Scientific correspondence
Published online Article Accepted on 19th August 2008
Alpha-synuclein pathology and Parkinsonism
associated with POLG1 mutations and multiple
mitochondrial DNA deletions
Mutations in the mitochondrial genome (mtDNA) accu-
mulate in a variety of tissues with age [1]. It has been
proposed that these somatic mtDNA mutations play a role
in ageing and age-related diseases, such as Parkinson’s
disease (PD) [2,3]. Recently, it was shown that high levels
of deleted mtDNA are present in substantia nigra (SN)
neurones from both aged controls and individuals with
PD, strengthening the suggestion that somatic mtDNA
deletions are involved in the selective neuronal loss ob-
served in brain ageing and in PD [2,4]. To further investi-
gate a possible association between multiple mtDNA
deletions and ageing, we performed post mortem examina-
tion of the brain from a patient with multiple mtDNA
deletions. Multiple mtDNA deletions usually develop
secondary to a mutation in one of the enzymes involved in
mtDNA maintenance, e.g. polymerase gamma, encoded
by the nuclear gene POLG1 [5]. Patients with multiple
mtDNA deletions show clinical heterogeneity [6],
although in our patient chronic progressive external
ophthalmoplegia (PEO) and Parkinsonism were both
prominent features.
The patient first presented at the age of 22 years with
left-sided ptosis, which slowly progressed over the next 20
years to an almost complete ophthalmoplegia. He devel-
oped proximal muscle weakness at age 50 and his first
Parkinsonian features at age 51. The features, including
rigidity, tremor, bradykinesia and difficulty performing
fine motor tasks, were initially unilateral and responded
well to dopamine agonists. Aged 54 years, he showed signs
of cognitive impairment and increasing dysphagia, dysar-
thria and dysphonia. His proximal myopathy also became
more prominent and there was evidence of sensorimotor
neuropathy at age 57. Dopamine agonists became less
effective, but he responded well to apomorphine. His dys-
phagia deteriorated and a PEG tube was inserted because
of recurrent aspiration. The patient died aged 59 years
because of pneumonia. There was no family history of
either mitochondrial disease or PD.
Examination of a post mortem skeletal muscle sample
revealed features of mitochondrial myopathy with fre-
quent muscle fibres deficient in the mtDNA-encoded cyto-
chrome c oxidase (COX), some of which were hyperactive
for nuclear DNA (nDNA) encoded succinate dehydroge-
nase (SDH) (Figure 1A). Multiple mtDNA deletions were
detected, both on Southern blot and long-range PCR assays
of total muscle homogenate DNA (Figure 1B). Genetic
analysis revealed that the patient was compound heterozy-
gous for mutations in the nDNA-encoded POLG1 gene,
coding for the catalytic subunit of the mtDNA-specific poly-
merase g [7]. These compound heterozygous mutations
were c.3311C>G (p.S1104C) and c.2542G>A (p.G848S).
Macroscopic examination of the brain did not reveal
any external abnormalities; however, the SN was almost
devoid of pigment throughout. Microscopic examination
of the midbrain revealed the extent of SN neuronal loss,
which was severe in the lateral group, moderately severe
in the middle group and moderately severe in the medial
group of the lower midbrain and also uniformly moderate
to moderately severe in the upper midbrain. With haema-
toxylin and eosin stain Lewy bodies (LBs) were seen in the
surviving SN neurones (Figure 2A). The red, oculomotor,
Edinger-Westphal and dorsal raphe nuclei appeared unaf-
fected. Within the pons there was mild loss of neurones in
the locus coeruleus and normal neuronal population
density (n.p.d.) within the superior raphe. Within the
medulla the n.p.d. of the dorsal nucleus of vagus and of
the olivary nuclei were relatively intact. The cerebellum
was affected with moderate focal loss of Purkinje cells and
focal mild neurone loss in the dentate nucleus. In the basal
forebrain the n.p.d. of the nucleus of Meynert appeared
intact. Immunohistochemistry using antibodies to alpha-
synuclein revealed a high proportion of neurones with
cytoplasmic immunoreactivity, neurites and immunore-
activity in the neuropil of the SN (Figure 2B) and the
nucleus of Meynert. The dorsal nucleus of vagus and
the other brainstem and subcortical nuclei showed only
mild LB pathology. Fewer than five alpha-synuclein-
immunoreactive LBs were present in the anterior cin-
gulate and the frontal cortex but none in the
transentorhinal, lateral temporal or parietal cortex. This
120 © 2009 Blackwell Publishing Ltd
Neuropathology and Applied Neurobiology (2009), 35, 120–124 doi: 10.1111/j.1365-2990.2008.00981.x

pattern is similar, but not identical, to that seen in idio-
pathic PD with cognitive impairment, as those patients
usually show more neurone loss and a-synuclein pathol-
ogy in the locus coeruleus, the medullary dorsal nucleus
of vagus and reticular formation, the nucleus of Meynert,
the limbic system and the neocortex [8]. Unlike PD
patients but similar to patients with mitochondrial
disease, the gracile fascicle in the cervical spinal cord
showed myelin loss (Figure 2C) and the gracile nucleus in
the lower medulla showed neurone loss and degeneration
with microvacuolation of the neuropil. The Alzheimer-
type pathology was minimal – scattered neurofibrillary
tangles restricted to the parahippocampal gyrus (Braak
stage 0) and no neuritic or amyloid plaques.
Dual COX/SDH histochemistry revealed that 21.2%
of the remaining SN neurones were COX-deficient
(Figure 2D). This level of COX deficiency is higher
than the levels recently observed in the SN of ageing
controls (<1%) and PD patients (<3%) [2]. Immu-
nohistochemistry for the mtDNA-encoded COX subunit
I revealed deficiency in individual nigral neurones
(Figure 2E), while all nigral neurones showed normal
activity for porin, a transmembrane mitochondrial
protein used as a reference marker for mitochondrial
mass (Figure 2F). From the remaining SN neurones, five
pooled, single neurones were laser-microdissected and
mtDNA deletion load was assessed using a previously
multiplex real-time PCR [9]. The results confirmed high
levels of mtDNA deletions in the SN neurones (~65%,
n = 5).
Parkinsonism has been previously described in PEO
patients with mtDNA deletions [5], as has severe neuronal
loss from the SN in patients with multiple mtDNA deletion
disorders [10,11]. However, we believe this to be the first
report of LB pathology in a patient with Parkinsonism,
PEO and multiple mtDNA deletions, secondary to a POLG1
mutation. Here, additional single-cell studies are required
to examine whether the LB pathology is specifically asso-
ciated with high levels of mtDNA deletions within indi-
vidual nigral neurones, or whether these represent a
coincidental pathology in a patient with mitochondrial
disease. Alpha-synuclein-defined Lewy pathology may
occur in neurologically impaired individuals and normal
aged individuals [12,13]. However, as our patient was
younger (59 years old) than the individuals previously
described, we do not believe that the observed LB pathol-
ogy is merely age-related. In addition, our patient demon-
strated Parkinsonism and signs of cognitive decline.
Interestingly, in a separate study of two patients with
autosomal dominant PEO and Parkinsonism, neurofibril-
lary tangles and plaques suggestive of the early stages of
Alzheimer’s disease were identified in one of the individu-
als who died at the age of 60 years [3]. The authors specu-
lated that high levels of mtDNA deletions present in that
patient may have caused premature ageing and the earlier
onset of AD, which usually occurs at a more advanced
age. Similarly, we believe that our finding of alpha-
synuclein immunoreactive LB-PD pathology associated
with mtDNA deletions strengthens the suggestion that
somatic mtDNA mutations contribute to the selective
neuronal involvement observed in brain ageing and in
ageing-related diseases [2,14].
1 kb Patient
- 9 kb
multiple mtDNA
deletions
A
B Control
Figure 1. Histochemical and mtDNA analyses. (A) Dual
histochemistry for the mtDNA-encoded cytochrome c oxidase (COX)
and the nDNA-encoded succinate dehydrogenase (SDH) on the
patient’s muscle biopsy demonstrates COX-deficient ragged-red
fibres (blue) and muscle fibres expressing both COX and SDH
(brown). Some freezing artefact is apparent in this muscle sample,
but the mitochondrial abnormality is clearly apparent;
bar = 40 mm. (B) Long-range PCR (10 kb amplimer) amplification
of skeletal muscle DNA extracted from a tissue homogenate. Lane
1, 1-kb ladder; lane 2, age-matched control muscle; lane 3, patient
muscle clearly revealing multiple mtDNA deletions in addition to
the full-length, wild-type molecule.
Scientific correspondence 121
© 2009 Blackwell Publishing Ltd, Neuropathology and Applied Neurobiology, 35, 120–124