Oxidative Damage in Nucleic Acids and
Yusaku Nakabeppu,*Daisuke Tsuchimoto, Hiroo Yamaguchi,
and Kunihiko Sakumi
Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience,
Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
Oxidative DNA lesions, such as 8-oxoguanine (8-oxoG),
accumulate in nuclear and mitochondrial genomes dur-
ing aging, and such accumulation can increase dramat-
ically in patients with Parkinson’s disease (PD). To co-
unteract oxidative damage to nucleic acids, human
and rodents are equipped with three distinct enzymes.
One of these, MTH1, hydrolyzes oxidized purine nu-
cleoside triphosphates, such as 8-oxo-20-deoxyguano-
sine triphosphate and 2-hydroxy-20-deoxyadenosine tri-
phosphate, to their monophosphate forms. The other
two enzymes are 8-oxoG DNA glycosylase encoded by
the OGG1 gene and adenine/2-hydroxyadenine DNA
glycosylase encoded by the MUTYH gene. We have
shown a significant increase in 8-oxoG in mitochondrial
DNA as well as an elevated expression of MTH1,
OGG1, and MUTYH in nigrostriatal dopaminergic neu-
rons of PD patients, suggesting that the buildup of
these lesions may cause dopamine neuron loss. We
established MTH1-null mice and found that MTH1-null
fibroblasts were highly susceptible to cell death caused
by H2O2characterized by pyknosis and electron-dense
deposits in the mitochondria, and that this was accom-
panied by an ongoing accumulation of 8-oxoG in nu-
clear and mitochondrial DNA. We also showed that
MTH1-null mice exhibited an increased accumulation of
8-oxoG in striatal mitochondrial DNA, followed by more
extreme neuronal dysfunction after 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine administration than that of
wild-type mice. In conclusion, oxidative damage in
nucleic acids is likely to be a major risk factor for Par-
kinson’s disease, indicating that a solid understanding
of the defense mechanisms involved will enable us to
develop new strategies for protecting the brain against
C 2007 Wiley-Liss, Inc.
Key words: oxidative damage; nucleic acids; Parkinson’s
The adult human brain represents only about 2%
of total body weight; however, it consumes about 20%
of the resting total body O2consumption, which totals
about 360 liters per day. In children, the brain takes up
an even larger fraction, as much as 50% in the middle of
the first decade of life. About 90% of total oxygen con-
sumption occurs in mitochondria, where oxidative phos-
phorylation allows cells to produce ATP as energy to
ensure viability and function. The brain requires an ex-
traordinarily large amount of energy to maintain active
transport of the ions required for neuronal excitation
and neurotransmission, and the adult brain utilizes about
10 mol of ATP per day (approximately 5 kg/day; Clarke
and Sokoloff, 1999).
In mitochondria, about 1–4% of consumed oxygen
molecules are partially reduced by electrons that have
leaked from the respiratory chain, generating reactive
oxygen species (ROS) such as superoxide, hydrogen per-
oxide and hydroxyl radicals (Kang et al., 1998). Reflect-
ing its extraordinarily large consumption of oxygen, the
brain is thought to produce large amounts of ROS con-
tinuously. ROS are so highly reactive that they can
readily oxidize macromolecules in living cells, including
lipids, proteins, and nucleic acids, thereby leading to var-
ious types of cellular dysfunction, including cell death
and mutagenesis (Ames et al., 1993).
Among the various types of oxidative damage in
cellular macromolecules, damage to nucleic acids is par-
ticularly hazardous, because it can alter the genetic infor-
mation present in both nuclear and mitochondrial
genomic DNA (Cooke et al., 2003). Neurons are post-
mitotic cells and thus undergo no replication of nuclear
genomes, however, we assume that genetic information
contained in nuclear genomes would be expected to be
*Correspondence to: Yusaku Nakabeppu, DrSc DVM, Division of Neu-
rofunctional Genomics, Department of Immunobiology and Neuro-
science, Medical Institute of Bioregulation, Kyushu University, 3-1-1
Maidashi Higashi-ku, Fukuoka 812-8582, Japan.
Received 3 September 2006; Revised 30 October 2006; Accepted 17
Published online 5 February 2007 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.21191
Journal of Neuroscience Research 85:919–934 (2007)
' 2007 Wiley-Liss, Inc.
well maintained in order to support proper expression of
genes whose functions are essential to brain activity.
Moreover, mitochondrial genomes, which are continu-
ously replicated even in the postmitotic neurons under
an increased oxidative stress, have to be maintained so
that the production of energy is secure.
We recently established that brains are equipped
with defense mechanisms against oxidative damage to
nucleic acids and found that Parkinson’s disease (PD)
brains contained a large buildup of oxidative DNA dam-
age, which was accompanied by an altered expression of
the genes involved in defense mechanisms (Nakabeppu
et al., 2006b). In this Mini-Review, our discussion cen-
ters on the implications of oxidative DNA damage in
neurodegeneration, especially with regard to an animal
model of PD.
DAMAGE TO NUCLEIC ACIDS UNDER
Among the five normal nucleobases—uracil, thy-
mine, cytosine, adenine, and guanine—guanine is the
most susceptible to oxidation, and, among guanine resi-
dues in nucleic acids, the C8 position of deoxyguanosine
(dG) or dGTP is the most effectively oxidized by
hydroxyl radicals. The lesion formed is 8-oxo-dG or 8-
oxo-dGTP (Kasai and Nishimura, 1984), and, in fact,
eight to nine times more 8-oxoguanine (8-oxoG) is
formed in nucleotide dGTP than in DNA. In the case
of dATP, however, the C2 position is under attack, and,
once it is effectively oxidized, the lesion formed is 2-
hydroxy-dATP (2-OH-dATP). However, the formation
of 2-OH-A residues in DNA is only about 1.5% of the
level of 2-OH-A residues that are formed from dATP
(Kamiya and Kasai, 1995). It is known that 8-oxo-dATP
is generated by g-ray irradiation (Fujikawa et al., 1999).
Free nucleotides are thus more susceptible to oxidation
by ROS than is DNA; however, it is very difficult to
detect these oxidized nucleotides in vivo, probably
because the dNTP precursors are newly synthesized just
prior to DNA replication. Ribonucleotides such as ATP
and GTP are also similarly oxidized to 2-OH-ATP and
8-oxo-GTP, respectively (Fujikawa et al., 2001).
8-OxoG generated in DNA by its direct oxidation
can pair with an incoming adenine as well as cytosine
during DNA replication, thus resulting in a G:C to T:A
transversion mutation after two rounds of replication
(Fig. 1; Shibutani et al., 1991). It has been established
that 8-oxo-dGTP and 2-OH-dATP are frequently mis-
inserted opposite an incorrect base in the template DNA
by various replicative DNA polymerases ranging from
bacterial to human (Maki and Sekiguchi, 1992; Kamiya
and Kasai, 2000). As summarized in Figure 1, 8-oxo-
dGTP is misinserted opposite adenine as well as cytosine
in template DNA, generally causing an A:T to C:G
transversion after two rounds of replication. 2-OH-
dATP tends mostly to be misinserted opposite guanine,
generally inducing a G:C to T:A transversion. It is also
known that 8-oxoGTP or 2-OH-ATP can be incorpo-
rated into RNA by RNA polymerases, thereby generat-
ing mutant proteins through translational errors (Taddei
et al., 1997; Hayakawa et al., 1999).
THE OXIDIZED PURINE NUCLEOSIDE
TRIPHOSPHATASE MTH1 SANITIZES
In human and rodent cells, an oxidized purine
nucleoside triphosphatase termed \MTH1" efficiently
hydrolyzes two forms of oxidized dATP, namely, 2-
OH-dATP and 8-oxo-dATP, to their monophosphates,
in addition to 8-oxo-dGTP. These monophosphates are
converted to nucleosides such as 8-oxo-dG, thus avoid-
ing their incorporation into DNA (Fig. 1B; Fujikawa
et al., 1999; Nakabeppu, 2001a). MTH1 also hydrolyzes
the oxidized ribonucleotides 2-OH-ATP, 8-oxo-ATP,
and 8-oxo-GTP (Fujikawa et al., 2001). Among sub-
strates, MTH1 has its highest affinity for 2-OH-ATP
(Km ¼ 4.3 lM), whereas its highest catalytic efficiency
is toward 2-OH-dATP (kcat/Km ¼ 1.68 sec?1lm?1).
We recently determined the structure of MTH1 in solu-
tion by multidimensional heteronuclear NMR spectros-
copy (Mishima et al., 2004). We generated models for
the substrate recognition of MTH1, based on the
arrangement of the pocket-forming residues, combined
with the mutagenesis data, and found that Asn-33 and
Asp-119 play pivotal roles in discriminating the oxidized
forms of purine, namely, 8-oxoG and 2-OH-A, whereas
Trp-117 is important in determining affinity with the
purine rings (Sakai et al., 2002).
The human MTH1 gene is located on chromo-
some 7p22, and consists of five major exons; two alter-
native exon 1 sequences, namely, exons 1a and 1b, and
three contiguous exon 2 segments (exons 2a, 2b, and
2c), which are alternatively spliced. Thus, the MTH1
gene produces seven types of mRNA that encode three
different human MTH1 isoforms, hMTH1b (p22),
hMTH1c (p21), and hMTH1d (p18; Oda et al., 1997).
There are two major single nucleotide polymorphisms
(SNPs) in the MTH1 gene: one (GC/GT) is located at
the beginning of exon 2c, whereas the other (GTG/
ATG) is located on exon 4. The GC/GT polymorphism
modifies patterns of alternative splicing and encodes a
fourth isoform, hMTH1a (p26), in addition to the three
known isoforms. The GTG/ATG polymorphism repla-
ces amino acid residue valine 83 (Val83) in hMTH1d
with methionine 83 (Met83; Oda et al., 1999). The sub-
stitution of Val83 with Met83 in hMTH1d was found
to increase the thermolability of this enzyme as well as
its a-helix content (Yakushiji et al., 1997). Furthermore,
molecular epidemiological studies have revealed that the
allele frequencies of Met83 with the GC polymorphism
in patients with hepatocellular carcinoma, gastric cancer,
and lung cancer were higher than those of healthy vol-
(Met83) may represent a functional defect (Nakabeppu,
2001a; Kimura et al., 2004; Kohno et al., 2006).
920 Nakabeppu et al.
Journal of Neuroscience Research DOI 10.1002/jnr
Fig. 1. Mutagenesis caused by the oxidation of nucleic acids and the
defense mechanisms in mammals. A: Altered base pairing of 8-oxo-
guanine and 2-hydroxyadenine. During DNA replication, 8-oxoG
and 2-OH-A can pair with adenine and guanine, respectively, in
template DNA. B: Mutagenesis caused by 8-oxoG and 2-OH-A. 8-
OxoG is accumulated in DNA as a result of the incorporation of 8-
oxo-dGTP from nucleotide pools or because of the direct oxidation
of DNA, and this buildup increases the likelihood of an A:T to C:G
or G:C to T:A transversion. On the other hand, 2-OH-A is derived
mainly from the incorporation of 2-OH-dATP from nucleotide
pools. The accumulation of 8-oxoG or 2-OH-A in DNA is mini-
mized through the coordinated actions of MTH1, OGG1, and
MUTYH. See text for details. GO, 8-oxoguanine (8-oxoG); AO, 2-
hydroxyadenine (2-OH-A). Bold lines: Nascent strands of DNA.
Oxidative Damage in Parkinson’s Disease921
Journal of Neuroscience Research DOI 10.1002/jnr
We reported earlier that most of the 18-kDa
hMTH1 protein is localized in the cytoplasm, with
about 5% in the mitochondrial matrix (Kang et al.,
1995). We recently demonstrated that all hMTH1 iso-
forms are capable of hydrolyzing both 2-OH-dATP and
8-oxo-dGTP to their monophosphates and that the 18-
amino acid sequence in the amino (N)-terminal region
of hMTH1a functions as a mitochondria-targeting signal.
hMTH1a is thus considered to be localized in the mito-
chondria to the same extent as hMTH1d. When the lat-
ter contains a Met83 substitution, it is less likely to local-
ize in mitochondria than is MTH1d with Val83 (Sakai
et al., 2006). These observations support the idea that
hMTH1 plays an important role in maintaining the qual-
ity of the nucleotide pools of both nuclear and mito-
chondrial genomes, as well as that of ribonucleotide
8-OXOGUANINE DNA GLYCOSYLASE,
OGG1, PREVENTS ACCUMULATION OF
8-OXOGUANINE IN BOTH NUCLEAR AND
Once 8-oxoG has formed in DNA, 8-oxoG DNA
glycosylase encoded by the OGG1 gene removes this
oxidized base to initiate base excision repair (BER). The
DNA glycosylase activity of OGG1 preferentially excises
8-oxoG or 2,6-diamino-4-hydroxy-5-formamidopyrimi-
dine (FapyG) opposite cytosine, and to a lesser extent
thymine, but not when either is opposite guanine or ad-
enine. In addition, OGG1 also possesses AP lyase activity
(Fig. 1B; Boiteux and Radicella, 2000).
The eight exons of the human OGG1 gene are
located on chromosome 3p25, a region showing a fre-
quent loss of heterozygosity in lung and kidney tumors
(Aburatani et al., 1997; Boiteux and Radicella, 2000).
There are more than seven alternatively spliced forms of
OGG1 mRNA, and these have been classified into two
types based on their last exons (type 1 with exon 7: 1a
and 1b; type 2 with exon 8: 2a to 2e; Nishioka et al.,
1999). Types 1a and 2a mRNA are the major OGG1
transcripts in various human tissues and encode hOGG1-
1a and hOGG1-2a isoforms of human OGG1 protein,
respectively. hOGG1-1a protein has a nuclear localiza-
tion signal (NLS) at its C-terminal end and thus is
located in the nucleus, whereas hOGG1-2a protein,
which has a unique C-terminal region consisting of two
distinct regions, namely, an acidic region (amino acid
residues from Ile345to Asp381) and a hydrophobic region
(the last 20 residues), is located exclusively in the mito-
chondria. Both hOGG1-1a and hOGG1-2a carry a rela-
tively poor mitochondrial targeting sequence (MTS) at
their N-terminal region. This sequence consists of resi-
dues 9–26, whose activity is not sufficient for localizing
nuclear hOGG1-1a with NLS within the mitochondria.
On the other hand, hOGG1-2a is likely associated with
the mitochondrial inner membrane and other BER ma-
chinery dependent on the unique C-terminal region
(Nishioka et al., 1999; Stuart et al., 2005).
MUTYH FUNCTIONS AS A BIFUNCTIONAL
DNA GLYCOSYLASE FOR
GUANINE AND ADENINE OPPOSITE
DNA polymerases may insert adenine into the nas-
cent strand when they encounter 8-oxoG in the tem-
plate strand during DNA replication, thus increasing the
likelihood of a G:C to T:A transversion (Fig. 1B; Miller,
1996; Maki, 2002). A DNA glycosylase encoded by the
MUTYH gene excises the adenine inserted opposite 8-
oxoG in the template strand (Slupska et al., 1999; Tomi-
naga et al., 2004). MUTYH protein also has the ability
to excise 2-OH-A incorporated opposite guanine in the
template (Fig. 1B; Ohtsubo et al., 2000; Ushijima et al.,
2005). It has been shown that the adenine base in DNA
is barely oxidized, whereas adenine nucleotides are easily
oxidized in vitro, suggesting that 2-OH-A is derived
mostly through the incorporation of 2-OH-dATP dur-
ing DNA replication (Kamiya and Kasai, 1995). It is
therefore likely that MUTYH has to recognize specifi-
cally adenine or 2-OH-A incorporated into the nascent
strand at this time. MUTYH has been demonstrated to
have a functional proliferating cell nuclear antigen
(PCNA) binding motif (Parker et al., 2001). We have
already shown that, in cultured cells, MUTYH repair
activity directed toward adenine incorporated opposite
8-oxoG in transfected plasmid DNA is dependent on
this motif (Hayashi et al., 2002). However, we recently
found that the PCNA-binding motif in MUTYH is not
essential for suppressing the increased spontaneous muta-
tion rate observed in MUTYH-null cells (Hirano et al.,
2003). MUTYH has been shown to interact with other
replication-associated proteins such as RPA or MSH2/
MSH6, which also can interact with PCNA, thus sug-
gesting that the interaction of MUTYH with these pro-
teins may support MUTYH function in the absence of
PCNA (Gu et al., 2002).
The human MUTYH gene is located on the short
arm of chromosome 1, between p32.1 and p34.3, and
consists of 16 exons (Slupska et al., 1996). We previ-
ously reported that there are three major MUTYH tran-
scripts in human cells, namely, types a, b, and g. Each
transcript has a different 50sequence or first exon, and
each is alternatively spliced, so multiple forms of human
MUTYH proteins are present in nuclei and mitochon-
dria (Ohtsubo et al. 2000). Human MUTYH protein
encoded by type a mRNA possesses a mitochondrial
targeting sequence (MTS) consisting of 14 amino-termi-
nal residues, which are required for its localization in the
encoded by type b and g mRNAs lack the MTS and
are localized in the nuclei (Ohtsubo et al., 2000). As a
result, the subcellular localization of MUTYH in human
cells indicates that mitochondrial DNA is an important
target for BER initiated by MUTYH, as well as OGG1,
probably because of increased oxidative stress (Naka-
al. 1998),whereas those
922Nakabeppu et al.
Journal of Neuroscience Research DOI 10.1002/jnr
CELLULAR DYSFUNCTION CAUSED BY
THE ACCUMULATION OF OXIDIZED
NUCLEOTIDES AND ITS PREVENTION
We reported that lung adenoma/carcinoma devel-
oped spontaneously in OGG1-null mice at about 1.5
years after birth and that 8-oxoG had accumulated in
their genomes because of the absence of BER of 8-
oxoG initiated by 8-oxoG DNA glycosylase encoded by
the Ogg1 gene (Sakumi et al., 2003). We then found
that no tumors had formed in the lungs of mice lacking
both OGG1 and MTH1 proteins, despite their increased
accumulation of 8-oxoG. This suggests that Mth1 gene
disruption resulted in suppression of the tumorigenesis
caused by OGG1 deficiency. We hypothesized, based on
these facts, that, because of these combined deficiencies,
a large accumulation of oxidized purine nucleoside tri-
phosphates in nucleotide pools and/or a buildup of oxi-
dized purine bases such as 8-oxoG or 2-OH-A in cellu-
lar DNA and RNA would necessarily result in cell
death. If this is the case, one could expect that cancer
stem cells lacking both OGG1 and MTH1 proteins
might not survive to produce progenitors with mutations
in either protooncogenes or tumor suppressor genes, as
was seen in a study in which carcinogenesis was sup-
pressed in mice lacking both OGG1 and MTH1 proteins
(Nakabeppu et al., 2004).
Indeed, MTH1-null mouse embryo fibroblasts
(MEF) were found to be highly susceptible to cell dys-
function and death caused by exposure to H2O2 and
showed morphological features of pyknosis and electron-
dense deposits in their mitochondria (Fig. 2; Yoshimura
et al., 2003). The cell death observed was not depen-
dent on either poly(ADP-ribose)-polymerase or caspases.
High-performance liquid chromatography-tandem mass
spectrometry analysis and immunofluorescence micros-
copy revealed an ongoing accumulation of 8-oxoG
in nuclear and mitochondrial DNA after exposure to
H2O2. All of the H2O2-induced changes observed in
MTH1-null MEF were effectively suppressed by the
expression of wild-type hMTH1, but were only partially
suppressed by the expression of mutant hMTH1 defec-
tive in either 8-oxo-dGTPase or 2-OH-dATPase activ-
ity. Thus, hMTH1 protects cells from H2O2-induced
cell death by hydrolyzing oxidized purine nucleotides,
Fig. 2. hMTH1 suppressed the H2O2-induced cell death of MTH1-
null MEF preceded by pyknosis and mitochondrial dysfunction.
A: MTH1-null MEF (MTH1-null) or hMTH1-expressing cells
(hMTH1) were examined by phase-contrast microscopy either with
or without (control) exposure to 500 lM H2O2for 24 hr. These
cells were also stained with Hoechst 33342 and propidium iodide
(PI) and examined under a fluorescence microscope. B: hMTH1
suppressed the morphological alterations of mitochondria in MTH1-
null MEF induced by exposure to H2O2. Mitochondria in MTH1-
null MEF (MTH1-null) and hMTH1-expressing cells (hMTH1)
were examined with an electron microscope 4–24 hr after exposure
to 500 lM H2O2. Control: untreated cells. C: The accumulation of
8-oxoG in cellular DNA of MTH1-null MEF after exposure to
H2O2 was suppressed by hMTH1. 8-OxoG that accumulated in
(hMTH1) exposed to 300 lM H2O2for 1 and 8 hr was detected by
laser scanning fluorescence microscopy with an anti-8-oxoG antibody
(green). The nuclei were counterstained with PI (red). Control, cells
not exposed to H2O2. (Adapted from Yoshimura et al., 2003).
and hMTH1-expressing cells
Oxidative Damage in Parkinson’s Disease 923
Journal of Neuroscience Research DOI 10.1002/jnr
Fig. 4. Altered expression of the mitochondrial form of hOGG1 in
PD brain. A–D: Immunohistochemistry for hOGG1-2a in the SN
(A,B) and pontine nuclei (PN; C,D) of representative subjects. Con-
trol (A,C), PD patient (B,D). Note the granular staining of hOGG1-
2a (arrow) in the cytoplasm of SN neurons of the PD patient.
Arrowheads indicate neuromelanin. E–J: Neurons in the SN of a PD
patient doubly stained with anti-hOGG1-2a antibody (red in E) and
antityrosine hydroxylase (TH) antibody (green in F) and merged (G),
or with anti-hOGG1-2a antibody (red in H) and anticytochrome ox-
idase subunit I antibody (green in I) and merged (J). Note the granu-
lar hOGG1-2a expression pattern in the cytoplasm of TH-positive
neurons and colocalization with cytochrome oxidase subunit 1. K:
Age-dependent increase in the percentage of hOGG1-2a-positive
neurons in the SN of control subjects. There was a significant corre-
lation with age (P < 0.05). L: Results of semiquantitative analysis of
hOGG1-2a in the SN of PD patients. Percentage of hOGG1-2a-pos-
itive neurons in short- and long-duration PD groups (mean 6 SEM).
The percentage of hOGG1-2a-positive neurons was significantly
higher in the short-duration group than in the long-duration group
(*P < 0.05). Scale bars ¼ 10 lm in A–D; 20 lm in E (applies to E–
G); 10 lm in H (applies to H–J). (Adapted from Fukae et al., 2005,
with permission from Springer.)
higher in the short-duration group relative to the age-
matched controls (Fig. 4L). In the long-duration group,
the number of hOGG1-2a-positive neurons was slightly
higher, although this was statistically insignificant com-
pared with the aged-matched controls. Western blot
analysis demonstrated that the level of hOGG1-2a (43
and 40 kDa) in the SN of PD brain was 1.6–2.9-fold
higher than that of the age-matched controls.
We also demonstrated up-regulation of MUTYH
in the mitochondria of the SN of PD patients (Fig. 5;
Arai et al., 2006). The dopaminergic neurons remaining
in the SN showed intense and diffuse immunostaining
for MUTYH in the cytoplasm but none in nuclei or
Lewy bodies (Fig. 5). On the other hand, glial cells,
including oligodendrocytes and astrocytes, were barely
immunoreactive. Western blot analysis of PD patients
revealed high MUTYH levels and the expression in PD
brains of a 47-kDa molecule as the major MUTYH iso-
form. The 47-kDa molecule was localized within the
mitochondria as confirmed by double staining with a
mitochondrial marker (Fig. 5C–E).
To confirmthe expression
MUTYH isoform, we performed RT-PCR with total
RNA isolated from the SN of PD and control brains
(Arai et al., 2006), and we detected four types of
MUTYH mRNA. Among these, type b4 mRNA
encoding the 47-kDa MUTYH isoform was identified
for the first time, although its existence had already been
theoretically considered. The level of type a3 mRNA
encoding a 54-kDa MUTYH isoform with an MTS,
was similar to that of type a4 mRNA also encoding the
47-kDa isoform. In contrast, the level of type b3
mRNA, encoding a 53-kDa nuclear form of MUTYH,
and that of type b4 mRNA were lower than those of
type a3 and a4 mRNAs. Given the size of the mole-
cules (47-kDa), the main MUTYH proteins detected in
the PD brains may be derived from type a4 mRNA.
On Western blot analysis, we could not detect the com-
mon forms of MUTYH, such as the 53- and/or 54-kDa
molecules encoded by b3 and a3 type mRNAs, suggest-
ing that these two molecules are unstable in these brains.
of the 47-kDa
Fig. 5. Altered expression of MUTYH in Parkinson’s brain. A,B:
Immunohistochemistry of MUTYH in the SN of a representative
control (A) and PD patient (B). Note the diffuse staining pattern for
MUTYH in the cytoplasm of SN neurons of PD. Arrowheads indi-
cate neuromelanin. C–H: Double-immunofluorescence study for
MUTYH. Neurons in the SN of PD brain were stained with anti-
MUTYH antibody (green in C) and anticytochrome oxidase subunit
Vb antibody (red in D; and E is the merged image) or were stained
with anti-MUTYH antibody (green in F) and DAPI (blue in G; and
H is the merged image). The dot-like signals of MUTYH in the
cytoplasm are colocalized with the cytochrome oxidase subunit
Vb(E), but not with DAPI (H). I: Semiquantitative analysis of
MUTYH-expressing neurons. The percentage of MUTYH-positive
neurons (mean 6 SEM) was significantly higher in the SN of PD
brain than in the control (CON; *P < 0.05). Scale bars ¼ 20 lm.
(Adapted from Arai et al., 2006, with permission from Springer.)
926 Nakabeppu et al.
Journal of Neuroscience Research DOI 10.1002/jnr
have a causative role in PD, and some of these toxins in-
hibit the mitochondrial respiratory chain (Mayeux,
2003). Among them, the 1-methyl-4-phenyl-1,2,3,6-tet-
rahydropyridine (MPTP) model constitutes the best-
characterized toxin paradigm for PD, faithfully replicat-
ing most of its clinical and pathological hallmarks (Javitch
et al., 1985; Nicklas et al., 1985; Speciale, 2002; Przedborski
Increased lipid peroxidation and chemical modifi-
cation of proteins are common features of the MPTP-
induced PD model, but there have been only a few
published studies reporting that MPTP induces the accu-
mulation of 8-oxoG in this model (Chen et al., 2005;
Yamaguchi et al., 2006). We clearly showed that the
amount of 8-oxoG in cellular DNA increased signifi-
cantly in both the SN and the striatum 12–24 hr after a
single administration of MPTP (30 mg/kg, ip; Fig. 6;
Yamaguchi et al., 2006). Of particular interest, 8-oxoG
accumulation in mitochondrial DNA of striatal dopami-
nergic nerve terminals was noted prior to their degener-
ation and earlier than the 8-oxoG buildup in nuclear
DNA of the SN, in which a loss of dopaminergic neu-
rons became apparent after the repeated administration
of MPTP. These observations strongly suggest that
MPTP triggers degeneration of the dopaminergic neu-
rons from their terminals in the striatum together with
mitochondrial dysfunction, and that this progresses in a
retrograde manner, as previously reported (Mitsumoto
et al., 1998; Nakai et al., 2003). We further observed
that microglia and astrocytes were significantly activated
in the striatum or SN 12–24 hr after a single administra-
tion of MPTP. It has been shown that the activated
microglia or astrocytes produce large amounts of extrac-
ellular superoxide as a result of NADPH oxidase activity,
further increasing oxidative stress in the nigrostriatal
pathway (Kurkowska-Jastrzebska et al., 1999; Song et al.,
MTH1 DEFICIENCY INCREASED
DEGENERATION OF STRIATAL NERVE
TERMINALS OF DOPAMINE NEURONS
AFTER MPTP ADMINISTRATION
Mth1 mRNA was detected in neurons throughout
mouse brain by in situ hybridization, and neurons in the
SN, including substantia nigra pars compacta (SNc), sub-
stantia nigra pars reticulata (SNr), and ventral tegmental
area (VTA), exhibited substantial levels of Mth1 mRNA
expression, as did neurons in the cerebral cortex and
hippocampus (Fig. 7; Yamaguchi et al., 2006).
To examine the role of MTH1 in the nigrostriatal
pathway, we assessed the neuroprotective role of MTH1
in the degeneration of dopaminergic neurons based on a
comparison of wild-type and MTH1-null mice. MPTP
(30 mg/kg, ip) or saline was administered to wild-type
and MTH1-null mice once a day for 5 consecutive days,
and then the degeneration of dopaminergic nerve termi-
nals in the striatum and the loss of dopaminergic neurons
in the SN were compared (Fig. 8A; Yamaguchi et al.,
2006). About a 30% reduction in the number of dopa-
minergic neurons across several levels of the SN was
observed in both wild-type and MTH1-null mice after
MPTP treatment, and there was no apparent difference
Fig. 7. Expression of Mth1 mRNA in the normal mouse brain. A:
The level of Mth1 mRNA in coronal midbrain sections prepared
from a C57BL/6J male mouse was visualized by in situ hybridization
with an antisense (a) and sense probe (b). Hybridization signals were
detected only with the antisense probe, and they were present in
neurons throughout the brain, including those of the SN and hippo-
campus. B: Hybridization signals for Mth1 mRNA were present in
neurons of the SNc (a), SNr (b), and VTA (c) as well as in neurons
scattered throughout the cerebral cortex (d), hippocampal CA1 (e),
and CA3 subfields (f). Scale bars ¼ 500 lm in A; 10 lm in B.
(Adapted from Yamaguchi et al., 2006).
928Nakabeppu et al.
Journal of Neuroscience Research DOI 10.1002/jnr
Fig. 8. MTH1-null mice display an
increased reduction in TH and DAT
immunoreactivities in striatal terminal
fibers of dopaminergic neurons after
chronic exposure to MPTP. A: MPTP
(30 mg/kg) or saline was administered
i.p. to wild-type and MTH1-null mice
once per day for 5 consecutive days.
Seven days after the last injection, the
mice were sacrificed for analyses. B:
Striatal immunoreactivities for TH,
DAT, and glial fibrillary acidic protein
(GFAP) were examined in sections
prepared from mice administered sa-
line (control, a–h) or MPTP (MPTP,
i–p). Wild type: a–d, i–l; MTH1-null
mice: e–h, m–p. The images d, h and
i, p are magnified images of c, g and k,
o, respectively. C: TH immunoreac-
tivities in the striatum, nucleus accum-
bens (NAc), and olfactory tubercle
(OT) were measured in the sections
shown in B, and TH indexes are
shown in a box-and-whisker plot. In
each plot, the boxes are drawn with
the ends at the quartiles, and the statis-
tical median is shown as a horizontal
line within the box. The whiskers
extend to the farthest points that are
not outliers (circles). TH indexes in
striatum, NAc, and OT of MTH1-null
mice after MPTP injection were sig-
nificantly lower than those of the wild
type (WT; **P < 0.01, *P < 0.02).
Control, open boxes; MPTP-treated,
shaded boxes. D: DAT immunoreac-
tivities in the striatum, NAc and OT
were measured in the sections shown
in B, and DAT indexes are shown in a
box-and-whisker plot (n ¼ 4 * 5), as
in C. DAT indexes in striatum and
OT of MTH1-null mice after MPTP
injection were also significantly lower
than those of the wild type (**P <
0.01, *P < 0.05). Scale bars ¼ 500 lm
in Ba (applies to Ba–c,e–g,i–k,m–o);
20 lm in Bd (applies to Bd,h,l,p).
(Adapted from Yamaguchi et al.,
Journal of Neuroscience Research DOI 10.1002/jnr
Oxidative Damage in Parkinson’s Disease 929
between the two groups. On the other hand, MTH1-
null mice displayed a more severe degeneration of the
dopaminergic nerve terminals in the striatum after chro-
nic exposure to MPTP, which was revealed by tyrosine
hydroxylase (TH) and dopamine transporter (DAT)
immunostaining (Fig. 8B–D). Furthermore, 12 hr after
MPTP injection, the accumulation of 8-oxoG in mito-
increased in MTH1-null mice compared with the wild-
type mice (Fig. 9A,B), and, at the same time, the stria-
tum of the former also showed strong 8-oxoG immu-
noreactivities (Fig. 9Ca,f). The 8-oxoG signals exhibited
a fiber-like shape and were distributed with TH-positive
fibers, and most of these were colocalized in MTH1-
null mice (Fig. 9Ci,j). It is noteworthy that there was
very little 8-oxoG immunoreactivitiy in the cytoplasm
of postsynaptic spiny neurons (their nuclei are in blue in
Fig. 9C). We thus concluded that, 12 hr after MPTP
injection, 8-oxoG in the MTH1-null mice accumulated
mostly in mitochondrial DNA in striatal nerve terminals
of dopaminergic neurons.
MPTP is converted to 1-methyl-4-phenylpyridi-
nium ion (MPPþ) in the brain, mostly by monoamine
oxidase B in glial cells, and MPPþcan then be specifi-
cally taken up by dopaminergic neurons through DAT,
which is mostly localized in dopaminergic nerve termi-
nals, as shown in Figure 10. MPPþthat accumulates in
dopaminergic nerve terminals binds to complex I of the
respiratory chain in mitochondria and blocks electron
transport. This leads to an energy failure and ATP deple-
tion as well as to an increase in electron leakage from the
respiratory chain, which results in the formation of ROS
(Nicklas et al., 1985; Ramsay et al., 1991). In addition,
dopaminergic nerve terminals are more likely to be
exposed to increased levels of oxidative stress caused by
the metabolic products of dopamine than are other parts
of the brain. MPPþitself induces a massive release of
vesicular dopamine into the cytosol (Lotharius and
O’Malley, 2000) or increased dopamine turnover. There-
fore, oxidative stress in dopaminergic nerve terminals is
suspected to increase significantly and specifically in
MPTP-administered animals (Schmidt and Ferger, 2001;
Przedborski and Vila, 2003). The oxidation of dopamine
stoichiometrically produces hydrogen peroxide, which
reacts with Fe2þion to form hydroxyl radicals as a result
of the Fenton reaction. Hydroxyl radicals are the most
powerful forms of ROS and easily oxidize almost all bio-
molecules, such as lipids, proteins, and nucleic acids,
resulting in cellular dysfunction or cell death (Karam
et al., 1991; Halliwell, 1992).
As indicated earlier, MPTP triggers mitochondrial
dysfunction and degeneration of the dopaminergic neu-
rons from their terminals in the striatum, and this degen-
eration progresses in a retrograde manner. Although we
expression of MTH1, OGG1, and MUTYH in striatal
regions of PD brains, it is likely that such retrograde
degeneration of dopaminergic neurons also takes place in
Fig. 10. MTH1 deficiency augments MPTP-induced accumulation
of 8-oxoG in mitochondrial DNA of striatal dopaminergic nerve ter-
minals and causes their dysfunction. Systemically administered MPTP
is converted to MPPþ, which can be specifically taken up by dopa-
minergic neurons through DAT. MPPþin dopaminergic neurons
binds to complex I of the respiratory chain in mitochondria and
blocks electron transport, resulting in the formation of ROS, which
oxidize various macromolecules, including nucleotides. MPPþfurther
increases ROS through the release of dopamine (DA) from synaptic
vesicles (SV). In wild-type mice, MTH1 efficiently sanitizes the nu-
cleotide pool by hydrolyzing 8-oxo-dGTP or other oxidized purine
nucleotides in the nucleotide pool, thereby avoiding accumulation of
8-oxoG in the mitochondrial DNA of the striatal dopaminergic nerve
terminals. On the other hand, 8-oxo-dGTP or other oxidized purine
nucleotides accumulated in MTH1-null mice is incorporated into
mitochondrial DNA in striatal dopaminergic nerve terminals, result-
ing in mitochondrial dysfunction and dopamine depletion (DA deple-
tion). L-DOPA, levodopa; DOPAC, 3,4-dihydroxyphenylacetic acid;
DA-Q, dopamine quinone. (Adapted from Nakabeppu et al., 2006b,
with permission from Elsevier.)
Oxidative Damage in Parkinson’s Disease931
Journal of Neuroscience Research DOI 10.1002/jnr
PD brain (Riederer and Wuketich, 1976; Mayeux,
2003). As shown in Figure 3, loss of dopaminergic neu-
rons in the SN is more evident in brains of chronic PD
patients and is accompanied by an increased accumula-
tion of 8-oxoG and expression of MTH1 in perikaryal
mitochondria. This indicates that these damaged mito-
chondria further contribute to the loss of dopaminergic
neurons in the SN, as in striatal nerve terminals, and
cause their degeneration.
In addition to 8-oxo-dGTP, MTH1 efficiently
hydrolyzes other oxidized purine nucleoside triphos-
phates (Nakabeppu et al., 2006a), so MTH1-null mice
accumulated a larger amount of these triphosphates in
dopaminergic nerve terminals after MPTP administra-
tion, resulting in an increased accumulation of oxidized
purines in mitochondrial DNA (Fig. 10). As a result, an
MTH1 deficiency augments MPTP-provoked degenera-
tion of dopaminergic nerve terminals in the striatum.
Take together, these findings strongly suggest that
increased levels of various oxidized purine nucleoside tri-
phosphates, including 8-oxo-dGTP, induce a dysfunc-
tion of dopaminergic nerve terminals (Yamaguchi et al.,
Observations in human PD patients together with
experimental analyses of a mouse model of PD indicated
that oxidative damage in nucleic acids might be a major
risk factor for a loss of dopaminergic neurons in the ni-
grostriatal system. We suggest that oxidative damage ini-
tially builds up in mitochondrial genomes of striatal
nerve terminals and triggers their dysfunction and that,
later, the damage is further accumulated in perikaryal
mitochondria of dopamine neurons in the SN, thereby
resulting in their loss.
It has been widely accepted that neurodegeneration
is tightly associated with mitochondrial dysfunction as
well as DNA damage (Ames et al., 1993). Our studies
reveal that oxidized purine deoxyribonucleotides are
misincorporated into mitochondrial DNA, causing mito-
chondrial dysfunction, which results in a loss of dopami-
nergic nerve terminals, as observed in human PD
patients (Yoshimura et al., 2003; Yamaguchi et al.,
2006). In this regard, we are very much interested in
what the 47-kDa isoform of MUTYH, which is highly
expressed in PD brain, actually does in the mitochondria
of dopamine neurons (Arai et al., 2006). Adenine can be
misinserted opposite 8-oxoG that has accumulated in
mitochondrial DNA of the PD brain. Therefore, it is
likely that MUTYH can excise the adenine opposite 8-
oxoG in DNA to initiate the process of base excision
repair. This BER may protect dopamine neurons from
the deleterious effects of 8-oxoG buildup in mitochon-
drial DNA, although there is no evidence showing
whether the 47-kDa isoform of MUTYH retains its
MTH1 efficiently hydrolyzes oxidized purine ribo-
nucleotides such as 2-OH-ATP, 8-oxo-ATP, and to a
lesser extent 8-oxo-GTP (Nakabeppu et al., 2006a). As a
result, cellular dysfunction might also be caused by their
incorporation into RNA. Furthermore, an increasing
concentration of 8-oxo-dGTP itself has been reported to
reduce the amounts of newly synthesized DNA in a
cell-free DNA replication system derived from Xenopus
egg lysates (Kai et al., 2002), indicating the possibility
that free forms of oxidized purine nucleotides independ-
ently exert a certain degree of cytotoxicity. To elucidate
further the biological significance of these oxidized nu-
cleotides in vivo, we are now developing transgenic
mice expressing human MTH1 protein. Extended analy-
ses of MTH1 using these materials will hopefully shed
some light on the molecular mechanisms of neurodegen-
eration as well as carcinogenesis.
We extend our special thanks to all other members
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and Y. Mizuno for their helpful discussions; and to Dr.
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Journal of Neuroscience Research DOI 10.1002/jnr