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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 505-512 |
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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 �C. They were then incubated at 37 �C for 2 h and 4 �C 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 �C for 30 min and avidin-biotin-peroxidase (1:200 dilution) at 37 �C 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'-TGGAG�GC�CATAGTGGAGTTTG-3' (CIN85-1) and 5'-CAGGT�A�GCTGAATGCCACCTG-3' (CIN85-2); and 5'-ATGAG�G�TA�GTCTGTCAGGT-3' (b-actin-1) and 5'-ATGGA�TG�ACGATATCGCT-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'-GCTAC�CTGC�C�C��C���AG�AATGACG�ATTCAAGAGAT�CGTCATTCTGG�G�G�C�AGGTAGCTTTTTT-3', 5'-GACG�TAGGCTGG�TG�G�GAAGG�ATTCAAGAGATCCT�TCCCACCAGCCTAC�G�T�CTTTTTT-3', or 5'-GCATTCAGCTACC�TG�CCC�CA�G�A�A���T��GACGATTCAAGAGATCG�TCATTCTGGGGCAG�G�TAGCTGAATGCTTTTTT-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 �C 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-immuno�positive 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.
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