Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00423.x |
Expression of Cbl-interacting protein of 85 kDa in MPTP mouse model of Parkinson’s
disease and 1-methyl-4-phenyl-pyridinium ion-treated dopaminergic SH-SY5Y cells
Minjuan Bian1#, Mei Yu1#, Shanzheng Yang1, Hui Gao1, Yalin Huang1, Chunguang Deng2, Yanqin Gao1, Fengyan Sun1, and Fang Huang1*
1 National Key Laboratory of Medical
Neurobiology, Shanghai Medical College, Fudan University, Shanghai 200032,
China
2 Shanghai Nan Fang Model Organism Research
Center, Shanghai 201203, China
Received: February 15, 2008
Accepted: April 9, 2008
This work was supported by the grants from the
National Natural Science Foundation (30570633) and the Shanghai Metropolitan
Fund for Research and Development (04BZ14005 and 07DJ14005)
# These authors contributed equally to this work
*Corresponding author: Tel, 86-21-54237296;
Fax, 86-21-64174579; E-mail: [email protected]
The newly
discovered Cbl-interacting protein of 85 kDa (CIN85) is involved in many cellular
processes, but its functions in the brain and in neurodegenerative diseases
remain unclear. In this paper, we investigated the distribution of CIN85
protein in different regions of adult mouse brain using Western blot analysis
and immunohistochemistry, and found that CIN85 was ubiquitously expressed in
mouse brain. In the striatum and substantia nigra, two regions most deeply
affected in Parkinson’s disease, the level of CIN85 protein was relatively
high. In the MPTP mouse model of Parkinson’s disease, the expression of CIN85
in the striatum and substantia nigra was complicated. But in
1-methyl-4-phenyl-pyridinium ion-treated human dopaminergic SH-SY5Y cells, the
expression of CIN85 increased dramatically. Knocking down of CIN85 by short
hairpin RNA reduced SH-SY5Y cell death. Therefore, CIN85 might play different
roles in the dopaminergic cell line and in the nigrostriatum of mouse brain
under neurotoxin challenge.
Keywords CIN85; shRNA; SH-SY5Y cell;
Parkinson’s disease
Cbl-interacting
protein of 85 kDa (CIN85), also known as SH3 domain-containing gene expressed
in tumorigenic astrocytes (SETA), regulator of ubiquitous kinase (Ruk),
SH3-domain kinase binding protein 1 (SH3BP1), or CD2 binding protein 3
(CD2BP3), is a multifunctional adaptor/scaffold protein [1,2]. Due to
differential promoter and alternative splicing, CIN85 has multiple transcripts
in cells. Some of the transcripts have the patterns of tissue-specific or
developmentally regulated expression [3-6]. Structurally, CIN85 protein
contains three SH3 domains, a proline-rich region, and a C-terminal coiled-coil
domain. These domains support its interaction with other signaling molecules,
including regulators of the cytoskeleton and modulators of apoptosis. More than
30 proteins have been proved to be CIN85 partners [1,2]. Functionally, CIN85 is
involved in many cellular processes, such as down-regulation of receptor
tyrosine kinase [7,8], and cytosolic protein sorting and turnover. But there is
growing evidence that CIN85 is involved in apoptosis. For example,
overexpression of CIN85 in cultured primary sympathetic neurons induces
apoptosis [5], and CIN85 is able to sensitize cultured primary astrocytes to
apoptosis [9]. In addition, CEM A301 human T leukemia cells ectopically expressing
CIN85 were 10-fold more susceptible to tumor necrosis factor a-induced
apoptosis than control cells [10]. Based on these studies, it is clear that
CIN85 regulates apoptosis in many different types of cells and strengthens the
effects of pro-inflammatory factors. Cell apoptosis and inflammatory reaction
are the common causes of neurodegenerative diseases, such as Parkinson’s
disease (PD), hallmarked by the loss of dopaminergic neuronal cells in the
substantia nigra (SN) [11,12]. We hypothesize that pro-apoptotic
characteristics of CIN85 might relate to PD.
N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) is a widely used neurotoxin to produce PD models in animals, such as
mice and monkeys. The 1-methyl-4-phenyl-pyridinium ion (MPP+),
a major metabolite of MPTP, is taken up by high-affinity dopamine transporter,
norepinephrine transporter, or serotonin transporter in catecholaminergic
cells. Once inside the cells, MPP+ mainly follows three
pathways: accumulating in the synaptic vesicles through the action of vesicular
monoamine transporter type 2; concentrating within the inner mitochondrial
membrane, where it inhibits complex I, releases reactive oxygen species, and
depletes ATP; or remaining in the cytoplasm, where it interacts with cytosolic
enzymes [11,13]. Another neurotoxin, 6-hydroxydopamine (6-OHDA), is also
conventionally used in PD models. It destroys catecholaminergic structures by a
combined effect of reactive oxygen species and quinines [14,15].
In this
paper, we investigate the distribution of CIN85 in different regions of adult
mouse brain and analyze the expression of CIN85 in MPTP-induced PD model mice.
Furthermore, on the cellular PD model using human dopaminergic cells SH-SY5Y
treated with MPP+ or 6-OHDA, we tried to
address whether CIN85 was involved in the cell death process of dopaminergic
neurons. Our findings indicated that CIN85 might be involved in the
pathogenesis of PD.
Materials and
Methods
Materials
C57BL/6 male mice (12–14 weeks old)
were purchased from Shanghai Slac Laboratory Animal Company (Shanghai, China).
MPTP, MPP+, and 6-OHDA were products of Sigma (St. Louis,
USA).
Protein
extraction and Western blot analysis
The method of protein extraction and Western blot analysis has been
described elsewhere [16], but was modified. Briefly, cells and dissected mouse
brain tissues were lysed in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl,
1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate] containing
complete protease inhibitor cocktail (Calbiochem, San Diego, USA). Proteins
were separated on 12.5% or 10% sodium dodecyl sulfate-polyacrylamide gels and
transferred to polyvinylidene difluoride membranes (Schleicher and Schuell,
Dassel, Germany). The membranes were blocked by 5% non-fat dried milk in TBS-T
[10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween] at room temperature for
3 h and incubated with the following primary antibodies at room temperature for
3 h: rabbit anti-CIN85 (C8116; 1:6000 dilution; Sigma); mouse anti-tyrosine
hydroxylase (T2928; 1:8000 dilution; Sigma); and mouse anti-actin (1:10,000
dilution; Sigma). Subsequently, the membranes were washed with TBS-T and
incubated for 1 h with goat peroxidase-conjugated anti-rabbit or anti-mouse
immunoglobulin G (1:10,000 dilution in TBS-T) at room temperature. They were
then washed three times with TBS-T and detected with a chemiluminescence
detection system (sc-2048; Santa Cruz Biotechnology, Santa Cruz, USA). The
protein levels were quantified by densitometry analysis using Quantity One
4.5.2 software (Bio-Rad, Hercules, USA).
Immunohistochemistry
Mice were anesthetized with 10% chloral hydrate and perfused
intracardially with 0.9% saline solution followed by 4% paraformaldehyde in 0.1
M phosphate buffer (pH 7.2). Brains were removed and post-fixed. Frozen
sections were cut at 30 mm on freezing microtome (Leica, Solms, Germany).
Immunohistochemistry of brain tissues was carried out according to the
previously published methods [17] with minor modifications. Briefly, sections
were placed in blocking buffer containing 10% goat serum with 0.3% Triton X-100
in 0.01 M phosphate-buffered saline (PBS; pH 7.2) for 30 min at 37 ºC. They
were then incubated at 37 ºC for 2 h and 4 ºC overnight with rabbit anti-CIN85
at 1:2000 dilution in PBS containing 1% goat serum and 0.1% Triton X-100. On
the second day, sections were incubated with biotinylated anti-rabbit secondary
antibody (1:200 dilution; Vector Laboratories, Burlingame, USA) at 37 ºC for 30
min and avidin-biotin-peroxidase (1:200 dilution) at 37 ºC for 45 min. The
peroxidase reaction was detected with 0.05% diaminobenzidine (Sigma) in 0.1 M
phosphate buffer and 0.03% H2O2. As
controls, adjacent sections received identical treatments except for incubation
with the primary antibody.
Real-time polymerase chain reaction (PCR)
Total RNA was extracted from SH-SY5Y cells using Trizol reagent
(Invitrogen, Carlsbad, USA). Reverse transcription was carried out using random
primer and Moloney murine leukemia virus reverse transcriptase (Promega,
Madison, USA), and real-time PCR was carried out for quantification of CIN85
mRNA on Rotor-Gene 3000 (Corbett Research, Sydney, Australia) based on
published methods [18,19]. For plotting a standard curve, serially diluted b-actin and CIN85
cDNA fragments were used in each experiment. Expression of CIN85 or b-actin was quantified to the standard curve, and the relative expression
value was calculated as the ratio of CIN85 to b-actin. The primers used in the real-time PCR were: 5‘-TGGAGGCCATAGTGGAGTTTG-3‘
(CIN85-1) and 5‘-CAGGTAGCTGAATGCCACCTG-3‘ (CIN85-2); and 5‘-ATGAGGTAGTCTGTCAGGT-3‘
(b-actin-1)
and 5‘-ATGGATGACGATATCGCT-3‘ (b-actin-2).
RNA interference
Three synthesized short hairpin RNAs (shRNA1, shRNA2, and shRNA3),
targeting different parts of the CIN85 mRNA sequence, were cloned into
lentiviral vector pLL3.7 (a cytomegalovirus -enhanced green fluorescent protein
expression cassette located downstream of the U6 promoter; a gift from Dr. Luk
Van Parijs, Massachusetts Institute of Technology, Cambridge, USA). The top
strands for shRNA1, shRNA2, or shRNA3 are: 5‘-GCTACCTGCCCCAGAATGACGATTCAAGAGATCGTCATTCTGGGGCAGGTAGCTTTTTT-3‘,
5‘-GACGTAGGCTGGTGGGAAGGATTCAAGAGATCCTTCCCACCAGCCTACGTCTTTTTT-3‘,
or 5‘-GCATTCAGCTACCTGCCCCAGAATGACGATTCAAGAGATCGTCATTCTGGGGCAGGTAGCTGAATGCTTTTTT-3‘,
respectively. The underlined sequences form the loop part of the shRNA. All
the final constructs were confirmed by DNA sequencing. RNA interference
experiments were carried out by transfecting the SH-SY5Y cells with
shRNA-expressing vectors using Lipofectamine 2000 (Invitrogen) and the
transfection efficiency was approximately 30%–95%.
Cell death assay
The SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s
medium (Invitrogen) supplemented with 10% fetal calf serum, 100 mg/ml
streptomycin, and 100 IU/ml penicillin in a water-jacketed incubator at 37 ºC
in a humidified atmosphere of 5% CO2/95% air. One day before
transfection, approximately 1.5´105 cells were plated in 24-well plates. On the
second day, cells were transfected with pLL3.7 or CIN85 shRNA with
Lipofectamine 2000 according to the manufacturer’s instructions. After
transfection for 12 h, the cells were cultured in normal medium for an
additional 20 h and collected for propidium iodide staining (BD Biosciences
Pharmingen, San Jose, USA) and fluorescence-activated cell sorting analysis
(FACSCalibur; BD Biosciences Pharmingen).
MPTP mouse model of PD
Adult male C57BL/6 mice were subcutaneously injected with MPTP at
the dose of 24 mg/kg (b.i.d.) every 12 h for 2 d, or 0.9% saline. The mice were
killed at 18, 36, or 72 h after the last injection.
Data analysis
Experiments were repeated at least three times. Data were analyzed
using spss software (version
11.5; SPSS, Chicago, USA). For comparison of statistical significance between
groups, Student’s t-test was used. P<0.05 was considered
significant.
Results
Distribution of CIN85 in different tissues of adult mouse
CIN85 is ubiquitously expressed in rats and mice [5,6]. Up to now, a
detailed expression pattern of CIN85 in the mouse brain has not been available,
and we were interested in whether CIN85 expresses in the striatum and SN,
regions that are rich of dopaminergic neurons and deeply affected in PD. The
antibody for CIN85 used in the study is specific to a C-terminal peptide
located between the proline-rich region and the coiled-coil domain, preserved
in most known isoforms of CIN85 protein. Western blot analysis revealed
expression patterns of CIN85 in mouse thymus, spleen, kidney, heart, and
testis, similar to those published [5,6]. Additionally, we found that the main
isoform of CIN85 protein is approximately 40 kDa in liver, kidney, heart, and
skeletal muscle [Fig. 1(A)]. In the cerebral cortex, hippocampus,
striatum, SN, and brain stem, CIN85 shows two major bands of approximately 85
kDa with similar densities. But in the cerebellum and spinal cord, the band of
lower molecular weight is dominant. There were more faint bands at
approximately 60 kDa and 40 kDa [Fig. 1(B)].
Furthermore, we detected the cellular distribution of CIN85 protein
in mouse brain by immunohistochemistry. Brain section without incubation with
the primary antibody was shown as a control [Fig. 2(A)]. Our results
showed that CIN85 could present in both neuron and glia-like cells based on the
cell morphology [Fig. 2(B,C)]. Throughout the cerebral cortex, CIN85
immunopositive cells were detected in all layers [Fig. 2(D)]. No
CIN85-immunopositive cellular fibers were found in the striatum, however, cells
between these fibers showed CIN85 immunostaining [Fig. 2(E)]. In the SN,
CIN85-immunopositive cells were in both substantia nigra pars compact and
substantia nigra pars reticulata [Fig. 2(F)]. The dorsal part of the
septal nuclei showed strong positive staining of CIN85, whereas the
intermediate part and ventral part showed moderate immunostaining [Fig. 2(G)].
Structures located in the mesencephalon presented moderate to strong
immunoreactivity. In the ventral part of the mesencephalon, strongly stained
large cells were shown in the red nucleus [Fig. 2(H)]. In the
hippocampus, CA1, CA2, and CA3 showed remarkable staining of CIN85 [Fig.
2(I)] and data not shown]; moreover, dentate gyrus displayed strong
CIN85 immunoreactivity [Fig. 2(I)]. In the corpus callosum, we detected
numerous CIN85-immunopositive glial cells (data not shown).
CIN85
expression in striatum and SN of mice with MPTP treatments
After detecting moderate to high level expression of CIN85 in the SN
and striatum, two regions tightly connected with PD, we were driven to
investigate if CIN85 protein played a role in the pathogenesis of PD. Adult
male C57BL/6 mice were challenged with MPTP (24 mg/kg b.i.d., every 12 h for 2
d). Mice were killed at 18, 36, or 72 h after the last dose, and the SN and
striatum were carefully microdissected. CIN85 protein was analyzed by Western
blot analysis. Tyrosine hydroxylase (TH) was used as a mark to evaluate the
mouse model. After MPTP treatments, TH protein in both SN and striatum had a
noticeable decrease [Fig. 3(A)]. Statistically, at 18, 36, and 72 h
after the last injection, the levels of TH protein were 38.16%±
5.71% (P<0.01), 45.53%±17.29% (P<0.05), and 31.32%± 5.92% (P<0.01)
of the saline group in the striatum, respectively, and 59.75%±11.29% (P<0.01),
38.36%±
17.24% (P<0.01) and 66.94%±9.64% (P<0.01) of the control in
the SN, respectively [Fig. 3(B)]. The results showed a dramatic loss of
TH neurons in the striatum and SN of toxin-challenged mice. Thus, these PD
animal models were valid. However, the alteration of CIN85 expression was
complex [Fig. 3(A)]. At 18, 36, and 72 h, the levels of CIN85 protein
were 68.36%±11.56% (P<0.05), 152.43%±36.10% and 141.18%±67.84% of the
saline group in the striatum, respectively, and 100.49%±14.07%, 42.01%±10.06% (P<0.01)
and 124.38%±27.83% of the control in the SN, respectively [Fig. 3(B)].
Gliogenesis is an obvious phenomenon in the MPTP mouse PD model. By
fluorescence immunolabeling, in the SN of PD model mice, GFAP-positive cells
increased dramatically (data not shown). To determine the expression of CIN85
in the remaining dopaminergic cells, double fluorescence labeling was carried
out. In the SN of PD mice, the immunoreactive signal of CIN85 could be clearly
detected in TH-positive cells (data not shown).
MPP+ treatment increased CIN85 gene
expression in SH-SY5Y cells
MPTP treatment elicits complex changes of CIN85 expression with time
in the striatum and SN. We were interested in the effect of MPP+ on the expression of the CIN85 gene in human dopaminergic SH-SY5Y
cells. After treatment with 1 mM MPP+ for 3, 8, 20, or 48 h,
real-time PCR was carried out. At early time points (3 or 8 h after MPP+ treatment), the expression of CIN85 mRNA had no significant
alteration compared with the controls [Fig. 4(A)]. But at 20 and 48 h
after exposure, the expression of CIN85 mRNA increased significantly
(P<0.05). The translation of differential mRNA levels to protein change is
capital to confirm the effect of MPP+ on CIN85
expression in SH-SY5Y cells. Therefore, we analyzed the CIN85 protein using
Western blot analysis. At 8, 20, and 48 h after MPP+
treatment, a significant increase of CIN85 protein was detected, but there was
no obvious change in cells treated for 3 h [Fig. 4(B,C)]. Therefore, MPP+ increased CIN85 gene expression in both mRNA and protein levels in
dopaminergic SH-SY5Y cells.
Furthermore, we tested the effect of 6-OHDA on CIN85 gene
expression. SH-SY5Y cells were exposed to 50 mM 6-OHDA or PBS for 20 h.
The expression of CIN85 mRNA could be increased by 10-fold in
6-OHDA-treated cells. CD2AP is the second member of the CIN85 adaptor protein
family. Similarly, 6-OHDA treatment increased the CD2AP mRNA level to
6-fold of that of the control (data not shown).
shRNA of CIN85 attenuated SH-SY5Y cell death
To investigate the possible significance of increasing CIN85 in MPP+-treated SH-SY5Y cells, we designed three shRNAs (shRNA1, shRNA2 and
shRNA3) targeting different parts of the CIN85 mRNA sequence that
contain a hairpin structure of 22 nt, 21 nt, or 29 nt in length respectively.
CIN85 is relatively highly expressed in SH-SY5Y cells (Figs. 4 and 5),
so to test the efficiency of these shRNA, shRNA-expressing vectors were
transfected into SH-SY5Y cells with Lipofectamine 2000. Cells were lysed for
protein extraction when green fluorescent protein-positive cells were over 85%,
and CIN85 protein was determined by Western blot analysis. We found that three
shRNAs had different effects on the expression of the CIN85 protein [Fig.
5(A,B)]. Two of them could efficiently knock down CIN85 protein, shRNA2
(69.4% of the control; P<0.05) and shRNA3 (34.6% of the control; P<0.01).
However, transfection using pLL3.7 or shRNA1 had no effect on CIN85 expression.
SH-SY5Y cells without transfection were set as controls.
Then the effect of CIN85 shRNA on autonomous cell death was carried
out. Among non-transfected SH-SY5Y cells, the percentages of dead cells from
shRNA1, shRNA2, and shRNA3, as well as the control were quite similar [Fig.
5(C) and data not shown]. The shRNA3, which most efficiently inhibited
CIN85 expression (34.6%±19.80% of the control), could attenuate cell death
significantly [Fig. 5(C)]. In shRNA3 transfected cells, the death rate
of green fluorescent protein-positive cells was reduced to 22.1%±1.3%, whereas
in the control it was 41.9%±8.1% [Fig. 5(D)]. The experiments were also
repeated in the CIN85 shRNA lentivirus-infected SH-SY5Y cells, in which both
autonomous apoptosis and death were decreased dramatically (data not shown).
Our results indicate that CIN85 could have a deleterious effect on the survival
of dopaminergic cells and CIN85 shRNA could be a neuronal protector.
Discussion
CIN85 protein shows a differential pattern of
expression in different regions of the adult mouse brain, but the dominant
isoform is approximately 85 kDa in size. Such a distribution is quite different
from other tissues (Fig. 1). In MPTP-treated PD mice, at 3 d after the last injection,
TH-positive neurons in the striatum and SN decreased to 31% and 67% of the
control, respectively. In the open field test, there was no effect on the
animals’ level of activity, regardless of the time spent in locomotion, or the
distance moved (data not shown). Similar results were shown by other
laboratories, summarized by Sedelis et al [20]. As early as 18 h after
the last dose of MPTP, TH protein reduced to 38% of the control in the striatum
and 59% in the SN, similar to other published data [21,22]. When tracing the
expression of CIN85, at the 18 h time point, CIN85 protein decreased to 68% in
the striatum, but did not change in the SN. At 36 and 72 h, CIN85 expression
was higher in the striatum in MPTP-treated mice than in the control mice,
although it was of no significance. In the SN, CIN85 reduced to 42% of the
control at 36 h, but recovered to 124% at 72 h after treatment (Fig. 3).
MPP+ challenge induces cell death in dopaminergic SH-SY5Y cells
[23,24]. In our experiments, CIN85 expression reached its peak at 20 h after
MPP+ treatment, and kept quite steady for 2 d (Fig.
4). We hypothesized that CIN85 might be involved in the activation of the
cell death pathway in SH-SY5Y cells, and down-regulation of CIN85 could have a
protective effect. In the same cell line, when shRNA3 was used to knock down
CIN85 expression, autonomous cell death was reduced to half of the control (Fig.
5). This reduction was the specific effect of shRNA3, as shRNA1 did not
provide any protection on the transfected cells. Recently, it was found that
the CD2AP/CIN85 protein balance played an important role in the normal
signaling response in podocytes [25]. In our 6-OHDA cellular PD model, the
relative ratio of CD2AP/CIN85 also reduced. CD2AP/CIN85 balance could have
general significance in the signaling response of different types of cells.
Therefore, the expression pattern of CIN85 in
the MPTP mouse PD model did not reproduce the increase of CIN85 expression in
MPP+-treated SH-SY5Y cells. There are several possible explanations for
this. First, a transient increase of CIN85 in damaged neurons might occur
before death, but the loss of dopaminergic neurons in the striatum and SN could
counteract the increasing effect and account for the decrease of CIN85 in the
same regions at the early time points. Second, as CIN85 is expressed in
astrocytes, which are expanded in MPTP challenged mice, the increasing
astrocytes in the SN or striatum might compensate for the loss of CIN85 in
degenerated neurons at the later time points. Finally, CIN85 functions
differently in the dopaminergic SH-SY5Y cells and the nigrostriatum of mouse
brain. To examine the role of CIN85 in PD, it will be necessary to conduct
experiments in CIN85 knockout mice. CIN85 and CD2AP comprise a new family of
adaptor proteins that plays important roles in animal development and diseases
[1,2], and we have provided the first evidence that CIN85 could play a role in
the pathogenesis of PD.
Acknowledgements
We thank Dr. Youqing Cai and Dr. Jinsong Yang for their assistance
in the experiments.
References
1
Dikic I, Giordano S. Negative receptor signaling. Curr Opin Cell Biol
2003, 15: 128–135
2
Huang F, Bian MJ. Structure and functions of CIN85. Chin J Cell Biol
2006, 28: 1–4
3
Take H, Watanabe S, Takeda K, Yu ZX, Iwata N, Kajigaya S. Cloning and
characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl.
Biochem Biophys Res Commun 2000, 268: 321–328
4
Finniss S, Movsisyan A, Billecke C, Schmidt M, Randazzo L, Chen B, B鰃ler O. Studying
protein isoforms of the adaptor SETA/CIN85/Ruk with monoclonal antibodies.
Biochem Biophys Res Commun 2004, 325: 174–182
5
Gout I, Middleton G, Adu J, Ninkina NN, Drobot LB, Filonenko V, Matsuka
G et al. Negative regulation of PI3-kinase by Ruk, a novel adaptor
protein. EMBO J 2000, 19: 4015–4025
6
Buchman VL, Luke C, Borthwick EB, Gout I, Ninkina N. Organization of
the mouse Ruk locus and expression of isoforms in mouse tissues. Gene 2002,
295: 13–17
7
Petrelli A, Gilestro GF, Lanzardo S, Comoglio PM, Migone N, Giordano S.
The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of
c-Met. Nature 2002, 416: 187–190
8
Soubeyran P, Kowanetz K, Szymkiewicz I, Langdon WY, Dikic I. Cbl-CIN85-endophilin
complex mediates ligand-induced downregulation of EGF receptors. Nature 2002,
416: 183–187
9
Chen B, Borinstein SC, Gillis J, Sykes VW, Bogler O. The
glioma-associated protein SETA interacts with AIP1/Alix and ALG-2 and modulates
apoptosis in astrocytes. J Biol Chem 2000, 275: 19275–19281
10
Narita T, Nishimura T, Yoshizaki K, Taniyama T. CIN85 associates with
TNF receptor 1 via Src and modulates TNF-a-induced
apoptosis. Exp Cell Res 2005, 304: 256–264
11
Dauer W, Przedborski S. Parkinson’s disease: mechanisms and models.
Neuron 2003, 39: 889–909
12
Gosal D, Ross OA, Toft M. Parkinson’s disease: The genetics of a
heterogeneous disorder. Eur J Neurol 2006, 13: 616–627
13
Jung TW, Lee JY, Shim WS, Kang ES, Kim JS, Ahn CW, Lee HC et al.
Adiponectin protects human neuroblastoma SH-SY5Y cells against MPP+-induced
cytotoxicity. Biochem Biophys Res Commun 2006, 343: 564–570
14
Yamamuro A, Yoshioka Y, Ogita K, Maeda S. Involvement of endoplasmic
reticulum stress on the cell death induced by 6-hydroxydopamine in human
neuroblastoma SH-SY5Y cells. Neurochem Res 2006, 31: 657–664
15
Nakamura M, Yamada M, Ohsawa T, Morisawa H, Nishine T, Nishimura O,
Toda T. Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during
response to 6-hydroxydopamine-induced oxidative stress. Biochim Biophys Acta
2006, 1763: 977–989
16
Ren JK, Wang L, Liu GX, Zhang W, Sheng ZJ, Wang ZG, Fei J. improved
method to raise polyclonal antibody using enhanced green fluorescent protein
transgenic mice. Acta Biochim Biophys Sin 2008, 40: 111115
17
Qiu MH, Zhang R, Sun FY. Enhancement of ischemia-induced tyrosine
phosphorylation of Kv1.2 by vascular endothelial growth factor via activation
of phosphatidylinositol 3-kinase. J Neurochem 2003, 87: 1509–1517
18
18 Ji XH, Jin YH, Chen YY, Li CY, Guo
LH. Existence of an endogenous glutamate and aspartate transporter in Chinese
hamster ovary cells. Acta Biochim Biophys Sin 2007, 39: 851-856
19
Liu HZ, Li Q, Yang XY, Liu L, Liu L, An XR, Chen YF. Expression of
basic fibroblast growth factor results in the decrease of myostatin mRNA in
murine C2C12 myoblasts. Acta Biochim Biophys Sin 2006, 38: 697703
20
Sedelis M, Schwarting RK, Huston JP. Behavioral phenotyping of the MPTP
mouse model of Parkinson’s disease. Behav Brain Res 2001, 125: 109–125
21
2Kurosaki R, Muramatsu Y, Kato H, Araki T. Biochemical, behavioral and
immunohistochemical alterations in MPTP-treated mouse model of Parkinson’s
disease. Pharmacol Biochem Behav 2004, 78: 143–153
22
Kim RH, Smith PD, Aleyasin H, Hayley S, Mount MP, Pownall S, Wakeham A et
al. Hypersensitivity of DJ-1-deficient mice to
1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) and oxidative stress. Proc
Natl Acad Sci USA 2005, 102: 5215–5220
23
Chen LJ, Gao YQ, Li XJ, Shen DH, Sun FY. Melatonin protects against
MPTP/MPP+-induced mitochondrial DNA oxidative damage in
vivo and in vitro. J Pineal Res 2005, 39: 34–42
24
Deng H, Jankovic J, Guo Y, Xie W, Le
W. Small interfering RNA targeting the PINK1 induces apoptosis in dopaminergic
cells SH-SY5Y. Biochem Biophys Res Commun 2005, 337: 1133–1138
25
Tossidou I, Kardinal C, Peters I, Kriz
W, Shaw A, Dikic I, Tkachuk S et al. CD2AP/CIN85 balance determines
receptor tyrosine kinase signaling response in podocytes. J Biol Chem 2007,
282: 7457–7464