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Introduction
nigra, is the second most common neurodegenerative disorder after Alzheimer's disease,
affecting people in mid and late
life[1]. Mutations of the parkin and α-synuclein genes have been associated with
autosomal recessive and dominant parkinsonism pedigrees,
respectively[2]. α-Synuclein is a highly-conserved, 140 amino acid
protein which is mainly expressed in neurons and
concentrated in presynaptic nerve
terminals[3,4]. It was first isolated as a constituent of protein aggregates from Alzheimer brains,
distinct from the β-amyloid
component[5]. Although the native function of
α-synuclein is still being investigated, evidence suggests that its potential roles are in neural
plasticity and the regulation of synaptic vesicle
pools[6], modulation of dopamine
release[7,8], alterations in dopamine
synthesis[9], and targeting the dopamine transporter to the plasma
membrane[10]. α-Synuclein pathology is involved in a large
number of neurodegenerative diseases, including PD,
dementia with LB, Lewy body variant of Alzheimer's disease,
neurodegeneration with brain iron accumulation type-1, and
multiple system atrophy, which is mostly due to the
remarkable discovery in dominantly inherited α-synuclein
substitutions. Missense mutations (A53T, A30P, and E46K)
in the α-synuclein gene were found to be the cause of
autosomal dominant PD in a small group of
Mediterranean[11],
German[12], and Spanish
families[13]. Among them, A53T was found in at least 12 families with familial PD, although these
families likely to share a common
ancestor[14,15] and A53T seems to induce more severe harm either
in vivo or in vitro than A30P. The pathological consequences of the A53T
α-synuclein mutation is closely associated with PD, whose
symptoms include reduced spontaneous movement, static
tremor, muscular rigidity, progressive inability to maintain
erect posture, and shortening of the step
length[16], and neurons in the substantia nigra are predominantly, but not
exclusively affected[17].
1-Methyl-4-phenylpyridinium (MPP+), the active
metabolite of 1-methyl-4-phenyl-2,3,6-tetrahydropyridine, can enter
the mitochondria to induce oxidative stress and impair
energy metabolism[18] by inhibiting mitochondrial complex
I[19,20], inducing a syndrome closely resembling PD. The
neurotoxicity of exogenous dopamine (DA) has been described in
in vivo primary cultures and several cell
lines[21,22]. In dopaminergic neurons, DA is oxidized easily
in vitro and in vivo to a variety of neurotoxic metabolites such as highly cytotoxic
quinine molecules[23,24], which participate in the generation
of reactive oxygen species. Many studies indicate that an
imbalance between cytoplasmic and vesicular DA may lead
to oxidative stress and cell
degeneration[25]. DA may cause apoptosis, but most studies on DA toxicity agree that
non-apoptotic mechanisms are also involved in DA-induced cell
death[26,27].
Because a slowly progressive brain disorder is difficult
to study in humans, a simple, reliable, and valid method is
needed for furthering our understanding of the role of
α-synuclein in PD and for screening compounds with
therapeutic potential. SH-SY5Y neuroblastoma is a widely and
extensively used target cell line in the assessment of
neurotoxicity and neuroprotection. So we sought to create a
cell-based model by overexpressing mutant A53T human
α-synuclein in SH-SY5Y cells.
Materials and methods
Cell culture Human dopaminergic neuroblastoma cells
SH-SY5Y (American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained at 37 °C in 5%
CO2 in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand
Island, NY, USA), supplemented with 10% fetal bovine
serum (Hyclone, Logan, UT, USA), 100 U/mL penicillin, and
0.1 g/L streptomycin (Gibco, USA).
Plasmid construction Total RNA was extracted from
cultured cells by the TRIzol (Invitrogen, Carlsbad, CA, USA)
extraction method. From 2 µg of total RNA, cDNA was
synthesized using 200 units of reverse transcriptase
(Super-scriptTM III RT, Invitrogen, USA) and oligo (dT) primers in a
final volume of 20 µL. The following primer pairs were used
when processing PCR with pfuUltra (Stratagen, La Jolla, CA,
USA): primers α-synuclein-F (5'-GAA CTC GAG GGA CTC
AGT GTG GTG-3' with the Xho I site) and α-synuclein-R
(5'-CT TCT AGA GGA TGG AAC ATC TGT CAG C-3' with the
Xbal I site) for α-synuclein (524 bp), and GAPDH-F (5'-CTC
ATG ACC ACA GTC CAT GC-3') and GAPDH-R (5'-CAC CAC
CCT GTT GCT GTA GC-3') for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH; 456 bp). PCR amplification was
conducted under the following conditions: for α-synuclein,
initial denaturation was at 94 °C for 2 min, 30 s at 94 °C, 30 s
at 57 °C, and 50 s at 72 °C for 30 cycles, followed by 10 min at
72 °C. For GAPDH, initial denaturation was at 94 °C for 2
min, 30 s at 94 °C, 30 s at 59 °C, and 30 s at 72 °C for 30 cycles,
followed by 10 min at 72 °C. After amplification, 50 µL aliquots
were electrophoresed in 1% agarose gel (Biowest, Miami,
FL, USA), followed by photographic recording of the gel
stained with ethidium bromide. Then, the product of the
PCR was obtained by agarose gel DNA purification (TaKaRa,
Tokyo, Japan) and ligated into the pMD18-T simple vector
(TaKaRa, Japan) after adding DeoxyAdenosine Triphosphate
(dATP) to the blunt terminal of the fragment. The positive
transformants were screened by bacteria PCR and DNA
sequencing after transforming to competent Escheria coli
TG-1. The A53T mutant, human α-synuclein gene was
procured by gene splicing and overlap extension (SOE) on
site-directed mutagenesis of wild-type human α-synuclein which
was inserted into a pMD18-T simple vector purified by
PureLinkTM Hipure plasmid DNA purification kits (Invitrogen,
USA). The following primers were used in the SOE: forward
(5'-TGC ATG GTG TGA CAA CAG TGG CTG AGA-3'),
reverse (5'-GCC ACT GTT GTC ACA CCA TGC ACC ACT C-3'). The A53T, mutant human
α-synuclein cDNA was sub-cloned into the pcDNA3.1(+) vector (Invitrogen, USA) after
double digestion of the Xho I and
Xbal I restriction enzymes to either insert DNA or the vector.
Transfection and selection One day before transfection,
2×105 cells were seeded in 500
µL of growth medium without antibiotics in a 24-well format so that cells would be
90%_95% confluent at the time of transfection. DNA was
diluted in 50 µL Opti-MEM I Reduced Serum Medium (Invitrogen,
USA) without serum, and the appropriate amount of Lipofectamine™ 2000 (Invitrogen, USA) was diluted
in 50 µL of Opti-MEM I Medium. After incubation for 5 min at room
temperature, the diluted DNA was combined with the diluted
Lipofectamine™ 2000, mixed gently, and incubated for 20
min at room temperature. We added 100 µL complexes to
each well containing the cells and medium. The cells were
incubated at 37 °C in a CO2 incubator for 18_48 h. During
incubation, the medium was changed after 4_6 h, and then
cells were passaged at 1:10 into fresh growth medium after
incubation. The cells were incubated in the medium
containing 0.6g/L G418 (Gibco, USA) for 14 d to select the
stably-transfected cells, and then the cells were collected for
monoclone screening. 100 µL medium containing 0.3 g/L
G418 was added to all of the wells in the 96-well plate except
well Al which was left empty. 200 µL cell suspension was
added to well A1, then 100 µL cell suspension was quickly
transferred from the first well to well B1 by gently pipetting
and repeating the same procedure as H1. 100
µL of cell suspension was discarded from H1 so that it
ended up with the same volume as the wells above it. An additional l00 µL
medium was added to each well in column 1 (A1_H1), and
then 100 µL cell suspension was quickly transferred from the
wells in column 1 to those in column 2 (A2_H2) with gently
pipetting and repeating the same procedure as that of
column 12. Cell suspension l00 µL was discarded from each
well in the last column (A12_H12) so that all of the wells
ended up with 100 µL of cell suspension. The final volume
of all the wells was brought to 200 µL by adding 100
µL medium to each well. Each well that contained just a single
cell was checked and marked so that these monoclones could
be subcultured from the wells into larger vessels. We did the
subsequent work listed below after the cells were cultured in
DMEM medium containing 0.3 g/L G418 for about 3 months.
Western blotting Protein concentration was determined
by the Bradford assay (Applygen, Beijing, China). Equal
amounts of protein were loaded onto each lane, and
electrophoresed on SDS-PAGE with Tris-glycine running buffer.
They were then transferred to nitrocellulose membranes by
wet electrotransfer for 90 min at 100 mA. The
blocked membranes were incubated overnight at 4 °C with the polyclonal
antihuman α-synuclein antibody (R&D, Minneapolis, USA
1:1000 dilution). Following 1 h of incubation at room
temperature with a horseradish-peroxidase (HRP)-coupled
secondary antibody (1: 5000 dilution), the blots were washed
and immunodetection was carried out using enhanced
chemiluminiscence detection reagents (Amersham
Bio-sciences, Piscataway, NJ, USA). The blots were then
stripped and reprobed with the monoclonal antibody against
β-actin (Sigma, St Louis, CA, USA, 1:1000 dilution) and
detected as described earlier.
Immunocytochemistry After fixation with 4%
paraformal-dehyde, the cell slides were rinsed in phosphate buffer
solution (PBS) and pre-incubated with 3%
H2O2 for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Then,
the cells were blocked with normal rabbit serum for 15 min at
room temperature. The following primary antibody (R&D,
USA, 1:100 dilution) was then added for overnight
incubation at 4 °C in a humidified chamber. After rinsing for 3×3 min
in PBS, the cells were incubated with secondary biotinylated
antibody at 37 °C for 15 min, followed by rinsing for 3×3 min
in PBS. Then, the cells were incubated with HRP-labeling
streptavidin/avidin working solution at 37 °C for15 min. The
final reaction product was visualized with chromogen
diaminobenzidine (DAB; Sigma, USA).
RT-PCR A total RNA of 2 µg from the transfected and
non-transfected cells as mixed with the RT reaction mixture
(Invitrogen, USA). After reverse transcription, Platinum Taq
DNA polymerase (Invitrogen, USA) and the samples (total
volume 50 µL) were placed in a thermal cycler. The following
primer pairs were used when processing PCR: primers
α-synuclein-F (5'-GAA GCA GAG GGA CTC AGT GTG GTG-3')
and α-synuclein-R (5'-CT TGT ACA GGA TGG AAC ATC TGT CAG C-3') for
α-synuclein (524 bp), β-actin-F (5'-CCT CGC CTT TGC CGA TCC-3'), and
β-actin-R (5'-GGA TCT TCA TGA GGT AGT CAG TC-3') for
β-actin (620 bp). PCR amplification of α-synuclein was conducted under the same
conditions listed above in plasmid construction, and the
amplification of β-actin was conducted under the following
conditions: initial denaturation at 94 °C for 2 min, 30 s at 94
°C, 30 s at 55 °C, and 60 s at 72 °C for 30 cycles, followed by 10
min at 72 °C. After PCR, 10 µL aliquots of the reaction
mixtures were resolved on 1% agarose gel containing ethidium
bromide to identify the DNA amplicons generated.
Cell viability MTT assay is a standard method used to
assess cell viability. Briefly, the cells
(5×103 cells/well) were seeded in 96-well plates. After exposure to various
concentrations (100, 200, and 500 µmol/L) of
MPP+ and DA for 24, 48, and 72 h, 10
µL MTT, (5 g/L in PBS, Sigma, USA) solution was added to each well and the plates were incubated for an
additional 4 h at 37 °C. Then, the MTT solution in medium
was aspirated off. To achieve solubilization of the formazan
crystals formed in the viable cells, 200 µL DMSO was added
to each well. The absorbance was read at 570 nm with DMSO
as the blank.
Flow cytometry (Fluorescence-activated cell sorter
analysis) To detect early apoptosis and late
apoptosis/necrosis, the cells were stained with fluorescein isothio-
cyanate-conjugated Annexin V and propidium iodide (PI, BD
Clontech San Jose, CA, USA) after being treated with 100
µmol/L MPP+ for 24 h. Approximately
1×106 cells were washed with cold PBS before being resuspended in 200
µL cold 1×binding buffer. 10 µL Annexin V-FITC and 5 µL PI were
added and incubated for 15 min at room temperature in the
dark. A further 300 µL binding buffer was added to terminate
the reaction, and flow cytometric analysis was conducted
immediately with 20 000 cells. The experiment was repeated 3
times and the results were averaged.
DNA fragmentation assay The cells were grown to about
80% confluence and then treated with 200 µmol/L
MPP+ for 48 h. After treatment, the cells were resuspended in 100 µL
DNA lysis buffer (TaKaRa, Japan), and then the supernatant
was moved to another tube. The above step was repeated,
and 200 µL of the final supernatant was collected. The liquid
was incubated at 56 °C for 1 h by adding 20 µL 10% SDS and
20 µL proteinase K, and then reacted at 37 °C for 1 h after
adding 20 µL RNase to allow complete RNA digestion. The
DNA was precipitated with 950 µL 100% ethanol and 130 µL
precipitant for 2 h at -70 °C. The DNA was pelleted at
12 000×g for 15 min and washed twice with 80% ethanol. The
DNA was dissolved in Tris-EDTA buffer, and analyzed on
1.5% agarose gel for electrophoresis. DNA bands were
visualized with ethidium bromide under ultraviolet light and
photographed.
Compound screening The procedures were done as the
part of cell viability, but the cytotoxin was 50 µmol/L DA for
24 h of incubation. Ninety-nine compounds were added to
the medium at the concentration of
1×10-5 mol/L, respectively. These compounds were extracted from
Fraxinus sielboldiana blume, belonging to the Oleaceae family, which is widely
distributed in the east of Asia, especially in the south of
China.
Statistical analysis All results were expressed as
mean±SD. Statistical analysis was performed between the 2
groups by Student's t-test. P<0.05 indicated significant
difference.
Results
Construction of the eukaryotic expression system of
mutant A53T human α-synuclein We obtained the
mutation using the SOE method and then identified it by DNA
sequencing blasted to GeneBank No BC013293 and named it
pcDNA3.1(+)-hmα-synuclein. After the double digestion
with the Xho I and Xbal I restriction enzymes, the fragments
of inserted DNA and pcDNA3.1(+) were 517 bp and 4911 bp
as expected.
Increased α-synuclein protein expression in the cell
culture model It was identified that α-synuclein expression
increased in human SH-SY5Y cells after 90 d of stable
transfection by Western blotting and immunocytochemistry. In
the normal control or pcDNA3.1(+)-alone transfected group,
the protein expression was maintained at a normal level.
How-ever, transfected monoclones showed different levels
(Figure 1). Through analysis by Gel-Pro Analyzer software
3.1 (Media Cybernetics, LR, USA), we found that clone 1
(P<0.05) and clone 3 (P<0.01) were significantly different
compared with the control, so clone 3 was used as a cell
model for our study due to the high level expression of
exogenous mutant α-synuclein. The other 3 transfected groups
seemed to only express the exogenous resistance gene
designed in the pcDNA3.1(+) vector for their survival in the
culture medium containing G418. Different clones showed
different expression levels, which suggested that random
integration of exogenous gene into genome was not the same
copies. We also used immunocytochemistry in cell slides to
characterize the cellular localization and expression of
α-synuclein. The results showed that positive
immunostaining of overexpression was widely localized in the cytoplasm and
synapse compared with the control. Almost no staining was
detected if the primary antibody was omitted (Figure 2).
Increased α-synuclein transcriptional level in the cell
model Given the change of protein expression, we sought to
validate the transcriptional level obtained by
semiquanti-tative RT-PCR. Clearly, α-synuclein was upregulated in the
transfected cells (Figure 3).
Comparison of MPP+ and DA-induced cytotoxicity
between normal and stably transfected cells All groups of
cells were treated with a range of concentrations (100,
200, and 500 µmol/L) of MPP+ and DA for different times (24, 48,
and 72 h), and cell viability was determined by MTT assay.
MPP+ and DA induced an evident dosage and
time-dependent loss in cell viability. The treatment of cells with 100,
200, and 500 µmol/L MPP+, resulted in a cell viability loss
of about 2.6%, 8.1%, and 11.7% for 24 h; 20.2%, 24.6%, and
28.9% for 48 h; and 40.3%, 49.2%, and 62.8% for 72 h in the
pcDNA3.1(+)-alone transfected group. However, cell
viability loss was about 9.3%, 14.8%, and 25.1% for 24 h; 27.3%,
29.4%, and 45.3% for 48 h; and 49.5%, 53.8%, and 75.1% for
72 h in the clone 3 group, respectively (Figure 4A_4C). The
incubation of cells with 100, 200, and 500
µmol/L DA resulted in a cell viability loss of about 56.1%, 57.6%, and 61.8% for
24 h; 69.7%, 77.1%, and 78.5% for 48 h; and 82.8%, 89.9%, and
95.3% for 72 h in the pcDNA3.1(+)-alone transfected group,
while there was a loss of about 65.2%, 70.3%, and 75.8% for
24 h; 77.6%, 78.9%, and 84.2% for 48 h; and 90.0%, 94.0%, and
98.0% for 72 h in the clone 3 group, respectively (Figure
4D_4F). It was found that more rapid loss of cell proliferation
occurred with the incubation of DA than
MPP+, and we concluded that there was significant difference between the
pcDNA3.1(+) group and the clone 3 group in cell viability
when treated with MPP+ and DA. However, there was no
statistical difference between normal cells and transfected
cells without MPP+ or DA, and it was indicated that the cell
model would not show the more severe cytotoxicity unless it
was exposed to an exogenous cytotoxin.
We identified early apoptotic cells, late apoptotic/necrotic
cells, and viable cells by double staining the cells with PI
and Annexin V. There was a significant difference between
the pcDNA3.1(+)-alone transfected group and the clone 3
group after the cells were treated with 100 µmol/L
MPP+ for 24 h. The percentage of both Annexin V+/PI_ cells and
Annexin V+/ PI+ cells increased from 0.98% to 1.56% and
from 2.98% to 4.45%, respectively (Figure 5).
MPP+ has been described as an effective inducer for cell
apoptosis, and a laddered pattern of DNA degradation is
considered a molecular hallmark of apoptosis. Here, we
proceeded to study the DNA from cells treated with 200
µmol/L MPP+ for 48 h by means of electrophoresis in agarose gels
(Figure 6). In the transfected cells, an apoptotic pattern of
DNA degradation was easily and reproducibly detected.
However, this apoptotic pattern could not be detected in
non-transfected cells.
Compounds screening In the primary screening
experiment, 12 of the 99 compounds showed anticytotoxic
activity at the concentration of
1×10-5 mol/L (Table 1). After further screening, we found
No 66 (F.mar-m-1), No 67 (F.mar-m-27), and
No 80 (F.mar-m-16) displayed definite anticytotoxic
activity in different concentration from
1×10-8 to 1×10-4
mol/L. The EC50 were
1.376×10-6,
8.409×10-7, and
2.842×10-6 mol/L, respectivity (Figure
7).
Discussion
In the search for a better approach to compound screens
for PD, which on the one hand targets a specifically related
protein, and on the other, demonstrates anticytotoxic effects
against the cytotoxin. In this study, we constructed a
cell-based model of α-synucleinopathy to successfully
develop a SH-SY5Y cell line expressing corresponding A53T mutant
α-synuclein, which could maintain the expression level
continually. In other studies[28_30], several kinds of models
were established for the research of α-synuclein, but not for
compound screens and drug development. With the
advantage of persistent expression, the cell model was suitable for
long-time research work without repeated transfection.
Overexpression or aggregates of α-synuclein are found
in a number of neurodegenerative disorders, now termed
synucleinopathies[31]. Despite the physiological localization
of α-synuclein in presynaptic terminals, the aggregates are
present throughout the cell body. In our study, similarly
altered localization of α-synuclein was widespread in the
cell model which expressed the A53T mutant version.
Effort in developing a therapy against neurodegeneration
is therefore aimed at the inhibition of overexpression or
aggregate formation[32,33], but the role of proteins in the
pathogenesis of human neurodegenerative disease has become
an issue of debate[34] because a number of studies have not
supported their toxicity[35,36]. It was demonstrated that
overexpression of wild-type human α-synuclein rescues
mesencephalic dopaminergic cells from
MPP+-induced apoptotic cell death by attenuating cytochrome c release,
caspase-3 activation, and proteolytic activation of Protein
Kinase C-δ (PKC-δ). However, overexpression of the A53T
human α-synuclein mutant exacerbates
MPP+-induced apoptosis by augmenting proteolytic cleavage of
PKCδ[37]. So by using this cell model, we can perform the studies from
elementary compound screens to mechanism research of the
selected compounds for drug development as this model
either imitates the reaction of human neurons in
vitro or simultaneously represents the interactions between
overexpression of mutant α-synuclein and active compounds.
Although it has been suggested that α-synuclein
aggregation may stimulate apoptosis, it remains elusive whether
programmed cell death itself is directly responsible for
neuronal loss, especially considering the observations that
α-synuclein may also exert anti-apoptotic
activity[38,39]. So its potential cytotoxic effects when encountering cytotoxin
were tested in this study between normal and
stably-transfected cells with MPP+ and DA on cell viability. The addition
of MPP+ or DA led to more severe α-synuclein-dependent
growth inhibition in transfected cells than in non-transfected
cells. It was suggested that the model overexpressing
mutant α-synuclein could show much more vulnerability to
cytotoxin compared with normal cells. When compound
screens were carried out, the candidates with anticytotoxic
activity would be screened out by the cell model.
Apoptosis and necrosis are 2 modes of cell death in
nucleated eukaryotic cells. Apoptosis is a programmed cell death
characterized by changes in condensation of nuclear
chromatin, cytoplasmic blebbing, and exposure of phosphatidylserine (PS) residues on the outside of the plasma
membrane[40]. Necrosis, on the other hand, is accidental cell
death and indicates the features of mitochondrial swelling,
rupture of the plasma membrane, and the release of
cytoplasmic constituents[41]. A key event of apoptosis in the
early stage is that PS of the inner
Leaflet of the cell membrane appears in the outer leaflet,
becoming a molecular marker for phagocytosis. Annexin V,
which preferentially binds to PS, is very useful in detecting
the expression of PS on the surface of apoptotic cells.
Although neuronal apoptosis is a normal event during
development, many neurodegenerative diseases are thought
to involve abnormal cell death that leads to the damage of
the nervous system. In our study, when
MPP+ existed, there was a significant difference between the control and the
transfected group, reflecting the increase of early apoptosis
and late apoptosis/necrosis in addition to the appearance of
the DNA "ladder". It was concluded that the cell model
showed much more sensitivity to cytotoxin than
non-transfected cells, which could demonstrated the validity of the
cell model.
After high throughput screening of 99 compounds, we
found that 12 could decrease DA-induced cytotoxicity, and
3 of the 12 had definite anticytotoxic activity. The
preliminary result indicated that the 3 compounds deserved further
research because of their therapeutic potential.
In summary, this study presented a cell-based model that
recapitulated pathological properties of mutant α-synuclein,
which is particularly associated with the severer phenotype
of familial PD[42], and demonstrated the potential
cytotoxicity of A53T mutant α-synuclein. This cellular model can be
used for further, fundamental studies associated with the
underlying mechanisms of overexpression and genetic or
environmental effects of α-synuclein-mediated cytotoxicity.
The model also can be applied to the screening of drugs with
therapeutic potential for synucleinopathies.
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