Biochemical and Biophysical Research Communications 309 (2003) 679–684

www.elsevier.com/locate/ybbrc

BBRC

Parkin suppresses wild-type a-synuclein-induced toxicityin SHSY-5Y cells

Yemisi Oluwatosin-Chigbu,a Alan Robbins,a Clay W. Scott,b Jeffrey L. Arriza,a

John D. Reid,a and John R. Zyska,*

a Department of Molecular Science, AstraZeneca Pharmaceuticals LP, 1800 Concord Pike, Wilmington, DE 19850-5437, USAb Department of Lead Discovery, AstraZeneca Pharmaceuticals LP, 1800 Concord Pike, Wilmington, DE 19850-5437, USA

Received 14 August 2003

Abstract

Current hypotheses concerning the mechanism of neuronal cell death in Parkinson’s disease (PD) and related synucleopathies

propose a functional interaction between parkin and a-synuclein (aS). Recently parkin was shown to suppress mutant aS-inducedtoxicity in primary neurons, providing a basis for an association between these proteins and neuronal loss [Neuron 36 (2000) 1007–

1019]. We have asked if a similar association could be made between wild-type (wt) aS and parkin. We examined inducible over-

expression of aS in SHSY-5Y cells through adenoviral infection under conditions which produce cellular toxicity through a

reduction in ATP levels. We demonstrate that parkin suppresses toxicity induced by mutant (A53T) and wt aS. Parkin over-ex-

pression was also associated with the appearance of higher molecular weight aS-immunoreactive bands by Western blot analysis.

These data, consistent with a protective role for parkin, extend previous findings to include a functional association between parkin

and the wt form of aS.� 2003 Elsevier Inc. All rights reserved.

Keywords: Parkin; a-Synuclein; Parkinson’s disease; Synucleopathies; Adenoviral gene delivery; Dopaminergic cell line

Parkinson’s disease is a neurodegenerative conditionresulting from a loss of dopaminergic neurons of the

substantia nigra [1]. Efforts to understand this mecha-

nism at the molecular level have involved the study of

genetic factors in rare familial forms of PD. One such

condition, autosomal recessive juvenile Parkinsonism

(ARJP), involves loss-of-function mutations in the E3

ubiquitin ligase parkin [2–5]. Another familial form of

PD results from autosomal dominant mutations in aS, aprotein of unknown function and a major constituent of

Lewy bodies (LB), a hallmark of the predominant,

sporadic disease [6,7, see 8 for review].

Studies involving parkin and aS suggest that an

association may exist between these proteins which

could be linked to a mechanism of cellular toxicity.

Over-expression of wt aS or its mutants is frequently

* Corresponding author. Fax: 1-302-886-4983.

E-mail address: john.zysk@astrazeneca.com (J.R. Zysk).

0006-291X/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2003.08.059

associated with cytotoxicity or an increased sensitivityto oxidative stress [9–11]. By contrast, parkin appears

to subserve a protective role at the cellular level,

ubiquitinating proteins destined for clearance by the

26S proteosome and responding to unfolded protein-

induced stress [4]. Parkin has also been shown to

moderate oxidative stress at the cellular level, whereas

ARJP mutations in parkin result in increased oxidative

cellular damage and sensitization of cells to death in-duced by other insults [12]. Co-localization studies

suggest an association between aS and parkin in PD,

and other synucleopathies [13,14]. Recently, Petrucelli

and co-workers demonstrated that parkin rescues pri-

mary neurons from mutant aS-induced toxicity and

insult by proteosome inhibitors [15].

In the present study, we have asked if parkin over-

expression affects wt aS-induced cellular toxicity in anacute cell-based model, since this is the form of syn-

uclein associated with the predominant sporadic form

of PD. In order to address this question, we have

680 Y. Oluwatosin-Chigbu et al. / Biochemical and Biophysical Research Communications 309 (2003) 679–684

employed adenoviral constructs to produce levels of aSexpression in dopaminergic SHSY-5Y cells sufficient to

induce cellular toxicity as measured by a reduction in

cellular ATP levels. We report that overexpression of

parkin suppresses mutant and wt aS-induced cellular

toxicity and that this cytoprotection is associated with

the appearance of higher molecular mass aS-immuno-

reactive proteins. These results are consistent with a

cytoprotective role for parkin, confirm earlier findingsbetween parkin and autosomal dominant synuclein,

and indicate that a functional association exists be-

tween parkin and the native form of the protein as

well.

Fig. 1. Adenoviral-induced over-expression of aS in SHSY-5Y cells in

the absence of doxycycline. (A) Western blot analysis time course with

monoclonal antibody against human aS (610787, Transduction Labs)

or actin as control (AB1501, Chemicon). Lane 1, b-gal; 2, wt aS; and 3,

A53T. (B) Time-dependent effect of wt aS and A53T expression on

cellular ATP compared to b-gal expressing controls. An MOI of 6 for

the tTA and 15 for the gene of interest was used in each of the above

experiments. Cellular measurements are described in Materials and

methods. *P < 0:001, compared to b-Gal controls (paired Student’s t

test), n ¼ 8 per bar and is representative of at least three experiments.

Materials and Methods

Antibodies. Monoclonal antibody 610787 against human aS was

purchased from Transduction Labs (Lexington, KY). A polyclonal

antibody against parkin, # 2132, was obtained from Cell Signaling

Technology (Beverly, MA) and the antibody for actin, AB1501, was

from Chemicon International (Temecula, CA).

Viral constructs, cell culture, transfection, and viral infection. In

order to attain a level of aS necessary to affect cellular ATP levels,

dopaminergic SHSY-5Y cells (ATCC) were infected with adenoviral

constructs in which aS expression is under the control of a tetra-

cycline (doxycycline) regulatory element (TRE, tet-off). The Adeno-

X Tet-Off Expression system from Clontech (Palo Alto, CA) was

used to construct adenoviruses expressing b-galactosidase (Adeno-X

b-gal), wt aS (Adeno-X aS), and the A53T familial mutation

(Adeno-X A53T). This system requires co-infection of an Adeno-X

virus expressing the tet-transactivator protein (tTA) and an Adeno-

X virus that will express the target gene. Briefly, aS wt and A53T

DNA sequences were PCR amplified using primers containing re-

striction enzyme sites to directionally clone them into the pTRE

shuttle vector. Subsequently, the cloned aS wt and A53T genes

were ligated into Adeno-X viral DNA which was then transfected

into HEK 293 cells. Cells were harvested after 7 days and a second

round of amplification was carried out by infecting fresh HEK 293

cells. Virus stocks derived from this second round of infection were

titered and then used to infect cells to assess aS expression. Large

scale amplification of the Adeno-X virus expressing aS was carried

out by Q-Biogene (Quebec, Canada). The integrity of the WT aSand A53T aS in the large-scale virus stocks was verified by direct

PCR amplification of the aS genes from the virus stocks followed

by DNA sequencing of the PCR products. Where indicated,

SHSY-5Y cells were transfected with episomal vector pCEP4 con-

taining the cDNA for wt parkin or the T240R mutant using

LipoFECTAMINE Plus and grown in hygromycin B (35 lg/ml for

4 weeks).

Cell viability. Cells seeded in 96-well plates at 10,000 cells per well

were infected the next day by replacing medium with a mixture of tTA

virus (6 MOI) and b-Gal or aS virus (15 MOI) in complete growth

medium containing 10% Tet-system approved FBS from Clontech.

Cells were assayed for total cellular ATP using the ViaLight HS kit

from BioWhittaker (Rockland, ME) according to the manufacturer’s

instructions and normalized for total cellular protein using the BCA

reagent from PIERCE (Rockford, IL). Statistical analyses were per-

formed with the aid of Prism software.

Western blot analysis and immunocytochemistry. Total cell lysates

from infected cells (10lg) were separated by 10% polyacrylamide/SDS

gel electrophoresis, transferred onto nitrocellulose filters, and blotted

with specifc antibodies described above.

Results

Protein expression in virally infected SHSY-5Y cells

In the tet-off configuration, protein expression in vi-

rally infected SHSY-5Y cells was maximally induced in

the absence of doxycycline. Under these conditions

maximal protein expression was attained by 48 hours

post infection for the control, b-gal (Adeno-X b-gal) aswell as for Adeno-X aS and Adeno-X A53T. The MOIs

resulting in this expression ranged from 1 to 6 for the tet

transactivator (tTA) and 3–15 for the gene-of-interest.

Maximal MOI administration resulted in protein ex-

pression in 100% of the cells in the tet-off configuration

when assessed by immunocytochemistry (data not

shown). In the absence of doxycycline, cells infected

with Adeno-X aS or A53T overexpressed these proteinsat levels significantly above the endogenous levels of aSfound in Adeno-X b-gal infected cells during the seven

day time course (Fig. 1A).

The effect of virally-induced protein over-expression on

cellular ATP levels

The intended use of the cellular ATP assay (ViaLight

HS) is for the rapid measurement of cytotoxicity or cell

proliferation. The assay was validated with SHSY-5Y

cells by showing that H2O2, a known oxidative stressor,

ig. 2. Effect of parkin co-expression on the ATP lowering effect

roduced by aS and A53T. (A) ATP levels at day 7 post-infection.

onditions are as described in Fig. 1. *P < 0:001, comparison between

roups (unpaired t test, two tailed), n ¼ 8 per bar and is representative

f three experiments. (B) Western blot analysis of parkin and a-syn-clein co-expressed in lysates from SHSY-5Y cells. Top panel, aS;iddle panel, parkin; and lower panel, actin. Lane 1, Adeno-X b-gal;ne 2, Adeno-X aS; and lane 3, Adeno-X A53T. Arrow indicates

cation of aS monomer.

Fig. 3. Effect of parkin T240R co-expression on ATP lowering pro-

duced by aS and A53T. Conditions are as described in Fig. 1.

*P < 0:005, comparison between groups (unpaired t test, two tailed),

n ¼ 8 per bar and is representative of two experiments. NS, no

significant difference.

Y. Oluwatosin-Chigbu et al. / Biochemical and Biophysical Research Communications 309 (2003) 679–684 681

reduced ATP levels in a concentration-dependent fash-ion (data not shown). During the seven day post infec-

tion time course, cellular ATP levels in Adeno-X b-galinfected cells were comparable to non-infected controls

(data not shown). Expression of aS in Adeno-X aS and

A53T in Adeno-X A53T had little, if any effect on cel-

lular ATP until 5 days post-infection. By day 7 there was

a consistent and significant decrease in cellular ATP

levels for Adeno-X aS and Adeno-X A53T-infected cells(Fig. 1B). Over-expression of A53T consistently pro-

duced greater reductions in cellular ATP than wt aSover the same time period. Achieving cytotoxicity

through wt aS over-expression was clearly dependent on

expression levels, since decreased cellular ATP was ob-

served at an MOI of 15, but not at an MOI of 5, whereas

A53T at an MOI of 5 produced a 25–28% decrease in

ATP (data not shown). In addition, cytotoxicity wasmeasured by the less facile XTT assay and similar results

were found albeit with slightly higher toxicity readings.

Over-expression of parkin by stable transfection of

SHSY-5Y cells suppresses aS-induced cytotoxicity

In order to determine if parkin affects the aS-inducedreduction in cellular ATP levels, SHSY-5Y cells were

transfected with the pCEP vector harboring the cDNA

for parkin. Morphologically, there was no difference

between cells expressing parkin (SHpark) and cells con-

taining vector alone (SHpCEP). Expression of parkin in

SHpark was significantly greater than for endogenouslevels in SHpCEP when analyzed by Western blot anal-

ysis (Fig. 2B). SHpCEP and SHpark infected with Ad-

eno-X b-gal produced no detectable effects on cellular

ATP levels over the seven day test period (control,

Fig. 2A). By contrast, SHpCEP infected with Adeno-X

aS or Adeno-X A53T exhibited a significant reduction in

ATP levels by day 7 post-infection (Fig. 2A). During the

same period and under the same conditions of infection,SHpark with either Adeno-X aS or Adeno-X A53T ex-

hibited significantly higher ATP levels than cells con-

taining empty vector (SHpCEP, Fig. 2A).

Parkin over-expression is associated with higher molecular

weight proteins recognized by aS antibody

Parkin expression in Shpark (Fig. 2B, right middle

panel) was significantly higher than the endogenous

parkin levels in SHpCEP (Fig. 2B, left middle panel).

When infected with Adeno-X aS or A53T, both SHp-

CEP and Shpark expressed similar levels of monomeric

synuclein (Fig. 2B, left and right top panels, arrow).However, SHpark infected with Adeno X aS or A53T

exhibited higher molecular weight bands recognized by

the synuclein antibody (Fig. 2B, top right panel) which

were not present in similarly infected SHpCEP (Fig. 2B,

top left panel).

F

p

C

g

o

u

m

la

lo

Over-expression of T240R parkin does not suppress aS-induced cytotoxicity

The T240R mutation in parkin is associated with

ARJP and was over-expressed in Adeno-X aS and

Adeno-X A53T. Unlike wt parkin, the T240R mutation

682 Y. Oluwatosin-Chigbu et al. / Biochemical and Biophysical Research Communications 309 (2003) 679–684

was not associated with suppression of synuclein-in-duced cytotoxicity (Fig. 3). Expression levels for T240R

were approximately three-fold higher than endogenous

parkin levels despite repeated attempts to further in-

crease expression. By contrast, expression levels for wt

parkin were at least four times higher than for T240R

(data not shown).

Discussion

The objectives of this communication were to de-

velop a dopaminergic, cell-based model of aS over-

expression with the capability of inducing wt and

mutant-dependent cytotoxicity and to determine if this

is affected by parkin co-expression. To this end, we

have devised a system using adenoviral infection, ca-pable of producing cytotoxicity with either mutant or

wt aS in a time and dose-dependent manner. Interest-

ingly, while maximal overexpression of aS was

achieved by day two post infection and remained stable

up to day seven, we did not detect significant cyto-

toxicity until day five, suggesting a cummulative effect

of synuclein overexpression. Cell viability was similar

in non-infected and control infected (b-gal-expressing)cells, indicating that toxicity in aS over-expressors is

specific and not virally-induced. We also demonstrated

that parkin co-expression provides cytoprotection

against both mutant and wt synuclein extending earlier

findings [15]. As in previous studies, we have observed

an enhancement in the toxicity of over-expressed aS in

the presence of stressors, notably MPPþ, rotenone,

and, to a lesser extent, lactacystin (data not shown).However, one of the goals of this study was to deter-

mine if over-expressed wt aS is capable of inducing

toxicity in the absence of external stressors and under

normal cell culture conditions.

It is generally accepted that the mechanism of toxicity

of aS in PD involves dopamine, a toxic compound in its

own right, which is linked to oxidative damage [16–18].

Indeed, in non-dopaminergic cortical neurons, aS ex-hibits neuroprotective characteristics [16]. So although

the absence of added stressors was an important objec-

tive of the toxicity model, a dopaminergic cell line

(SHSY-5Y) was deemed relevant to this study. Mito-

chondrial dysfunction is also associated with PD

through a decrease in complex I activity and an atten-

dant diminution in cellular ATP [11,19]. In fact, inhib-

itors of complex I, such as MPTP and rotenone areknown inducers of Parkinsonism [20,21]. Thus, total

cellular ATP was chosen as a readout for cellular tox-

icity because of its relevance to the disease process as

well as the central role it plays in cellular metabolism

and maintenance of cell viability [22]. The biolumines-

cent assay was also implemented because of its facility,

wide dynamic range, high sensitivity, and validation

against other assays for cell viability and cytotoxicity[23,24].

ARJP-associated loss-of-function mutations in par-

kin and the attendant loss of dopaminergic neurons in

the disease state suggest that parkin plays an important

role in neuronal viability. By itself, the stable, over-ex-

pression of wt parkin in SHSY-5Y cells had no overt

effect on cell viability reflecting similar findings in SK-N-

MC and NT-2 cells [12]. The positive effect associatedwith parkin on cell viability was only observed during

aS over-expression. A similar parkin-associated cyto-

protective effect was found in SHSY-5Y cells over-ex-

pressing Pael-receptor, a parkin substrate and

constituent of LBs [25] and more recently in a Dro-

sophila model [26]. The failure of the A240T form of

parkin to provide cytoprotection from aS-induced tox-

icity reflects the findings of Petrucelli and coworkerswho showed that R42P parkin, another loss-of-function

mutant, was ineffective against insult by lactacystin [15].

Another possible reason for a lack of protection is the

lower fold-expression of the mutant relative to wt over-

expression.

The tendency of aS to aggregate is associated with the

formation of LBs in sporadic PD and familial forms of

the disease involving autosomal dominant mutations[6,7]. Parkinsonism resulting from ARJP is character-

ized by a lack of inclusions, suggesting that functional

parkin is necessary for LB formation [2,3,27]. Thus, the

argument has been made that LBs are protective in

nature or at least not inherently cytotoxic [28]. There is

also evidence to suggest that aS exists as a protofibril

multimer which exists in equilibrium with LBs [9,21].

We found no evidence of aS-immunosensitive LB-likeinclusions in SHpark or SHpCEP cells over-expressing

either wt or mutant synuclein by immunocytochemistry

(although diffuse cytoplasmic staining of aS was evident,

data not shown). However, Lewy-like inclusions in a

chronic disease state would probably take longer to

form than in the acute cell model used in our study. We

therefore examined whole cell extracts in order to de-

termine if aS aggregation occurs and if parkin co-ex-pression affects this phenomenon. In fact, detectable,

higher molecular weight a-synuclein immunoreactive

bands were only found in SHpark-infected cells, sug-

gesting that parkin plays a role in regulating synuclein

aggregation and/or processing. If these bands are ag-

gregates of aS, they could represent precursors to in-

clusion formation [9,11]. Although speculative, a lack of

higher molecular weight aS immunoreactive bands incells not over-expressing parkin is consistent with a role

for parkin in processing synuclein as a less toxic species.

It should be mentioned that the SHSY-5Y cells in this

study express low levels of endogenous parkin (Fig. 2B)

which would be overwhelmed by virally induced aSexpression. The higher molecular weight species of aSdescribed in the present study were not recognized with

Y. Oluwatosin-Chigbu et al. / Biochemical and Biophysical Research Communications 309 (2003) 679–684 683

ubiquitin or parkin antibodies, suggesting that thesebands are neither parkin substrates nor parkin–synuc-

lein complexes (data not shown). The further charac-

terization of these higher molecular weight species will

probably be necessary in defining the nature of the

parkin/aS association and attendant mechanisms of

toxicity and protection.

In this study, we have used virally transfected SHSY-

5Y cells as a starting point to understand synucleopa-thies at the molecular level. Adenoviral delivery offers

the advantages of inducible, high level expression and

high efficiency of transfection without inherent toxicity

under the conditions described. Adenoviral vectors also

enable the delivery of up to 8 kb of DNA lending flexi-

bility to the size of the gene under consideration. The

facile nature of this system may be useful in defining

pathways and identifying targets involved in aS-relatedtoxicity. Co-expression of selected candidate genes and

screening of enhancers or supressors of cytotoxicity

provides a platform for examining protein-protein in-

teractions and cell-based mechanisms. Parkin co-ex-

pression and suppression of wt aS-induced cytotoxicity

as a proof-of-principle indicates that this is a viable

approach to understanding the nature of synucleopa-

thies at the molecular level. The validation of potentialtargets derived from this system may be accomplished

using suitable transgenic and genetic models currently

being developed [29].

Acknowledgments

We thank Steve Culp for aiding in the statistical analyses, and Jude

Prosser, Dee Wilkins, Helen Manley, and Jeanine Olie for technical

assistance.

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