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Ataxin-7 aggregation and ubiquitination in infantile SCA7 with 180 CAG repeats.

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Ataxin-7 Aggregation and
Ubiquitination in
Infantile SCA7 with
180 CAG Repeats
Olaf Ansorge, MD,1 Paola Giunti, MD,2
Andrej Michalik, PhD,3 Christine Van Broeckhoven, DSc,3
Brian Harding, DPhil,4 Nicholas Wood, PhD,2
and Francesco Scaravilli, PhD1
Extremely long (>150) CAG repeats are often used to
create models of polyglutamine diseases yet are very rare
in humans where they manifest as pediatric multisystem
syndromes of little specificity. Here, we describe an infant with 180 CAG repeats in the spinocerebellar ataxia
type 7 gene and focus on systemic ataxin-7 aggregation.
This was found in many organs, including the cardiovascular system. In the brain, the hippocampus emerged as a
principal site of ataxin-7 aggregation without cell loss.
We note differential ubiquitination of aggregates and discuss how this may relate to selective vulnerability.
Ann Neurol 2004;56:448 – 452
Spinocerebellar ataxia type 7 (SCA7) is one of several
neurodegenerative diseases that are caused by an expansion of unstable CAG repeats coding for polyglutamine
(polyQ) residues (for review, see Michalik and colleagues1). The gene products of these diseases are unrelated except for the polyQ tract and are expressed
throughout the body, yet each disease displays a distinct pattern of neuronal degeneration and nuclear inclusions (NIs) of aggregated mutated protein. However, the clinical phenotypes become less distinctive as
the length of the polyQ tract increases. Generally, diseases manifest in adulthood at thresholds of 36 to 40
Qs and in adolescence or even infancy if the repeat
number exceeds 60 to 100 Qs. Infantile SCA7 may be
unique in that extreme expansions may even result in
systemic disease. Particularly cardiovascular abnormali-
ties have been documented clinically2– 4 but not investigated pathologically. CAG expansions of more than
150 repeats are very rare in humans but often are used
to create models of polyQ diseases.5,6 Because many of
the current hypotheses concerning the pathogenesis of
polyQ diseases are derived from these models, it is important to document the effects of such extreme expansions in humans. Here, we present a detailed clinicopathological investigation of infantile SCA7 with 180
CAG repeats with a focus on ataxin-7 protein expression throughout the body. We observed ataxin-7 aggregates in the cardiovascular system but also in other
nonneuronal tissues. In the brain, the hippocampus,
generally not implicated in SCA7 pathophysiology,
emerged as a principal site of ataxin-7 aggregation
without neuronal loss. Finally, we note that nonneuronal inclusions, in contrast with many neuronal ones,
are not detected by an ubiquitin antibody. We discuss
the possibility that differential ubiquitination may contribute to selective cellular vulnerability.
Case Report
Patient and Family History
The patient was a 29-month-old girl born into a family
with known SCA7 (see pedigree, Fig 1).7 The pregnancy, birth, and first few months postpartum were
uneventful. A generalized limb tremor was noted at approximately 9 months of age and developmental mile-
From the 1Division of Neuropathology and 2Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London,
United Kingdom; 3Department of Molecular Genetics VIB8,
Flanders Interuniversity Institute for Biotechnology, University of
Antwerp, Belgium; and 4Department of Pathology, Great Ormond
Street Hospital for Sick Children, London, United Kingdom.
Received Mar 8, 2004, and in revised form May 27 and Jun 14.
Accepted for publication Jun 15, 2004.
Address correspondence to Dr Ansorge, Department of Neuropathology, The Radcliffe Infirmary, Oxford OX2 6HE, England. Email:
Published online Aug 31, 2004, in Wiley InterScience
( DOI: 10.1002/ana.20230
Fig 1. Anonymized four-generation tree of the SCA-7 family.
The 180Q-allele of the patient with infantile onset (IV:4) was
paternally inherited. The patient’s father (III:3) is confirmed
carrier of the SCA-7 mutation with a pathological allele of
39Q, but was clinically asymptomatic at age 54 years. (open
symbols) Clinically asymptomatic; (filled symbols) symptomatic individuals.
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
stones were delayed. Soon after, marked dysphagia developed and there was a general failure to thrive.
Neurological examination at 19 months showed pigmentary degeneration in both retinae. There was also
downbeat nystagmus and general muscle hypotonia
with head lag. Reflexes, however, were brisk and there
was a positive Rossolimo sign. There was marked cerebellar ataxia. A computed tomography scan showed
cerebellar and brainstem atrophy. Routine laboratory
tests were unremarkable. Analysis of blood DNA revealed an expansion of 180 CAG repeats in the SCA7
gene. The pathological allele was inherited from the
father who had 39 repeats but was clinically asymptomatic when last examined at age 54 years (III:3, see
pedigree). The patient died 20 months after clinical
onset. With consent of next of kin, a postmortem examination was conducted.
Genetic Analysis
DNA was extracted from peripheral blood lymphocytes
by standard methods. Analysis of the SCA7 (CAG)n
expansion was done by polymerase chain reaction.8 Allele repeat size was determined by polyacrylamide gel
electrophoresis using an ABI 377 automatic sequencer
and Genescan software (PE Applied Biosystems, Foster
City, CA).
Formalin-fixed, paraffin-embedded tissue samples were
cut into 5␮m sections and stained with hematoxylin
and eosin or Luxol fast blue. Tissue from a 27-monthold female patient who died of encephalitis was used as
a control. Microwave antigen retrieval (8 minutes) was
used for polyclonal ataxin-7 antibody CM1899
(1:2,000) and polyclonal ubiquitin antibody (1:400;
Dako, Glostrup, Denmark). Primary antibodies were
incubated for 1 hour at room temperature. Appropriate
biotinylated secondary antibodies were applied for 30
minutes, followed by avidin-biotin complex and 3⬘,3diaminobenzidine as chromogen. Sections were photographed with a digital camera mounted on an Olympus microscope.
General Pathological Findings
The body was small for age (height, 82.5cm; weight,
7.4kg). The head circumference for age was below the
tenth percentile, and individual organ weights were low
for age: brain, 850gm (normal mean, 1,064gm); kidneys, 23 and 26gm (normal, 50gm); liver, 306gm (normal, ⬎400gm);unfortunately, the weight of the heart
was not recorded. Peripheral organs were macroscopically normal; there was no evidence of a cardiac malformation or patent ductus arteriosus. The brain
showed macroscopically severe olivopontocerebellar at-
rophy and thinning of the spinal cord. In contrast, the
neocortex, hippocampi, and central gray nuclei appeared relatively preserved.
Distribution of Inclusions in Neural Tissue and
Relationship to Neuronal Loss
Ataxin-7 NIs were seen throughout the central, peripheral, and autonomous nervous system (for summary
and examples, see Table 1 and Fig 2). Very large nuclear inclusions could be detected on routine hematoxylin and eosin preparations (see Fig 2D). Inclusions
were clearly not limited to areas of severe cell loss such
as the retina, olive, or cerebellum. In fact, NIs tended
to be most frequent in areas not affected by neurodegeneration. This was particularly remarkable in the
hippocampus which showed neither obvious neuronal
loss nor gliosis despite the presence of ataxin-7–positive
NIs in 93% of pyramidal cells (see Fig 2A, B). Ubiquitinated NIs were detected in only 52% of hippocampal pyramidal neurons compared with 93% of
surviving olivary neurons.
Table 1. Summary of Nervous System Distribution of
Ataxin-7 Protein Neuronal Nuclear Inclusions in
Relation to Neuronal Loss
Nervous System Region
Frontal neocortex
Anterior cingulate
Deep gray nuclei
Caudate nucleus
Globus pallidus
Lateral geniculate
Substantia nigra
Pontine nuclei
Inferior olive
Oculomotor nucleus
Purkinje cells
Golgi cells
Granule cells
Dentate nucleus
Spinal cord
Anterior horn cells
Sensory ganglia
Sympathetic ganglia
with NIs
100 pyramidal neurons were assessed for NIs: 93 showed ataxin-7
NIs, 52 contained ubiquitinated NIs.
Of 60 surviving olivary neurons 58 (97%) contained ataxin-7 NIs
and 56 (93%) ubiquitinated NIs.
Hardly any Purkinje cell was left for assessment.
Semiquantitative rating: ⫺ ⫽ absent; ⫹ present at low frequency/
degree; ⫹⫹ present at moderate frequency/degree; ⫹⫹⫹ present at
high frequency/degree.
Ansorge et al: Ataxin-7 in 180Q SCA7
Fig 2. Ataxin-7 inclusions in neuronal and nonneuronal cells in infantile (Q180) SCA7. There is no loss of hippocampal neurons
(A, Luxol fast blue cresyl violet) despite ataxin-7 nuclear inclusions (NIs) in virtually all pyramidal cells (B); however, an ubiquitin
antibody labels fewer NIs (C). Some NIs are so large that they can easily be detected as paranucleolar eosinophilic spheroids on routine stains: (D) olivary neuron (hematoxylin and eosin); compare with E, anterior horn cell (ataxin-7). (F) Oligodendroglial
ataxin-7 NIs in the brainstem. Nonneuronal ataxin-7 NIs are present in endothelial cells (G), cardiac (H) and skeletal (I) muscle,
exocrine pancreas (J), and epithelial cells of Brunner’s glands of the duodenum (L) (K, hematoxylin and eosin for comparison).
Original magnifications ⫻40 (A), ⫻400 (B, C, K, L), ⫻600 (D–J).
Distribution of Inclusions in Nonneural Tissues
We found widespread yet regionally selective nuclear
aggregation of ataxin-7 protein in nonneuronal cell
Annals of Neurology
Vol 56
No 3
September 2004
types (summarized in Table 2, examples in Fig 2G–L).
The presence of nuclear ataxin-7 inclusions was the
only distinguishing feature in peripheral organs be-
Table 2. Summary of Peripheral Organ Distribution of Ataxin-7 Protein NIs
Anterior pituitary
Adrenal gland
Thyroid gland
GI system
Skeletal muscle
Cardiac muscle
Smooth muscle (gut)
Vascular system
Nuclear Inclusions
Cell Types
Endocrine epithelium
Exocrine epithelium, islet cells
Cortex ⬎ medulla
Chief and neuroendocrine cells
Brunner’s gland epithelium, Auerbach and Meissner plexus
Endothelial cells
Tubular epithelium, glomeruli
Semiquantitative rating as in Table 1.
n/a ⫽ non applicable.
tween patient and control material. However, skeletal
muscle showed occasional atrophic fibers, some of
which were angulated. In contrast with neuronal inclusions, we could not detect ubiquitin epitopes in any of
the nonneuronal inclusions.
The CAG repeat associated with SCA7 is extremely
unstable in the male germline which may result in
massive intergenerational expansions not commonly
seen in other CAG/polyQ diseases.7,8 Therefore, a pediatric neurologist may be confronted with an
infantile-onset, rapidly progressive, complex neurological syndrome in a child of an apparently healthy father,
as illustrated in this study, where we observed an intergenerational expansion of 141 CAG repeats (see Fig
1). Although the salient diagnostic features of SCA7
(retinal degeneration, cerebellar ataxia) are usually
present in the infantile form, nonspecific neurological
as well as systemic symptoms have been reported.2– 4
Our proband with 180 CAG repeats showed a failure
to thrive, muscle weakness, and internal organs small
for age. When we probed the peripheral tissues with
antibody CM189, which preferentially detects aggregated (N-terminal) ataxin-7,9 we found widespread
NIs. Particularly intriguing was the finding of very frequent NIs in endothelial cells (see Fig 2G), as capillary
leakage syndrome and multiple hemangiomas have
been reported in SCA7 infants with even higher repeat
numbers (⬎325 CAGs).4 Ataxin-7 aggregates were also
present in cardiac and skeletal muscle, tissues with high
transcript levels,8 that are also implicated clinically in
infantile SCA7 (atrial septum defect, patent ductus ar-
teriosus and congestive heart failure were noted in children with ⬎230 repeats).2– 4 These tissues showed no
evidence of NI formation in a patient with 60 CAG
repeats.10 Skeletal muscle weakness in SCA7 therefore
may reflect loss of anterior horn cells compounded by a
direct myopathic effect of mutated ataxin-7 in cases
with extremely large repeat expansions.11
The most interesting finding in the brain of our patient was the presence of ataxin-7 NIs in almost all
pyramidal neurons of the hippocampus in the absence
of obvious cell loss. This has not been reported before
in human SCA7 to our knowledge but was a feature in
a recently created 266Q knock in model of the disease,6 where it was associated with mild impairment of
short-term synaptic plasticity. Hippocampal NI formation is a feature that only emerges with extreme CAG
expansions, because even with a relatively high number
of 85 CAGs only very rare NIs (⬍1%) were seen,12
and even fewer repeats were associated only with diffuse nuclear staining.10
The role of the large visible NIs in the pathogenesis
of the polyQ disorders is still debated.13 The dynamics14 and toxicity15 of NI formation may vary considerably not only between different cell types but also in
homogeneous populations. Postmitotic cells (neurons,
myocytes) appear to be more vulnerable than proliferating cells15; however, NIs may induce cell cycle arrest
in the latter.16 An attractive hypothesis postulates that
NIs may engage the ubiquitin-proteasome system in a
futile attempt of refolding and degradation, leading to
demise of the cell.16 In this context, it is, however,
noteworthy that not all NIs (as defined by staining for
the disease protein) are ubiquitinated, and that ubiq-
Ansorge et al: Ataxin-7 in 180Q SCA7
uitination of neuronal NIs is a late process6,17 that may
vary between brain regions.18 An intriguing observation in our study as well as previous12 studies is that
ubiquitination of neuronal NIs appears to be more frequent in areas with severe cell loss (olive) than in those
without (hippocampus). This may suggest that a cell
survives the presence of a large NI as long as it is not
ubiquitinated, or, more likely, only monoubiquitinated
or oligoubiquitinated19 (which may be below the immunohistochemically detectable threshold, see Hicke20),
because it is only a polyubiquitin chain of four or more
residues that recruits the proteasome (see Hicke20).
Data concerning differential monoubiquitination or
oligoubiquitination versus polyubiquitination of proteins within the NIs are not yet available but may yield
important insights into the mechanisms of selective cellular vulnerability in this group of diseases.
This work was supported by a grant from the Joint Research Advisory Committee of The National Hospital for Neurology and Neurosurgery and Institute of Neurology (O.A.) and by a research grant
from the Fund for Scientific Research Flanders, Belgium (C.V.B.).
The technical expertise of L. Martinian is greatly appreciated.
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repeat, ataxia, cag, sca7, infantile, ubiquitination, 180, aggregation
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