodies have been observed [ 61 ] . Additionally, neuron loss has been detected within the gracile and cuneate nuclei, Clarke’s column within the spinal cord, the sensory neurons within the dorsal root ganglia, and less in the basal ganglia and thalamus. Myelin loss, likely secondary to neuronal/axonal loss, is an additional degenerative feature documented in the superior cerebellar peduncle and posterior spinal columns [ 62 ] . The selective vulnerability to degeneration of specifi c neuronal populations in MERRF remains under investigation. An immunohistochemical investigation showed selective reduced expression of COX II within remaining neurons of the cerebellar dentate nucleus, inferior olivary nuclei, and frontal cortex [ 63 ] . Also, electron chain abnormalities of complex I, III, and IV have been reported in MERRF [ 64 ] . However, a microdissection study conducted on a patient harboring the m.8344A>G mutation demonstrated an apparent lack of correlation between extent of pathology and mutation load within individual CNS neurons [ 65 ] . This suggests that in MERRF there are important factors other than proportion of mutant mtDNA that determine the degree of mitochondrial dysfunction and the fate of neurons.

2.4.3 KSS

White matter abnormalities are often described as being the neuropathological hall-mark of KSS, however gray matter involvement, in particular of the cerebellar cortex and brainstem nuclei, has also been documented (Fig. 2.1c ). The defi ning characteristic


is the presence of widespread spongiform degeneration of white matter tracts (spongi-form leukoencephalopathy) in the cerebrum, brain stem, spinal cord, cerebellum, thala-mus, and basal ganglia [ 62 ] . The degree of spongiform degeneration can vary from slight myelin pallor at early stages through mild spongy changes to coarse vacuolation, resulting in a “sieve-like appearance.” The selective involvement of white matter pathol-ogy observed in KSS is proposed to be a consequence of a specifi c vulnerability of the myelin-producing glia, oligodendrocytes [ 66 ] .

Although white matter abnormalities are the predominant feature in KSS, gray matter pathology has also been documented, particularly within the cerebellum and brainstem. Evidence of Purkinje cell loss, formation of axonal torpedos (Fig. 2.1d ), and abnormal dendrites (Fig. 2.1e ) have all been described in the cerebellar cortex of patients with KSS. The deep cerebellar nuclei, including the dentate nucleus, show intact neuronal cell density despite evidence of reduced ETC protein expres-sion, a loss of synaptic contact from Purkinje cells and spongy changes [ 67 ] .

2.4.4 Leigh Syndrome

The symmetrical lesions identifi able by MRI in the brainstem, basal ganglia, and thalamus of patients with Leigh syndrome are microscopically characterized by “vasculonecrotic” microcystic degeneration with neuron loss, astrogliosis, myelin loss, and vascular proliferation [ 26 ] . In addition, more subtle microscopic lesions are widespread, commonly involving the dorsal pons, inferior olives in the medulla, roof nuclei in the cerebellum, and posterior columns and anterior horns in cervical spinal cord [ 58, 68 ] . In the gray matter nuclei, the main features are neuronal ischemic-type change and neuronal loss, whilst in the white matter there is both myelin and axonal loss. Subacute lesions show neuropil edema and swollen cell processes. The cerebral cortex and cerebral white matter are generally only mildly affected. Given that Leigh syndrome has been associated with mutations in a multitude of nuclear and mtDNA genes encoding mitochondrial enzymes, including pyruvate dehydrogenase, and respiratory complexes I, II, IV, and V the molecular mechanisms involved in the pathogenesis of these lesions may vary, depending on the underlying mutation.

2.4.5 LHON

In general, neuropathology in LHON is limited to the optic system, in particular to neurodegeneration of the retinal ganglion cell (RGC) layer, with sparing of the reti-nal pigment epithelium and photoreceptors. Within the RGC layer, there is pro-nounced cell body and axonal degeneration, with associated myelin loss and atrophy observed from the optic nerves to the lateral geniculate bodies [ 69 ] . There is evidence of impaired glutamate transport, increased mitochondrial reactive oxy-gen species (ROS), and apoptosis within the RGCs [ 70 ] . Rare LHON patients have been described with additional distinct spinal cord pathologies: an infl ammatory MS-like disorder – “classic” Harding disease [ 71 ] or a subacute metabolic axonop-athy of posterior columns with associated features of Leigh-like syndrome; the


pathogenesis of the subacute selective axonopathy is unclear as the patient was homoplasmic for m.3460G>A in all tissues [ 72 ] .

2.4.6 Mitochondrial Diseases Associated with Multiple mtDNA Deletions

The neuropathology in a patient harboring two recessive heterozygous mutations, p.A467T and a novel p.X1240T in POLG with secondary multiple mtDNA dele-tions, was initially described in 2000 [ 73 ] . In this patient, the inferior medullary olives were the most severely affected region, suffering massive neuronal loss. Moderate to extensive neuronal loss was observed throughout the cerebellum and to a lesser extent in the dentate nucleus and the red nuclei, while the pons remained unaffected. This pattern is indicative of cerebello-olivary atrophy and probably underlies the cerebellar ataxia observed in patients with multiple deletions. The spi-nal cord tissues demonstrated evidence of severe myelin and axonal loss within the posterior columns and in dorsal spinal roots. Severe neuronal loss and ETC abnor-malities were also present in dorsal root ganglia and in paraspinal sympathetic gan-glia (Fig. 2.2 ). Ventral and lateral spinal tracts, motor roots, and motor neurons were intact. There was also severe depletion of neurons from the substantia nigra without

a b c

fed

Fig. 2.2 Evidence of prominent defi ciencies of ETC proteins within the sensory neurons of the dorsal root ganglia in a patient harboring two primary heterozygous mutations, p.A467T and p.X1240C, in POLG1 and secondary multiple deletions of the mtDNA. ( a ) Unusually reduced mitochondrial density in one neuron ( arrow ) and uneven cellular distribution within sensory neu-rons (anti-porin immunohistochemistry). ( b ) Profound neuronal defi ciency of the 15 kDa subunit of complex I with focal cytoplasmic clumping of immunoreactivity. ( c ) Reduced expression of the 70 kDa subunit of complex II within the neurons of the dorsal root ganglia, similar to the distribu-tion observed with porin. ( d ) Dual COX/SDH histochemistry reveals a high level of complex IV (COX) defi ciency in some neurons ( blue ), whilst in other neurons the COX activity is relatively unaffected ( brown ). ( e ) Markedly reduced expression and focal cytoplasmic clumping of subunit I of complex IV in all neurons. ( f ) Reduced expression and focal cytoplasmic clumping of subunit IV of complex IV in some neurons ( arrow ). Scale bar represents 100 m m


Lewy body formation. Throughout the brain of this patient there was lack of correla-tion between the distribution of COX-defi cient neurons and the degree of neuro-pathological damage, similar to the fi ndings in patients with MELAS and MERRF.

More recently, a study on a patient harboring multiple deletions due to two heterozygous POLG mutations revealed loss of pigmented neurons in the substantia nigra (SN) with alpha-synuclein-reactive Lewy body formation. In addition, unlike in patients with Lewy body Parkinson’s disease (PD), but similar to patients with some mitochondrial diseases, there was loss of Purkinje cells, loss of neurons from the cerebellar dentate nucleus, profound loss of myelin and axons from posterior columns of spinal cord, and loss of neurons from gracile nucleus. In the patient with the POLG mutations, the remaining SN neurons had high levels of mtDNA dele-tions (~65%) associated with COX-defi ciency affecting 21% of the neurons. This was in contrast to the percentage of COX-defi ciency observed in SN neurons in PD and ageing at <3% and <1%, respectively [ 74 ] .

Multiple mtDNA deletions and/or mtDNA depletion also underlie severe degenera-tion of the cerebellum, brainstem, and spinal cord that occur in mitochondrial inherited recessive ataxia syndrome (MIRAS) and in infantile-onset spinocerebellar ataxia with sensory neuropathy (IOSCA) [ 75 ] . In both of these diseases, neuronal complex I pro-tein expression is either absent or dramatically reduced in the cerebellum and frontal cortex. In MIRAS, the brain shows multiple deletions of mtDNA and mtDNA deple-tion due to a primary mutation in POLG , whilst in IOSCA, caused by a primary muta-tion in C10orf2 gene that encodes the mitochondrial DNA helicase Twinkle, the brain shows only mtDNA depletion but no multiple deletions of mtDNA [ 35 ] .

2.5 Mechanisms of Neurodegeneration in Mitochondrial Diseases

Understanding molecular processes that lead to neuronal loss in the various mito-chondrial diseases remains elusive. In particular, it is unclear why particular neu-ronal types/systems show greater susceptibility to neurodegeneration in association with certain mtDNA mutations. There appears to be little correlation between the degree of neuropathological damage, intraneuronal level of defi cit in mtDNA-encoded enzymes, and intraneuronal levels of mutated mtDNA (heteroplasmy) in patients with many mitochondrial diseases, including multiple mtDNA deletions, MELAS, and MERRF. This suggests involvement of additional factors. There is evidence for mitochondrial ETC protein defi ciency and dysfunction in cerebral vas-culature and possibly regional dysfunction of astroglia in MELAS patients. In addi-tion, existence of differences in the patterns of neuronal activity/physiological properties, and neurotransmitter metabolism between neurons in different brain ana-tomical areas is well documented. It is likely that particular patterns of neurodegen-eration can be accounted for by interactions between energy demand and variable types of defi ciencies in the mitochondrial ETC proteins, back-up mechanism, and calcium handling at the levels of individual neurons, supporting astroglia and cere-bral vasculature. The complexities of these interactions are yet to be unraveled.


2.5.1 Mitochondrial Homeostasis

There is evidence for a mitochondrial cause of neurodegeneration in patients har-boring mtDNA mutations or defects in nuclear-encoded components involved in mitochondrial homeostasis. Although mitochondria are essential for ATP genera-tion through the ETC, mitochondria also play additional key roles in cellular homeo-stasis, including production of iron-sulfur clusters, calcium handling, regulation of apoptosis ( Chap. 2 ), and oxidative stress ( Chap. 6 ). To some extent, all of these processes have been implicated in neurodegenerative disease.

2.5.2 Mitochondria and Calcium Handling

Mitochondria are well-recognized intracellular calcium buffers with increased cal-cium uptake during oxidative phosphorylation [ 76 ] mediated by activation of calcium-sensitive dehydrogenases from the Krebs cycle and pyruvate dehydrogenase [ 77 ] . Calcium homeostasis is particularly important for neuronal function where calcium is intricately linked to neurotransmitter vesicle release, synaptic plasticity, and organelle movement (see Chap. 7 ). Calcium is critical for the regulation of mitochondrial distri-bution throughout the brain, particularly for mitochondrial movement along axons (see Chap. 10 ). Neuronal mitochondria transition between mobile and stationary states in response to intracellular calcium [ 78 ] . It is thought that this mechanism facilitates mitochondrial retention in presynaptic terminals and postsynaptic dendritic spines, where calcium infl ux is dynamic and homeostasis maintenance is heavily required. Whilst there have been a limited number of studies investigating the effect of mtDNA defects on calcium homeostasis, these have often focused upon non-neuronal cell lines where physiological differences are manifold. Fibroblasts obtained from patients har-boring the m.3243A>G mutation associated with MELAS tend to reveal higher basal calcium levels and a sustained elevation of calcium in response to stimulation with potassium [ 79 ] . Furthermore, fi broblasts from patients harboring complex I defi ciency due to nuclear subunit defi ciencies show evidence of a prolonged increase in calcium following stimulation with bradykinin [ 80– 82 ] . Clearly, there does seem to be some abnormalities of calcium homeostasis due to mtDNA mutations. Indeed, a recent study on neurons derived from mouse embryonic stem cells has revealed a progressive calcium handling defi cit in those cells harboring mtDNA mutations [ 83 ] .

2.5.3 Mitochondria and Cell Death

There are at least three cell death pathways which might become induced in ener-getically compromised neurons. These processes include: apoptosis, autophagy, and necrosis and each are morphologically distinct and are induced differentially. The process of regulated cell death takes place via the process of apoptosis, and mitochondria are intricately involved in this pathway through the intrinsic mito-chondrial apoptotic pathway.


Although the mechanism of neuronal cell death in patients with mitochondrial disease has not been investigated, there are few studies which examine the apopto-sis in muscle in association with ETC defects. However, the results from these reports are inconsistent and contradictory, with some papers suggesting the pres-ence of molecular executioners of apoptosis [ 84– 87 ] whilst others do not detect any evidence of these proteins [ 88 ] . Therefore, the role of apoptosis in the demise of muscle fi bers due to mtDNA defects is highly contentious. It remains to be determined how cell death might become initiated in neurons containing dysfunc-tional mitochondria.

2.5.4 Oxidative Stress

Mitochondrial metabolism is also responsible for the majority of the ROS produc-tion in cell. The formation of ROS occurs when unpaired electrons escape the ETC and react with molecular oxygen, generating superoxide. Complexes I and III of the ETC are the main sites implicated in superoxide generation. Although ROS can be very damaging to the cell as they can react with DNA, proteins, and lipids leading to oxidative stress, they also play an important role in intracellular signaling [ 89 ] . Excessive ROS generation is associated with both neurodegenerative diseases and ageing (see Chap. 6 ). The maintenance of physiological ROS levels is critical to normal cellular functioning. Exploration of ROS generation as a consequence of mtDNA defects stems mainly from animal models or cell culture. An increase in ROS production has been reported in neuronal NT2 cybrid cells that harbor a muta-tion causing LHON [ 90 ] . A study conducted on patient cybrid cells harboring the m.8883T>G mutation associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa revealed that antioxidant treatment conferred cellular protec-tion. However, contrasting results have been observed in transgenic mice harboring a POLG mutation with no evidence of increased ROS [ 91, 92 ] .

2.5.5 Cell Culture and Animal Models of Mitochondrial Disease

The recent development of transgenic animal models and cell culture systems has proved to be invaluable in exploring mechanisms of neurodegeneration in mito-chondrial disease (see Chap. 12 ).

Recent work conducted in differentiated neurons from mouse embryonic stem cells harboring mtDNA defects has allowed a more mechanistic approach to under-standing neurodegeneration in mitochondrial disease. Four cell lines have been gen-erated to date and these consist of: wild-type parental cells, polymorphic cells, mild complex IV mutant cells, and severe complex I mutant cells. Those neurons harbor-ing severe complex I mutations revealed the most dramatic alterations, with a reduced ability to undergo neuronal differentiation and delayed development [ 93 ] . Despite this, differentiated neurons showed maintenance of a high mitochondrial membrane potential with a reversal of the ATP synthase and marked ROS production [ 94 ] .


Subsequent analysis following repetitive neuronal stimulation demonstrated that the ability of the neurons to respond to calcium transients was impaired [ 83 ] .

Recently, the development of an animal model, the “mutator” mouse, harboring a proofreading defi ciency of polymerase g has shown the accumulation of high level of mtDNA point mutations and deletions [ 92, 95 ] . The phenotype of the “mutator” mouse show evidence of accelerated ageing, curvature of the spine, weight loss, alopecia, and reduced lifespan. Whilst no neuropathological studies have been con-ducted in this mouse, neurological changes would be interesting to investigate.

2.5.6 Other Neurodegenerative Disorders

Deciphering the mechanisms of neurodegeneration in patients with mtDNA dis-eases poses a diffi cult challenge to neuroscientists because of the diversity in clini-cal presentations, different types of mtDNA mutations, and variable pattern of brain involvement. However, increased understanding of the processes underlying CNS degeneration in patients with mtDNA diseases will be incredibly informative for other neurodegenerative disorders where a mitochondrial etiology is suspected.

There is some evidence that mutations within the mitochondrial genome or mito-chondrial dysfunction play a role in a number of neurodegenerative disorders including PD, Huntington’s disease (HD), Alzheimer’s disease (AD), and Multiple sclerosis [ 96– 99 ] (see Chap. 5 ). Whilst the role of these reported mitochondrial abnormalities, whether causative for the onset of neurodegeneration or arising sec-ondary to other neurodegenerative mechanisms, it is clear that mitochondrial dys-function would act to exacerbate disease pathogenesis. However, much like the mitochondrial encephalomyopathies, our understanding of the pathophysiological mechanisms involved in these diseases remains incomplete.

Acknowledgments This work was in part supported by the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Hospitals NHS foundation Trust, UK.

Glossary

Ataxia Progressive loss of coordination and balance in hands, arms, and legs often due to dysfunction of the cerebellum.

Bradykinesia An abnormal slowness of movement, often associated with the clin-ical symptoms seen in Parkinson’s disease.

Cardiomyopathy The deterioration of heart muscle resulting in compromised heart function.

Chronic progressive external ophthalmoplegia Slowly progressive paralysis of the extraocular muscle which results in bilateral, symmetrical, progressive ptosis (drooping of the eyelids) followed by ophthalmoparesis (paralysis) months to years later.


Dysarthria A speech disorder characterized by poor articulation. Often present in patients suffering from cerebellar ataxia.

Dysphagia Swallowing problems often present in conjunction with dysarthria in patients suffering from cerebellar ataxia.

Dystonia Dystonia is characterized by involuntary and uncontrollable muscle spasms which can force affected parts of the body into abnormal, sometimes painful, movements or postures.

Endocrinopathy A disorder in the function of the endocrine gland which can often result in hormone imbalances.

Epilepsy Recurrent seizures (often described as fi ts). Extrapyramidal features Maybe defi ned as an inability to initiate movements or

an inability to remain motionless. Hypotonia A state of low muscle tone resulting in reduced muscle strength. Lipoma A benign tumor consisting of brown fat cells that forms under the skin. Myoclonus Brief involuntary twitching of a muscle or a group of muscles. Nystagmus A form of uncontrolled eye movements. Peripheral neuropathy Damage to the nerves comprising the peripheral nervous

system, which can result in a combination of weakness, autonomic dysfunction, and sensory abnormalities.

Proximal myopathy Weakness of those muscles located closely (proximal) to the body.

Ptosis Droopy eyelids. Retinitis pigmentosa Retinitis pigmentosa is a disorder of the retina which is char-

acterized by dysfunction of the photoreceptors resulting in incurable blindness or tunnel vision.

Stroke-like episodes Neurological defi cits often described as resembling stroke however do not conform to a vascular territory. They are often accompanied by cortical blindness, hemianopsia, or hemiparesis.

Wolff–Parkinson–White syndrome This syndrome is a heart condition caused by extra electrical activity and can lead to increased heart rate.

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Neurodegeneration in Primary Mitochondrial Disorders 2 · 2.2.2 Chronic Progressive External Ophthalmoplegia Chronic progressive external ophthalmoplegia (CPEO) is deÞ ned by a slowly