DJ-1 can inhibit microtubule associated protein 1 B formed aggregates
DJ-1 can inhibit microtubule associated protein 1 B formed aggregates
Zhiquan Wang 1
Yu Zhang 0
Shi Zhang 0
Qianqian Guo 2
Yuyan Tan 0
Xinyi Wang 0
Ran Xiong 0
Jianqing Ding 0
Shengdi Chen 0 1
0 Department of Neurology & Institute of Neurology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Shanghai 200025 , China
1 Laboratory of Neurodegenerative Diseases & key Laboratory of Stem Cell Biology, Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Science & Shanghai Jiao Tong University School of Medicine. Shanghai 200025 , China
2 Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Science , Shanghai 200025 , China
Background: Abnormal accumulation and aggregation of microtubule associated proteins (MAPs) plays an important role in the pathogenesis of neurodegenerative diseases. Loss-of-function mutation of DJ-1/Park7 can cause early onset of PD. DJ-1, a molecular chaperone, can inhibit a-synuclein aggregation. Currently, little is known whether or not loss of function of DJ-1 contributes to abnormal MAPs aggregation in neurodegenerative disorders such as PD. Results: We presented evidence that DJ-1 could bind to microtubule associated protein1b Light Chain (MAP1bLC). Overexpression of DJ-1 prevented MAP1b-LC aggregation in HEK293t and SH-SY5Y cells while DJ-1 knocking down (KD) enhanced MAP1b-LC aggregation in SH-SY5Y cells. The increase in insoluble MAP1b-LC was also observed in the DJ-1 null mice brain. Moreover, in the DJ-1 KD SH-SY5Y cells, overexpression of MAP1B-LC led to endoplasmic reticulum (ER) stress-induced apoptosis. Conclusion: Our results suggest that DJ-1 acts as a molecular chaperone to inhibit MAP1B aggregation thus leading to neuronal apoptosis. Our study provides a novel insight into the mechanisms that underly the pathogenesis of Parkinson's disease (PD).
PD is a common neurodegenerative disease which affects
approximately 1% of individuals of 65 years and 5% of
those 85 years or older. The featured pathological
changes of PD are the selective and progressive loss of
dopaminergic (DA) neurons as well as protein
aggregation and Lewy body formation [
]. Lewy bodies mainly
constitute of aggregated a-synuclein protein and they
also contain cytoskeletal components and other proteins.
Although the role of protein aggregation in the
pathogenesis of neurodegenerative diseases remains
controversial, many studies have shown that protein aggregation
contributes to neurodegeneration [
]. Failure to clear
misfolded proteins leads to protein aggregation, which
may in turn lead to the pathogenesis of
It has been reported that cytoskeletal proteins are
involved in the pathology of neurodegenerative diseases
]. For example, tau has been linked to both
Alzheimer’s disease (AD) and PD [
]. MAP1b has also been
reported to participate in the pathogenesis of Fragile X
] and Giant axonal neuropathy [
MAP1b plays a principal role in the development of the
nervous system and is essential for normal development
of the murine nervous system [
]. It has been
reported that MAP1b co-localized with a-synuclein in
the Lewy body , which provides a hint that insoluble
MAP1b may contribute to the pathogenesis of PD.
Abnormal accumulation of MAP1B-LC leads to
neuronal death in Giant Axonal Neuropathy (GAN) knockout
(KO) mice [
]. So it is important to explore whether
there is any link between MAP1b aggregation and PD
Loss of function mutation of Park7/DJ-1 contributed
to the pathogenesis of early-onset Parkinsonism [
Several PD-causing mutations have been identified
including exon deletions, truncations, homozygous and
heterozygous point mutations, which are all
predominantly in the loss of function manner [
]. DJ-1 belongs
to the ThiJ/PfpI superfamily and expresses in both
neurons and astrocytes [
]. DJ-1 could function as a
molecular chaperone [
] and inhibit the aggregation
of a-synuclein [
]. However, the exact role of DJ-1
in the cytotoxic process induced by MAPs aggregation
is poorly understood. Here we reported that DJ-1 could
directly bind to MAP1b-LC and inhibit its aggregation.
Aggregation of MAP1b-LC was exacerbated when DJ-1
was deficient. Furthermore, we also showed that the
excessive aggregation of MAP1b-LC could lead to
apoptosis in DJ-1 KD SH-SY5Y cells. Therefore, DJ-1 may
act as a molecular chaperone to suppress the neuronal
death caused by protein aggregation.
DJ-1 interacted with MAP1b-LC
MAP1b-LC has been shown to be a potential DJ-1
binding protein [
]. To study whether DJ-1 can interact
with MAP1b-LC, GST-DJ-1 fusion protein and
6xHisMAP1b-LC were expressed in E. coli BL21 cells and
purified respectively. The pull down assay showed that
MAP1b-LC interacted with GST-DJ-1 but not with
GST, suggesting that DJ-1 could bind to MAP1b directly
in vitro (Figure 1A). Flag tagged MAP1b-LC and HA
tagged DJ-1 were co-transfected into HEK293T cells for
36 hours. Cells were lysed and immunoprecipitated with
either anti-Flag M2 beads or HA antibody-conjugated
beads. The results showed that MAP1b-LC and DJ-1
could be immunoprecipitated reciprocally (Figure 1, B).
Co-localization of DJ-1 and MAP1b-LC in HEK293t,
SH-SY5Y cell lines (Figure 1, C and 1D) and cultured
primary neurons (Figure 1F) was also observed.
Furthermore, endogenous MAP1b-LC in the wild-type mice
brain could also be immunoprecipitated by DJ-1
antibody-conjugated beads (Figure 1, E). These results all
demonstrated that DJ-1 could form a complex with
MAP1b-LC to regulate the physiological activities of
DJ-1 could inhibit the aggregation of MAP1b-LC
It has been reported that MAP1b-LC is a component of
cortical Lewy bodies [
], and abnormal accumulation
of MAP1B-LC in the animal model of GAN could lead
to neuronal death [
]. DJ-1 is thought to be a
molecular chaperone that can inhibit the aggregation of
]. To explore whether DJ-1 can affect
the aggregation of MAP1b, The Flag tagged MAP1b-LC
construct was transfected into the HEK293t cells for 48
hrs. Cells were lysed and the lysates were separated into
the detergent soluble and insoluble fractions . These
fractions were analyzed by SDS-PAGE/immunoblotting.
The results showed that MAP1b-LC formed aggregates
were in the insoluble fraction (Figure 2A) and overexpressed
DJ-1 decreased the insoluble MAP1b-LC (Figure 2A).
To further evaluate the effect of DJ-1 on MAP1b-LC
aggregation, MAP1b-LC construct was transfected into
HEK293t cells with either DJ-1 or the empty pcDNA3
vector as a control. We observed less MAP1b-LC
aggregates in the DJ-1 overexpressed cells compared with the
control (Figure 2B).
The relationship between DJ-1 and MAP1B-LC
aggregation was also confirmed in the dopaminergic SH-SY5Y
cells. Overexpression of DJ-1 decreased the insoluble
MAP1b-LC fraction and inhibited the formation of
MAP1b-LC aggregates when they were co-transfected into
SH-SY5Y cells (Figure 2 C, D). Moreover, overexpressed
DJ-1 also decreased endogenous insoluble MAP1b-LC in
SH-SY5Y cells (Figure 2 E). Taken together, these data
revealed that DJ-1 could act as a chaperone to inhibit the
abnormal aggregation of MAP1b-LC in both HEK293t
cells and SH-SY5Y cells.
Malfunction of DJ-1 exacerbated aggregation of MAP1b-LC
L166P, the most common form of DJ-1 mutation, can
prevent the dimer formation. The DJ-1 mutant is
unstable and is degraded rapidly [
observations have suggested that the DJ-1 mutation could be a
loss-of-function mutation. To examine whether or not
the DJ-1 mutation impairs the ability of DJ-1 to inhibit
MAP1B-LC aggregation, Flag-MAP1b-LC was
co-transfected into HEK293t or SH-SY5Y cells with either
pEGFP-DJ-1 L166P or pEGFP vector. The results
showed that L166P mutation of DJ-1 failed to suppress
the accumulation of insoluble MAP1b-LC (Figure 3 A, B).
DJ-1 KD SH-SY5Y cell line was also established to
further investigate whether the malfunction of DJ-1
could induce the aggregation of MAP1b-LC. Western
blot results confirmed that DJ-1 was effectively knocked
down in the DJ-1 shRNA stable cells compared with
scramble shRNA control (Figure 4A). To examine the
effect of down regulation of DJ-1 on the aggregation of
MAP1b-LC, the Flag-MAP1b-LC was transfected into
DJ-1 KD cells or scrambled control cells. The result
showed that there was more insoluble Flag-MAP1b-LC
in the DJ-1 KD cells compared with the controls (Figure
4B). Similarly, increased Flag-MAP1b-LC aggregation
was also observed in the DJ-1 KD cell lines (Figure 4C).
Furthermore, we also observed the increased endogenous
insoluble MAP1b-LC (Figure 4D) and the endogenous
MAP1b-LC aggregates was observed in DJ-1 KD
SHSY5Y cells (Figure 4E).
The relationship between MAP1b-LC aggregation
and DJ-1 deficiency was also studied in vivo.
Sixmonth-old DJ-1 KO mice or wild type littermates were
used. The brain lysates of 3 KO or wild type mice were
extracted and separated into Triton-X100 soluble and
insoluble components. The Western blot results showed
an increase in insoluble MAP1b-LC in the DJ-1 KO
mouse compared with that of the wild type (Figure 4F,
G). Taken together, our results showed that DJ-1
abolishment enhanced MAP1b-LC aggregation both in vitro
and in vivo.
DJ-1 abolishment did not alter the ubiquitination of
MAP1b-LC and the activity of proteasome
MAP1b-LC is degraded through the ubiquitin
proteasome system (UPS) and impairments of the UPS in the
GAN-null mice may lead to the accumulation of
]. DJ-1 has also been shown to form a
complex with Pink1 and Parkin to promote
degradation of unfolded or misfolded proteins [
failure of UPS has been thought to play a critical role in
the pathogenesis of PD, we explored whether
ubiquitination of MAP1b-LC was altered in the DJ-1 KD cells.
Flag-tagged MAP1b-LC was transfected into the DJ-1
KD SH-SY5Y cells or scrambled control cells. Cells
were lysed and the lysates were immunoprecipated
with anti-Flag antibody and probed with anti-ubiquitin
antibody. The result showed that the ubiquitination of
MAP1b-LC was unchanged when DJ-1 was knocked
down (Figure 5A and 5B).
Down-regulation of DJ-1 can enhance the death of
proteasome inhibitor-treated Neuro2A cells [
we attempted to assess whether DJ-1 deficiency may
affect the proteasome activity. Proteasome
chymotrypsin-like, caspase-like and trypsin-like activities were
measured with succinyl-Leu-Leu-Val-Tyr-AFC,
Z-LeuLeu-Glu-AMC and Boc-Leu-Arg-Arg- AMC,
]. The results did not show any significant
difference between the proteasome activity of DJ-1 KD
cells and that of scrambled control (Figure 5C). Using
the same method, we did not find the impairment of
proteasome activity in DJ-1 KO mice brain either
(Figure 5D). These results suggest that the DJ-1 deficiency
did not affect the activity of UPS and ubiquitination of
MAP1b-LC. DJ-1 may work as a molecular chaperone
to regulate the folding of MAP1b-LC but not its
MAP1b-LC aggregation caused ER stress dependent apoptosis in the DJ-1 KD SH-SY5Y cells
Since protein aggregation is a major cause of
neurodegeneration and malfunction of DJ-1 can lead cells to
produce more aggregated MAP1b-LC, we next studied
whether MAP1b-LC aggregation induced apoptosis of
DJ-1 KD SH-SY5Y cells. We transfected the MAP1b-LC
into DJ-1 KD SH-SY5Y cells or scrambled control,
pDsRed2 being co-transfected with MAP1b-LC for the
selection of the transfected cells. After 60 hours, the
cells were harvested for the Annexin-V apoptosis assay.
We observed more Annexin-V positive cells in the
MAP1b-LC transfected group (Figure 6A, d) compared
to that of the scramble cells (Figure 6A, c) (Figure 6 B).
Since apoptosis caused by protein aggregation is mainly
through the ER stress dependent pathway [
examined the phosphorylated eIF2a, an ER stress
marker. Phosphorylation of eIF2a will only happen at the
early stage of ER stress to counteract the insult and is
thought to be protective for the cells from ER stress.
However, when the insult continues, activated eIF2a
will be dephosphorylated and the protection will be
abolished, leaving the cells to undergo apoptosis [
Our results showed that phosphorylated eIF2a was
much higher in MAP1b-transfected DJ-1 KD cells at 48
hrs after transfection compared with that in the
scrambled control DJ-1 KD cells (Figure 7A). However,
eIF2a dephosphorylation in the MAP1b-LC transfected
DJ-1 KD cells was increased at 60 hrs after transfection
(Figure 7B). These results showed that phosphorylation
of eIF2a was induced upon MAP1b-LC aggregation to
protect the cells against ER stress and increased
MAP1b-LC aggregation induced severer ER stress in
DJ1 KD cells. However, the protection was abolished as
the DJ-1 deficient cells failed to decrease the aggregated
proteins in the DJ-1 KD cells, which finally led to ER
stress induced apoptosis. Particularly, Salubrinal, the
specific inhibitor of dephosphorylation of eIF2a 
suppressed the MAP1b-LC induced apoptosis in DJ-1
KD SH-SY5Y cells (Figure 7C and 7D). Taken together,
these results suggested that excessive MAP1b-LC
aggregation caused by DJ-1 ablation may induce apoptosis in
the ER stress dependent manner.
Mutations of DJ-1 have been linked to early onset
Parkinsonism. However, the molecular mechanism
underlying the pathogenesis is still obscure. DJ-1 is thought to
be a molecular chaperone and oxidative sensor,
participating in both familial and sporadic PD [
researches have been focused on the anti-oxidative
stress function of DJ-1 [
] but the molecular
chaperone function of DJ-1 was hardly noticed . Our
results showed that DJ-1 interacted with MAP1b-LC
both in vitro and in vivo. Furthermore, aggregates
formed by overexpressed MAP1b-LC in HEK293t and
SH-SY5Y cells could be inhibited by the overexpression
Protein aggregation and insoluble inclusion bodies are
one of the main causes for the pathogenesis of many
neurodegenerative diseases [
]. MAP1b is essential for normal
development of the murine nervous system [
] and its
abnormal accumulation has been linked to
neurodegenerative disease. Moreover, MAP1b-LC has been observed
in the insoluble Lewy bodies in the brain of PD patients
]. Allen et al. reported that MAP1b-LC accumulation
could lead to the neuron death in GAN KO mouse [
Since DJ-1 has been shown to interact with MAP1b-LC
and inhibit its aggregation, we hypothesized that the
formation of MAP1b-LC aggregation may be suppressed by
the molecular chaperone DJ-1.
Mutations of DJ-1 are thought to be loss function
mutations that can lead to autosomal recessive familial
PD. The most frequently DJ-1 mutant L166P fails to
form dimers and monomeric mutant DJ-1 is unstable
and degraded rapidly [
]. Therefore DJ-1 KD SH-SY5Y
stable cell lines and DJ-1 KO mice were used to study
the effect of DJ-1 on the aggregation of MAP1b-LC. In
the DJ-1 KD cells, MAP1b-LC aggregation was
increased compared with that of the scramble control
cells. Moreover, the level of insoluble MAP1b-LC was
also increased in the DJ-1 KO mice. So it implicated
that DJ-1 may work as a molecular chaperone to control
the normal state of MAP1b-LC and loss function of
DJ1 may lead to increased aggregation of MAP1b-LC.
It has been proposed that the impairments of
ubiquitin proteasome system (UPS) play an important role in
the pathogenesis of PD. DJ-1 has also been reported to
participate in regulating the activity of UPS [
has been found that inhibition of MAP1b-LC
ubiquitination leads to neuronal death in the GAN-null mice
. Therefore, we examined whether increased
aggregation of MAP1b-LC results from the failure of its UPS
dependent degradation. However, our results did not
show any change of the ubiquitination of MAP1b-LC
(Figure 5A). Neither was there any change of the total
protein (soluble plus insoluble) level of MAP1b-LC
(Figure 4 G). Moreover, there was no impairment of
proteasome activity in DJ-1 KD cells and DJ-1 KO mice.
All of these observations have indicated that the
loss-offunction of DJ-1 does not affect the UPS dependent
degradation of MAP1b-LC. The increase in insoluble
MAP1b-LC in the DJ-1 KD cells and DJ-1 KO mice
suggests that DJ-1 may work as a molecular chaperone to
promote correct folding of MAP1b-LC or maintain the
normal state of MAP1b-LC.
Protein aggregation has been implicated to play an
important role in the pathogenesis of neurodegenerative
diseases. Therefore, we analyzed whether excessive
MAP1b-LC aggregation can cause cell apoptosis. The
data suggested that overexpression of MAP1b-LC in the
DJ-1 KD SH-SY5Y cells, which produced overburdened
MAP1b-LC aggregates, increased cell apoptosis. It has
been reported that abnormal protein aggregation can
induce apoptosis mainly through the ER stress pathway
]. Previous studies have implicated that
phosphorylation of eIF2a was a protective cell response to
counteract the ER stress and the failure of the phosphorylation
of elf2a will lead to apoptosis [
]. Our results
revealed that the enhancement of MAP1b-LC
aggregation can induced more phosphorylated eIF2a in
DJ-1 KD cells than that in scramble controls. The
persistent existence of MAP1b-LC aggregation increased
dephosphorylated eIF2a and led the cells to ER stress
dependent apoptosis at 60 hrs. The fact that the eIF2a
dephosphorylation inhibitor Salubrinal can partially
inhibit the apoptosis supported our hypothesis.
DJ-1 has been shown to be a molecular chaperone
that can inhibit a-synuclein aggregation [
However, Ramsey et. al showed that DJ-1-deficient mice had
similar vulnerability to pathogenic Ala53Thr human
asynuclein toxicity . Based on our experimental
results, we cannot make the conclusion that chaperone
activity of DJ-1 is unrelated to alpha-synuclein
aggregation. It is possible that compensatory mechanisms exist
in DJ-1 null mice which act to mimic the function of
DJ-1 protein just as the author of the paper claimed
We observed increased aggregation of MAP1b-LC in
DJ-1 KD SH-SY5Y cells, as well as an increased level of
insoluble MAP1b-LC in DJ-1 KO mice. In contrast, two
previous studies did not observe the formation of
inclusion bodies in either adult or aged DJ-1 null mice
]. It suggested that DJ-1 abolishment produced
more MAP1b-LC aggregation in vitro and more
insoluble MAP1b-LC in vivo. There are two potential
explanations for the difference between our in vitro and in vivo
experiments: 1) In vitro experiment showed the acute
responses of the cells to MAP1b overexpression or DJ-1
KD, in contrast, the in vivo study showed the chronic
responses of animals to DJ-1 KO; and 2) both
environmental and genetic factors are responsible for the
pathogenesis of PD. The absence of MAP1b aggregation in
DJ-1 KO mice may be due to the absence of certain
non-genetic factors such as aging or neurotoxins in our
In summary, we report that DJ-1 is a molecular
chaperone that can inhibit the aggregation of MAP1b-LC in
vitro as well as the formation of insoluble MAP1b-LC in
vivo. Our findings have provided the first evidence that
links DJ-1 deficiency to MAPs aggregation, which may
improve our understanding regarding the role of DJ-1 in
the pathogenesis of PD.
Materials and methods
Antibodies and Reagents
The following antibodies were used: DJ-1 Monoclonal
Antibody (3E8) (Assay Designs, ADI-KAM-SA100-E),
mono- and polyubiquitinylated conjugates, monoclonal
Antibody (FK2) (Biomol, BML-PW8810R), DJ-1
polyclonal antibody (Abcam, ab18257), Rabbit polyclonal
antiFlag (F7425), mouse monoclonal anti-Flag (F1804),
mouse monoclonal anti-beta-actin (A5441)
(SigmaAldrich), mouse anti-HA (clone 12C5) (Covance,
MMS101R), mouse anti-EGFP (Roche, 11814460001),
Phospho-elF2alpha (Ser51) antibody (Cell Signaling, 9721),
Goat polyclonal anti-MAP1b (c-20) (Santa Cruz
Biotechnology, sc-8971). EIF-2a inhibitor Salubrinal was
purchased from Calbiochem. All the Chemicals were
purchased from Sigma-Aldrich except noted elsewhere.
Human DJ-1 and MAP1B-LC cDNA were amplified
from the human fetal brain cDNA library (Invitrogen)
and ligated to the pcDNA3 vector with an N terminal
HA tag and pCMV-3xflag (sigma), respectively. To
knock down DJ-1, a DNA fragment and a scramble
fragment were synthesized and ligated to the
pSilencer-3.1Hygro (Ambion). The sequence of the inserted DJ-1
DNA fragments is
GTTTTTTGGAAA, and the scramble sequence is
the constructions were confirmed by sequencing.
PCRbased site directed mutagenesis was used to construct
the L166P mutation of DJ-1, which was cloned to the
Cell culture and transfection
HEK293T and SH-SY5Y cells were purchased from
American Type Culture Collection and maintained in
DMEM with 10% Fetal Bovine Serum and 100 U/ml
penicillin/streptomycin. All the culture materials were
purchased from Invitrogen. HEK293t cells were
transfected by calcium phosphate precipitation and SH-SY5Y
cells were transfected with lipofectamine 2000
(Invitrogen). Transfection efficiency was neutralized by
cotransfected with a pRL-tk plasmid. Because DJ-1 L166P is
unstable, the amount of the plasmid used for its
transfection was 3 times more than that used for the control
vector. pSilencer-Hygro-DJ-1 used for DJ-1 KD was
transfected into SH-SY5Y and the stable clones were
selected with 300ug/ml Hygromycin.
Co-immunoprecipitation and Western blotting
For co-immunoprecipitation, HEK293T cells or the mice
brain were lysed in the buffer A containing 50 mM
Tris·HCl pH 7.6, 150 mM NaCl, 0.5% NP-40, 1%
sodium deoxycholate and protease inhibitor cocktail
(Roche). To detect the ubiquitination of MAP1b-LC, the
cells were harvested and boiled in the lysis buffer B (50
mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1%
sodium deoxycholate, 1% SDS) for 10 min. Then the
concentration of SDS in Buffer B was diluted to 0.1% by
RIPA buffer. The lysates were pre-cleared with protein
A sepharose (GE bioscience) for 30 min. The
supernatants were incubated with the primary antibody for 4
hours at 4°C. Then protein A sepharose (GE Bioscience)
was added and the mixture was further incubated for 2
h at 4°C. For the Flag fusion protein IP, Flag M2 beads
(Sigma-Aldrich) were used. The beads with bound
proteins were washed for 6 times with lysis buffer and were
boiled in 2X SDS sample buffer, then the samples were
detected by immunoblotting.
For Western blotting the cells were lysed in RIPA
buffer (50 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40,
1% sodium deoxycholate, 0.1% SDS) with protease
inhibitor cocktail (Roche). To separate the detergents
soluble and insoluble proteins, the homogenized mice brain
or the cells were lysed in 0.2% Triton X-100 lysis buffer
(25 mM Tris·HCl pH 7.6, 150 mM NaCl, 0.2% Triton
X-100, 1% sodium deoxycholate) on ice for 20 min.
Triton X-100-soluble and -insoluble fractions were
separated via centrifugation at 13,000 rpm for 15 min [
The samples were boiled in 2XSDS sample buffer and
detected by immunoblotting. For all the western blot
results, at least 3 independent experiments were done
and the most representative result was shown.
HEK293T or SH-SY5Y cells were grown on glass
coverslips, fixed with 4% PFA, permeabilized with 0.2% Triton
X-100, and blocked with 20% goat serum or 5%BSA in
0.2% PBST and then incubated with primary antibody.
Cells were washed and Alexa 594 or Alexa 488 goat
anti-mouse or rabbit IgG antibody (Invitrogen) was
added. After washed 3 times using PBS, anti-fade
mounting medium with DAPI (Vector Laboratory) was
added and the stained cells were analyzed with a
confocal microscopy (Leica SP5).
Apoptosis detection by Annexin V assay
Cells were seeded in 6-well plates and transfected with
the indicated plasmids and pDsRed2 for 48 hours. Then
cells were stained with Annexin V using the Annexin
VFITC apoptosis detection kit (BD bioscience) as the
instructions of the manufacturer. Cells
(30,000/treatment) were analyzed using a flow cytometer (Becton
Dickinson LSR II).
The DJ-1 knock-out mice were kindly provided from Dr.
Jie Shen (Harvard Medical School) [
] and crossed with
C57bl/6 mice at least 6 generations after arriving in our
lab. Throughout the experiments, the animals were kept
in stainless-steel cages in a controlled environment
(2225°C, 40-60% relative humidity, 12-h light-dark cycle),
with food and water available freely. All animal
experiments were performed in accordance with guidelines of
the laboratory animal ethical standards of Shanghai Jiao
Tong University School of medicine. For animal studies,
in each group 3 littermate mice brains were used, which
was defined as one independent experiment. Statistical
analyses were conducted on the results from three
Paired or unpaired Student’s t-test was used for
statistical analyses. Statistical significance was set at a P value
of less than 0.05 and there was no statistical correction
was used for all the values.
List of Abbreviations
PD: Parkinson’s disease; MAP1b LC: microtubule associated protein 1b; ER:
endoplasmic reticulum; KD: Knocking down; KO: Knock out; SDS-PAGE:
sodium dodecyl sulfate polyacrylamide gel electrophoresis; GST:
GlutathioneS-transferase; UPS: ubiquitin proteasome system.
We thank Dr. Jie Shen of Harvard medical school for her kindly providing of
the DJ-1 Knock-out Mice. This work was supported by the National Program
of Basic Research (2007CB947900, 2010CB945200, 2011CB504104) of China,
the Natural Science Fund (30700888, 30770732, 30872729, 30971031), Key
Discipline Program of Shanghai Municipality (S30202), Shanghai Key Project
of Basic Science Research (10411954500), Shanghai Pujiang Program
(08PJ1407900) and Program for Outstanding Medical Academic Leader of
Shanghai (LJ 06003).
ZQW designed and performed the experiments. YZ contributed to the
protein expression and purification. SZ, XR and XYW helped to perform the
animal studies. QQG helped to perform the flow cytometry analysis and YYT
provided essential advice to the project. SDC and JQD supervised the
project and edited the manuscript. All authors read and approved the final
The authors declare that they have no competing interests.
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