www.elsevier.com/locate/ynbdi

Neurobiology of Disease 19 (2005) 419–426

Synaptophysin enhances the neuroprotection of VMAT2 in

MPP+-induced toxicity in MN9D cells

Carol X.-Q. Chen, Steven Y. Huang,1 Limei Zhang, and Yong-Jian LiuT

Departments of Neurology and Neurobiology, University of Pittsburgh School of Medicine, W958 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh,

PA 15213, USA

Received 4 August 2004; revised 10 January 2005; accepted 20 January 2005

Available online 5 March 2005

The use of the potent neurotoxin MPTP in producing a model for

Parkinson’s disease (PD) has allowed us to dissect the cellular processes

responsible for both selective neuronal vulnerability and neuroprotec-

tion in idiopathic PD. It has been suggested that vesicular monoamine

transporters (VMATs) play a critical neuroprotective role in MPP+

toxicity. However, little is known about how this detoxificative

sequestration in dopaminergic (DAergic) neurons is regulated at the

molecular and cellular levels. Using the DAergic cell line MN9D as an

in vitro model, we found that overexpression of VMAT2 (a neuronal

isoform of VMATs) protects the transformants from MPP+-induced

toxicity, consistent with the previous work on fibroblastic CHO cells.

We further found that the MN9D cells displayed lower expression levels

of secretory vesicle proteins such as synaptophysin. Overexpression of

synaptophysin in MN9D cells can significantly increase the resistance

of the transformants to MPP+ toxicity. The co-expression of VMAT2

and synaptophysin has shown synergistic protection for the trans-

formants, suggesting a role of synaptophysin in the biogenesis of

secretory vesicles and in influencing the targeting of VMAT2 to these

vesicles. Our work indicates that both the expression level of VMAT2

and capacity of vesicular packaging of DA are important in protecting

DAergic cells from MPP+ toxicity.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Parkinson’s disease; Neuroprotection; MPP+ toxicity; MN9D;

VMAT2; Synaptophysin

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.01.014

Abbreviations: DAT, dopamine transporter; DA, dopamine; DAergic,

dopaminergic; EGFP, enhanced green fluorescent protein; AMPT, a-

methyl-p-tyrosine; PNS, postnuclear supernatant; Syn, synaptophysin;

TH, tyrosine hydroxylase; TBZ, tetrabenazine; VMAT2, vesicular mono-

amine transporter 2.

* Corresponding author. Fax: +1 412 624 9914.

E-mail address: yjliu@pitt.edu (Y.-J. Liu).1 Present address: AEMTEK, Inc., 46309 Warm Springs Blvd., Suite A,

Fremont, CA, USA.

Available online on ScienceDirect (www.sciencedirect.com).

Introduction

In several animal models, the neurotoxin MPTP induces a

selective loss of dopamine (DA) neurons in the substantia nigra of

the midbrain, and this loss resembles a clinical symptom

occurring in idiopathic Parkinson’s disease (PD) (Kopin and

Markey, 1988; Langston, 1987; Przedborski et al., 2001). The in

vitro neurotoxicity study on cell lines or primary cultures using

MPP+, an active form of MPTP, has allowed dissection of the

cellular processes responsible for both selective neuronal vulner-

ability and neuroprotection in idiopathic PD. The vesicular

sequestration of MPP+ mediated by overexpressed vesicular

monoamine transporters (VMATs) has provided a mechanism

for protecting fibroblastic CHO transformants from MPP+-induced

toxicity (Liu et al., 1992a) (for a review, see Ahnert-Hilger et al.,

2003; Gainetdinov and Caron, 2003; Liu and Edwards, 1997b;

Uhl et al., 2000). However, little has been done to directly

confirm whether similar neuroprotection of VMATs occurs in

dopaminergic (DAergic) cell lines or DAergic neurons. More

importantly, little is known about how this protection is regulated

at the molecular and cellular levels. As a neuronal isoform of the

vesicular transporters, VMAT2 normally transports monoamines

from both de novo synthesis and reuptake from the synapse into

secretory vesicles for regulated release (Kanner and Schuldiner,

1987). VMAT2 in DAergic neurons thus appears to play a dual

role: neuroprotection by detoxification and signaling in synaptic

transmission. In support of their potential role in detoxification,

the transporters share sequence similarity with a group of bacteria

multidrug resistant genes (Liu et al., 1992b; Schuldiner et al.,

1995). Nonetheless, the loss of DAergic neurons in the MPTP

model in the presence of VMAT2 has raised a reasonable doubt

concerning whether VMAT2 is sufficient to protect DAergic

neurons in vivo.

Over the past decade, there has been limited clinical evidence in

support of the neuroprotective function of VMAT2 although the

transporter has in fact served as a marker for assessment of PD

progression (Brooks et al., 2003). However, several recent studies

on VMAT2 knockout mice strongly suggest that impaired DA

packaging may induce DAergic neuron susceptibility to neurotoxins

C.X.-Q. Chen et al. / Neurobiology of Disease 19 (2005) 419–426420

(Fon et al., 1997; Gainetdinov et al., 1998; Takahashi et al., 1997;

Wang et al., 1997). These independent studies have consistently

shown that heterozygote VMAT2 knockout mice display increased

MPTP neurotoxicity and increased methamphetamine neurotoxic-

ity, suggesting that VMAT2-mediated protection correlates with the

expression level of the transporter. The VMAT2 knockdown model

has also shown consistent results with increased vulnerability to

MPTP toxicity (Mooslehner et al., 2001). More recently, Staal and

his colleagues suggested that higher expression of VMAT2 and

greater ability to sequester MPP+ in rat than in mouse may

contribute to the reduced vulnerability of rat DAergic neurons to

MPP+ neurotoxicity (Staal et al., 2000). The recent report on the

correlation between lower VMAT2 expression and higher neuro-

melanin pigment accumulation in human ventral substantia nigra

neurons also indicates a protective role for the transporter (Liang et

al., 2004). Interestingly, the significant resistance of the mutant

mouse Tottering to MPTP partially results from hyperinnervation of

noradrenergic terminals in the brain, which presumably offers

higher vesicular storage capacity in MPP+ sequestration (Kilbourn

et al., 1998). Furthermore, the neuroprotection of the transporter has

also been observed in CHO cells in dopamine toxicity (Weingarten

and Zhou, 2001). However, lack of an in vitro model for VMAT2

neuroprotection prevents further study of molecular mechanisms

involved in the protection and its regulation.

Secretory vesicles provide a reducing environment for the

function of VMAT2 during the storage of monoamines (Liu and

Edwards, 1997b). Thus, the availability and the function of

secretory vesicles might determine the capacity by which VMAT2

mediates sequestration of MPP+ and other neurotoxins. It has been

suggested that the impaired function of secretory vesicles at nerve

terminals might be associated with the pathogenesis caused by

overexpression of mutant proteins such as a-synuclein and parkin

in PD (Lotharius and Brundin, 2002; Zhang et al., 2000). However,

little is known about how vesicular proteins such as VMAT2 and

synaptophysin target to these specified vesicles and permit them to

perform normal function in synaptic transmission. On the other

hand, the size and contents of secretory vesicles may also regulate

the function. One such example is that the secretory granules (120

nm in diameter, counterparts of large dense core vesicles in brain)

in PC12 cells can concentrate monoamines more than 10,000 times

within the granules (~5 mM in lumen of vesicles) over the

cytoplasmic levels (Winkler, 1976). In contrast, synaptic vesicles

(40 nm in diameter) display less than 1000 times concentration.

This might contribute to the higher capacity for MPP+ detoxifica-

tion in PC12 cells than in other neuronal cell lines.

In this study, we used a DAergic cell line, MN9D, to establish

an in vitro model for MPP+ toxicity study. We aimed to determine

whether VMAT2 expression can indeed protect DAergic cells, as

suggested by the study with fibroblast CHO cells. Furthermore, our

study explored some unknown features of MN9D cells such as the

transport activity for dopamine transporter (DAT) and VMAT2,

and secretory vesicle properties.

Materials and methods

Chemicals

General chemicals used in this work were purchased from

Sigma (St. Louis, MO) unless otherwise noted. The following

antibodies were used in the experiments: polyclonal anti-synapto-

physin (1:6000 for blotting and 1:200 for immunofluorescent

staining, Zymed, South San Francisco, CA); monoclonal anti-

synaptophysin (1:4000 for blotting and 1:200 for immunofluor-

escent staining, Sigma); polyclonal anti-secretogranin (1:6000 for

blotting, Biodesign, Saco, ME); monoclonal anti-SV2 (1:3000 for

blotting, Hybridoma Bank, Iowa City, IA); monoclonal anti-

synapsin I (1:3000 for blotting, Chemicon, Temecula, CA), and

monoclonal anti-h-actin (1:5000 for blotting, Sigma).

Cell culture and transfection

All cells were maintained in 5% CO2 at 378C in medium

containing penicillin and streptomycin. PC12 cells were grown in

high-glucose Dulbecco’s modified Eagle’s-H21 medium (Invitro-

gen, San Diego, CA) supplemented with 5% Cosmic calf serum

and 10% equine serum (Hyclone, Logan, UT) as described

previously (Liu et al., 1994). MN9D and SY5Y cells were grown

in high-glucose DMEM supplemented with 10% fetal bovine

serum. CHO cells were grown in F12 supplemented with 10%

Cosmic calf serum. For making stable MN9D transformants, the

cells were transfected with VMAT2-EGFP-N2, synaptophysin/

pcDNA3.1/hygro, or pcDNA3.1/hygro vector alone using a

modified calcium phosphate transfection method. The transfected

cells were then treated for selection by either G418 (500 Ag/ml) or

hygromycin B (300 Ag/ml, Invitrogen, San Diego, CA). Positive

clones for EGFP-VMAT2 were selected by FACS (MoFlo high-

performance cell sorter, Cytomation, Inc., Fort Collins, CO). Other

positive transformants were screened by both Western blot analysis

and immunofluorescence staining. Several synaptophysin trans-

formants were further transfected with HA-VMAT2 and selected

by G418 to obtain double transformants.

cDNA plasmid constructs

An HA-tagged VMAT2 cDNA (Tan et al., 1998) was recovered

from a pCDNA3 vector by HindIII/XbaI digestion and was

subcloned into pEGFP-N2 vector (BD Clontech, CA). Synaptophy-

sin was amplified from a PC12 cell library using PCR with a pair of

primers (5V-ACTAAGCTTCATTGCTGCTGCTGCTGCTGCT-3Vand 5V-ATCTCGAGAGGCCTTCTC TTGAGCTGTT-3V) contain-ing restriction sites for HindIII and XhoI, respectively. Amplified

PCR fragment was digested and subcloned into pcDNA3.1/hygro

expression vector (Invitrogen). cDNA plasmids were prepared using

Qiagen Endo-free Midiprep kits (USA-Qiagen, Valencia, CA).

Cytotoxicity assay

About 3 � 104 cells to be tested were plated in each well of 24-

well plates for 24 h before adding the neurotoxin MPP+ at

concentrations between 10 and 1000 AM. After 3 days of

treatment, the cells were harvested with trypsin–EDTA (0.05%)

and stained with trypan blue. Live cells were then counted using a

hemacytometer. The percent viability of cells was calculated based

on the number of live cells divided by cells from the non-treated

control.

Western blotting analysis

Cells were harvested in buffer A (150 mM NaCl, 10 mM Hepes

(pH 7.4), 1 mM EGTA, and 0.1 mM MgCl2) with protease

inhibitors (0.4 nM PMSF, 2 ng/ml leupeptin, and 1 Ag/ml pepstatin).

Fig. 1. MN9D cell line is more sensitive to MPP+ toxicity than other

monoaminergic cell lines. MPP+ toxicity assay was performed on MN9D,

PC12, SY5Y, and CHO cells. Cells were subsequently treated with different

concentrations of MPP+ (0–1 mM) for 3 days. Cells were harvested with

0.025% trypsin–EDTA and stained with trypan blue. The cell viability (%)

is expressed as percentage of survival cells compared with that in non-

MPP+-treated control. Data are mean F SD (bars) values from four

experiments, each one of which was performed with duplicate incubations.

C.X.-Q. Chen et al. / Neurobiology of Disease 19 (2005) 419–426 421

The cells were then homogenized by sonication and centrifuged at

3000 rpm for 5 min at 48C to obtain postnuclear supernatant (PNS).

The PNS was further sedimented at 100,000 � g for 30 min at 48Cto obtain membrane protein fraction. The membrane pellet was

further resuspended in 3� SDS sample buffer (New England

Biolab, Beverly, MA) and incubated at room temperature for 5 min.

A 10–100 Ag protein sample was separated by electrophoresis

through 10% SDS–PAGE. After electrophoresis, the proteins were

transferred to nitrocellulose (BA-85, Schleicher-Schuell Bio-

science, Keene, NH), and HA-VMAT2 and synaptophysin were

visualized by enhanced chemiluminescence (Supersignal West

Pico, Pierce, Rockford, IL) using monoclonal anti-HA.11 (Babco,

Berkeley, CA) at a 1:3000 dilution and anti-synaptophysin at

1:6000 dilution, respectively. The secondary HRP-conjugated anti-

mouse antibody was used at a dilution of 1:5000 (Amersham,

Arlington Heights, IL). Protein concentration was determined by

protein assay kit (Bio-Rad, Hercules, CA). To quantify the intensity

of the detected bands, a densitometer was used (Personal

Densitometer S1, Molecular Dynamics, Amersham Biosciences,

Piscataway, NJ).

Subcellular fractionation

For cell fractionation by equilibrium sedimentation, the PNS

was layered onto a linear 0.65 to 1.55M sucrose gradient for

sedimentation in an SW41 rotor (Beckman Instruments, Palo Alto,

CA) at 30,000 rpm for 6 h at 48C. Fractions were collected from

the bottom of the tube and analyzed by Western blot.

Uptake assays

For the DAT uptake assay, intact cells cultured in 24-well

plates were used. 1 � 104 cells per well were plated on poly-d-

lysine pre-coated 24-well plates 1 day before the assay. Cells

were pretreated with or without 1 AM mazindol or 2 AMGBR12909 for 20 min, followed by a brief rinse with 0.5 ml of

ice-cold PBS solution supplemented with 1 mM MgCl2 and 0.1

mM CaCl2. Cells were then incubated in 300 Al of 10 nM [3H]-

MPP+ with or without mazindol at room temperature at intervals

of 0, 5, 10, 30, 60, and 90 min. The uptake was terminated by

rapid rinse with ice-cold PBS w/Ca2+, Mg2+ three times. Cell

extracts were prepared by solubilizing cells by 1% SDS, and the

radioactivity was determined by liquid scintillation counting. A

VMAT2 uptake assay was performed as described previously (Liu

and Edwards, 1997a). Briefly, PNS of cells were prepared as

described above. Since VMAT2 showed similar affinity to

monoamine substrates (Peter et al., 1994), for measuring the

transport activity of VMAT2 we chose [1,2-3H]-serotonin (NEN,

Boston, MA) over other monoamines because of its availability

and stability (substrates such as dopamine have a higher tendency

toward auto-oxidation; Stokes et al., 1999). In this transport

activity assay, 10 Al (1 Ag protein/Al) of PNS were incubated with

20 nM of [1,2-3H]-serotonin at 308C for the indicated times. The

uptake was terminated by filtration separation through a 0.45-AmMillipore filter (Millipore, Billerica, MA). The dried filters were

then used for scintillation counting.

Tetrabenazine binding assay

The binding assay was performed essentially as previously

described (Scherman and Henry, 1981). Membrane proteins

(50–100 Ag) from PNS preparation were incubated in 300 Al ofbinding buffer (0.3 M sucrose, 10 mM HEPES, pH 7.2) with 3

nM of [2-3H] a-dihydrotetrabenazine (3H-TBZ, ART-496, Amer-

ican Radiolabeled Chemicals, Inc., St. Louis, MO) at room

temperature. After 15–30 min incubation, the reaction was

stopped by the addition of 4 ml of ice-cold binding buffer

containing 10 AM TBZ and filtered through 0.45 Am HAWP

Millipore filters pretreated with 0.3% polyethylenimine. The

filters were then washed once more with 4 ml of the same buffer,

and the radioactivity was measured by liquid scintillation counter

(Beckman 9000, Beckman Coulter, Inc., Fullerton, CA). Non-

specific binding was determined by the addition of 2 AM cold

TBZ to the binding buffer.

Results

The MN9D cell line is more sensitive to MPP+ toxicity than are

other monoaminergic cell lines

To identify a cell line that may resemble the DAergic neurons

sensitive to MPP+, four cell lines, CHO, MN9D, PC12, and SH-

SY5Y, were chosen for the MPP+-induced toxicity assay. PC12

cells were more resistant to MPP+ than other cell lines with

EC50 of MPP+ about 500 AM (Fig. 1). SH-SY5Y cells also

showed responses to MPP+ similar to that of PC12 cells. As a

dopaminergic cell line, MN9D cells were more susceptible to

MPP+ than other cell lines with EC50 of ~60 AM. The

fibroblastic CHO cells showed a level of sensitivity between

those of the MN9D and the other two cell lines (Fig. 1). To

examine if accumulation of MPP+ was mediated by dopamine

transporter (DAT) and responsible for the selectivity, a [3H]-

MPP+ uptake assay was performed. All cell lines except the CHO

cell line showed similar time-dependent uptake of MPP+ in intact

cells (Fig. 2a). The uptake was inhibited by the DAT-specific

antagonists Mazindol (Fig. 2a) and GBR12909 (data not shown).

The results indicate that selective DAT-mediated uptake of MPP+

Fig. 3. Overexpression of VMAT2 protects MN9D transformants from

MPP+ toxicity. Cells cultured for 1 day in 24-well plates were treated with

different concentrations of MPP+ (0–200 AM) in the presence or absence of

2 AM reserpine for 3 days. The viable cells were counted, and the viability

was expressed as percentage of survival cells compared with that in non-

MPP+-treated control. Each value represents three to five independent

experiments in duplicate for each cell line. Overexpression of VMAT2

protects MN9D transformants from MPP+ toxicity, which is abolished by

reserpine.

Fig. 2. Vesicular transport activity of VMAT2 contributes partially to the

susceptibility of MN9D cells to MPP+ toxicity. (a) DAT transport activity of

different cell lines. 1 � 104 cells per well of different cell lines were

pretreated with or without 1 AM mazindol, followed by incubation with

[3H]-MPP+ for indicated time intervals. Cell lysates were collected and

measured by liquid scintillation counting. (b) Vesicular transport activity of

the monoaminergic cell lines. PNS of four different cell lines was used for

vesicular transport activity of VMAT using [3H]-serotonin as a substrate

with a filtration method (see Materials and methods section for details).

Data are presented with 5- and 15-min time points, both in the linear range

of the time course. Data are mean F SD (bars) values from three

experiments, each of which was performed with duplicate incubations.

C.X.-Q. Chen et al. / Neurobiology of Disease 19 (2005) 419–426422

was not responsible for the supersensitivity of MN9D cells to the

neurotoxin. To further examine if protective mechanisms such as

VMAT activity were involved in the selectivity of MPP+ toxicity

among these cell lines, vesicular transport activity was analyzed

using cell homogenates. As shown in Fig. 2b, for all cell lines

tested, PC12 cells expressed the highest vesicular uptake activity

for MPP+. Both MN9D and SH-SY5Y cells showed relatively

lower uptake activity compared with PC12 cells. The results

suggest that VMAT2 activity may partially contribute to the

susceptibility of MN9D cells to MPP+ toxicity, of which the

EC50 is similar to that seen in dopaminergic neurons from

primary culture studies (Lotharius and O’Malley, 2000).

VMAT2 protects MN9D cells

To examine if overexpression of VMAT2 protects MN9D

cells from MPP+ toxicity, enhanced GFP-tagged VMAT2 was

heterogeneously expressed in MN9D cells. The EGFP was

chosen as an indicator to facilitate the selection and maintenance

of positive transformants through FACS (fluorescent-activated

cell sorter). The normal transport activity and subcellular

localization of N-terminal EGFP-tagged VMAT2 (VMAT2-

EGFP-N2) were confirmed previously in the laboratory (data

not shown). The toxicity assay following 3 days of treatment of

MPP+ showed that the viability of MN9D transformants was

2.5-fold higher than that of wild-type cells (Fig. 3). The

enhanced resistance seen in MN9D transformants could be

abolished by the treatment with 2 AM reserpine, a specific

inhibitor for both VMAT1 and VMAT2, indicating a role of

VMAT2 in neuroprotection. Furthermore, a VMAT2 uptake

activity assay was performed on either wild-type or stably

transfected MN9D cells. Consistent with the neuroprotection of

VMAT2 in the transformants, MN9D cells that stably overex-

pressed VMAT2 showed increased transport activity compared

with wild-type cells (Fig. 8). Although the MN9D cell line is

dopaminergic, Western analysis failed to show detectable

amounts of VMAT2 expression in the wild-type MN9D cells

(data not shown). However, tetrabenazine binding did show a

very low level of VMAT2 expression at 0.16 pmol/mg protein,

which is consistent with that in dopaminergic neurons in the

brain (Fon et al., 1997).

Lower expression of secretory vesicle markers is present in MN9D

cells

To examine if overexpressed VMAT2 is localized to

secretory vesicles for which VMAT2 plays a role for sequestra-

tion of MPP+, we used both immunofluorescent staining and

subcellular localization. Surprisingly, several secretory vesicle

markers such as synaptophysin, secretogranin II, and SV2 were

expressed at a substantially lower level in MN9D cells than in

Fig. 5. Overexpression of synaptophysin attenuates MPP+ toxicity in MN9D

cells. (a) Synaptophysin-stable transformants were used to perform the

toxicity assay. pcDNA3.1/hygro vector-transfected MN9D transformants

were used as control. (b) Synergistic neuroprotective effect by double

overexpression of VMAT2 and synaptophysin in MN9D cells. The experi-

ments were performed essentially the same way as in Fig. 1.

Fig. 6. VMAT2 colocalizes with synaptophysin on synaptic vesicles in

MN9D cells. Density sucrose gradient fractionation was performed by sedi-

mentation of PNS of MN9D transformants through an equilibrium gradient

composed of 0.6–1.6 M sucrose. Fractions were collected and analyzed by

Western blot. VMAT2 was co-migrated with synaptophysin enriched in fracQ

tions 8–10 for secretory vesicles. Fractions were labeled from top to bottom.

Fig. 4. Low expression of secretory vesicle marker proteins in MN9D cells.

Membrane proteins from PNS (10 Ag for PC12 cells and 100 Ag for MN9D

cells) were loaded for PAGE and Western analyzed for secretory vesicle

marker proteins such as synaptophysin, secretogranin II, and SV2. To detect

synaptophysin in MN9D cell PNS, 100 Ag protein was used. Protein

samples were further normalized by h-actin.

C.X.-Q. Chen et al. / Neurobiology of Disease 19 (2005) 419–426 423

PC12 cells (Fig. 4). More specifically, the expression level of

synaptophysin was about 30- to 50-fold less than that in PC12

cells (Fig. 4, quantitative data not shown), suggesting that the

relatively small number of secretory vesicles in MN9D cells

might be responsible for their supersensitivity to the neurotoxin

MPP+.

Overexpressed synaptophysin attenuates MPP+ toxicity

Previous evidence has suggested that synaptophysin may play

a role in the biogenesis of secretory vesicles (Thiele et al., 2000).

We hypothesized that a low expression of synaptophysin

contributes to the reduced biogenesis of secretory vesicles in

MN9D cells. Therefore, we tested whether heterogeneous over-

expression of synaptophysin would increase the generation of

secretory vesicles and then the capacity of detoxification by

sequestration of MPP+ in MN9D cells. Determined by toxicity

assay, the synaptophysin-overexpressed cells showed increased

viability, with EC50 of ~92 AM in MPP+ treatment (Fig. 5a). This

result is comparable to that of VMAT2 transformants in the

MPP+ toxicity study (Fig. 3, EC50 = ~115 AM). The transport

activity assay also suggested an increased endogenous expression

of VMAT2 in secretory vesicles (refer to Discussion). Further-

more, the tetrabenazine binding assay on the synaptophysin

transformants has shown a consistent increase in endogenous

VMAT2 expression, from 0.27 pmol/mg protein to 0.41 pmol/mg

protein. Similar results on decreased sensitivity to MPP+ were

obtained from transient synaptophysin-transfected MN9D cells

(data not shown).

Co-expression of VMAT2 and synaptophysin provides synergistic

protection of MN9D transformants

To examine if overexpression of synaptophysin can facilitate

the neuroprotection of VMAT2 in MPP+ toxicity, we co-

expressed two proteins in MN9D cells. The transformants that

overexpress both proteins showed a ~2-fold increase in EC50 of

MPP+ compared with the overexpression of either protein alone

in the MPP+ toxicity assay (Fig. 5b). Both proteins also co-

localized to secretory vesicles as shown by double immuno-

fluorescent staining (data not shown) and subcellular fractionation

(Fig. 6). The results suggest a role for vesicular sequestration of

Fig. 9. Inhibition of dopamine synthesis attenuates MPP+ toxicity. MN9D

transformants transfected by either pcDNA3/RSV or VMAT2-EGFP-N2

were pretreated with or without 100 AM MPT for 24 h followed by the

addition of 50 or 200 AM MPP+. The viability of cells was measured using

essentially the same procedure as that in Fig. 1. MPT protects MN9D/

pcDNA3 from MPP+ toxicity but does not enhance VMAT2 protection.

Data are presented as meanF SD (bars) values from four experiments, each

of which was performed with quadruplicate incubations.

Fig. 7. Increased expression of secretory vesicle proteins in synaptophysin

and VMAT2 double-transfected MN9D cells. Lane 1, PC12/WT; Lane 2,

MN9D/WT; Lane 3, MN9D/Syn+VMAT2; Lane 4, MN9D/Syn. Membrane

proteins from PNS (10 Ag for PC12 cells and 100 Ag for MN9D cells) were

loaded for PAGE and Western blotting. The protein concentration was

further normalized by h-actin.

C.X.-Q. Chen et al. / Neurobiology of Disease 19 (2005) 419–426424

the neurotoxin MPP+ by secretory vesicles in which VMAT2 is

localized. Furthermore, several secretory vesicle markers also

showed increased expression levels. The increase was about

twofold for secretogranin II and fourfold for SV2 (Fig. 7),

indicating an increased biogenesis of secretory vesicles in the

transformants. In addition, the transport activity also suggests an

increased uptake of [3H]-serotonin, an indication of VMAT2

function (Fig. 8).

Inhibition of dopamine synthesis attenuates MPP+ toxicity

To test whether dopamine toxicity is involved inMPP+-mediated

toxicity and VMAT2 is sufficient for protection, we used a-methyl-

p-tyrosine (AMPT) as a TH-specific blocker to inhibit the synthesis

of dopamine in MN9D cells. As shown in Fig. 9, the inhibition of

dopamine synthesis can significantly reduce MPP+ toxicity by 30%.

Fig. 8. Neuroprotection of MN9D transformants correlates with the

transport activity of VMAT2. PNS of MN9D transformants was used for

transport activity assay with a filtration method (see Materials and methods

section for detail). Wild-type MN9D cells were used as control. Data are

presented as mean F SD (bars) values from at least three experiments, each

of which was performed with duplicate incubations. *P b 0.05, vs. control

(MN9D/WT), **P b 0.01 vs. control (MN9D/WT), #P b 0.05 vs. MN9D/

VMAT2.

This neuroprotection does not synergize with that by VMAT2

overexpression.

Discussion

We have shown that VMAT2 protects a DAergic cell line,

MN9D, from MPP+-induced toxicity. Our results also indicate that

the sensitivity of MN9D cells to MPP+ toxicity is relatively higher

than that of other dopamine-producing cell lines. This feature of

MN9D cells is consistent with that observed in primary dopamine

neurons. We have reported DAT-like activity in MN9D cells, which

is believed to be important for MPP+ toxicity. We also found that

the MN9D cells displayed lower expression levels of secretory

vesicle markers, suggesting a low amount of secretory vesicles in

this cell line. In our studies, the overexpression of either VMAT2

or synaptophysin in MN9D cells has shown significant increases in

resistance to MPP+ toxicity. Furthermore, the co-expression of two

proteins that may promote the vesicular targeting of VMAT2 and

the biogenesis of secretory vesicles has also shown synergistic

protection for the transformants.

Dopaminergic cell lines and MPP+ toxicity

Our results suggest that dopaminergic cell lines vary dramati-

cally in their response to MPP+ toxicity. This variation may be

determined by differences in DAT expression, expression level of

VMATs, and vesicle type (secretory granules versus synaptic

vesicles) and amount. Previously, we used the CHO cell line for

molecular cloning of VMAT2 (Liu et al., 1992b). Our present data

are consistent with previous unpublished work in which no

catecholamine transport activity was detected in wild-type CHO

cells. The MPP+-induced cell death might result from low

cytoplasmic accumulation of MPP+ upon exposure to high

concentrations of MPP+ (EC50 of 150 AM). In contrast, higher

cytoplasmic MPP+ (shown in Fig. 2) mediated by the amine

transporter system may occur in DAergic cell lines when they are

C.X.-Q. Chen et al. / Neurobiology of Disease 19 (2005) 419–426 425

exposed to much lower concentrations of MPP+ (e.g., EC50 of 60

AM for MN9D cells). However, DAT expression is not the only

deciding factor given that SH-SY5Y cells express high levels of

DAT but are resistant to MPP+ toxicity. Our results confirm the

notion that the capacity of detoxification can also be determined by

the expression of VMATs and the amount of secretory vesicles

where VMATs are localized. Early studies on PC12 cells have

demonstrated a relative resistance to the toxicity of MPP+ (Denton

and Howard, 1987; Greene and Rein, 1978). The vesicular

accumulation of [3H]-MPP+ in chromaffin cell homogenates

indicated a specific mechanism in this neuroprotection, which

further led to identification of the VMAT gene family (Liu et al.,

1992a; Reinhard et al., 1987). Additional in vitro pharmacological

studies have clearly supported a role of VMATs in the vesicular

sequestration of neurotoxin (Liu et al., 1992b; Peter et al., 1994).

Finally, differences in type and amount of secretory vesicles may

play a role. The lower expression level of VMAT2 in SH-SY5Y

determined by our uptake assay may indicate that VMAT2

expression does not explain resistance in this cell line. One possible

explanation is that this resistance may be attributed to the higher

detoxification capacity mediated by secretory granules which are

abundant in SY5Y cells (unpublished data). This secretory vesicle

profile represents a neuroendocrine phenotype similar to that of

PC12 cells, which are also relatively resistant. Thus, all these factors

must be considered in modeling MPP+ toxicity.

MN9D cells have demonstrated their potential as an in vitro

system for studying MPP+ toxicity because of their dopamine

neuron origin (Choi et al., 1991, 1992). Their sensitivity to MPP+

toxicity from our study is within the range of that seen in primary

dopamine neuronal cultures (Lotharius et al., 1999). Our character-

ization of this cell line has also indicated that both DAT-like

activity and low levels of VMAT2-like activity were detected. Our

RT-PCR analysis also indicates the expression of VMAT2 but not

VMAT1 in this cell line (data not shown). On the other hand, our

pharmacological analysis using both mazindol and GBR12909

inhibitors may not exclusively rule out whether other amine

transporters such as serotonin transporter are expressed. As MPP+

is a substrate for all monoamine transporters with similar affinity,

MPP+ toxicity may be also mediated by transporters other than

DAT. Analysis of the neurotransmitter transporter profile for

MN9D cells might provide some important information about

molecular mechanisms underlying MPP+ toxicity. However, it

seems inevitable that for any of the cell lines used in PD studies,

certain limitations may exist. In the case of MN9D cells, low

expression of secretory vesicle marker proteins may suggest a

relatively low amount of secretory vesicles as well as lower

capacity for detoxification. Overexpression of synaptophysin

increases the resistance of the transformants, which may result

from increased vesicular capacity for detoxification, an important

aspect in understanding the cellular mechanism underlying MPP+

toxicity.

VMAT2 detoxification and dopamine toxicity

As indicated by previous studies, vesicular sequestration of the

neurotoxin MPP+ may play a critical role in the neuroprotection of

fibroblast CHO transformants by VMAT2 (Liu et al., 1992a).

However, normal function of VMAT2 in the brain is mainly

involved in the packaging of monoamines, including dopamine,

into secretory vesicles (e.g., synaptic vesicles and large dense-core

vesicles). This raises the possibility that VMAT2 may be essential

in DAergic neurons to prevent cytoplasmic DA oxidation by

sequestering DA into vesicles. In contrast, under pathological

situations such as MPP+ insult, endogenous expression of the

transporter might not be sufficient to protect the cells. Interestingly,

a recent report indicated that DA may mediate MPP+ toxicity

through its intracellular oxidation and through production of non-

mitochondrial reactive oxygen species (ROS) (Lotharius and

O’Malley, 2000). Our result also supports this observation in that

the inhibition of DA synthesis attenuated MPP+ toxicity. More

importantly, depletion of DA did not enhance VMAT2 protection,

suggesting that vesicular sequestration of neurotoxins such as

MPP+ or DA may be sufficient for executing VMAT’s neuro-

protection. It would be interesting to examine whether vesicular

DA efflux occurs during MPP+-induced toxicity and how this

process is related to the pathogenesis of PD.

Synaptophysin and biogenesis of secretory vesicles

Although the function of synaptophysin remains unknown,

evidence has suggested that this abundant synaptic vesicle protein

may be involved in the biogenesis of secretory vesicles (Daly et al.,

2000; Thiele et al., 2000). Furthermore, it has also been speculated

that synaptophysin may play a role in stabilizing and modifying the

function of other synaptic proteins (Becher et al., 1999; Daly et al.,

2000; Edelmann et al., 1995; Galli et al., 1996). Our studies have

shown that VMAT2 may require dimerization in order to function

(unpublished data). It would be interesting to test whether

synaptophysin increases VMAT2 function through its interaction

with the transporter or other mechanisms. On the other hand, the

overexpression of synaptophysin may promote the formation of

secretory vesicles as suggested by the increased expression level of

other membrane proteins such as secretogranin II and SV2.

Therefore, the increased amount of secretory vesicle may enhance

the function of VMAT2 by providing membrane surface for protein

targeting and vesicular capacity for sequestering the neurotoxin

molecules.

Acknowledgments

We gratefully acknowledge Dr. Alfred Heller for providing

MN9D cells. We thank Ms. Carol Culver for reviewing the

manuscript. This work was supported by grants from the National

Institutes of Health, the Scaife Charitable Foundations and CMRF

of the University of Pittsburgh Medical Center (to Y. L.).

References

Ahnert-Hilger, G., Holtje, M., Pahner, I., Winter, S., Brunk, I., 2003.

Regulation of vesicular neurotransmitter transporters. Rev. Physiol.,

Biochem. Pharmacol. 150, 140–160.

Becher, A., Drenckhahn, A., Pahner, I., Margittai, M., Jahn, R., Ahnert-

Hilger, G., 1999. The synaptophysin–synaptobrevin complex: a hall-

mark of synaptic vesicle maturation. J. Neurosci. 19, 1922–1931.

Brooks, D.J., Frey, K.A., Marek, K.L., Oakes, D., Paty, D., Prentice, R.,

Shults, C.W., Stoessl, A.J., 2003. Assessment of neuroimaging

techniques as biomarkers of the progression of Parkinson’s disease.

Exp. Neurol. 184 (Suppl. 1), S68–S79.

Choi, H.K., Won, L.A., Kontur, P.J., Hammond, D.N., Fox, A.P., Wainer,

B.H., Hoffmann, P.C., Heller, A., 1991. Immortalization of embryonic

C.X.-Q. Chen et al. / Neurobiology of Disease 19 (2005) 419–426426

mesencephalic dopaminergic neurons by somatic cell fusion. Brain Res.

552, 67–76.

Choi, H.K., Won, L., Roback, J.D., Wainer, B.H., Heller, A., 1992. Specific

modulation of dopamine expression in neuronal hybrid cells by primary

cells from different brain regions. Proc. Natl. Acad. Sci. U. S. A. 89,

8943–8947.

Daly, C., Sugimori, M., Moreira, J.E., Ziff, E.B., Llinas, R., 2000.

Synaptophysin regulates clathrin-independent endocytosis of synaptic

vesicles. Proc. Natl. Acad. Sci. U. S. A. 97, 6120–6125.

Denton, T., Howard, B.D., 1987. A dopaminergic cell line variant

resistant to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-

dine. J. Neurochem. 49, 622–630.

Edelmann, L., Hanson, P.I., Chapman, E.R., Jahn, R., 1995. Synaptobrevin

binding to synaptophysin: a potential mechanism for controlling the

exocytotic fusion machine. EMBO J. 14, 224–231.

Fon, E.A., Pothos, E.N., Sun, B.C., Killeen, N., Sulzer, D., Edwards, R.H.,

1997. Vesicular transport regulates monoamine storage and release but

is not essential for amphetamine action. Neuron 19, 1271–1283.

Gainetdinov, R.R., Caron, M.G., 2003. Monoamine transporters: from

genes to behavior. Annu. Rev. Pharmacol. Toxicol. 43, 261–284.

Gainetdinov, R.R., Fumagalli, F., Wang, Y.M., Jones, S.R., Levey, A.I.,

Miller, G.W., Caron, M.G., 1998. Increased MPTP neurotoxicity

in vesicular monoamine transporter 2 heterozygote knockout mice.

J. Neurochem. 70, 1973–1978.

Galli, T., McPherson, P.S., De Camilli, P., 1996. The V0 sector of the

V-ATPase, synaptobrevin, and synaptophysin are associated on

synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensi-

tive, complex. J. Biol. Chem. 271, 2193–2198.

Greene, L.A., Rein, G., 1978. Release, storage and uptake of catechol-

amines by a clonal cell line of nerve growth factor (NGF) responsive

pheochromocytoma cells. Brain Res. 129, 247–263.

Kanner, B.I., Schuldiner, S., 1987. Mechanisms of storage and transport of

neurotransmitters. CRC Crit. Rev. Biochem. 22, 1–38.

Kilbourn, M.R., Sherman, P., Abbott, L.C., 1998. Reduced MPTP

neurotoxicity in striatum of the mutant mouse tottering. Synapse 30,

205–210.

Kopin, I.J., Markey, S.P., 1988. MPTP toxicity: implications for research in

Parkinson’s disease. Annu. Rev. Neurosci. 11, 81–96.

Langston, J.W., 1987. Parkinson’s disease: current view. Am. Fam.

Physician 35, 201–206.

Liang, C.L., Nelson, O., Yazdani, U., Pasbakhsh, P., German, D.C., 2004.

Inverse relationship between the contents of neuromelanin pigment and

the vesicular monoamine transporter-2: human midbrain dopamine

neurons. J. Comp. Neurol. 473, 97–106.

Liu, Y., Edwards, R.H., 1997a. Differential localization of vesicular

acetylcholine and monoamine transporters in PC12 cells but not CHO

cells. J. Cell Biol. 139, 907–916.

Liu, Y., Edwards, R.H., 1997b. The role of vesicular transport proteins in

synaptic transmission and neural degeneration. Annu. Rev. Neurosci.

20, 125–156.

Liu, Y., Roghani, A., Edwards, R.H., 1992a. Gene transfer of a reser-

pine-sensitive mechanism of resistance to MPP+. Proc. Natl. Acad.

Sci. U. S. A. 89, 9074–9078.

Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G.G., Eisenberg, D.,

Brecha, N., Edwards, R.H., 1992b. A cDNA that supresses MPP+

toxicity encodes a vesicular amine transporter. Cell 70, 539–551.

Liu, Y., Schweitzer, E.S., Nirenberg, M.J., Pickel, V.M., Evans, C.J.,

Edwards, R.H., 1994. Preferential localization of a vesicular mono-

amine transporter to dense core vesicles in PC12 cells. J. Cell Biol. 127,

1419–1433.

Lotharius, J., Brundin, P., 2002. Pathogenesis of Parkinson’s disease:

dopamine, vesicles and alpha-synuclein. Nat. Rev., Neurosci. 3,

932–942.

Lotharius, J., O’Malley, K.L., 2000. The parkinsonism-inducing drug

1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation.

A novel mechanism of toxicity. J. Biol. Chem. 275, 38581–38588.

Lotharius, J., Dugan, L.L., O’Malley, K.L., 1999. Distinct mechanisms

underlie neurotoxin-mediated cell death in cultured dopaminergic

neurons. J. Neurosci. 19, 1284–1293.

Mooslehner, K.A., Chan, P.M., Xu, W., Liu, L., Smadja, C., Humby, T.,

Allen, N.D., Wilkinson, L.S., Emson, P.C., 2001. Mice with very low

expression of the vesicular monoamine transporter 2 gene survive into

adulthood: potential mouse model for parkinsonism. Mol. Cell. Biol.

21, 5321–5331.

Peter, D., Jimenez, J., Liu, Y., Kim, J., Edwards, R.H., 1994. The

chromaffin granule and synaptic vesicle amine transporters differ in

substrate recognition and sensitivity to inhibitors. J. Biol. Chem. 269,

7231–7237.

Przedborski, S., Jackson-Lewis, V., Naini, A.B., Jakowec, M., Petzinger,

G., Miller, R., Akram, M., 2001. The parkinsonian toxin 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine (MPTP): a technical review of its

utility and safety. J. Neurochem. 76, 1265–1274.

Reinhard Jr., J.F., Diliberto Jr., E.J., Viveros, O.H., Daniels, A.J., 1987.

Subcellular compartmentalization of 1-methyl-4-phenylpyridinium

with catecholamines in adrenal medullary chromaffin vesicles may

explain the lack of toxicity to adrenal chromaffin cells. Proc. Natl.

Acad. Sci. U. S. A. 84, 8160–8164.

Scherman, D., Henry, J.P., 1981. The catecholamine carrier of Bovine

chromaffin granules. Form of the bound amine. Mol. Pharmacol. 23,

431–436.

Schuldiner, S., Shirvan, A., Linial, M., 1995. Vesicular neurotransmitter

transporters: from bacteria to humans. Physiol. Rev. 75, 369–392.

Staal, R.G., Hogan, K.A., Liang, C.L., German, D.C., Sonsalla, P.K., 2000.

In vitro studies of striatal vesicles containing the vesicular monoamine

transporter (VMAT2): rat versus mouse differences in sequestration of

1-methyl-4-phenylpyridinium. J. Pharmacol. Exp. Ther. 293, 329–335.

Stokes, A.H., Hastings, T.G., Vrana, K.E., 1999. Cytotoxic and genotoxic

potential of dopamine. J. Neurosci. Res. 55, 659–665.

Takahashi, N., Miner, L.L., Sora, I., Ujike, H., Revay, R.S., Kostic, V.,

Jackson-Lewis, V., Przedborski, S., Uhl, G.R., 1997. VMAT2 knockout

mice: heterozygous display reduced amphetamine-conditioned reward,

enhanced amphetamine locomotion and enhanced MPTP toxicity. Proc.

Natl. Acad. Sci. U. S. A. 94, 9938–9943.

Tan, P.K., Waites, C., Liu, Y., Krantz, D.E., Edwards, R.H., 1998. A

leucine-based motif mediates the endocytosis of vesicular monoamine

and acetylcholine transporters. J. Biol. Chem. 273, 17351–17360.

Thiele, C., Hannah, M.J., Fahrenholz, F., Huttner, W.B., 2000. Cholesterol

binds to synaptophysin and is required for biogenesis of synaptic

vesicles. Nat. Cell Biol. 2, 42–49.

Uhl, G.R., Li, S., Takahashi, N., Itokawa, K., Lin, Z., Hazama, M., Sora, I.,

2000. The VMAT2 gene in mice and humans: amphetamine responses,

locomotion, cardiac arrhythmias, aging, and vulnerability to dopami-

nergic toxins. FASEB J. 14, 2459–2465.

Wang, Y.-M., Gainetdinov, R.R., Fumagalli, F., Xu, F., Jones, S.R.,

Bock, C.B., Miller, G.W., Wightman, R.M., Caron, M.G., 1997.

Knockout of the vesicular monoamine transporter 2 gene results in

neonatal death and supersensitivity to cocaine and amphetamine.

Neuron 19, 1285–1296.

Weingarten, P., Zhou, Q.Y., 2001. Protection of intracellular dopamine

cytotoxicity by dopamine disposition and metabolism factors. J. Neuro-

chem. 77, 776–785.

Winkler, H., 1976. The composition of adrenal chromaffin granules: an

assessment of controversial results. Neuroscience 1, 65–80.

Zhang, Y., Dawson, V.L., Dawson, T.M., 2000. Oxidative stress and

genetics in the pathogenesis of Parkinson’s disease. Neurobiol. Dis. 7,

240–250.