JBC Papers in Press. Published on January 4, 2012 as Manuscript M111.291179 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.291179
Glutathione S-Transferase Omega 1 Activity is Sufficient to Suppress Neurodegeneration in a Drosophila Model of Parkinson’s Disease Kiyoung Kim, Song-Hee Kim, Jaekwang Kim†, Heuijong Kim, and Jeongbin Yim* School of Biological Sciences, Seoul National University, Seoul 151-742, Korea *To whom correspondence should be addressed: Jeongbin Yim, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea, Tel: 82-2-880-6702; Fax: 82-2-871-4315; E-mail:
[email protected] Running title: Function of Glutathione S-Transferase Omega 1 CAPSULE 1. Background: Glutathione S-Transferase Omega has been shown to be associated with the
2. Result: Drosophila GSTO1 regulates mitochondrial ATP synthase activity in parkin mutants. 3. Conclusion: Drosophila GSTO1 plays a protective role in a Drosophila model of Parkinson's Disease. 4. Significance: These findings may lead to a better understanding of the molecular mechanism of neuroprotection due to GSTO in Parkinson's Disease.
A loss-of-function mutation in the gene
DmGSTO1. We found that glutathionylation
parkin causes a common neurodegenerative
of the ATP synthase β subunit is rescued by
disease that may be caused by mitochondrial
DmGSTO1, and that the expression of
dysfunction.
S-transferase
DmGSTO1 partially restores the activity, and
omega (GSTO) is involved in cell defense
assembly of the mitochondrial F1F0-ATP
mechanisms, but little is known about the
synthase in parkin mutants. Our results
role
of
suggest a novel mechanism for the protective
Parkinson’s Disease (PD). Here, we report
role of DmGSTO1 in parkin mutants,
that
GSTO1
through the regulation of ATP synthase
(DmGSTO1), which is downregulated in
activity, and provide insight into potential
parkin mutants, alleviates some of the parkin
therapies for PD neurodegeneration.
of
Glutathione
GSTO
restoration
in of
the
progression
Drosophila
pathogenic phenotypes, and that the loss of DmGSTO1 function enhances parkin mutant
Parkinson’s Disease (PD) is a progressive
phenotypes. We further identified the ATP
neurodegenerative disorder characterized by the
synthase β subunit as a novel in vivo target of
degeneration of dopaminergic neurons. Previous
1
Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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Parkinson's Disease.
mitochondrial
(DmGSTS1), which is another member of the
dysfunction, oxidative stress, and ER stress
GST family in Drosophila, suppresses the
induced by the aggregation of abnormal proteins
phenotypes of parkin mutants and α-synuclein-
can contribute to the pathogenesis of PD (1,2).
expressing mutants (11-13). To elucidate the
However, the molecular mechanisms of PD
protective role of GSTO in neurodegenerative
pathogenesis have not yet been fully elucidated.
diseases, we investigated the biological function
A loss-of-function mutation in the parkin gene
of D. melanogaster GSTO (DmGSTO) in a
is a major cause of autosomal recessive, early
Drosophila model of Parkinson’s disease. We
onset PD.
found that one of the DmGSTOs, DmGSTO1, is
reports
suggested
that
able to rescues some phenotypes of parkin
Glutathione S-transferases (GSTs) constitute a superfamily of enzymes that are grouped into at
mutants,
least ten classes; some superfamily members are
dopaminergic neurons and muscle. The ability
known
of DmGSTO1 to rescue these phenotypes was
to
be
involved
in
cell
defense
including
DmGSTO1. Furthermore, tubulin accumulation,
that can form a disulfide bond with GSH, while
and ER stress caused by the parkin mutation
other eukaryotic GSTs have tyrosine or serine
were also significantly reduced by ectopic
residues in their active sites (4). The biological
expression of DmGSTO1. We discovered that
functions
been
the Drosophila ATP synthase β subunit (14) is a
modulates
novel target of DmGSTO1 and upregulation of
calcium channels (5), has a role in the activation
DmGSTO1 restored glutathionylation levels of
of interleukin-1β (6), and interacts with a serine
the ATP synthase β subunit in parkin mutants.
protease inhibitor (7). Recently, our group
The mitochondrial F1F0-ATP synthase is a
reported that one of the four GSTO genes in
membrane protein complex that couples the
Drosophila, CG6781, is the structural gene of
synthesis of ATP to the hydrogen ion gradient
the Drosophila eye color mutant, sepia, which
generated by the respiratory chain (15,16). The
encodes pyrimidodiazepine (PDA) synthase, a
upregulation of DmGSTO1 also restored ATP
key enzyme in the drosopterin biosynthetic
synthase activity, complex assembly, and ATP
pathway (8). In humans, GSTOs are mapped to
levels in parkin mutants. Our findings suggest
the linkage region correlated with the age at
that DmGSTO1 plays a protective role in parkin
onset of Alzheimer’s disease (9). Variations in
mutants by regulating mitochondrial ATP
human GSTO1 genes that modify the age at
synthase activity.
determined.
Human
GSTO1-1
have
activity
of
onset of Alzheimer’s and Parkinson’s diseases
EXPERIMENTAL PROCEDURES
have been reported (10). However, the in vivo function of GSTOs, and its target protein have not yet been fully identified.
Drosophila Stocks
Recently, Pallanck’s group reported that expression
of
Drosophila
GST
sigma
To generate DmGSTO1 mutants, we obtained
1
the GE26508 P-element insertion line from the
2
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omega (GSTOs) have a unique cysteine residue
GSTOs
catalytic
of
dependent
several
the
degeneration
mechanisms (3,4). The active sites of GST
of
on
the
GenExel Drosophila library (KAIST, Korea).
Total protein (20 µg) was separated by 8% or
The P-element was mobilized using transposase,
10% SDS-PAGE and transferred to PVDF
and GE26508 was imprecisely excised to
membranes (Millipore, USA). Membranes were
null
(Fig. S1A). The UAS-
blocked by tris-buffered saline (TBS) with 4%
mitGFP line was a gift from H. J. Bellen
non-fat dry milk or 4% BSA for 1 h. We used
(Baylor
We
the following primary antibodies: rabbit anti-p-
generated four transgenic lines: DmGSTO1A,
JNK (1:1000; Promega, USA), rabbit anti-JNK
generate DmGSTO1 College
of
Medicine)
(17).
C31A
, and CG6662.
(1:1000; Santa Cruz Biotechnology, USA),
The coding sequences for these four genes were
rabbit anti-β-actin (1:5,000; Sigma-Aldrich,
amplified by PCR. The four PCR products were
USA), mouse anti-α-tubulin (1:4,000; Sigma-
ligated into the pUAST expression vector and
Aldrich, USA), mouse anti-β-tubulin (1:3,000;
introduced into the germ line by microinjection.
Sigma-Aldrich, USA), rabbit anti-phospho-
All PCR products were confirmed by DNA
eIF2α
sequencing. Drosophila stocks were maintained
Technology, USA), rabbit anti-eIF2α (1:200;
on standard food conditions at 25℃. The parkin
Abcam, USA), rabbit anti-HSP60 (1:1,000;
DmGSTO1B, DmGSTO1A
B9
(1:1,000;
Cell
Signaling
were gifts
Stressgen Bioreagents, Belgium), mouse anti-
from J. Chung (Seoul National University)
HSP/HSC70 (1:1,000; Stressgen Bioreagents,
(18,19). The TH-Gal4 line was a gift from S.
Belgium), mouse anti-ATP synthase α subunit
Birman (CNRS-Université de la Méditerranée)
(1:20,000; Mitosciences, USA), rabbit anti-
(20). The Tub-Gal4 fly line and the 24B-Gal4
Prohibitin (1:500; Abcam, USA), and rabbit
line were obtained from the Bloomington Stock
anti-Drosophila
Center. We obtained the ATP synthase β subunit
(1:20,000; a kind gift from Rafael Garesse,
RNAi lines, CG11154R-1, and R-3, from the
Universidad Autonoma de Madrid). Detection
NIG-FLY stock center (National Institute of
of the primary antibodies was carried out with
Genetics, Japan).
HRP-conjugated secondary antibodies and an
Exposure to Paraquat
ECL-Plus detection kit (Amersham Biosciences,
null mutant line, park , and PINK1
Sweden).
One- to two-day-old male flies were starved
ATP
Polyclonal GSTO1A
synthase
β
antibodies were
subunit
against
for 5 h and then kept in vials with 3M filter
Drosophila
produced
by
paper soaked with 20 mM paraquat (Methyl
immunizing rabbits with a C-terminal synthetic
Viologen, Sigma-Aldrich, USA) in 5% sucrose.
peptide, 231-EFQKSKTLGNPQY-243, as the
Flies were kept in the dark all times.
antigen (Abfrontier, Korea).
Immunoblot Analysis
Muscle Histology Muscle section analysis was carried out as
Protein extracts for immunoblot analysis were prepared by homogenizing 10 fly thoraces from
previously
3-day-old male flies in lysis buffer (50 mM Tris-
modifications. For muscle tissue analyses,
HCl, pH 7.5; 150 mM NaCl; and 0.5% NP-40)
whole thoraces from 3-day-old male fly were
containing a protease inhibitor cocktail (1X;
fixed with 4% formaldehyde overnight at 4℃.
Calbiochem-Merck4Biosciences,
After fixation, the samples were oxidized with
Germany).
3
described
(21)
with
some
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1
Ser51
1% OsO4 for 2 h at room temperature and then
reations were performed using Ex Taq (Takara
dehydrated in a series of acetone:water mixtures
Bio, Japan) and Taq DNA polymerase (Bioneer,
of increasing acetone concentration (50, 70, 80,
Korea) with a PTC-100 Programmable Thermal
90, and 100% acetone). The samples were
Controller (MJ Research, USA). We used the
embedded in Spurr’s resin. The thoraces were
previously reported Drosophila GSTO primers
then trimmed and sectioned from the transverse
for
side of the thorax. The sections were stained
DmGSTO1B (8). The following dparkin, and
with a toluidine blue dye and observed by light
dPINK1 primers were used: dparkin-For (CAT
microscopy (Carl Zeiss, Axio Imager A1).
ATG AGT TTT ATT TTT AAA TTT ATT
Immunohistochemistry and TUNEL Assay
GCC ACT TTT GTA C), dparkin-Rev (CTC
CG6776,
CG6662,
DmGSTO1A,
and
GAG TTA GCC GAA CCA GTG GG),
old, 5-day-old and 20 day-old male flies were
dPINK1-For (TTC TGC CAC CAC CGC CCC
fixed with 4% formaldehyde in a fixative buffer
CAC ACT TC), and dPINK1-Rev (CCG CAG
(100 mM PIPES, 1 mM EGTA, 1% Triton X-
CAC ATT GGC AGC GGT GG).
100, and 2 mM MgSO4; pH 6.9) and blocked in
The comparative cycle threshold (Ct) method
a wash buffer (50 mM Tris, 150 mM NaCl,
was adapted to estimate transcript levels using
0.1% Triton X-100, and 0.5 mg/ml BSA; pH
an ABI7300 system (Applied Biosystems, USA).
6.8) with 10 mg/ml BSA. The following
The transcript levels were calculated as the
antibodies were used: rabbit anti-TH (1:100;
relative fold-change over rp49 mRNA. We used
Pel-freeze, USA), mouse anti-TH (1:100;
the following primers for α-tubulin, and β-
Immunostar, USA), rabbit anti-phospho-JNK
tubulin: α-tubulin-For (ACA ACG AGG CTA
(1:100; Promega, USA), mouse anti-GFP
TCT ACG ACA TCT), α-tubulin-Rev (TTT
(1:500; Roche, Switzerland), and mouse anti-α-
TCA GTG TTG CAG TGA ATT TTT) (22), β-
tubulin (1:500; Sigma-Aldrich, USA). Alexa
tubulin-For (CAA GGC TTC CAA CTC ACA
488-conjugated streptavidin (1:100; Invitrogen,
CAC TC), and β-tubulin-Rev (AGG TGG CGG
USA) was used to identify mitochondria.
ACA TCT TCA GAC) (23).
Rhodamine phalloidin (Invitrogen, USA) was
Site-Directed Mutagenesis and Expression of
used to visualize actin. All images were
Mutant Proteins
obtained on a Carl Zeiss confocal microscope
The DmGSTO1AC31A mutant was generated by
(DE/LSM510 NLO). For the TUNEL assay,
site-directed mutagenesis (Cosmo Genetech,
apoptosis in the IFMs of 3-day-old flies was
Korea). Cysteine 31 at the active site was
detected using the In Situ Cell Death Detection
mutated to alanine by changing the TGC codon
Kit, Fluorescein (Roche, Switzerland).
encoding cysteine 31 to GCC. Mutated DNA
Quantitative RT-PCR and real-time qRT-PCR
was sequenced to confirm the single codon
Total RNA was extracted with a Trizol reagent
change. The DmGSTO1AC31A mutant was
(Invitrogen, USA), and cDNA was prepared
expressed in Escherichia coli strain BL21 after
from 2 µg of total RNA using M-MLV reverse
cloning the cDNA into a pET15b expression
transcriptase
vector (Novagen-Merck4Biosciences, Germany).
(Promega,
USA).
The
PCR
4
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Adult brains and thoraces of 1-day-old, 3-day-
conjugated secondary antibodies.
In Vitro Glutathionylation Assay Recombinant ATP synthase β subunit (5 µg)
Mitochondrial ATP Synthase Activity Assay
was incubated at 37℃ in 50 mM potassium
To isolate mitochondria from fly thoraces, a
phosphate (pH 7.6), and 10 mM GSH in the
mitchondria isolation kit (PIERCE, USA) was
presence of either recombinant DmGSTO1A or
used according to the manufacturer’s protocol.
DmGSTO1B protein. After 30 min, the samples
Freshly prepared total mitochondrial protein
were placed on ice, and 5X non-reducing SDS
was used for the ATP synthase activity assay.
loading buffer was added to the mixtures.
ATP synthase activity was measured by ATP
Samples were separated by 12% SDS-PAGE,
hydrolysis using a spectrophotometric method
transferred and probed with mouse anti-GSH
described previously (24). ATP synthase was
(1:1,000; Virogen, USA), and rabbit anti-
assayed in 20 mM Hepes (pH 7.5), 5 mM KCl,
Drosophila ATP synthase β subunit (1:4,000)
5
antibodies.
phosphoenolpyruvate (Sigma-Aldrich, USA), 15
Immunoprecipitation and Glutathionylation
units pyruvate kinase (Sigma-Aldrich, USA), 15
assay
units lactate dehydrogenase (Sigma-Aldrich,
mM
MgCl2,
5
mM
KCN,
2.5
mM
USA), 300 µM NADH (Sigma-Aldrich, USA),
homogenized in lysis buffer containing 1X
and 20 µg of total mitochondrial proteins. After
protease
(Calbiochem-
2 min of pre-incubation at 37℃, the reaction
Merck4Biosciences, Germany). Thorax lysates
was initiated by the addition of 2 mM ATP. The
were incubated with mouse anti-GSH antibodies
initial velocity of the reaction was followed for
(Virogen, USA) for 2 h at 4℃ and incubated
2 min at 340 nm at 37℃. The molar extinction
overnight with 60 µl of a solution of 50%
coefficient of NADH is 6220 M-1.
protein
ATP Assay
inhibitor
cocktail
G-Sepharose
beads
(Amersham
Biosciences, Sweden) at 4℃. The resins were
Five thoraces from 3-day-old male flies were
collected by centrifugation at 1,000 x g for 20 s.
homogenized in 100 µl 1X Reporter lysis buffer
Bound proteins, which were glutathionylated,
(Promega, USA) on ice. The homogenized
were eluted by boiling in a 2X non-reducing
samples were quickly frozen in liquid nitrogen
SDS loading buffer for 5 min. Materials were
to inhibit ATP synthase activity. The frozen
subjected to SDS-PAGE and visualized with
samples were boiled for 8 min to destroy
Coomassie blue or silver staining.
endogenous ATP synthase and then centrifuged
After immunoprecipitation with mouse anti-
at 20,000 x g for 15 min. ATP was quantified in
GSH antibodies (Virogen, USA), proteins bound
the supernatant using the ATP bioluminescent
to resins were separated by 8% SDS-PAGE and
assay kit (Sigma-Aldrich, USA) according to
transferred to PVDF membranes (Millipore,
the manufacturer’s protocol.
USA) for the glutathionylation assay. Western
Blue Native Electrophoresis (BN-PAGE)
blot analysis was carried out with rabbit anti-
Mitochondria were isolated from 3-day-old
Drosophila ATP synthase β subunit antibodies
male fly thoraces using the mitochondrial
(1:4,000). Signals were detected by HRP-
isolation kit (PIERCE, USA). Mitochondrial
5
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Thoraces from 3-day-old male flies were
in
mutant was viable and fertile, and it exhibited
sample buffer (Invitrogen, USA),
no obvious defects in adult morphology. In the
1% digitonin (Invitrogen, USA), and 2%
DmGSTO1 null mutant fly, there were no
dodecylmaltoside (Invitrogen, USA). Samples
detectable levels of either DmGSTO1A or
were incubated for 15 min on ice and
DmGSTO1B transcripts (Fig. S1B). GSTO
centrifuged at 12,000 x g for 25 min. BN-PAGE
enzymes exhibit higher glutathione-dependent
proteins
(10
NativePAGE
TM
ug)
were
dissolved
was performed on NativePAGE
TM
thiol
Novex 3-12%
transferase,
and
dehydroascorbate
reductase (DHAR) activity than any other class
Bis-Tris Gels (Invitrogen, USA).
of GSTs (27). We have shown that DmGSTO1B
RESULTS
has
much
higher
DHAR
activity
than
DmGSTO1A in vitro (8). The DHAR activity in DmGSTO1null flies was dramatically decreased
stress. We previously reported that there are
to approximately 5% of the DHAR activity
four different GSTO genes in Drosophila
found in wild type flies (Fig. S1C). Furthermore,
melanogaster:
DmGSTO1null
CG6781
(sepia),
CG6776,
mutants
were
sensitive
to
CG6673, and CG6662. CG6781 is the structural
paraquat, an oxidative stress inducer (Fig. 1A).
gene for sepia, which encodes PDA synthase
Using a ubiquitous driver, Tub-Gal4, we
and is expressed exclusively in Drosophila
directed
heads
(8).
CG6673
is
also
called
D.
expression
DmGSTO1B
in
the
of
DmGSTO1A
DmGSTO1
null
or
mutant
melanogaster GSTO1 (DmGSTO1), and has the
background. The paraquat sensitivity was
highest thiol transferase and DHA reductase
rescued by expression of DmGSTO1B, which
activity among the GSTO genes, reminiscent of
exhibited more oxidative stress protection than
thioredoxin and glutaredoxin (8). CG6776
DmGSTO1A (Fig. 1A). These results suggest
responds to heat stress in Drosophila (25).
that the paraquat-sensitive phenotype in the
Although the function of CG6662 is not known,
DmGSTO1null mutant is primarily due to the loss
CG6662 transcripts are primarily expressed in
of DmGSTO1B function. Because DHAR
the ovary and testes (26). Thus, we focused our
catalyzes the conversion of dehydroascorbate
study on CG6673, DmGSTO1, and excluded
(DHA) to ascorbate (AsA) using glutathione as
other Drosophila GSTO genes.
a reducing agent, we investigated the level of
We generated both loss-of-function and gain-
DHA and AsA in the mutant flies. As shown in
of-function DmGSTO1 mutant flies. Because
Fig.
DmGSTO1 encodes two alternatively spliced
DmGSTO1B rescued the AsA/DHA ratio in
transcripts,
both
DmGSTO1null mutants. These results indicate
DmGSTO1A- and DmGSTO1B-overexpressing
that DmGSTO1B has a protective role in
null
response to paraquat-induced stress and plays an
was generated by imprecise P-element excision,
important role in the in vivo conversion of DHA
which resulted in partial deletion of the
to AsA.
A and
B,
we
created
fly lines. Loss-of-function mutant DmGSTO1
DmGSTO1 gene (Fig. S1A). The DmGSTO1
null
1B,
only
the
overexpression
of
DmGSTO1 partially rescues park1 mutant
6
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DmGSTO1 mutants are sensitive to oxidative
phenotypes. Although it has been reported that
by demonstrating that CG6662 overexpression
GSTs are involved in neurodegenerative disease
in park1 mutants had no effect on the park1
(13,28,29), the molecular function of GSTOs
mutant phenotype (Fig. 2A). These results
remains unknown. To investigate the biological
indicate that CG6662 is not involved in the
function of DmGSTO1 in PD, we conducted
suppression of parkin mutant phenotypes.
genetic
studies
1
with
park
As
The downturned wing, and thorax disruption
mutants
phenotypes of parkin mutant flies are caused by
showed collapsed thorax, and downturned wing
the degeneration of the indirect flight muscles
phenotypes.
(IFMs) (21,30). Therefore, we investigated
mutants. 1
previously reported (18,19), park Surprisingly,
increasing
whether
DmGSTO1A expression using the muscle-
DmGSTO1
prevented
muscle
1
significantly
degeneration in park mutants. As determined
suppressed both thorax and wing phenotypes in
by histological analysis of thoracic IFMs, the
specific
24B-Gal4
driver
1
integrity of IFMs in dorsal longitudinal muscles
expression showed no effects (Fig. 2A, Figs.
(DLMs), which regulate adult wing posture was
S2A and B). Of the GSTO genes, only the
clearly disrupted in park1 mutants (Fig. 2E).
transcriptional
DmGSTO1,
Although DmGSTO1null mutants showed a
(CG6673) was decreased in park1 mutants (Figs.
normal muscle phenotype, park1/DmGSTO1null
2B and S1D). Because the level of DmGSTO1
double mutants showed dramatically enhanced
expression
of
1
mRNA decreased in park mutants (Fig. 2B), we
degeneration of IFMs compared to park1 single
investigated whether the protein levels were
mutants (Fig. 2E). Furthermore, overexpression
1
also decreased in park mutants. We generated a
of DmGSTO1A using a ubiquitous driver, Tub-
specific antibody against DmGSTO1A. As
Gal4, suppressed the degeneration of IFMs in
shown in Fig. 2C, the DmGSTO1A protein level
park1 mutants. Overexpression of DmGSTO1A
was dramatically decreased in park1 mutants
in park1 mutants resulted in regular and compact
compared to wild type flies. These results
muscle tissues in the dorsal longitudinal IFMs,
indicate that parkin regulates transcriptional
which were similar to those of wild type flies
expression of the DmGSTO1 gene. We further
except for occasional vacuoles (Fig. 2E). These
examined the genetic interactions between
data
parkin and DmGSTO1 by introducing a
partially rescues the morphological defects, and
1
suggest
that
DmGSTO1
expression
muscle degeneration in park1 mutants.
DmGSTO1 null mutation in the park mutants. We found that the loss of function of DmGSTO1
Phospho-JNK
signal
and
apoptosis
are
1
further enhanced the downturned wing and
suppressed by DmGSTO1 in park mutants.
collapsed thorax phenotypes in the one-day-old
Many studies have suggested that neuronal cell
1
null
death in various neurodegenerative diseases is
CG6662
closely related to JNK activation. Cha et al.
transcripts were detected (Fig. S1B). We could
(2005) reported that parkin inhibits the JNK
eliminate the possibility that CG6662 played a
pathway (18). As shown in Fig. 2F, phospho-
park mutants (Fig. 2D). In the DmGSTO1 mutant,
neither
DmGSTO1
nor
1
JNK was dramatically increased in IFMs of the
role in suppressing the park mutant phenotype
7
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park mutants, whereas increased DmGSTO1B
park1 mutant, whereas there was no change in phospho-JNK in the DmGSTO1
null
in
neuronal 1
park /DmGSTO1
mutant
degeneration null
between
double mutants and park1
compared to wild type. The expression of
single mutants in 1-day-old flies (Fig. 2J).
DmGSTO1A suppressed activation of phospho-
These results indicate that DmGSTO1 protects
1
JNK in park mutants (Fig. 2F), and the degree
the
DA
neurons
from
age-dependent
1
degeneration in park mutants.
of suppression was confirmed by western blot
DmGSTO1 restores the accumulation of
parkin mutants occurs through an apoptotic
tubulin in IFMs in park1 mutants. Parkin
mechanism (30). The IFMs in park1 mutants
functions as an E3 ubiquitin ligase and has
were
terminal
important roles in the degradation of many
dUTP
substrates (32,33). Recent in vitro studies
end labeling (TUNEL) assay. As shown in Fig.
demonstrated that parkin binds to microtubule
1
2H, the increased TUNEL signal in park
and tubulin proteins with high affinity and that
mutants
parkin
examined
by
a
deoxynucleotidyltransferase-mediated
was
suppressed
by
DmGSTO1A
ubiquitinates,
and
promotes
the
expression, similar to the suppression in the
degradation of α/β-tubulin (34). It remains
phospho-JNK signal. These data suggest that
controversial whether putative in vitro substrates
DmGSTO1 prevents degeneration of IFMs by
are relevant in vivo. Therefore, we investigated
blocking the activation of JNK and apoptosis in
the α/β-tubulin protein levels in park1 mutants,
park1 mutants.
in vivo. Levels of α-tubulin were increased in
DmGSTO1 suppresses dopaminergic neuronal
park1 mutant muscles (Fig. 3A). Interestingly,
degeneration in park1 mutants. Park1 mutants
the accumulation of α-tubulin in park1 mutant
show
muscles
an
age-dependent
dopaminergic
neurons,
degeneration especially
in
of
was
dramatically
reduced
by
the
DmGSTO1A expression, and it was enhanced in
protocerebral posterior lateral 1 (PPL1) cluster
park1/DmGSTO1null double mutant muscles (Fig.
(18,31). To further clarify the effect of
3A). The levels of actin filaments in IFMs were
1
DmGSTO1 on park mutants, we examined the
unchanged in all mutants (Fig. 3B). These
degeneration of dopaminergic neurons that is
changes were confirmed by western blot
characteristic of PD. As shown in Fig. 2I,
analysis (Figs. 3C and D). We found that tubulin
DmGSTO1A expression under the control of a
was slightly increased in DmGSTO1null mutants
dopaminergic
by western blot analysis (Fig. 3D). We also
hydroxylase
neuron-specific (TH)-Gal4
driver
tyrosine resulted
in
detected tubulin accumulation in dopaminergic
significant restoration of the lost dopaminergic
neurons
1
1
neurons in 20-day-old park mutant flies. In 1
addition, 20-day-old park /DmGSTO1
null
park1
in
park /DmGSTO1
null
mutants
and
double mutants (Fig. S3).
double
These data indicate that parkin does not directly
increased
regulate the level of tubulin in Drosophila in
dopaminergic neuron degeneration in the PPL1
vivo. In contrast to the change in total protein
mutants
showed
significantly 1
cluster compared to the park single mutants of
levels (Fig. 3C), there were no detectable
the same age, whereas there was no difference
changes in the α/β-tubulin transcript levels in
8
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analysis (Fig. 2G). Degeneration of IFMs in
park1
DmGSTO1A
DmGSTO1A was critical for the rescue the
expression level (Fig. 3E). Thus, the increased
park1 mutant phenotypes. GSTOs have a unique
α/β-tubulin levels were not caused by increased
cysteine at their active site that binds to GSH
transcription, but by protein accumulation.
(27). We constructed a catalytically inactive
These results indicate that parkin is required for
form of DmGSTO1A, DmGSTO1AC31A, in
the regulation of tubulin levels and that
which cysteine 31 was mutated to alanine. In
DmGSTO1 suppresses the accumulation of
contrast to wild type DmGSTO1A, expression
mutants
regardless
of
1
of DmGSTO1AC31A in muscle did not rescue the
tubulin in park mutants.
collapsed
DmGSTO1 suppresses activation of the UPR in 1
park
thorax,
phenotypes
mutant muscles. Many studies have
claimed that ER stress is involved in the
Moreover,
in
and 1
park
tubulin
downturned mutants
accumulation C31A
progression of neurodegenerative diseases such
(Fig.
suppressed by DmGSTO1A
was
wing 4A). not
expression in
1
park mutants (Fig. 4B). Our data demonstrate
a signaling pathway that is activated in response
that DmGSTO1 catalytic activity is required to
to ER stress. UPR activation has been observed
rescue the defective phenotypes of park1
in DA neurons of PD patients and is exemplified
mutants.
by an increase in phospho-PERK and eIF2α
Although the ability of DmGSTO1B to
(35). To determine whether the parkin mutation
respond to oxidative stress was higher than that
induces UPR activation, we examined eIF2α,
of
which is one of the components of the UPR
DmGSTO1A
signaling pathway and is activated by the
phenotypes in parkin mutants (Figs. 1A and 2A).
upstream kinase PERK (36). The level of active
Therefore, DmGSTO1A may suppress parkin
1
phospho-eIF2α was highly increased in park
mutant phenotypes by other mechanisms. The
mutants
by
catalytic detoxification functions of the GST
DmGSTO1A expression (Fig. 3F). The ER and
family have been studied by several research
mitochondrial UPR share similar pathways that
groups. However, some members of the GST
increase chaperone levels to promote protein
family have physiological functions unrelated to
homeostasis in the cytoplasm and mitochondria
detoxification (38). Previous reports revealed
(37). We also examined heat shock proteins
that the rate of protein glutathionylation, a post-
(HSPs), including Hsp60, and Hsp/Hsc70. As
translational modification that regulates the
shown in Fig. 3G, the levels of HSPs were
function of proteins, is enhanced by the
and
dramatically
restored
1
DmGSTO1A,
only
suppressed
upregulation the
of
defective
increased in park mutants and reduced by
presence of active GSTP (GSTpi family) (39).
DmGSTO1A expression. These data indicate
Based on the result that DmGSTO1 enzyme
that DmGSTO1 suppresses UPR activation in
activity is required to suppress the parkin
1
mutant phenotypes, we screened in vivo targets
park mutants. ATP
of DmGSTO1 by immnunoprecipitation with an
synthase activity in park mutants. We next
anti-GSH antibody in adult thorax extracts.
examined whether the catalytic activity of
Only one glutathionylated protein showed
DmGSTO1
restores
mitochondrial 1
9
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as PD. The unfolded protein response (UPR) is
altered
abundance
park1
in
Glutathionylation
mutants.
is
a
reversible
DmGSTO1A expression increased the level of
posttranslational modification that can lead to
glutathionylation
alteration of protein or enzyme function, such as
DmGSTO1A
of
C31A
this protein,
whereas
Ca2+-uptake
the
did not. The protein was
activity
of
the
identified by MALDI-TOF mass spectrometric
sarco/endoplasmic reticulum calcium ATPase
analysis
(40).
as
the
mitochondrial
F1F0-ATP
To
test
the
hypothesis
that
synthase β subunit. We also confirmed that
glutathionylation of the ATP synthase β subunit
DmGSTO1A can directly glutathionylate the
by DmGSTO1A in park1 mutants regulates ATP
ATP synthase β subunit in a dose-dependent
synthase activity, we isolated mitochondria from
manner in the presence of GSH (Fig. 5A).
the thorax and measured F1F0-ATP synthase
Furthermore, the endogenous levels of the
activity. As shown in Fig. 5C, the level of
glutathionylated form of the ATP synthase β
mitochondrial F1F0-ATP synthase activity was
subunit in thorax extracts were decreased in
partially rescued by DmGSTO1A expression in park1
park mutants and decreased even more in 1
park /DmGSTO1
null
mutants,
1
park /DmGSTO1
double mutants, whereas the
and null
was
decreased
in
double mutants. Indeed, the
total expression levels of the ATP synthase β
change in mitochondrial F1F0-ATP synthase
subunit were unchanged in all fly lines.
activity was correlated to the change in
DmGSTO1A expression
1
in
park
mutants
glutathionylation of the ATP synthase β subunit.
restored the levels of glutathionylated ATP
Consistent with the ATP synthase activity results,
synthase β subunit to wild type levels, whereas
we observed a change in the total ATP levels in
C31A
or
all the mutant lines (Fig. 5C). Impaired
DmGSTO1B in park mutants had no effect on
mitochondrial respiration caused by decreased
the
ATP
the
expression
of
DmGSTO1A
1
level
of
ATP
synthase
β
subunit
has
been
reported
in
Parkinson’s disease, and agents that improve
glutathionylation (Fig. 5B). However, in the null
production
of
mitochondrial respiration can exert beneficial
glutathionylation of the ATP synthase β subunit
effects in animal models of PD (2). These
was only slightly decreased compared to control.
reports are consistent with our findings: ATP
This finding suggests that a compensatory
synthase
DmGSTO1
mutant,
the
level
activity,
and
ATP
levels
are
1
mechanism related to glutathionylation of the
significantly decreased in park mutants and can
ATP synthase
be modulated by DmGSTO1 expression.
DmGSTO,
β
subunit
CG6662,
did
exists. not
Another affect
Decreased
ATP
synthase
β
subunit
in
1
glutathionylation of the ATP synthase β subunit
Drosophila muscle leads to a park mutant-like
(Fig. S4). These data indicate that the ATP
phenotype. Mutations of ATP synthase subunits
synthase β subunit is a novel and specific target
are known to exhibit mitochondrial dysfunction
of DmGSTO1A in Drosophila and that only
and neuromuscular impairment (41,42). To
1
DmGSTO1A expression in park mutants is
determine whether decreased ATP synthase
sufficient to partially restore glutathionylation
activity was directly linked to the phenotypes
of the ATP synthase β subunit.
found in parkin mutants, we downregulated the
10
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1
Blue Native (BN)-PAGE, and western blot
muscle using a UAS-RNAi line (obtained from
analysis with an anti-ATPsyn α subunit antibody.
the NIG-FLY stock center) together with the
Three bands (super complex, > 800 kDa;
muscle-specific 24B-Gal4 driver. Because the
assembled ATP synthase, Complex V, > 600
ATP synthase β subunit knockdown flies reared
kDa; F1 subcomplex, > 400 kDa) were detected
at 25℃ displayed pupal lethality, we performed
(Fig. 6). All three bands associated with the
all knockdown experiments at 18℃ to increase
mitochondrial F1F0-ATP synthase complex
adult viability. When ATP synthase β subunit
were dramatically decreased by RNAi knock
knockdown was induced at 18℃ in muscle
down of the ATP synthase β subunit (Fig. S5).
using 24B-Gal4, fewer than 20% of the flies
The amount of assembled ATP synthase was
were able to eclose. We observed abnormal
normalized using mitochondrial prohibitin. In
muscle structure in the knockdown mutant flies,
comparison to the wild type, the amount of
similar to the phenotypes found in parkin
assembled ATP synthase complex was decreased
mutants (Fig. 5D). Moreover, the ATP synthase
in park1 mutants. Expression of DmGSTO1A in
β subunit RNAi flies showed accumulation of
park1
total α/β-tubulin (Fig. 5E). These results
assembled ATP synthase complex (Fig. 6A,
demonstrate that loss of the ATP synthase β
Complex V). Interestingly, park1/DmGSTO1null
subunit induces some of the parkin mutant
double mutants tend to exhibit lower amounts of
phenotypes, including muscle degeneration, and
assembled ATP synthase than those in park1
accumulation of tubulin.
mutants, although the effect was not statistically
mutants
increased
the
amount
of
Next, to examine whether the ATP synthase β
significant (Fig. 6B). BN-PAGE analysis is not
subunit RNAi enhances the parkin mutant
sensitive to small changes; nevertheless, the
phenotype, the ATP synthase β subunit was
change in the amount of assembled ATP
1
knocked down in park mutant muscles. As
synthase complex correlated to the change in
shown in Fig. 5F, we observed an increase in
ATP synthase activity. These results indicate
α/β-tubulin accumulation. Moreover, the levels
that DmGSTO1A affects mitochondrial ATP
of Hsp70/Hsc70, and phosphorylated eIF2α in
synthase activity by regulating the assembly
1
efficiency of ATP synthase in park1 mutants.
the park mutant were further increased by RNAi knock down of the ATP synthase β
DISCUSSION
subunit (Fig. 5G). These data indicate that downregulation of ATP synthase activity in the park1 mutant results in a more severe phenotype
In this study, we suggest that DmGSTO1 is a
1
compared to that of park single mutants.
novel genetic suppressor of parkin dysfunction
DmGSTO1 rescues mitochondrial ATP synthase
and has a protective role in a model of PD.
1
assembly in park mutants. To investigate how
Moreover, we showed that the ATP synthase β
expression of DmGSTO1A affects ATP synthase
subunit is a novel target of DmGSTO1 in
activity, we examined the assembly level of the
Drosophila. The ATP synthase β subunit is an
F1F0-ATP synthase complex (Complex V) by
essential catalytic core component of the F1F0-
11
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ATP synthase β subunit in Drosophila thorax
ATP synthase complex in mitochondria. We
mechanism between glutathionylation of the
found that levels of glutathionylation of the ATP
ATP synthase β subunit and ATP synthase
synthase β subunit were significantly decreased
activity is not known. It is technically difficult
1
in park mutants, whereas its total protein level
to
remained unchanged. Glutathionylation is a
glutathionylation of the ATP synthase β subunit
mechanism of post-translational regulation of
and
several proteins, including protein tyrosine
Drosophila, mitochondrial ATP synthase is a
phosphatase 1B (PTP1B), and MEKK1. The
large multi-protein complex composed of eight
glutathionylation
channel,
different subunits. Further studies will be
ryanodine receptor 1 (RyR1), activates it and
required to determine how glutathionylation of
enhances calcium release (43). Our results
the
of
a
calcium
1
show
a
ATP
direct
synthase
ATP
synthase
relationship activity
β
mitochondrial
mutants increases the level of glutathionylated
assembly at the molecular level.
synthase
mitochondrial thereby
β
subunit
and
F1F0-ATP synthase
partially
rescuing
park
However,
restores 1
DmGSTO1
activity,
ATP synthase the
null
exhibited
regulates
activity,
loss-of-function no
in
and
mutant obvious
mutant
morphological defects and slightly reduced
phenotypes, including the degeneration of
glutathionylation of the ATP synthase β subunit,
dopaminergic neurons.
and ATP synthase activity. It is not clear why
The increase in F1F0-ATP synthase activity
DmGSTO1null single mutants show a weak effect
and assembly by DmGSTO1A in the park1
on the glutathionylation, and activity of ATP
mutant led to a recovery of ATP depletion.
synthase. One possible explanation is that
Interestingly, because mitochondrial F1F0-ATP
compensatory
synthase
glutathionylation of the ATP synthase β subunit
has
a
role
in
maintaining
mechanisms
related
to
exist in vivo.
innermembrane morphology, and mitochondrial
Whereas our current study focused on the
membrane potential (41,44) and mutation of F1F0-ATP synthase ε subunit can cause
specific
mitochondrial dysfunction (42), the restoration
neurodegeneration, previous studies have shown
1
of F1F0-ATP synthase activity in the park
that GSTs have protective functions against
mutant is important for rescuing the parkin
oxidative stress in neurodegenerative diseases.
mutant phenotypes. Moreover, RNAi mutants of
Loss-of-function of the yeast GSTSigma1
the ATP synthase β subunit exhibit phenotypes
homolog, gtt-1, enhances α-synuclein toxicity
1
target
of
GSTO
related
to
similar to park mutants, including α/β-tubulin
(45), and mouse GSTpi contributes to the
accumulation, locomotor dysfunction, UPR
sensitivity to xenobiotics in idiopathic PD (29).
activation, and muscle degeneration.
Additionally, protected
Although the change in mitochondrial F1F0-
increased cells
from
GSTpi
expression
rotenone-induced
ATP synthase activity, and ATP levels correlated
neurotoxicity (46). However, the various roles
with the degree of glutathionylation of the ATP
of GSTs remain controversial because of the
synthase β subunit, the exact regulatory
diversity and complexity of these proteins.
12
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ATP
because
subunit
suggest that expression of DmGSTO1A in park
between
Because the expression levels of several GSTs
individual
GSTs
remain
unknown,
the
increase due to oxidative stress, we measured
functional diversity of GSTs might explain why
1
endogenous DmGSTO1A expression in park
they have different substrates against various
mutants and found that the protein and mRNA
stresses in vivo.
significantly
DmGSTO1B more strongly protects against
mutants. Transcriptional
oxidative stress than DmGSTO1A, but only the
profiling of parkin mutants showed that the
upregulation of DmGSTO1A improved the
oxidative stress response genes are upregulated
defective
and overexpression of GSTS1 in dopaminergic
Although DHAR activity and reduced form of
neurons suppressed neurodegeneration in parkin
ascorbate have protective roles under oxidative
mutants
also
stress conditions (50), they are not sufficient to
upregulated by the 4E-BP1-mediated stress
rescue parkin mutant phenotypes. As shown in
1
response (47). Therefore, DmGSTS1 in park
Fig. 5A and B, DmGSTO1B was not able to
mutants may be increased as an effort to
glutathionylate the ATP synthase β subunit.
decrease the stress induced by the parkin
Thus, we propose that these two isoforms,
mutation, but its increased level is not sufficient
DmGSTO1A and DmGSTO1B, have different
levels
of
DmGSTO1A were 1
lowered in park
(11,12).
DmGSTS1
was
in
parkin
mutants.
to rescue the park phenotype. Both DmGSTO1
substrates and act differently on the stress
mRNA and protein levels were decreased in
response pathway in vivo.
1
mutants, suggesting that the normal
Mitochondrial defects have been detected in
function of parkin is critical for the regulation of
PD causes with parkin mutations. Recent
DmGSTO1 expression. Little is known about
studies
the
mitochondrial
park
factors
regulating
DmGSTO1
gene
suggest
that fission
parkin
promotes
and/or
inhibits
expression, but we can make the following
mitochondrial fusion in muscle tissues and DA
speculation. Because the DmGSTO1 gene
neurons (51-53). Staining mitochondria with
contains a potential NF-κB-like transcription
streptavidin
factor binding motif (48), and parkin stimulates
mitochondrial integrity in the IFMs of park1
NF-κB-dependent
mutants. Interestingly, these defects were not
transcription
(49), 1
DmGSTO1 may be downregulated in the park
restored
by
revealed
park /DmGSTO1
1
defects
DmGSTO1A expression,
1
mutant. Thus, the mechanism underscoring the
severe
null
in
and
double mutants were not
protective role of DmGSTO1 in park mutants
distinguishable from park1 single mutants (Fig.
is distinct from the mechanisms used by general
S6). These findings were further confirmed by
antioxidants, and the detoxifying enzyme
expressing mito-GFP in thorax muscles (Fig.
DmGSTS1. Furthermore, GSTO1 has an active-
S6). In contrast to the park1 mutants, the ATP
site cysteine residue, which is distinct from the
synthase β subunit RNAi mutants did not show
tyrosine residue found in GSTS1. This cysteine
disrupted mitochondrial morphology (Fig. S6).
residue could enable GSTO1 to modulate the
These results suggest that DmGSTO1 is not
disulfide status of cysteine residues on substrate
important
for
restoring 1
proteins (27). Although the specific substrate of
mitochondrial
morphology in park mutants, and might act
13
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1
phenotypes
downstream or in parallel to the mitochondrial
DmGSTO1
is
linked
to
the
pathogenic
dynamics pathway.
phenotypes displayed in parkin mutants. We
PTEN-induced putative kinase 1 (PINK1) is a
found that DmGSTO1 is a novel genetic
Ser/Thr kinase containing a mitochondrial
suppressor of parkin dysfunction. The two
targeting motif (54). Previous studies reported
isoforms of DmGSTO1 have different functions.
that mutations of PINK1 lead to muscle
DmGSTO1B in Drosophila is required to
degeneration,
and
protect the flies against oxidative stress.
mitochondrial dysfunction. PINK1 and Parkin
Although the exact molecular mechanism is not
are linked in the same pathway and PINK1 acts
clear, glutathionylation of the ATP synthase β
upstream of Parkin (19,51-53,55). We therefore
subunit by DmGSTO1A regulates mitochondrial
hypothesized
would
F1F0-ATP synthase activity, and the restoration
genetically interact with the PINK1 null mutant
of ATP synthase activity by DmGSTO1A
DA
neuron
that
loss,
DmGSTO1A
B9
PINK1 . Interestingly, consistent with park
expression is critically important for partial
mutants, we found that DmGSTO1 and Parkin
rescue of the mitochondrial function in park1
mRNA were decreased in PINK1B9 mutants (Fig.
mutants. These findings present a novel
S7A). Glutathionylation of the mitochondrial
mechanism of regulation of ATP synthase by
ATP synthase β subunit was significantly
DmGSTO1 in parkin mutants. Our results
B9
mutants (Fig. S7B).
strongly suggest that promoting DmGSTO1
Therefore, it seems possible that DmGSTO1A
activity could alleviate neurodegeneration in
upregulation
parkin mutants. These findings will lead us to a
decreased in PINK1 may
also
contribute
prevention of degeneration in PINK1 A detailed study of PINK1
B9
B9
to
the
better
mutants.
understanding
of
the
molecular
mechanism of neuroprotection due to GSTO in
mutants is
PD, and could help in developing new
currently underway in our group.
therapeutic approaches for PD.
Our results support the hypothesis that
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FOOTNOTES †Present address. Dept. of Neurology, Washington University School of Medicine, St. Louis, United States of America We would like to thank Drs. J. Chung, S. Birman, H. J. Bellen, the Bloomingtion Stock Center, the KAIST GenExel Drosophila library, and the NIG-FLY Stock Center for Drosophila stocks. We also thank Dr. Rafael Garesse for providing us with the rabbit anti-Drosophila ATP synthase β subunit antibody. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (MEST) (KRF-2008-313-E00068) and by the Brain Korea 21 Research Fellowships from the Ministry of Education, Science and Technology of Korea. The abbreviations used are: GSTO, glutathione S-transferase omega; GSTS, GST sigma; PD, Parkinson’s disease; DmGSTO1, Drosophila melanogaster GSTO1; PDA, pyrimidodiazepine; UAS, upstream activation sequence; DHA, dehydroascorbate; DHAR, DHA reductase; AsA, ascorbate; IFMs, indirect flight muscles; GSH, glutathione; RT-PCR, polymerase chain reaction after reverse transcription of RNA; DA, dopaminergic; TH-Gal4, tyrosine hydroxylase promoter-dependent Gal4 transgene; UPR, unfolded protein response; GSTP , GST pi
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FIGURE LEGENDS FIGURE 1. Sensitivity of DmGSTO1 mutants to oxidative stress. A, Survival rates of paraquattreated (20 mM) flies overexpressing DmGSTO1A, and DmGSTO1B under the control of TubulinGal4, a ubiquitously expressed driver (n ≥ 80). DmGSTO1null mutants were sensitive to oxidative stress. Overexpression of DmGSTO1B in a DmGSTO1null mutant background reduced sensitivity to treatment with paraquat. Error bars indicate standard deviation. The significance was determined by one-way ANOVA (** is P < 0.05, and * is P < 0.01). B, Ascorbic acid, and dehydroascorbic acid content in fly extracts. The level of AsA was lower in DmGSTO1null flies than in WT flies, whereas DHA was higher in DmGSTO1null flies than in WT flies. The AsA/DHA ratio decreased from 7.29 in WT to 3.28 in DmGSTO1null. Overexpression of DmGSTO1B in a DmGSTO1null mutant background restored the ratio of AsA/DHA to 6.35. Error bars indicate standard deviation. Experimental were performed in triplicate. FIGURE 2. Upregulation of DmGSTO1 suppresses phenotypes caused by parkin loss of function. A, Statistical analysis of the percentage of collapsed thorax (n > 120) and downturned wing (n > 90) phenotypes in 3-day-old flies. DmGSTO1A overexpression by the 24B-Gal4 muscle-specific driver suppresses the thorax, and wing defects of parkin mutant flies. Experimental significance was determined by one-way ANOVA (** is P < 0.05, and ns, not significant). B, DmGSTO1 mRNA levels determined by RT-PCR were also reduced in park1 mutants. Error bars represent standard deviation. Significance was determined by one-way ANOVA (* is P < 0.01). Experiments were performed in triplicate. C, Endogenous DmGSTO1A levels (black arrow) were dramatically reduced in park1 mutants. Error bars indicate standard deviation. The significance was determined by one-way ANOVA (** is P < 0.0001, and * is P < 0.01). D, Percentage of collapsed thorax (n > 300), and downturned wing (n > 110) phenotypes in 1-day-old park1/DmGSTO1null double mutants. The DmGSTO1 mutation enhanced the thorax, and wing defects exhibited by parkin mutants. Error bars indicate standard deviation. Statistical analysis was carried out with one-way ANOVA (** is P < 0.001, and * is P < 0.05). E, Light microscopy was used to examine indirect flight muscle morphology (red arrow, muscle degeneration). Magnified views of the dorsal longitudinal muscle (X200). Tubulin-Gal4 driven DmGSTO1A expression rescues muscle degeneration of park1 mutant flies. The park1/DmGSTO1null double mutants showed enhanced degeneration of muscles compared to park1 single mutants. F, Muscle-specific expression of DmGSTO1A in park1 mutants reduced phosphorylated JNK. Activated JNK (p-JNK) is visualized in green, phalloidin-labeled muscle tissues in red. G, Western blot analysis of p-JNK. Error bars represent standard deviation. Experimental significance was determined by one-way ANOVA (* is P < 0.05). Experiments were performed in triplicate. H, Merged images of apoptotic cells (TUNEL, green), and nuclei (DAPI, blue).
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significance was determined by one-way ANOVA (** is P < 0.01, and * is P < 0.05). Experiments
Upregulation of DmGSTO1A resulted in fewer apoptotic cells in the muscle. I-J, Quantification of TH-positive neurons in PPL1 clusters in 1- and 20-day-old flies (n > 10). Upregulation of DmGSTO1A by the TH-Gal4 dopaminergic neuron-specific driver can rescue dopaminergic neuron loss in parkin mutants. Error bars represent standard deviation. Experimental significance was determined by one-way ANOVA (*** is P < 0.0001). FIGURE 3. Upregulation of DmGSTO1 in muscle decreases tubulin accumulation and UPR activation in park1 mutants. Representative images of flight muscle stained with anti-α-tubulin antibody, and phalloidin. A, α-tubulin accumulates in park1 mutant muscle. The accumulation of tubulin in the park1 mutant was suppressed by the muscle-specific upregulation of DmGSTO1A. B, The levels of actin filaments in muscle did not change in any mutants as visualized with phalloidin. C, The increased levels of α/β-tubulin in park1 mutants was rescued by DmGSTO1A expression. Error bars represent standard deviation. The experimental significance was determined by one-way ANOVA park1 single mutants. Error bars represent standard deviation. The significance was determined by one-way ANOVA (*** is P < 0.01, ** is P < 0.05). E, mRNA levels of male thoraces were determined by real-time PCR analysis. Relative amounts of tubulin mRNA were unchanged in all mutants. F, Western blot analysis of phosphorylated, and total eIF2α. G, Increased HSPs in park1 mutants were decreased with the upregulation of DmGSTO1A. F-G, Error bars represent standard deviation. The significance was determined by one-way ANOVA (* is P < 0.05). β-actin was used as a loading control. Experiments were performed in triplicate. FIGURE 4. The catalytic activity of DmGSTO1 is critical for rescue of the defective phenotypes of the park1 mutant. A, Expresstion of DmGSTO1AC31A in the park1 mutant background did not suppress the collapsed thorax (n > 200), and downturned wing (n > 160) phenotypes of parkin mutants. Error bars represent standard deviation. The experimental significance was determined by one-way ANOVA (* is P < 0.05). B, Western blot analysis of adult thorax extracts using anti-β-tubulin antibodies. DmGSTO1AC31A expressed in the park1 mutants did not rescue the tubulin accumulation phenotypes. Error bars represent standard deviation. The significance was determined by one-way ANOVA (** is P < 0.05, and * is P < 0.01). β-actin was used as a loading control. Experiments were performed in triplicate. FIGURE 5. DmGSTO1 partially restored mitochondrial F1F0-ATP synthase activity in park1 mutants. A, In the presence of GSH, recombinant ATP synthase β subunit was glutathionylated by DmGSTO1A in a dose-dependent manner. B, Glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody, and were immunobloted with an anti-ATPsyn β antibody. Glutathionylation of endogenous ATP synthase β subunit in park1 mutants was regulated by the GSH-conjugating catalytic activity of DmGSTO1A but not by DmGSTO1B. The endogenous
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(** is P < 0.05, and * is P < 0.01). D, Tubulin levels were higher in the double mutants than in the
levels of the glutathionylated form of the ATP synthase β subunit were decreased even more in park1/DmGSTO1null double mutants. Error bars represent standard deviation. The experimental significance was determined by one-way ANOVA (* is P < 0.05). C, Mitochondrial F1F0-ATP synthase activity, and ATP levels in Drosophila thoraces. DmGSTO1A expression enhances ATP synthase activity, and ATP level of park1 mutants. Error bars represent standard deviation. One-way ANOVA was used for statistical analysis (ATP synthase activity: * is P < 0.05, and ATP level: ### is P < 0.001). D, Muscle morphology of wild-type and ATP synthase β subunit RNAi mutant flies (arrow, muscle degeneration). Muscle-specific ATP synthase β subunit RNAi resulted in degeneration of the flight muscles. E, Western blot analysis of adult thorax extracts from the ATP synthase β subunit RNAi mutant flies. α-, β-tubulin showed significant accumulation in the RNAi mutants muscle. F, Western blot analysis of adult thorax extracts from the ATP synthase β subunit RNAi in a park1 mutant background. Accumulation of tubulin was increased more in the RNAi mutants than in the park1 single mutants. Error bars represent standard deviation. The significance was determined by phosphorylated eIF2α, and total eIF2α. (Asterisk indicates the nonspecific band). Error bars represent standard deviation. The significance was determined by one-way ANOVA (* is P < 0.01). Experiments were performed in triplicate. FIGURE 6. Mitochondrial F1F0-ATP synthase (Complex V) assembly is affected by DmGSTO1 expression in park1 mutants. A-B, Mitochondrial protein extracts from the thorax of mutant fly lines were subjected to Blue Native-PAGE, followed by western blot analysis with anti-ATPsyn α subunit antibody. Three bands were detected: super complex (> 800 kDa), assembled ATP synthase (complex V, > 600 kDa), and F1 subcomplex (> 400 kDa). Prohibitin was used as a mitochondrial loading control. A, The assembly of ATP synthase (Complex V) was significantly decreased in park1 mutants. The amount of assembled ATP synthase was restored by DmGSTO1A upregulation in park1 mutants. Error bars represent standard deviation. The experimental significance was determined by one-way ANOVA (** is p < 0.01, and * is P < 0.0001). Experiments were performed in quintuplicate. B, The amount of assembled ATP synthase tended to decrease more in park1/DmGSTO1null double mutants than park1 mutants; however, the effect was not statistically significant. Error bars represent standard deviation. The experimental significance was determined by one-way ANOVA (** is p < 0.0001, * is P < 0.001, and ns, not significant). Experiments were performed in quadruplicate.
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one-way ANOVA (** is P < 0.01, and * is P < 0.05). G, Western blot analysis of Hsp70/Hsc70,
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Supporting Information Glutathione S-Transferase Omega 1 Activity is Sufficient to Suppress Neurodegeneration in a Drosophila Model of Parkinson’s Disease Kiyoung Kim, Song-Hee Kim, Jaekwang Kim, Heuijong Kim, and Jeongbin Yim FIGURE S1. Generation of DmGSTO1 mutants in Drosophila. A, Genomic structures of DmGSTO1 (CG6673). Transposon insertion sites are indicated above the map by an inverted triangle. The GE26508 P-element was imprecisely excised to generated DmGSTO1null. DmGSTO1null was a 593 bp deletion, which removed the DmGSTO1, and CG6662 coding regions. DmGSTO1 codes for two transcripts, A and B, which share the first exon. B, Quantitative RT-PCR analysis on extracts from mutant and control flies. DmGSTO1null mutants showed loss of the two transcripts (DmGSTO1A and DmGSTO1B), and CG6662. GAPDH was used as a control. C, GSH-dependent DHA reductase activity in wild type and DmGSTO1null. Error bars indicate standard deviation. The experimental significance was determined by one-way ANOVA (* is P < 0.0001). Experiments were performed in triplicate. D, CG6776 and CG6662 mRNA levels in park1 mutants are the same as in WT. GAPDH was used as a control. FIGURE S2. DmGSTO1 suppresses the defective thorax, and downturned wing phenotypes of park1 mutants. A and B, Upregulation of DmGSTO1A suppressed the collapsed thorax (white arrows), and downturned wing phenotypes of parkin mutant flies. FIGURE S3. Park1 mutants display accumulation of tubulin in DA neurons. Immunostaining with anti-α-tubulin, and anti-TH antibodies in DA neurons in Drosophila brains. Accumulation of αtubulin was observed in DA neurons from park1 mutants and park1/DmGSTO1null double mutants (white arrowheads). Immunostaining with anti-TH antibody was performed to identify DA neurons. FIGURE S4. CG6662, another GSTO in Drosophila, was unable to rescue glutathionylation of the ATP synthase β subunit in park1 mutants. Glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody, and were immunoblotted with an anti-ATPsyn β antibody. CG6662 was unable to glutathionylate the endogenous ATP synthase β subunit in park1 mutants. Experiments were performed in triplicate. FIGURE S5. Mitochondrial F1F0-ATP synthase (Complex V) assembly in the ATP synthase β subunit RNAi mutants. Mitochondrial protein extracts from the thorax were subjected to BlueNative-PAGE, followed by western blot analysis with anti-ATPsyn α subunit antibody. All bands were decreased in the ATP synthase β subunit RNAi mutants. Prohibitin was used as a mitochondrial loading control.
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FIGURE S6. DmGSTO1A is not important for the suppression of mitochondrial morphological defects in park1 mutants. Mitochondria in flight muscle were stained with Alexa 488-conjugated streptavidin. Compared with WT, park1 mutants displayed large clumps of intense signal. Upregulation of DmGSTO1A in park1 mutants did not suppress either the park1 mutant phenotype or the mitochondrial morphological defects (first panel). ATP synthase β subunit RNAi mutants also displayed normal mitochondrial morphology (second panel). Mitochondria in IFM tissues of Drosophila thorax were labeled by mito-GFP. Compared with park1 mutant, DmGSTO1A or DmGSTO1AC31A expressing lines in a park1 mutant background also showed mitochondrial morphological defects (third panel). FIGURE S7. The endogenous levels of the glutathionylated form of the ATP synthase β subunit in thorax extracts were decreased in PINK1B9 mutants. A, parkin and DmGSTO1 mRNA levels were also reduced in the PINK1B9 mutants. Error bars represent standard deviation. Experimental significance was determined by one-way ANOVA (* is P < 0.05). Experiments were performed in triplicate. B, The levels of the glutathionylated ATP synthase β subunit in thorax extracts were decreased in PINK1B9 mutants. The total level of the ATP synthase β subunit was unchanged in PINK1B9 mutants. β-actin was used as a loading control.
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Glutathione S-transferase Omega 1 Activity is Sufficient to Suppress Neurodegeneration in a Drosophila Model of Parkinson's Disease Kiyoung Kim, Song-Hee Kim, Jaekwang Kim, Heuijong Kim and Jeongbin Yim J. Biol. Chem. published online January 4, 2012
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