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Mitochondrial dysfunction has been associated with late onset neurodegenerative disorders, among which is Machado-Joseph disease (MJD/SCA3). In a previous study, using a transgenic mouse model of MJD, we reported a decrease in mitochondrial DNA (mtDNA) copy number and an accumulation of the 3876-bp deletion with age and with phenotype development. We extended this study by analyzing the pattern of mtDNA depletion and the accumulation of the 3876-bp deletion in 12 older transgenic (TG) and 4 wild-type (wt) animals, and by investigating the accumulation of somatic mutations in the D-loop region in 76 mice (42 TG and 34 wt). mtDNA damage was studied in TG and wt mice at different ages and tissues (blood, pontine nuclei, and hippocampus). Results for older mice demonstrate an accumulation of the mtDNA 3867-bp deletion with age, which was more pronounced in TG animals. Furthermore, the tendency for mtDNA copy number decrease with age, in all analyzed tissues of TG and wt animals, was also confirmed. No point mutations were detected in the D-loop, neither in TG nor wt animals, in any of the tissues analyzed. Due to the absence of mtDNA somatic mutations, we can suggest that mtDNA point mutation accumulation cannot be used to monitor the development and progression of the phenotype in this mouse model and likely in any MJD mice model. The present results further confirm not only the association between mtDNA alterations (copy number and deletions) and age, but also between such alterations and the expression of the mutant ataxin-3 in TG mice.
1 23
Journal of Molecular Neuroscience
ISSN 0895-8696
J Mol Neurosci
DOI 10.1007/s12031-014-0360-1
Differential mtDNA Damage Patterns in
a Transgenic Mouse Model of Machado–
Joseph Disease (MJD/SCA3)
Amanda Ramos, Nadiya Kazachkova,
Francisca Silva, Patrícia Maciel, Anabela
Silva-Fernandes, Sara Duarte-Silva,
Cristina Santos, et al.
1 23
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Differential mtDNA Damage Patterns in a Transgenic Mouse
Model of MachadoJoseph Disease (MJD/SCA3)
Amanda Ramos & Nadiya Kazachkova & Francisca Silva &
Patrícia Maciel & Anabela Silva-Fernandes &
Sara Duarte-Silva & Cristina Santos & Manuela Lima
Received: 15 April 2014 /Accepted: 19 June 2014
Springer Science+Business Media New York 2014
Abstract Mitochondrial dysfunction has been associated with
late onset neurodegenerative disorders, among which is
MachadoJoseph disease (MJD/SCA3). In a previous study,
using a transgenic mouse model of MJD, we reported a de-
crease in mitochondrial DNA (mtDNA) cop y number and an
accumulation of the 3876-bp deletion with age and with phe-
notype development. We extended this study by analyzing the
pattern of mtDNA depletion and the accumulation of the 3876-
bp deletion in 12 older transgenic (TG) and 4 wild-type (wt)
animals, and by investigating the accumulation of somatic
mutations in the D-loop region in 76 mice (42 TG and 34
wt). mtDNA damage was studied in TG and wt mice at differ -
ent ages and tissues (blood, pontine nuclei, and hippocampus).
Results for older mice demonstrate an accumulation of the
mtDNA 3867-bp deletion with age, which was more pro-
nounced in TG animals. Furthermore, the t endency for
mtDNA copy number decrease with age, in all analyzed tissues
of TG and wt animals, was also confirmed. No point mutations
were detected in the D-loop, neither in TG nor wt animals, in
any of the tissues analyzed. Due to the absence of mtDNA
somatic mutations, we can suggest that mtDNA point mutation
accumulation cannot be used to monitor the development and
progression of the phenotype in this mouse model and likely in
any MJD mice model. The present results further confirm not
only the association between mtDNA alterations (cop y number
and deletions) and age, but also between such alterations and
the expression of the mutant ataxin-3 in TG mice.
Keywords Mitochondrial DNA
mtDNA damage
MachadoJoseph disease
Transgenic mouse model
Neurodegenerative disorder
Polyglutamine disorder
Over the past two decades, multiple studies have provided
evidence on the relevance of mitochondrial biology in neuro-
logical disorders (Martin 2010)aswellasintheagingprocess
(Larsson 2010). Mitoch ondrial dysfunction is being extensively
studied in patients with adult onset neurodegen erative disorders
such as poly-Q-related ataxias, among which is Machado
Joseph disease (MJD). MJD/SCA3 (OMIM 109150;
ORPHA98757) is caused by a mutation in ataxin-3, a protein
encoded by the ATXN3 gene (for a revision on MJD see
Bettencourt and Lima (201 1)). Although some studies have
reported the presence of mitochondria l DNA (mtDNA) damage
in MJD patients (Liu et al. 2008;Yuetal.2009; Zheng et al.
2012), the way by which mitochondrial impairment and oxida-
tive stress are actually involved in the onset and progression of
the disease is not clear. A previous study from our group
Electronic supplementary material The online version of this article
(doi:10.1007/s12031-014-0360-1) contains supplementary material,
which is available to authorized users.
A. Ramos (*)
N. Kazachkova
F. Silva
M. Lima
Centre of Research in Natural Resources (CIRN),
Department of Biology, University of the Azores,
Ponta Delgada, Portugal
A. Ramos
N. Kazachkova
F. Silva
M. Lima
Institute for Molecular and Cell Biology (IBMC),
University of Porto, Porto, Portugal
P. Maciel
A. Silva-Fernandes
S. Duarte-Silva
Life and Health Sciences Research Institute (ICVS), School of Health
Sciences, University of Minho, Braga, Portugal
P. Maciel
A. Silva-Fernandes
S. Duarte-Silva
ICVS/3Bs-PT Government Associate Laboratory,
Braga/Guimarães, Portugal
C. Santos
Unitat dAntropologia Biològica, Departament (BABVE),
Universitat Autònoma de Barcelona, Cerdanyola del Vallès(
Barcelona, Spain
J Mol Neurosci
DOI 10.1007/s12031-014-0360-1
Author's personal copy
(Kazachkova et al. 2013b), using 8-, 16-, and 24-week old
transgenic (TG) mice of MJD expressing the mutated ataxin-3
and displaying a motor phenotype (Silva-Fernandes et al.
2010), reported mtDNA depletion and an increase in the level
of the 3867-bp deletion (the homolog of the 4977-bp deletion in
humans, considered a marker of aging). Aiming to better un-
derstand the pattern of mtDNA damage in MJD, we extended
this previous study by (1) analyzing the pattern of mtDNA
depletion and the accumula tion of the 3876-bp deletion in older
TG animals and comparing them with wild-type (wt) litter-
mates and (2) sequencing the D-loop, a mutation-prone region
of mtDNA (the most variable segment in mammalian mito-
chondrial genomes (Attardi and Schatz 1988; Druzhyna et al.
2008)), to investigate the pattern of accumulation of somatic
mutations in both TG and wt animals.
Material and Methods
Mouse Model and Experimental Design
A TG mouse model of the early stages of MJD (Mus musculus,
strain C57B1/6, line CMVMJD94) developed in the Lab of P.
Maciel was used in the present study (Silva-Fernandes et al.
2010). These TG mice ubiquitously express the full-length
mutant human ataxin-3 and display a motor phenotype . The
mouse model used also mimics some key features that are
common in MJD patients, namely CAG repeat instability , neu-
rological damage, and brain pathology (Silva-Fernandes et al.
2010). The sample selection of TG and wt mice is shown in
Table 1. Affected tissue corresponded to pontin e nuclei (Pn) and
non-affected corresponded to hippocampus (Hp) as well as
blood (Bl) (Table 1). Overall, 180 samples were analyzed for
early age groups (90 TG and 90 wt) and 32 samples for late age
groups (24 TG and 8 wt) (T able 1). A total of 76 mice were used
to study the accumulation of somatic mutations (42 TG and 34
wt); the late age groups were analyzed to study the mtDNA copy
number and the 3867-bp deletion (12 TG and 4 wt).
The animals used in the present work were maintained in
accordance with Eur opean regulations (Europea n Union
Directive 86/609/EEC). Animal facilities and the people di-
rectly involved in animal experiments were certified by the
Portuguese regulatory entityDirectorate General for
Veterinary Medicine. All protocols were approved by the joint
Animal Ethics Committee of the Life and Health Sciences
Research Institute, University of Minho and the Institute for
Molecular and Cell Biology, University of Porto, Porto,
DNA Isolation, PCR Amplification, and Sequencing
DNA was extracted using the Puregene DNA isolation kit
(Gentra Systems), and the size of the CAG tract was assessed
as previously described (Silva-Fernandes et al. 2010).
Mitochondrial D-loop (positions 15423 to 16299) was ampli-
fied using a new designed primer pair L15349 and H133
based on the M. musculus mitochondrion complete genome
reference sequence (NC_005089.1). As previously demon-
strated, low heteroplasmy levels can be detected with confi-
dence using an automated sequencing system, provided that a
Table 1 Sample selection of
transgenic (TG) and wild-type
(WT) mice used in the present
study (Pn pontine nuclei; Hp
Mice Age (weeks) Number of
Number of samples
Pn Hp Blood Total samples
TG Early age group 8 10 10 10 10 30
16 10 10 10 10 30
24 10 10 10 10 30
Total 30 30 30 30 90
Late age group 60 6 6 6 12
72 6 6 6 12
Total 12 12 12 24
Total number of mice 42 Total number of samples 114
WT Early age group 8 10 10 10 10 30
16 10 10 10 10 30
24 10 10 10 10 30
Total 30 30 30 30 90
Late age group 60 2 2 2 4
72 2 2 2 4
Total 4 4 4 8
Total number of mice 34 Total number of samples 98
J Mol Neurosci
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good sequencing strategy and an accurate procedure of
heteroplasmy detection and validation are used (Ramos et al.
2013). Therefore, all samples were fully sequenced and puri-
fied using the BigDye® Terminator v3.1 Cycle Sequencing
Kit (Applied Biosystems) according to the manufacturers
instructions. Sequences were run in an ABI 3130XL sequenc-
er (Applied Biosystems) at the Servei de Genòmica,
Universitat Autònoma de Barcelona. Sequences were aligned
with the M. musculus mtDNA reference sequence
(NC_005089.1), using SeqScape v2.5 software (Applied
Biosystems). To exclude sequencing errors, heteroplasmic
authentication criteria previously described by Santos et al.
Quantitative Real-Time PCR
Determination of the mtDNA copy number and quantitative
detection of the 3867-bp deletion were performed by
fluorescence-based quantitative real-time PCR (FQ-PCR) as
described by Kazachkova et al. (2013b).
Results and Discussion
mtDNA Depletion and 3867-bp Deletion Accumulation
To obtain a more complete picture of the pattern of mtDNA
damage (copy number and deletions), data published by our
group (Kazachkova et al. 2013b) were extended. In our pre-
viously published w ork, th ree age groups (8 , 16, and
24 weeks) were analyzed for each tissue (Pn, Hp, and Bl), in
both TG and wt animals (Kazachkova et al. 2013b). In the
present work, we have analyzed Pn and Hp samples from
older animals, namely 60- and 72-week old TG and wt mice
(Table 1). The results obtained (Fig. 1a) confirmed the global
tendency for mtDNA copy n umber decrease with age .
Interestingly, TG animals presented a significantly more evi-
dent accumulation of the 3867-bp deletion than wt (Mann
Whitney U Test: Z = 2.74; p=0.0062) (Fig. 1b). Although not
significant, correlation between brain tissues in TG and wt
animals demonstrated a more evident copy number decrease
as well as a more evident accumulation of deletion in Pn, the
affected brain region (Fig. 2). The present results allow us to
confirm the presence of a statistically significant accumulation
of the 3867-bp deletion in TG mice; furthermore, we corrob-
orate the tendency for mtDNA copy number decrease in TG
mice previously reported by Kazachkova et al. (2013b).
Fig. 1 Correlation of mtDNA copy number and mtDNA deletion per-
centage with age (weeks). The line in the graph represents a polynomial
trend line. a mtDNA copy number versus age; b mtDNA deletion
percentage % versus age
Fig. 2 Correlation of mtDNA
copy number (a) and mtDNA
deletion percentage (b)with
mtDNA origin (tissue)
J Mol Neurosci
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Analysis of Somatic Mutations in the Mitochondrial D-Loop
Sequence analysis of the mitochondrial D-loop of the
CMVMJD94 mice and their wt littermates revealed the absence
of point mutations in all samples (Supplemental material).
Neither fixed mutations nor mutations in heteroplasmy were
observed in any of the affected or unaffected t issues
(Supplemental material). No differential pattern of mtDNA
point somatic mutation accumulation was thus observed be-
tween TG and wt mice. The results obtained indicate that the
murine pattern of mtDNA somatic mutation accumulation is
less pronounced than in humans, a finding in accordance with
most previous studies, which, with the exception of the work of
Khaidakov et al. (2003), reported an absence of mtDNA point
mutations in mice (Goios et al. 2007; Kazachkov a et al. 2013a;
Song et al. 2005; Dai et al. 2005; Ameur et al. 201 1;Ferrisetal.
1983). In the study of Song et al. (2005), the authors analyzed
the D-loop in samples of brain, skeletal muscle, heart, and other
tissues from aged mice, but were unable to find point
mutations. Similar results were presented by Goios et al.
(2007 ); these authors found no mutations in the D-loop of 32
complete mitochondrial genomes of the 16 inbred strains
In humans, for the whole mtDNA molecule, the frequency
of point heteroplasmic individuals exceeds 24 % (Ramos et al.
2013). Specifically, in the D-loop, 8.2 % of individuals present
point heteroplasmy. Thus, in humans, one point heteroplasmy
is expected in each 12 individuals. Using this expectation, the
sample size needed to observe the presence of at least one
heteroplasmic individual, with 95 % of confidence, is 34. In
this sense, at least two heteroplasmic mice would be expected,
since 76 mice have been analyzed to study the accumulation
of somatic mutations (Table 1). Moreover, and given the
putative negative effect of the mutant ataxin-3 on mtDNA
integrity (Yu et al. 2009), higher frequencies of mtDNA point
heteroplasmy in TG animals compared to wt mice would be
expected. We can therefore conclude that the accumulation of
point mutations in the D-loop of the mtDNA is not an indica-
tor of mtDNA damage in the present and likely in any MJD
mice model, and therefore its utility in the study of the
involvement of mtDNA in MJD development is limited.
Final Remarks
Despite the differences between humans and mice, namely on
what concerns physiological properties, disease pathogenesis,
and life history, mouse models have been frequently used in
the understanding of the process of mitochondrial alterations,
mainly because they share genomic similarities. Kazachkova
et al. (2013a) performed a comparative revision of studies
focused on mtDNA damage (copy number alterations, accu-
mulation of deletions, and of point mutations) carried out in
humans and mice. The compilation showed consistent results
among studies with a similar pattern of mtDNA deletions for
humans and mice. Contradictory results, however, were re-
ported for copy number and mtDNA point mutations accu-
mulation (Kazachkova et al. 2013a).
Results from our study evidenced a pattern of mtDNA
damage consistent with that reported by Kazachkova et al.
(2013a). The presence of a statistically significant accumula-
tion of the 3867-bp deletion was evidenced in a mouse model
of MJD, being in accordance with the tendency reported in the
revision of Kazachkova et al. (2013a). The absence of a
significant mtDNA copy number decrease would be in line
with the discrepancies observed among studies (Kazachkova
et al. 2013a); thus, further studies would be necessary to
elucidate this pattern. mtDNA point mutations accumulation
has been clearly associated with age in humans, but not in
mice (revised in Kazachkova et al. (2013a)); our study reports
the lack of mtDNA somatic mutations, either at fixed or
heteroplasmic level in TG mice, suggesting that mtDNA point
mutation accumulation is not a useful indicator to monitor the
development of the phenotype in the CMVMJD94 TG model.
We can postulate that a similar pattern could be observed in
mouse models of other neurodegenerative disorders, this hy-
pothesis requiring validation.
The present results further confirm not only the association
between mtDNA alterations (copy number and deletions) and
age, but also between such alterations and the expression of
Acknowledgments NK and AR are a Fundo Regional para a Ciência
postdoctoral fellow (M3.1.7/F/002/2008 and M3.1.7/F/031/2011). This
work was partially supported by Generalitat de Catalunya (SGR 2009-566).
Conflict of Interest The authors declare that they have no conflict of
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J Mol Neurosci
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... Mitochondrial DNA (mtDNA) depletion and an increased number of large deletions, namely the common deletion (m.8482_13460del4977, hereafter named as del4977), previously associated with neurodegeneration, have been observed in Machado-Joseph disease (MJD) cell lines and transgenic (TG) animal models [1][2][3], as well as in blood samples from MJD patients [1,4,5]. MJD, also known as spinocerebellar ataxia type 3 (SCA3; MIM#109150; ORPHA98757), is an autosomal dominant late-onset proteinopathy, which is caused by an abnormal number of coding CAG repeats in the gene encoding for ataxin-3 -ATXN3 (reviewed in [6]). ...
... Also, the behaviour of both mtDNA alterations during the natural history of MJD, including the preclinical stage, remains therefore unknown. Previous work from our group using a MJD TG mouse model in different stages of the disease has shown a decrease in the mtDNA copy number with age that was more pronounced in TG mice than in wild-type controls [2,3]. The same study has described an accumulation of the 3867-bp deletion (homologue of the human mtDNA del4977) in the stage prior to disease phenotype establishment. ...
... As previously demonstrated in several studies with human subjects, there is an association between mitochondrial alterations and ageing (see, amongst others, [19,20]), implying that the effect of age needs to be accounted for when looking for associations between such alterations and disease. The increase in the percentage of the del4977 in the presence of mutated ataxin-3 observed by us is in agreement with a previous report that analysed MJD patients [1], as well as with our own data from a MJD TG mouse model [2,3]. In this TG model, the amount of deletions was consistently higher than the observed in wild-type control mice; in particular, the affected brain area analysed (pontine nuclei) presented the highest percentage of deletions [2,3]. ...
Full-text available
Molecular alterations reflecting pathophysiologic changes thought to occur many years before the clinical onset of Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3), a late-onset polyglutamine disorder, remain unidentified. The absence of molecular biomarkers hampers clinical trials, which lack sensitive measures of disease progression, preventing the identification of events occurring prior to clinical onset. Our aim was to analyse the mtDNA content and the amount of the common deletion (m.8482_13460del4977) in a cohort of 16 preataxic MJD mutation carriers, 85 MJD patients and 101 apparently healthy age-matched controls. Relative expression levels of RPPH1, MT-ND1 and MT-ND4 genes were assessed by quantitative real-time PCR. The mtDNA content was calculated as the difference between the expression levels of a mitochondrial gene (MT-ND1) and a nuclear gene (RPPH1); the amount of mtDNA common deletion was calculated as the difference between expression levels of a deleted (MT-ND4) and an undeleted (MT-ND1) mitochondrial genes. mtDNA content in MJD carriers was similar to that of healthy age-matched controls, whereas the percentage of the common deletion was significantly increased in MJD subjects, and more pronounced in the preclinical stage (p < 0.05). The BCL2/BAX ratio was decreased in preataxic carriers compared to controls, suggesting that the mitochondrial-mediated apoptotic pathway is altered in MJD. Our findings demonstrate for the first time that accumulation of common deletion starts in the preclinical stage. Such early alterations provide support to the current understanding that any therapeutic intervention in MJD should start before the overt clinical phenotype.
... Changes of mitochondrial genomes can be associated with the mitochondrial dysfunction because 13 subunits of complexes Ⅰ, III, IV and V of the oxidative phosphorylation (OXPHOS) system are encoded by genes located in the mitochondrial genome ( Supplementary Fig. 1) (Suzuki et al., 2011;Lott et al., 2013;Stewart and Chinnery, 2015).For now, quantities of studies have been carried out to show that there was a relationship between the mitochondrial DNA and the pathogenesis of SCA3/MJD (Kazachkova et al., 2013;Ramos et al., 2015;Raposo et al., 2019). Conversely, some studies disapprove that mitochondrial DNA can affect the pathogenesis of SCA3/MJD (Lee et al., 2007;Zeng et al., 2012). ...
... A research found out that there was a higher percentage of the common deletion (m.8482_13460del4977) in SCA3/MJD preataxic carriers and clinical patients . And a decrease in mtDNA copy number and an accumulation of the 3876-bp deletion with age and phenotype development were confirmed in a SCA3/MJD mouse model (Kazachkova et al., 2013;Ramos et al., 2015). ...
Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), is the most common type of autosomal dominant cerebellar ataxias. Few studies focused on the changes of the whole mitochondrial genomes of SCA3/MJD patients and its relationship with the pathogenesis of SCA3/MJD. We adapted one-step long-range PCR to amplify the entire mitochondrial DNA (mtDNA) followed by next-generation sequencing technology to investigate the information of whole mitochondrial genomes in 38 SCA3/MJD patients and 31 healthy controls from mainland China. Compared to the healthy control group, the mitochondrial variations in SCA3/MJD patients were more concentrated in the tRNA-transcribed genes which were further found to be potentially associated with the pathogenesis of SCA3/MJD by SKAT-O analysis. However, owning variations in tRNA-transcribed genes could not affect the age of onset (AO) of SCA3/MJD patients. We also noticed that the variant loads greater than 90% took up more in SCA3/MJD patients than in controls. Moreover, from our preliminary study, compared to the patients whose ages of onset were elder than 20, the mitochondrial genomes showed no difference in those AO less than 20. This is the first study to demonstrate the feasibility of using the next-generation sequencing technology for mtDNA variant analysis of SCA3/MJD patients from mainland China. And this research enriches the genetic information of SCA3/MJD and provides a direction for further investigations about the mitochondrial genomes in SCA3/MJD.
... Furthermore, the mitochondrial phenotype in SCA3 is various and, thus, offers other promising points of action explaining neuronal cell loss. Mitochondrial damage indicated by increased mitochondrial DNA (mtDNA) deletions in symptomatic and preclinical SCA3 patients as well as mtDNA deletions and decreased mitochondrial copy numbers in SCA3 mouse models were reported [16][17][18]. Ataxin-3 itself was shown to localize to mitochondria [19,20], and a calpain-mediated cleavage fragment of ataxin-3 is potentially responsible for mitochondrial fragmentation in SCA3 [7,21]. Additionally, mass spectrometry analyses revealed new mitochondrial proteins as potential interaction partners of ataxin-3 under normal and also disease conditions [20]. ...
Full-text available
Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.
... This overlong polyQ chain grants the protein a toxic gain of function and the propensity to aggregate [32]. Consequently, the mutated proteins impair several physiological pathways, such as cell waste clearance (autophagy and ubiquitin-proteosome system [UPS]), transcriptional functions, calcium homeostasis, and mitochondria functions [33][34][35][36][37]. Ultimately, these diseases are characterized by extensive neurodegeneration in multiple brain regions and patients' symptoms are mostly related to neuromotor impairments. ...
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Recent research demonstrated pathological spreading of the disease-causing proteins from one focal point across other brain regions for some neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease. Spreading mediated by extracellular vesicles is one of the proposed disease-spreading mechanisms. Extracellular vesicles are cell membrane-derived vesicles, used by cells for cell-to-cell communication and excretion of toxic components. Importantly, extracellular vesicles carrying pathological molecules, when internalized by “healthy” cells, may trigger pathological pathways and, consequently, promote disease spreading to neighboring cells. Polyglutamine diseases are a group of genetic neurodegenerative disorders characterized by the accumulation of mutant misfolded proteins carrying an expanded tract of glutamines, including Huntington’s and Machado–Joseph disease. The pathological spread of the misfolded proteins or the corresponding mutant mRNA has been explored. The understanding of the disease-spreading mechanism that plays a key role in the pathology progression of these diseases can result in the development of effective therapeutic approaches to stop disease progression, arresting the spread of the toxic components and disease aggravation. Therefore, the present review’s main focus is the disease-spreading mechanisms with emphasis on polyglutamine diseases and the putative role played by extracellular vesicles in this process.
... Interestingly, a subset of those mitochondrial proteins, namely the cytochrome C oxidase subunit NDUFA4 (NDUFA4), the complex II succinate dehydrogenase (ubiquinone) iron-sulfur subunit (SDHB), and cytochrome C oxidase assembly factor 7 (COA7), was found to be enriched in the expanded polyQ ATXN3 samples compared to wild-type, suggesting a stronger interaction [139]. Although much of the evidence described above come from cellular fractionation studies that will need validation, these findings may explain the mitochondrial dysfunction reported in in vitro and in vivo SCA3 models, namely mtDNA deletions and reduced copy number that are often found in SCA3 cells, transgenic mice, and patients; the reduced complex II activity in SCA3 patient lymphoblast cell lines and cerebellar granule cells from transgenic mice; the altered mitochondrial morphology and respiration; the increased oxidative stress and mutant ATXN3-mediated cell death; as well as the metabolic disruption detected in the cerebrospinal fluid of SCA3 patients [140][141][142][143][144][145][146][147]. Consistent with this, creatine administration, which increases the concentration of the energy buffer phosphocreatine exerting protective effects in the brain, slowed disease progression and improved motor dysfunction and neuropathology of SCA3 mice [148]. ...
Spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease (MJD), is a neurodegenerative disorder caused by a polyglutamine expansion in the ATXN3 gene. In spite of the identification of a clear monogenic cause 25 years ago, the pathological process still puzzles researchers, impairing prospects for an effective therapy. Here, we propose the disruption of protein homeostasis as the hub of SCA3 pathogenesis, being the molecular mechanisms and cellular pathways that are deregulated in SCA3 downstream consequences of the misfolding and aggregation of ATXN3. Moreover, we attempt to provide a realistic perspective on how the translational/clinical research in SCA3 should evolve. This was based on molecular findings, clinical and epidemiological characteristics, studies of proposed treatments in other conditions, and how that information is essential for their (re-)application in SCA3. This review thus aims i) to critically evaluate the current state of research on SCA3, from fundamental to translational and clinical perspectives; ii) to bring up the current key questions that remain unanswered in this disorder; and iii) to provide a frame on how those answers should be pursued.
... Some lines of evidence demonstrate the involvement of mitochondrial dysfunction in the pathogenesis of MJD [29][30][31][32][33][34]. However, the way in which mitochondrial impairment and oxidative stress are actually involved in the onset and progression of the disease remains incompletely understood. ...
Background Mitochondrial dysfunction has been implicated in the pathogenesis of several neurodegenerative disorders, namely of Machado‐Joseph disease, an autosomal dominant late‐onset polyglutamine ataxia that results from an unstable expansion of a CAG tract in the ATXN3 gene. The size of the CAG tract only partially explains age at onset, highlighting the existence of disease modifiers. Mitochondrial DNA haplogroups have been associated with clinical presentation in other polyglutamine disorders, constituting potential modifiers of Machado‐Joseph disease phenotype. Methods To investigate if mtDNA haplogroups contribute to age at onset of Machado‐Joseph disease, a cross‐sectional study, using 235 unrelated patients from Portugal, Brazil, India and Japan was performed. mtDNA haplogroups were obtained after sequencing the mtDNA hypervariable region I. Patients were classified in 15 phylogenetically related haplogroup clusters. Results Age at onset was significantly different among populations, implying the existence of another non‐CAG factors, which seem to be population‐specific. In the Portuguese population, patients classified as JT haplogroup presented the earliest onset (estimated onset of 34.6 years). W and X haplogroups seem to have a protective effect, causing a delay in onset (estimated onset of 47 years). No significant association between haplogroup clusters and age at onset was detected in the other populations or when all patients were pooled. Although JT haplogroup has already been implicated in other neurodegenerative disorders, no previous reports of an association between haplogroups W and X and disease were found. Conclusions These findings suggest that JT, W and X haplogroups seem to modify age at onset in Machado‐Joseph disease; replication studies should be performed in European populations, where the frequency of the candidate modifiers is similar. This article is protected by copyright. All rights reserved.
... Recent mass spectrometry analyses revealed several new mitochondrial proteins as confirmed or potential interaction partners of wildtype and/ or mutant ataxin-3, including cytochrome C oxidase subunit NDUFA4 (NDUFA4), succinate dehydrogenase (ubiquinone) iron-sulfur subunit (SDHB) and cytochrome C oxidase assembly factor 7 (COA7) (Kristensen et al., 2018). Mitochondrial DNA (mtDNA) deletions were frequently found in SCA3 transgenic mice as well as in most SCA3 patients and were more pronounced in the preclinical stage but not present in healthy individuals or SCA3 mutation carriers (Yu et al., 2009;Kazachkova et al., 2013;Ramos et al., 2015;Raposo et al., 2018). Additionally, the protein mitochondrial genome maintenance exonuclease 1 (MGME1) which is linked to mitochondrial DNA repair was found enriched in ataxin-3 overexpressing HEK293 cells by mass spectrometry analysis. ...
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Alterations in mitochondrial morphology and function have been linked to neurodegenerative diseases, including Parkinson disease, Alzheimer disease and Huntington disease. Metabolic defects, resulting from dysfunctional mitochondria, have been reported in patients and respective animal models of all those diseases. Spinocerebellar Ataxia Type 3 (SCA3), another neurodegenerative disorder, also presents with metabolic defects and loss of body weight in early disease stages although the possible role of mitochondrial dysfunction in SCA3 pathology is still to be determined. Interestingly, the SCA3 disease protein ataxin-3, which is predominantly localized in cytoplasm and nucleus, has also been associated with mitochondria in both its mutant and wildtype form. This observation provides an interesting link to a potential mitochondrial involvement of mutant ataxin-3 in SCA3 pathogenesis. Furthermore, proteolytic cleavage of ataxin-3 has been shown to produce toxic fragments and even overexpression of artificially truncated forms of ataxin-3 resulted in mitochondria deficits. Therefore, we analyzed the repercussions of expressing a naturally occurring N-terminal cleavage fragment of ataxin-3 and the influence of an endogenous expression of the S256 cleavage fragment in vitro and in vivo. In our study, expression of a fragment derived from calpain cleavage induced mitochondrial fragmentation and cristae alterations leading to a significantly decreased capacity of mitochondrial respiration and contributing to an increased susceptibility to apoptosis. Furthermore, analyzing mitophagy revealed activation of autophagy in the early pathogenesis with reduced lysosomal activity. In conclusion, our findings indicate that cleavage of ataxin-3 by calpains results in fragments which interfere with mitochondrial function and mitochondrial degradation processes.
... All rights reserved. mouse model (Kazachkova et al., 2013;Ramos et al., 2015). In line with these results, Laço and colleagues (2012) reported a significant decrease in mitochondrial complex II activity, which might underlie the cell death observed in MJD/SCA3 cell models (Laço et al., 2012). ...
Machado‐Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an incurable disorder, widely regarded as the most common form of spinocerebellar ataxia in the world. MJD/SCA3 arises from mutation of the ATXN3 gene, but this simple monogenic cause contrasts with the complexity of the pathogenic mechanisms that are currently admitted to underlie neuronal dysfunction and death. The aberrantly expanded protein product – ataxin‐3 – is known to aggregate and generate toxic species that disrupt several cell systems, including autophagy, proteostasis, transcription, mitochondrial function and signalling. Over the years, research into putative therapeutic approaches has often been devoted to the development of strategies that counteract disease at different stages of cellular pathogenesis. Silencing the pathogenic protein, blocking aggregation, inhibiting toxic proteolytic processing, and counteracting dysfunctions of the cellular systems affected have yielded promising ameliorating results in studies with cellular and animal models. The current review analyses the available studies dedicated to the investigation of MJD/SCA3 pathogenesis and the exploration of possible therapeutic strategies, focusing primarily on gene therapy and pharmacological approaches rooted on the molecular and cellular mechanisms of disease. This article is protected by copyright. All rights reserved.
... More recently, mitochondria from a MJD mouse model were found dysfunctional, particularly on complex II of the respiratory chain [76]. Lately, it was also reported that mtDNA is damaged in blood and brain samples from a transgenic MJD mouse model further suggesting a compromised of mitochondrial function [103,104]. ...
Machado-Joseph disease (MJD) is a dominantly inherited disorder originally described in people of Portuguese descent, and associated with the expansion of a CAG tract in the coding region of the causative gene MJD1/ATX3. The CAG repeats range from 10 to 51 in the normal population and from 55 to 87 in SCA3/MJD patients. MJD1 encodes ataxin-3, a protein whose physiological function has been linked to ubiquitin-mediated proteolysis. Despite the identification of the causative mutation, the pathogenic process leading to the neurodegeneration observed in the disease is not yet completely understood. In the past years, several studies identified different molecular mechanisms and cellular pathways as being impaired or deregulated in MJD. Autophagy, proteolysis or post-translational modifications, among other processes, were implicated in MJD pathogenesis. From these studies it was possible to identify new targets for therapeutic intervention, which in some cases proved successful in models of disease.
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Recently, NPY overexpression has been proposed to alleviate motor deficits and neuropathy in Machado-Joseph disease (MJD) mouse models, indicating its neuroprotective role in the pathogenesis of MJD. We aimed to evaluate the association between SNPs in NPY and its receptors and the susceptibility of MJD in the Chinese population. Moreover, we investigated whether these SNPs modulate the age at onset (AO) of MJD. In total, 527 MJD patients and 487 healthy controls were enrolled in the study, and four specific selected SNPs (rs16139, rs3037354, rs2234759, and rs11100494) in NPY and its receptor genes were genotyped. In this study, the genotypic frequency using the dominant model and the allelic distribution of rs11100494 in NPY5R revealed a significant difference between the MJD and control group during the first-stage analysis ( P = 0.048 and P = 0.024, respectively). After we expanded the sample size, significant differences were observed between the two groups using the dominant model in genotypic and allelic distribution ( P = 0.034, P = 0.046, and P = 0.016, respectively). No significant differences in genotypic and allelic distribution were found between the MJD and control groups for the other three SNPs. All selected SNPs had no significant effect on the AO of MJD. The association of rs11100494 in the NPY5R gene and susceptibility of MJD suggested that the NPY system might be implicated in the pathogenesis of MJD. Our study demonstrated the existence of other genetic modifiers in MJD, along with CAG expansion and known genetic modifier factors, which might lead to a better understanding of MJD pathogenesis.
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A significant body of work, accumulated over the years, strongly suggests that damage in mitochondrial DNA (mtDNA) contributes to aging in humans. Contradictory results, however, are reported in the literature, with some studies failing to provide support to this hypothesis. With the purpose of further understanding the aging process, several models, among which mouse models, have been frequently used. Although important affinities are recognized between humans and mice, differences on what concerns physiological properties, disease pathogenesis as well as life-history exist between the two; the extent to which such differences limit the translation, from mice to humans, of insights on the association between mtDNA damage and aging remains to be established. In this paper we revise the studies that analyze the association between patterns of mtDNA damage and aging, investigating putative alterations in mtDNA copy number as well as accumulation of deletions and of point mutations. Reports from the literature do not allow the establishment of a clear association between mtDNA copy number and age, either in humans or in mice. Further analysis, using a wide spectrum of tissues and a high number of individuals would be necessary to elucidate this pattern. Likewise humans, mice demonstrated a clear pattern of age-dependent and tissue-specific accumulation of mtDNA deletions. Deletions increase with age, and the highest amount of deletions has been observed in brain tissues both in humans and mice. On the other hand, mtDNA point mutations accumulation has been clearly associated with age in humans, but not in mice. Although further studies, using the same methodologies and targeting a larger number of samples would be mandatory to draw definitive conclusions, the revision of the available data raises concerns on the ability of mouse models to mimic the mtDNA damage patterns of humans, a fact with implications not only for the study of the aging process, but also for investigations of other processes in which mtDNA dysfunction is a hallmark, such as neurodegeneration.
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Determining the levels of human mitochondrial heteroplasmy is of utmost importance in several fields. In spite of this, there are currently few published works that have focused on this issue. In order to increase the knowledge of mitochondrial DNA (mtDNA) heteroplasmy, the main goal of this work is to investigate the frequency and the mutational spectrum of heteroplasmy in the human mtDNA genome. To address this, a set of nine primer pairs designed to avoid co-amplification of nuclear DNA (nDNA) sequences of mitochondrial origin (NUMTs) was used to amplify the mitochondrial genome in 101 individuals. The analysed individuals represent a collection with a balanced representation of genders and mtDNA haplogroup distribution, similar to that of a Western European population. The results show that the frequency of heteroplasmic individuals exceeds 61%. The frequency of point heteroplasmy is 28.7%, with a widespread distribution across the entire mtDNA. In addition, an excess of transitions in heteroplasmy were detected, suggesting that genetic drift and/or selection may be acting to reduce its frequency at population level. In fact, heteroplasmy at highly stable positions might have a greater impact on the viability of mitochondria, suggesting that purifying selection must be operating to prevent their fixation within individuals. This study analyses the frequency of heteroplasmy in a healthy population, carrying out an evolutionary analysis of the detected changes and providing a new perspective with important consequences in medical, evolutionary and forensic fields.
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Mitochondrial DNA (mtDNA) is believed to be particularly susceptible to oxidative damage during aging, resulting in mtDNA point mutations, duplications, and deletions. Although mtDNA deletions have been reported in various human tissues, e.g., the brain, heart, and skeletal muscle, little is known about the occurrence in hair. Therefore, we screened for the presence of mtDNA 13162 bp, 10422 bp, 7663 bp, 7436 bp, 4989 bp, and 4977 bp deletions in 90 hair samples from subjects aged 5 days to 91 years by using polymerase chain reaction (PCR) and investigated the deletion load by TaqMan probe-based real-time PCR. We detected the mtDNA 4977 bp deletion in hair samples, but none of the other deletions that were screened for. The proportion of mtDNA 4977 deletion carriers was 98.3% (89/90) and the deletion loads increased from 0 to 1.436 ± 0.2086% of the total mtDNA with an exponential increase with age (r = 0.677, p < 0.05). These results suggest that mtDNA 4977 bp deletion is a common phenomenon in hair and increases with age. These findings expand our understanding of the tissue-specific distribution of mtDNA deletions.
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Background: Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia caused by a CAG tract expansions in the ATXN3 gene. Patterns of mitochondrial damage associated with pathological findings of brain tissues could provide molecular biomarkers of this disorder. Objective: The potential of mitochondrial DNA (mtDNA) damage as a biomarker of MJD progression was investigated using a transgenic mouse model. Methods: DNA was obtained from affected (pontine nuclei) and nonaffected tissues (hippocampus and blood) of transgenic animals of three distinct age groups: 8 weeks, before onset of the phenotype; 16 weeks, at onset, and 24 weeks, at well-established phenotype. Wild-type littermate mice, serving as controls, were analyzed for the same tissues and age groups. mtDNA damage was studied by fluorescence-based quantitative PCR in 84 transgenic and 93 wild-type samples. Results: A clear pattern of decrease in mtDNA copy number with age and accumulation of 3,867-bp deletions at the initial stages (both being more pronounced in transgenic mice) was observed. Pontine nuclei, the affected tissue in transgenic mice, displayed 1.5 times less copies of mtDNA than nonaffected brain tissue hippocampus (odds ratio = 1.21). Pontine nuclei displayed the highest percentage of mtDNA deletions (6.05% more in transgenic mice). Conclusion: These results suggest that mtDNA damage is related to the initiation of the phenotype in transgenic mice; mtDNA 3,867-bp deletions may be a biomarker of the initial stages of the disease.
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Machado-Joseph Disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), represents the most common form of SCA worldwide. MJD is an autosomal dominant neurodegenerative disorder of late onset, involving predominantly the cerebellar, pyramidal, extrapyramidal, motor neuron and oculomotor systems; although sharing features with other SCAs, the identification of minor, but more specific signs, facilitates its differential diagnosis. MJD presents strong phenotypic heterogeneity, which has justified the classification of patients into three main clinical types. Main pathological lesions are observed in the spinocerebellar system, as well as in the cerebellar dentate nucleus. MJD's causative mutation consists in an expansion of an unstable CAG tract in exon 10 of the ATXN3 gene, located at 14q32.1. Haplotype-based studies have suggested that two main founder mutations may explain the present global distribution of the disease; the ancestral haplotype is of Asian origin, and has an estimated age of around 5,800 years, while the second mutational event has occurred about 1,400 years ago. The ATXN3 gene encodes for ataxin-3, which is ubiquitously expressed in neuronal and non-neuronal tissues, and, among other functions, is thought to participate in cellular protein quality control pathways. Mutated ATXN3 alleles consensually present about 61 to 87 CAG repeats, resulting in an expanded polyglutamine tract in ataxin-3. This altered protein gains a neurotoxic function, through yet unclear mechanisms. Clinical variability of MJD is only partially explained by the size of the CAG tract, which leaves a residual variance that should be explained by still unknown additional factors. Several genetic tests are available for MJD, and Genetic Counseling Programs have been created to better assist the affected families, namely on what concerns the possibility of pre-symptomatic testing. The main goal of this review was to bring together updated knowledge on MJD, covering several aspects from its initial descriptions and clinical presentation, through the discovery of the causative mutation, its origin and dispersion, as well as molecular genetics aspects considered essential for a better understanding of its neuropathology. Issues related with molecular testing and Genetic Counseling, as well as recent progresses and perspectives on genetic therapy, are also addressed.
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Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice) have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.
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Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) are the most common human adult-onset neurodegenerative diseases. They are characterized by prominent age-related neurodegeneration in selectively vulnerable neural systems. Some forms of AD, PD, and ALS are inherited, and genes causing these diseases have been identified. Nevertheless, the mechanisms of the neuronal cell death are unresolved. Morphological, biochemical, genetic, as well as cell and animal model studies reveal that mitochondria could have roles in this neurodegeneration. The functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations, triggering neurodegeneration according to a cell death matrix theory. In AD, alterations in enzymes involved in oxidative phosphorylation, oxidative damage, and mitochondrial binding of Aβ and amyloid precursor protein have been reported. In PD, mutations in putative mitochondrial proteins have been identified and mitochondrial DNA mutations have been found in neurons in the substantia nigra. In ALS, changes occur in mitochondrial respiratory chain enzymes and mitochondrial cell death proteins. Transgenic mouse models of human neurodegenerative disease are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria and the mitochondrial permeability transition pore. This review summarizes how mitochondrial pathobiology might contribute to neuronal death in AD, PD, and ALS and could serve as a target for drug therapy.
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Machado-Joseph disease (MJD) is a late-onset neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the ataxin-3 protein. We generated two transgenic mouse lineages expressing the expanded human ataxin-3 under the control of the CMV promoter: CMVMJD83 and CMVMJD94, carrying Q83 and Q94 stretches, respectively. Behavioral analysis revealed that the CMVMJD94 transgenic mice developed motor uncoordination, intergenerational instability of the CAG repeat and a tissue-specific increase in the somatic mosaicism of the repeat with aging. Histopathological analysis of MJD mice at early and late stages of the disease revealed neuronal atrophy and astrogliosis in several brain regions; however, we found no signs of microglial activation or neuroinflammatory response prior to the appearance of an overt phenotype. In our model, the appearance of MJD-like symptoms was also not associated with the presence of ataxin-3 cleavage products or intranuclear aggregates. We propose the transgenic CMVMJD94 mice as a useful model to study the early stages in the pathogenesis of MJD and to explore the molecular mechanisms involved in CAG repeat instability.
Mitochondrial dysfunction is heavily implicated in the multifactorial aging process. Aging humans have increased levels of somatic mtDNA mutations that tend to undergo clonal expansion to cause mosaic respiratory chain deficiency in various tissues, such as heart, brain, skeletal muscle, and gut. Genetic mouse models have shown that somatic mtDNA mutations and cell type-specific respiratory chain dysfunction can cause a variety of phenotypes associated with aging and age-related disease. There is thus strong observational and experimental evidence to implicate somatic mtDNA mutations and mosaic respiratory chain dysfunction in the mammalian aging process. The hypothesis that somatic mtDNA mutations are generated by oxidative damage has not been conclusively proven. Emerging data instead suggest that the inherent error rate of mitochondrial DNA (mtDNA) polymerase gamma (Pol gamma) may be responsible for the majority of somatic mtDNA mutations. The roles for mtDNA damage and replication errors in aging need to be further experimentally addressed.
Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by polyglutamine expansion in the ataxin-3 protein that confers a toxic gain of function. Because of the late onset of the disease, we hypothesize that the accumulated oxidative stress or/and defective antioxidant enzyme ability may be contributory factors in the pathogenesis of MJD. In this study, we utilized SK-N-SH and COS7 cells stably transfected with full-length MJD with 78 polyglutamine repeats to examine any alterations in the antioxidant activity. We demonstrated a significant reduction in the ratio of GSH/GSSG and total glutathione content (GSH + 2x GSSG) in mutant MJD cells compared with the wild-type cells under normal or stressful conditions. We also showed that both SK-N-SH-MJD78 and COS7-MJD78-GFP cell lines have lower activities of catalase, glutathione reductase, and superoxide dismutase compared with the wild-type cell lines. In addition, it is known that, when cells are under oxidative stress, the mitochondrial DNA is prone to damage. Our results demonstrated that mitochondrial DNA copy numbers are decreased in mutant cells and SCA3 patients' samples compared with the normal controls. Furthermore, the amount of common mitochondrial DNA 4,977-bp deletion is higher in SCA3 patients compared with that in normal individuals. Overall, mutant ataxin-3 may influence the activity of enzymatic components to remove O(2)(-) and H(2)O(2) efficiently and promote mitochondrial DNA damage or depletion, which leads to dysfunction of mitochondria. Therefore, we suggest that the cell damage caused by greater oxidative stress in SCA3 mutant cells plays an important role, at least in part, in the disease progression.