Tài liệu Báo cáo khoa học: P25a ⁄ TPPP expression increases plasma membrane presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity pptx

P25a
⁄
TPPP expression increases plasma membrane
presentation of the dopamine transporter and enhances
cellular sensitivity to dopamine toxicity
Anja W. Fjorback
1,2,
*, Sabrina Sundbye
2
, Justus C. Da
¨
chsel
2,
, Steffen Sinning
1
, Ove Wiborg
1
and Poul H. Jensen
2
1 Centre for Psychiatric Research, Aarhus University Hospital, Denmark
2 Department of Medical Biochemistry, Aarhus University, Denmark
Keywords
dopamine transporter; p25a; Parkinsons
disease; toxicity; TPPP; tubulin
polymerization promoting protein
Correspondence
P.H. Jensen, Department of Medical
Biochemistry, Aarhus University, Ole
Worms Alle 1170, DK-8000 Aarhus C,
Denmark
Fax: +45 86131160

Tel: +45 89422856
E-mail:
*Present address
Stereology and EM Research Laboratory,
Aarhus University, Denmark
Present address
Division of Neurogenetics, Department of
Neuroscience, Mayo Clinic Florida,
Jacksonville, FL 32224, USA
(Received 21 June 2010, revised 1
November 2010, accepted 20 November
2010)
doi:10.1111/j.1742-4658.2010.07970.x
Parkinson’s disease is characterized by preferential degeneration of the
dopamine-producing neurons of the brain stem substantia nigra. Imbal-
ances between mechanisms governing dopamine transport across the
plasma membrane and cellular storage vesicles increase the level of toxic
pro-oxidative cytosolic dopamine. The microtubule-stabilizing protein p25a
accumulates in dopaminergic neurons in Parkinson’s disease. We hypothe-
sized that p25a modulates the subcellular localization of the dopamine
transporter via effects on sorting vesicles, and thereby indirectly affects its
cellular activity. Here we show that co-expression of the dopamine trans-
porter with p25a in HEK-293-MSR cells increases dopamine uptake via
increased plasma membrane presentation of the transporter. No direct
interaction between p25a and the dopamine transporter was demonstrated,
but they co-fractionated during subcellular fractionation of brain tissue
from striatum, and direct binding of p25a peptides to brain vesicles was
demonstrated. Truncations of the p25a peptide revealed that the require-
ment for stimulating dopamine uptake is located in the central core and
were similar to those required for vesicle binding. Co-expression of p25a

and the dopamine transporter in HEK-293-MSR cells sensitized them to
the toxicity of extracellular dopamine. Neuronal expression of p25a thus
holds the potential to sensitize the cells toward dopamine and toxins
carried by the dopamine transporter.
Structured digital abstract
l
MINT-8055798: DAT (uniprotkb:Q01959) and p25 alpha (uniprotkb:O94811) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-8054201: p25 alpha (uniprotkb:B1Q0K1), bip (refseq:GI:194033595), Synaptophysin
(uniprotkb:
Q62277), Alpha-synuclein (uniprotkb:Q3I5G7) and DAT (uniprotkb:C6KE31)
colocalize (
MI:0403)bycosedimentation in solution (MI:0028)
l
MINT-8055878: Synaptophysin (uniprotkb:Q62277), bip (refseq:GI:194033595) and p25-alpha
(uniprotkb:
O94811) colocalize (MI:0403)bycosedimentation through density gradient
(
MI:0029)
Abbreviations
a-syn, a-synuclein; DA, dopamine; DAT, dopamine transporter; NET, norepinephrine transporter; PBSCM, phosphate-buffered saline
supplemented with Ca and Mg; PD, Parkinson’s disease; SERT, serotonin transporter; VMAT-2, vesicle monoamine transporter-2.
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 493
Introduction
Parkinson’s disease (PD) is a progressive neurodegen-
erative disorder that is characterized by motor dys-
functions, including resting tremor, postural
imbalance, slowness of movement and muscle rigidity.

The motor dysfunctions are preferentially caused by
loss of dopamine (DA)-producing neurons in the sub-
stantia nigra pars compacta, which results in a deple-
ting of DA in their projection area in the striatum
[1,2]. The loss of dopaminergic neurons plays an essen-
tial role in causing motor dysfunction, as demonstrated
by complete reversal of the symptoms in newly diag-
nosed PD patients when treated with DA receptor
agonists or the DA precursor l-Dopa (l-3,4-dihydroxy-
phenylalanine) [3–6]. DA is a neurotransmitter that is
released into the synaptic cleft by fusion of DA storage
vesicles with the plasma membrane. DA is then trans-
ported back into the pre-synaptic neuron via the plasma
membrane DA transporter (DAT). The cytosolic level
remains low, as DA is subsequently transported into
storage vesicles by the vesicle monoamine transporter-2
(VMAT-2) [7,8]. However, the selective vulnerability of
dopaminergic neurons is hypothesized to be caused by
oxidative stress produced by DA metabolism. Normal
catabolism of DA by monoamine oxidase generates
hydrogen peroxide that can be broken down into highly
reactive hydroxyradicals in the presence of iron [3,4].
The cytosolic DA represents an additional risk of
oxidative stress, as DA can auto-oxidize at the neutral
pH of the cytosol and thereby form toxic DA-quinone
species, superoxide radicals and hydrogen peroxide.
The low pH in the DA storage vesicles stabilizes DA
against oxidative breakdown. Thus it is important to
maintain a low cytosolic concentration of DA. The low
level of DA is thought to be maintained by a regulated

balance between the functional levels of plasma mem-
brane DAT and vesicular VMAT-2.
The protein a-synuclein (a-syn) is known to play a
central role in PD because mutations in its gene cause
familial PD, and it is a major component of Lewy
bodies in the sporadic forms of the disease [9]. All
though little is known about events that triggers the
death of dopaminergic neurons, it has been shown that
a-syn affects cellular DA homeostasis as cellular a-syn
decreases DAT-mediated DA uptake via mechanisms
inhibited by PD-causing mutations [10,11]. Because of
the a-syn-mediated effect on DA uptake, abnormal
function or aggregation of a-syn in dopaminergic neu-
rons may affect the DA balance, and thereby increase
oxidative stress in the cell.
The brain-specific protein p25a is normally only
expressed in oligodendrocytes, where it is thought to
affect myelin metabolism [12], possibly via modulation
of microtubule dynamics [13]. However, p25a is abnor-
mally expressed in neurons in a range of neurodegener-
ative diseases, where it is found in the cytoplasm and
nucleus and is often associated with inclusions contain-
ing aggregated a-syn [12,14–16]. It has been shown
that p25a directly stimulates the aggregation of a-syn,
and thereby induces cytotoxicity [16,17]. We hypothe-
sized that expression of p25a in dopaminergic neurons,
in addition to its putative effects on a-syn aggregation,
may increase the sensitivity to dopaminergic stress,
thus contributing to cell vulnerability. We demonstrate
that p25a increases DAT-mediated DA uptake in tran-

siently transfected HEK-293-MSR cells, an increase
that is caused by elevated DAT expression at the cell
surface. This increase in DA uptake subsequently leads
to increased sensitivity towards cellular DA. No direct
interaction between DAT and p25a was demonstrated,
but both co-fractionated with light vesicle fractions
from porcine striatum, and direct binding of recombi-
nant p25a protein to brain vesicles was demonstrated
for the first time. Abnormal p25a expression in dopa-
minergic neurons thereby has the potential to increase
the sensitivity to DA-dependent oxidative stress in PD
via actions on DA-containing vesicles.
Results
p25a stimulates the membrane expression of
DAT
A potential effect of p25a on DAT function was
addressed by expressing DAT alone and in the presence
of p25a in HEK-293-MSR cells. To validate the specific-
ity of the C-20 goat polyclonal IgG, against C-terminal
cytoplasmic domain of DAT, HEK-293-MSR cells were
either transfected with the DAT pcDNA.3.1 vector or
the empty control vector for 48 h before analysis by
immunoblotting (Fig. 1A) and immunofluorescence
microscopy (Fig. 1B). The immunoblot analysis showed
the presence of a strong broad band centered around
75 kDa in the DAT-expressing cells that was absent in
the mock-transfected cells. The broad band is probably
due to the presence of glycosylated and non-glycosylat-
ed DAT species. The mock-transfected cells lacked
DAT-reactive bands but exhibited a weak immunoreac-

tive smear at  150 kDa that was also faintly visible for
DAT-expressing cells. The immunofluorescence analysis
demonstrated strong DAT immunoreactivity on the
plasma membrane of the DAT-expressing cells, with
essentially no signal for the mock-transfected control
p25a regulates dopamine transporter function A. W. Fjorback et al.
494 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
(Fig. 1B). Hence, the C-20 antibody binds specifically
and selectively to DAT in our cellular system, in agree-
ment with previous reports [18].
Figure 1C shows that expression of DAT caused a
cocaine-inhibitable and saturable uptake of [
3
H]-DA in
HEK-293-MSR cells, and this was enhanced by
co-expression with p25a. As a control, we also
A
C
D
E
B
Fig. 1. p25a increases dopamine uptake by increasing the surface
expression of the dopamine transporter. (A) HEK-293-MSR cells
were transiently transfected with DAT expression vector or empty
control vector for 48 h, and extracted for immunoblot analysis. Pro-
tein aliquots (20 lg) were resolved by 8–16% SDS ⁄ PAGE followed
by electroblotting. The filter was first probed with the C-20 goat
antibody, after which the membrane was stripped and reprobed
with an anti-actin antibody. Top panel, anti-DAT immunoreactivity;
molecular size markers indicated on the left. Lower panel, actin

immunoreactivity demonstrating equal loading. Lanes marked by
DAT and MOCK represent cells transfected with DAT expression
vector and empty control vector. (B) Cells transfected as in (A)
were analyzed by confocal laser scanning microscopy using the
C-20 antibody. Left panels show anti-DAT immunoreactivity and
right panels show phase contrast images of the same cells. Top
row, transfected with empty vector; lower row, transfected with
DAT vector. (C) Uptake of increasing concentrations of
3
[H]-DA was
measured in HEK-293-MSR cells transiently transfected with DAT
and p25a, a-syn or the empty vector as a negative control. The
x axis shows the total concentration of DA and the y axis shows
the DA uptake after 10 min. The y axis demonstrates specific
uptake, defined as total uptake subtracted the uptake in the pres-
ence of the DAT inhibitor cocaine (200 l
M) and represents the
mean ± 1 SEM of four experiments. Uptake in vector control trans-
fected cells was determined for 5 l
M dopamine and found to be
negligible (open square). The effect of p25a on DAT-mediated DA
uptake was compared to that in the presence of the empty vector
control, and was found to be different at V
max
values (*P < 0.05,
Student’s t test), as was the a-syn-mediated decrease in DAT
uptake at V
max
(*P < 0.05, Student’s t test). (D) Expression of p25a
and a-syn affects surface but not total DAT expression. HEK-293-

MSR cells transiently transfected with DAT and p25a, a-syn or the
empty vector were either extracted directly in lysis buffer or sub-
jected to cell surface biotinylation with EZ-Link Sulfo-NHS-SS-Biotin
followed by extraction. This cross-linker allows cleavage between
biotin and the target by reducing the disulfide bridge. The biotinyla-
ted proteins were captured by incubating with NeutraAvidin beads.
The total and surface-bound protein fractions were analyzed by
reducing SDS ⁄ PAGE followed by Western blotting with antibodies
toward DAT, p25a, a-syn and b-actin (as loading control). Total cel-
lular DAT is present as two bands representing glycosylated and
non-glycosylated DAT, and was slightly lower in the double trans-
fected cells compared to those expressing DAT alone. By contrast,
surface-bound DAT was increased in p25a-expressing cells and
decreased in a-syn-expressing cells compared to control-transfected
cells. (E) Quantification of the surface DAT bands in (B) normalized
against b-actin as analyzed by densitometry and shown as a per-
centage of the control. The columns represent means ± 1 SEM of
three experiments. Comparison of the three conditions by one-way
ANOVA was significant (P < 0.05). Individual comparison of p25a or
a-syn to control by Student’s t test was also significant (*P < 0.05).
A. W. Fjorback et al. p25a regulates dopamine transporter function
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 495
co-expressed DAT with a-syn, which is known to
decrease DAT activity [10], and noted a decrease in
DAT-mediated uptake. Kinetic analysis showed that
p25a increased the V
max
without affecting the K
m
(Table 1). This effect was not caused by increased DAT

expression, as total cellular extracts showed a minor
decrease in DAT expression as determined by C-20 anti-
DAT IgG binding when co-expressed with either p25a
or a-syn, compared to the empty control vector
(Fig. 1D, top row). Biotinylation of surface proteins
was performed to determine whether the increased V
max
represents a redistribution of DAT from intracellular
vesicles to the plasma membrane. HEK-293-MSR cells
expressing DAT alone and in the presence of p25a or
a-syn were incubated with the membrane-impermeable
cross-linker EZ-Link Sulfo-NHS-SS-Biotin, followed by
detergent extraction and isolation of biotinylated pro-
tein by incubation with streptavidin-coated beads. The
second row of Fig. 1D shows that the level of biotinylat-
ed plasma membrane DAT was highest in p25a-express-
ing cells. Quantification of the data showed that p25a
stimulated a significant ( 50%) increase in plasma
membrane DAT, in contrast to the significant decrease
of  50% when co-expressed with a-syn (Fig. 1E).
Therefore, p25a stimulates cellular DA uptake by trans-
locating intracellular DAT to the plasma membrane.
Structural requirements for p25a-stimulated
dopamine uptake
p25a has a folded dynamic structure [19], and NMR
spectroscopic data of p25a homologs from mice,
Caenorhabditis elegans and humans [20–22] demon-
strate a folded central core and unfolded N- and C-ter-
minal extensions of  45 and 70 amino acid residues,
respectively. We constructed two deletion mutants:

p25aDN, which lacked N-terminal residues 3–43, and
p25aDC, which lacked C-terminal residues 156–219
(Fig. 2A). The truncated p25a proteins were compared
with full-length p25a with respect to their ability to
induce increased DA uptake when co-expressed with
DAT. Figure 2B demonstrates that the full-length and
two truncated p25a proteins all induce a similar
increase in DA uptake when expressed in the HEK-
293-MSR cells expressing DAT alone. This suggests
that the central folded core domain is responsible for
the stimulatory effect on DAT.
DAT belongs to the family of neurotransmitter
transporters that includes the norepinephrine and sero-
tonin transporters (NET and SERT), which share a
range of structural characteristics. The expression of
a-syn has been shown to decrease the membrane
expression of all three transporters [10,23,24]. Table 1
shows that p25a selectively stimulates uptake via DAT
Table 1. Determination of V
max
and K
m
for DAT, NET and SERT in
the presence and absence of p25a. HEK-293-MSR cells were tran-
siently transfected with DAT, NET or SERT and with p25a or mock
vector. The cells were subsequently used for analysis of DA and
serotonin uptake, respectively.
3
[H]-DA was used to determine V
max

curves for DAT and NET, as
3
[H]-DA can also be used by NET, and
3
[H]-serotonin was used for determination of V
max
curves for SERT.
The V
max
curve was obtained by 10 min incubation with
3
[H]-DA
and
3
[H]-serotonin diluted 20 times with unlabeled DA and seroto-
nin, respectively, at eight concentrations ranging from 0 to 10 l
M.
V
max
and K
m
values are given as means ± 1 SEM (n = 3).
V
max
(pmolÆmin
)1
per well)
K
m
(lM)

SERT ⁄ pcDNA3 0.648 ± 0.04 3.2 ± 1.3
SERT ⁄ p25a 0.588 ± 0.06 3.81 ± 0.96
NET ⁄ pcDNA3 0.157 ± 0.01 1.25 ± 0.14
NET ⁄ p25a 0.135 ± 0.007 1.85 ± 0.46
DAT ⁄ pcDNA3 0.591 ± 0.013 2.49 ± 0.80
DAT ⁄ p25a 0.792 ± 0.016 2.47 ± 0.63
A
B
Fig. 2. Effect of p25a deletions on dopamine uptake. (A) p25a con-
tains a folded central core (gray box) and unfolded termini. Expres-
sion vectors for deletion mutants p25aDN, lacking residues 3–43,
and p25aDC, lacking residues 156–219, were constructed. (B) [
3
H]-
DA (2.5 l
M) uptake in HEK-293-MSR cells transfected with DAT
and control vector or DAT in combination with wild-type p25a,
p25aDM or p25aDC vector. Bars represent mean ± 1 SEM of three
independent experiments performed in triplicate, and are normal-
ized against a control expressing DAT alone. Comparison of the
four conditions by one-way ANOVA indicates significant differences
(P < 0.05). *P < 0.05 as compared to control by Student’s t test.
p25a regulates dopamine transporter function A. W. Fjorback et al.
496 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
but not via SERT and NET. Hence, the central folded
core of p25a selectively increases the surface expression
of DAT.
p25a and DAT are associated with light brain
vesicles
Immunoprecipitations and subcellular fractionations

were performed to identify a possible interaction
between p25a and DAT. Immunoprecipitations in cel-
lular detergent extracts previously demonstrated an
interaction between a-syn and DAT [10], but we were
unable to co-immunoprecipitate p25a and DAT (data
not shown). A series of protocols and antibodies were
tested for co-immunoprecipitating p25a with DAT and
DAT with p25a, and various antibodies were tested
for precipitation of DAT, including epitope-specific V5
antibody for a tagged DAT fusion protein.
DAT is an integral membrane protein, so we tested
whether p25a was present in subcellular brain fractions
enriched in DAT-containing vesicles. We performed
the subcellular fractionation on tissue from the nucleus
caudatus of the porcine striatum because this tissue is
enriched in dopaminergic nerve terminals.
Figure 3A shows that DAT was highly enriched in
the light fraction LP2, together with the pre-synaptic
vesicle marker synaptophysin. Lower concentrations of
DAT were present in the vesicular fractions P3 and LP1,
together with the endoplasmic reticulum marker, the
78 kDa glucose regulated protein/BiP (GRP78) BIP.
p25a was primarily localized in two fractions (S3
and LP2), with minor amounts in the remaining frac-
tions. Its presence in the cytosolic fraction S3 was
expected as it has hitherto been considered a soluble
cytosolic constituent. The even larger p25a concentra-
tion in the light vesicular LP2 fraction, together with
DAT, was unexpected and suggests that p25a may
have previously unknown functions related to vesicular

biology. For comparison, the presence of a-syn was
also investigated because this protein is known to be
both cytosolic and vesicle-associated. a-syn showed a
less distinct localization, being present in most frac-
tions in significant concentrations. For instance, when
the synaptosomal fraction P2 is subjected to hypotonic
lysis, a-syn appear in the cytosolic fraction LS2 and
the vesicular fraction LP2 in almost equal amounts.
By contrast, p25a appears almost exclusively in the
light vesicular fraction LP2, with no protein in the
cytosolic fraction LS2. This indicates some specificity
in vesicle binding when compared to the membrane-
associated proteins DAT and synaptophysin.
Having established for the first time that p25a is a
vesicle-associated protein in subcellular brain fractions,
we investigated whether purified recombinant p25a can
bind directly to brain vesicles. A vesicle-binding experi-
ment comprising a vesicle-flotation assay was used,
as described previously for a-syn [25], wherein brain
homogenate was supplemented with 55% sucrose,
overlaid with a sucrose density gradient, and subjected
to ultracentrifugation. Lipid-containing vesicles have a
low density and float up into the gradient, whereas
A
B
Fig. 3. Co-fractionation of DAT and p25a in subcellular fractions of
porcine striatal tissue and localization in cells. (A) Isolated porcine
striatal tissue (nucleus caudatus) was fractionated as described in
Experimental procedures. The fractions were crude pellet (P1),
microsomal fraction (P3), cytosolic fraction (S3), lysed dense synap-

tosomal pellet (LP1), lysed light synaptosomal pellet (LP2) and
lysed synapsomal cysosol (LS2). Protein aliquots (20 lg) for each
fraction were subjected to SDS ⁄ PAGE followed by immunoblotting;
the membrane was probed using antibodies against BIP (endoplas-
mic reticulum marker), DAT, synaptophysin (synaptic vesicle mar-
ker), p25a and a-synuclein. Molecular size markers are shown on
the left, and the antigens are indicated on the right. (B) Localization
of DAT and p25a transiently expressed in SH-SY5Y cells was ana-
lyzed by confocal laser scanning microscopy. A representative cell
is shown with granular and membrane staining of DAT and more
diffuse cytoplasmic p25a staining. To better visualize the possible
co-localization of p25a and DAT, the neurites indicated by the inset
in the lower left panel are enlarged in the lower right panel. It is
evident that some co-localization occurs, although this is not com-
plete.
A. W. Fjorback et al. p25a regulates dopamine transporter function
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 497
cytosolic proteins remain in the dense bottom frac-
tions. Figure 4B shows that endogenous p25a is dis-
tributed in both the dense cytosolic bottom fraction
and the light vesicle-containing fractions, in agreement
with the subcellular fractionation into vesicular and
cytosolic fractions (Fig. 3A).
For the binding analysis, we used the following
recombinant human p25a peptides: full-length p25 and
truncated p25aDN and p25aDC (Fig. 4A). The purified
p25a core peptide corresponding to residues 44–156
was highly insoluble except in 10 mM acetic acid.
However, its insolubility in the neutral binding buffers
made it impossible to study in the binding assay (data

not shown). The full-length p25a protein was subse-
quently biotinylated to allow it to be distinguished
from endogenous porcine brain p25a. Incubation of
brain vesicles with biotinylated p25a prior to vesicle
flotation resulted in the recovery of biotinylated p25a
in light fractions 2–6. As a control, a sample was
supplemented with 1% Triton X-100 and 1% SDS to
A
B
Fig. 4. p25a binds to porcine brain vesicles. (A) The wild-type and deletion constructs of p25a p25aDN, p25aDC and the p25a core, corre-
sponding to residues 44–156, were cloned into pET11d vector, expressed in E. coli and purified. The purified proteins were subjected to
reducing SDS ⁄ PAGE and Coomassie blue staining. Molecular size markers are shown on the left. (B) The association of p25a with brain ves-
icles was demonstrated by subjecting a porcine brain homogenate to a vesicle flotation assay. The assay is based on the light vesicles float-
ing in the density gradient and the cytosolic proteins remaining in the bottom fractions. The homogenate was supplemented with sucrose to
55%, overlaid with a sucrose density gradient of 48–20%, and subjected to ultracentrifugation, after which nine fractions were isolated from
the top of the gradient. The isolated fractions were subjected to reducing SDS ⁄ PAGE and immunoblotting for detection of endogenous p25a
(E-p25a), BIP and synaptophysin. BIP and synaptophysin immunoreactivity were detected on the filter where biotinylated p25a (B-p25a) had
been resolved. To ensure that the flotation was due to binding to light vesicles, 1% Triton X-100 and 1% SDS were added to dissolve the
membranes, as demonstrated for BIP, synaptophysin and biotinylated p25a (B-p25a + TX). As a positive control for the immunoblot, purified
recombinant p25a is shown on the left (R-p25a). To measure direct binding of recombinant full-length p25a to the brain vesicles, purified
protein was biotinylated (B-p25a) and incubated with the brain homogenate prior to vesicle flotation, followed by visualization with horse-
radish peroxidase-conjugated streptavidin. Similarly, the truncated peptides p25aDN and p25aDC were incubated with the homogenate and
treated like B-p25a, except they were detected with using p25a-1 antibody. Representative data from one of three independent experiments
are presented.
p25a regulates dopamine transporter function A. W. Fjorback et al.
498 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
dissolve the vesicles prior to analysis. This caused the
biotinylated p25a tracer to remain in the bottom frac-
tions, thus demonstrating that the flotation was due to
binding to vesicles. Because the vesicle-binding profile

of biotinylated p25a differed from that of the endoge-
nous p25a, the filter used for biotinylated p25a was
probed for the ER marker BIP and the synaptic vesicle
marker synaptophysin. Both showed a profile essen-
tially identical to that of biotinylated p25a, thus
confirming its vesicle-binding properties. Moreover,
solubilization of the vesicles caused BIP, synaptophy-
sin and biotinylated p25a to remain in the bottom
fractions, thus confirming that vesicle binding was
responsible for their flotation. The electrophoretic
mobility of the two truncated p25a species differed
from that of full-length p25a (Fig. 4A), so biotinyla-
tion was not needed for their detection by immuno-
blotting. Figure 4B shows that both truncated proteins
were distributed in the density gradient similar to the
endogenous p25a. Detergent treatment completely
abrogated their flotation (data not shown) as demon-
strated for the biotinylated p25a. This indicates the
termini are dispensable for vesicle binding and suggests
a critical role for the core domain.
To visualize the putative cellular co-localization of
DAT and p25a, laser scanning confocal microscopic
imaging of SH-SY5Y cells transiently transfected with
DAT and p25a was performed (Fig. 3B). SH-SY5Y
cells were chosen because they have a more flattened
morphology, with several thin processes, thus allowing
improved visualization of subcellular co-localization
compared to the epithelial morphology of HEK-293-
MSR cells. DAT was found to localize in the cellular
processes, together with granular intracellular staining

compatible with an association with vesicles and the
plasma membrane. p25a showed cytosolic staining, was
localized in the cytosol with a less granular appearance
than DAT. Focusing on the finer neurites revealed that
p25a and DAT co-localize in some instances, but it
should be remembered that both proteins are highly
over-expressed, and this may saturate specific inter-
actions and reduce the signal-to-noise ratio (Fig. 3B).
Therefore, we conclude that p25a is a brain vesicle-
binding protein that may associate with DAT-contain-
ing brain vesicles.
p25a increases dopamine toxicity
Oxidative stress caused by cytoplasmic DA may con-
tribute to PD-associated cell death. To investigate a
putative role for aberrant neuronal p25a expression in
this scenario, we incubated HEK-293-MSR cells with
0.5 mm DA for 24 h, and quantified their viability
using the MTT assay. Expression of DAT, p25a and
a-syn alone, or p25 or a-syn together with DAT
respectively, did not affect survival (data not shown),
but the presence of 0.5 mm DA for 24 h caused a 20%
reduction in the survival of DAT-expressing cells but
had no effect on p25a- and a-syn-expressing cells
(Fig. 5). Co-expression of DAT and p25a enhanced
the DA toxicity to 50%, in agreement with an
increased DA uptake (Fig. 5). By contrast, co-expres-
sion of DAT with a-syn was used as a control for
protein over expression. a-syn eliminated the toxicity
produced by addition of DA (Fig. 5).
Discussion

Degeneration of the dopaminergic neurons of the sub-
stantia nigra pars compacta is responsible for the
major motor symptoms of PD. The neurotransmitter
DA has been hypothesized to play a major role in this
selective loss of dopaminergic neurons, and the cyto-
solic fraction of DA is considered particularly toxic
[3,7,26]. This highlights the importance of a controlled
balance between the two transport systems that
regulate cytosolic DA: the DAT, which transports
Fig. 5. Expression of p25a increases DAT-mediated DA toxicity.
HEK-293-MSR cells transfected with DAT + empty vector,
p25a + empty vector, a-syn + empty vector, DAT ⁄ P25a and
DAT ⁄ a-syn to obtain equal concentrations of vector DNA were
incubated in the absence and presence of 0.5 m
M DA for 24 h and
subjected to analysis of viability by the MTT assay. Co-transfection
of DAT with empty vector served as a control for background
sensitivity to DA toxicity in the absence of p25a. Displayed is the
percentage survival of DA-treated cells relative to cells not treated
with dopamine. Cells not expressing DAT were insensitive to DA,
but DAT expression caused a minor but significant loss of survival.
Co-expression of DAT and p25a increased this cell loss, but
co-expression with AS attenuated it. Bars represents means ± 1 SD
of triplicates in one representative experiment of three performed,
except for AS, which was only included in two experiments.
*P < 0.05 compared to controls (Student’s t test).
A. W. Fjorback et al. p25a regulates dopamine transporter function
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 499
extracellular DA into the cytosol, and VMAT-2, which
transports cytosolic DA into storage vesicles. The criti-

cal role of these systems in degeneration of dopaminer-
gic neurons has been shown through both genetic and
chemical evidence. DAT is an import molecule for
environmental toxins causing PD [27], and there is an
increased sensitivity toward DAT-dependent toxins in
VMAT-2 heterozygous mice [28]. The instability of
DA at the neutral pH of the cytosol causes the forma-
tion of toxic aminochromes, and these species are not
formed when DA is protonated at the acidic pH of the
storage vesicles. Factors that increase the surface
expression of DAT holds the potential to increase
cytosolic DA by disturbing the balance between DAT
and VMAT-2 and causing chronic oxidative stress due
to an increased concentration of toxic DA metabolites
[26]. Abnormal expression of p25a in nerve cells has
been shown in the Parkinson’s disorders PD, multiple
systems atrophy and Lewy body dementia [16,29,30],
in which it is principally associated with a-syn-contain-
ing inclusions but also with other a-syn-negative cyto-
plasmic and nuclear inclusions [14].
The p25a protein is normally expressed in myelinat-
ing oligodendrocytes [12,31]. p25a is a microtubule-
associated protein that stimulates the aggregation of
tubulin in vitro [13], but cellular experiments have also
shown an effect on the actin system via (LIMK1) a
cytoplasmic serine/threonine kinase [32]. The impor-
tance of vesicular transport in regulating DAT sorting
to the plasma membrane, combined with ectopic
expression of the microtubule regulator p25a in degen-
erating dopaminergic neurons, suggested that p25a

could be a regulator of DAT activity and a contribut-
ing factor in sporadic parkinsonistic syndromes.
We have shown that co-expression of DAT with
p25a in HEK-293-MSR cells increased DA uptake
compared to controls via an increased plasma mem-
brane presentation of DAT, and this sensitizes the cells
to DA-mediated toxicity. Hence, pathological expres-
sion of p25a in DAT-expressing neurons in PD may
potentiate the toxic effects of dopamine.
Here we show for the first time that p25a binds to
brain vesicles, and demonstrate co-fractionation of p25a
with DAT-containing vesicle fractions. By contrast, no
direct interaction with DAT could be demonstrated by
immunoprecipitation, as was previously demonstrated
between a-syn and DAT [33,34]. To investigate the
co-fractionation, we used porcine striatal tissue from the
nucleus caudatus that are innervated by dopaminergic
axonal projections from the substantia nigra and which
are rich in DAT. Surprisingly, the highest concentration
of p25a was present in the LP2 light synaptosomal
vesicles, which were also enriched in DAT and synapto-
physin. However, whereas DAT and synaptophysin
were both enriched in vesicle-containing fractions P3,
LP1 and LP2, p25a was predominantly present in LP2.
The second major location of p25a was in the cytosolic
S3 fraction, and only minor amounts of p25a were pres-
ent in the other fractions. The partial co-fractionation
was confirmed by confocal laser scanning microscopical
detection of human p25a and DAT transiently
expressed in SH-SY5Y cells, showing partial overlap of

the two antigens. We performed a vesicle binding experi-
ment to confirm that the association of p25a with brain
vesicles could be mediated by direct binding, and also to
investigate structural requirements for an association.
The assay is based on subjecting a brain homogenate to
density gradient centrifugation, whereby the lower den-
sity of vesicles makes them float in the gradient whereas
soluble proteins remain at the bottom. Biotinylated full-
length p25a did bind to vesicles, and addition of deter-
gents to solubilize the vesicles abolished the flotation of
biotinylated p25a. Non-biotinylated N- and C-termi-
nally truncated p25a proteins showed a vesicular bind-
ing pattern similar to that of endogenous p25a,
suggesting that the folded core domain possesses the
structure necessary for the vesicle binding function.
However, direct evidence could not be obtained because
the recombinant p25a core protein was insoluble at neu-
tral pH. The similarity between the core sequences of
the a, b and c p25 gene products suggests that the vesi-
cle-binding function may be a common property for this
protein family [35]. It should be remembered that p25a
is preferentially expressed in oligodendrocytes, and is
present in both the cell body and myelin sheets [12,31],
and its putative functions related to microtubules and
vesicle transport warrant further investigations. The
specificity of vesicle association in combination with
the direct binding of p25a to vesicles suggest the pres-
ence of vesicular p25a receptors, and this is now under
investigation.
The functional role of the vesicle interaction was

confirmed by cellular DA-uptake experiments, because
both the N- and C-terminally truncated p25a proteins
stimulated DA uptake to the same extent as the full-
length p25a when co-expressed with DAT in HEK-
293-MSR cells. The selective effect of p25a on DAT
activity but not NET and SERT may reflect the fact
that DAT is present in specific sorting vesicles that are
targeted by p25a, and thus resembles the apical DAT
sorting in MDCK cells, in contrast to the basolateral
sorting of NET and SERT in these cells [36]. This
interpretation is corroborated by the increased sensitiv-
ity to dopamine of stable SH-SY5Y clones expressing
human p25a. However, variability in DAT expression
due to clonal effects not attributed to the transgene
p25a regulates dopamine transporter function A. W. Fjorback et al.
500 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
cannot be excluded, because anti-DAT immunoblot
analyses failed to detect the low endogenous level.
We conclude that p25a has vesicle-binding proper-
ties and facilitates cellular DA uptake via increased
plasma membrane presentation of DAT. Determina-
tion of the biological role of the vesicle binding and its
potential role in relation to PD pathogenesis requires
further experimentation, but may be exploited in creat-
ing PD models in which p25a expression in DA
neurons sensitizes them to oxidative damage and pre-
pare them for environmental toxins that use DAT for
cellular entry.
Experimental procedures
Plasmids and antibodies

pcDNA3 vectors expressing DAT and NET were a kind
gift from Dr Susan Amara (Center for Neuroscience, Uni-
versity of Pittsburgh, PA, USA). SERT was cloned into the
pcDNA3 vector as previously described [37]. cDNA encod-
ing a-syn or p25a was cloned into the pcDNA3 vector as
described previously [16,38].
Prokaryotic expression vectors containing inserts for
human p25a, N-terminally truncated by amino acid resi-
dues 3–43 (p25aDN) and C-terminally truncated by residues
156-219 (p25aDC) were used, and were purified as described
previously [17,19,22,39]. The central folded core of p25a,
corresponding to amino acid residues 44–156, was amplified
and tagged with six histidine residues by PCR using for-
ward primers 5¢-CACCATCACGGAGCATCCCCTGAG-
3¢,5¢-TCGCATCACCATCACCATCACGGAGCA-3¢ and
5¢-CACCCATGGGATCGCATCACCAT-3¢, and reverse
primer 5¢-CACGGATCCCTACGTCACCCCTGA-3¢. After
digestion with NcoI and BamHI restriction enzymes, the
insert was ligated into pET11d vector (Novagen, Rodovre,
Denmark). Correct insertion was verified by DNA sequenc-
ing (Eurofins-MWG, Martinsried, Germany). The protein
was expressed in Escherichia coli BL21 (DE3) cells (Strata-
gene, La Jolla, CA, USA), and extracted by sonication on
ice in 50 mm NaH
2
PO
4
, pH 7.0. Cell debris was removed
by centrifugation at 1000 g and the supernatant was fil-
trated. The hexahistidine-tagged p25a core protein was

purified using a TALON metal affinity resin (Clontech,
Mountain View, CA, USA) according to the manu-
facturer’s instructions.
The eukaryotic full-length human p25a expression vector
has been described previously [17]. The eukaryotic p25aDN
and p25aDC vectors were constructed by PCR using cDNA
coding for human p25a and primers 5¢-CACTCTAGAC-
CATGGCTGCATCCCCTGAGCTCAGT-3¢ and 5¢-CAC-
GGATCCCTACTTGCCCCCTTGCAC-3¢ for P25aDN,
and 5¢-CACTCTAGACCATGGCTGACAAGG-3¢ and
5¢-CACGGATCCCTACGTCACCCCTGA-3¢ for P25aDC.
These fragments were then inserted into pcDNA3.1 ⁄ Zeo())
vector (Invitrogen, Carlsbad, CA) using XbaI and BamHI
restriction enzymes (New England Biolabs, Denmark).
Correct insertion was verified as stated above.
Rabbit polyclonal anti-a-syn ASY1 antibody and poly-
clonal rabbit anti-p25a1 were all homemade in our labora-
tory [16,40]. Polyclonal goat anti-DAT antibody (C-20) and
horseradish peroxidase-conjugated secondary anti-goat and
anti-rabbit antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). The monoclonal
mouse anti-b-actin antibody (clone AC15) was purchased
from Sigma-Aldrich (Denmark). The mouse monoclonal
anti-synaptophysin IgG was purchased from DAKO
(Glostrup, Denmark). Mouse monoclonal anti-BIP IgG
against the endoplasmic reticulum marker glucose-regulated
protein 78 (BIP) was purchased from BD Transduction
Laboratories (Denmark).
Cell culture and transfection
HEK-293-MSR cells were maintained in Dulbecco’s modi-

fied Eagle’s medium supplemented with 10% fetal calf
serum, 2 mm glutamine, 100 lgÆmL
)1
streptomycin and
100 UÆmL
)1
penicillin at 37 °C and 5% CO
2
. The cells were
transfected 48–72 h prior to the experiments with appropri-
ate amounts of plasmid and Genejuice (Novagen) mixed
with medium according to the manufacturer’s recommenda-
tions. SH-SY5Y cells were grown in Dulbecco’s modified
Eagle’s medium supplemented with 5% FCS and 100 lg/mL
pen ⁄ strep, and zeocin (25 lgÆmL
)1
) was added to the med-
ium for selection of stably transfected cell lines. The cells
were not differentiated prior to experimentation.
Immunofluorescence microscopy
For cellular localization studies, HEK and SH-SY5Y cells
were transiently transfected with human DAT and p25a.At
36 h after transfection, the cells were fixed in 4% parafor-
maldehyde for 10 min, and subsequently permeabilized for
30 min (50 mm glycine, 0.1% Triton-X-100, 3 mm CaCl
2
,
2mm MgCl
2
). After blocking for 30 min with 3% BSA in

NaCl ⁄ P
i
, the transfected cells were stained with the respec-
tive primary antibodies, diluted in NaCl ⁄ P
i
with 1% BSA
for 60 min at room temperature (anti-DAT, 1 : 100; poly-
clonal rat anti-p25a1, 1 : 1000). The secondary antibodies
were Alexa Fluor 568 donkey anti-goat or Alexa Fluor 488
donkey anti-rat IgG conjugates (1 : 1000, Molecular
Probes ⁄ Invitrogen). The cells were investigated using a Zeiss
confocal LSM 510 META microscope with a 40 · NA 1.2
C-Apochromat objective (Zeiss, Jena, Germany).
Western blotting
Protein samples were prepared by incubation of transfected
HEK-293-MSR cells with 300 lL lysis buffer [NaCl ⁄ P
i
:
A. W. Fjorback et al. p25a regulates dopamine transporter function
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 501
137 mm NaCl, 2.7 mm KCl, 4.3 mm Na
2
HPO
4
, 1.4 mm
KH
2
PO
4
, pH 7.4, supplemented 1 mm EDTA, 1% Tri-

ton X-100, 0.1% SDS and protease inhibitor cocktail
(Complete EDTA-free tablets, Roche, Denmark)] for
30 min at 4 °C under gentle shaking. The cell lysate was
cleared by centrifugation at 12 000 g at 4 °C for 15 min,
and the remaining supernatant was mixed with SDS sample
buffer (250 mm Tris ⁄ HCl, pH 6.8, 5% SDS, 0.25% brom-
ophenol blue, 25% glycerol) and left at 37 °C for 30 min.
Fractions of the cell samples were analyzed by 8–16%
SDS ⁄ PAGE and transferred to nitrocellulose membrane.
The membrane was blocked for 2 h in 5% dry milk in
TBST buffer (pH 8.0, 50 mm Tris ⁄ HCl, 150 mm NaCl,
0.5% Tween-20), and then probed overnight with primary
antibody (1 : 1000, except ASY1 at 1 : 500), followed by
incubation with horseradish peroxidase-conjugated second-
ary antibody: anti-goat antibody (1 : 2500) or anti-rabbit
antibody (1 : 1000). Proteins were visualized using an ECL
Advance Western blotting detection kit (GE Healthcare,
Denmark) and developed on a Kodak Image station 440
(Denmark).
Uptake activity
Dopamine or serotonin uptake assays were performed 48 h
after transfection of the cells. The medium was removed and
the cells were washed with NaCl ⁄ P
i
(137 mm NaCl, 2.7 mm
KCl, 4.3 mm Na
2
HPO
4
, 1.4 mm KH

2
PO
4
, pH 7.4) supple-
mented with 0.1 mm CaCl
2
and 1 mm MgCl
2
(PBSCM). To
distinguish total binding from non-specific binding, cells were
incubated for 10 min in either PBSCM alone or PBSCM
containing 200 lm cocaine for inhibition of DAT or 200 lm
S-citalopram for inhibition of SERT. Then the cells were
incubated with radioactive labeled [
3
H]-dopamine or [
3
H]-
serotonin, respectively. For K
m
and V
max
determinations, the
cells were incubated with increasing concentrations of [
3
H]-
dopamine or [
3
H]-serotonin diluted 20 times with unlabeled
dopamine or serotonin, respectively. The final concentration

of dopamine and serotonin ranged from 0–10 lm. For single
V
max
determinations, only one concentration (5 lm)of[
3
H]-
dopamine diluted 20 times with unlabeled dopamine was
used. Washing twice with PBSCM terminated the uptake. All
washing steps were performed using an automated plate
washer. Following uptake, cells were solubilized in scintilla-
tion solution (MicroScient-20, Packard Bell, Denmark), and
plates were counted in a Packard Top counter. Values are the
mean of six replicates. The specific uptake was determined by
subtracting the uptake counts in the absence of inhibitor
from the uptake counts in the presence of cocaine or S-cita-
lopram, respectively. We also confirmed that no uptake
occurred in mock-transfected HEK cells. Assuming Michal-
is–Menten kinetics, the data were plotted and analyzed by a
non-linear-squares curve fit (GraphPadPrism, Denmark).
The amount of cellular protein per well for each condition
was determined as the mean values for the contents of three
individual wells extracted in 0.1 m NaOH and measured
using the bicinchoninic acid method.
DA toxicity assay
Dose-dependent DA toxicity was evaluated in the
0.1–2.5 mm DA concentration range, and a concentration
of 0.5 mm was used for further survival studies. HEK-
MSR-293 cells were transfected in a solution with a fixed
concentration of vector DNA (empty vector or the DAT,
AS or p25a plasmids), and 5000 cells ⁄ well were plated in

96-well, flat-bottomed microtiter plates in a final volume of
200 lL of complete cell culture medium. After 48 h, the
cells were incubated in the absence or presence of 0.5 mm
DA. After 24 h of incubation at 37 °C, 5% CO
2
, the med-
ium containing the DA was removed and the cells were
washed in PBSCM to remove DA. Cell viability was then
measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) non-radioactive cell prolifera-
tion assay (Promega, Madison, WI, USA).
Biotinylation of surface-expressed DAT
Surface biotinylation was performed essentially as previ-
ously described [41]. HEK-293-MSR cells transfected with
DAT in the presence or absence of p25a and a-syn, respec-
tively, were grown in six-well plates and used at 80% conflu-
ence. Cells were washed twice with ice-cold PBSCM, and
incubated for 30 min on ice with PBSCM containing
1mgÆmL
)1
EZ-Link sulfo-NHS-S-S-biotin (Pierce, Rock-
ford, IL, USA). After incubation with biotin, the cells were
washed twice in PBSCM supplied with 100 mm glycine, and
incubated for 20 min to quench further cross-linking. The
quenching buffer was removed by washing twice in PBSCM,
and the cells were then lysed in lysis buffer (PBSCM, 1%
Triton X-100, 0.1% SDS) supplemented with Complete pro-
teinase inhibitor (Roche). The cell lysate was transferred to
Eppendorf vials, and centrifuged for 15 min at 16 000 g and
the supernatant was transferred to new vials. A fraction was

retained for determination of total protein. The remainder
of the supernatant was incubated with NeutraAvidin
(Pierce, Rockford, IL, USA) beads to precipitate the bioti-
nylated proteins. The beads were washed four times in
PBSCM before elution with 50 lLof20mm dithioerythrei-
tol-containing SDS sample buffer, and incubated for 30 min
at 37 °C. The dithioerythreitol reduces the disulfide bridge
in the cross-linker between biotin and the target proteins,
and thus allows their analysis by SDS ⁄ PAGE.
Subcellular fractionation of porcine striatal brain
tissue
Porcine brain cut in half in the saggital plane was obtained
fresh from a local abattoir and immediately cooled on ice.
Within 2 h, the nucleus caudatus, i.e. the part of the
p25a regulates dopamine transporter function A. W. Fjorback et al.
502 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
striatum facing the lateral ventricle, was dissected and fro-
zen in liquid nitrogen followed by storage at ) 80 °C. A
method originally described for rat brain tissue was used
for subcellular fractionation [40]. All procedures were per-
formed on ice or at 4 °C. Frozen tissue (2 g) was thawed in
10 mL ice-cold homogenization buffer [320 mm sucrose,
4mm HEPES ⁄ NaOH, pH 7.4, 2 mm EDTA, Complete
proteinase inhibitor (Roche)]. The brain was homogenized
using a glass-Teflon Dounce homogenizer, and the homoge-
nate was centrifuged for 10 min at 1000 g. The resulting
pellet (P1) was frozen. The supernatant (S1) was collected
and centrifuged for 15 min at 12 000 g, yielding S2 and P2.
P2 was washed by resuspension in 8.5 mL of homogeniza-
tion buffer, and re-centrifuged for 15 min at 10 200 g,

yielding P2¢ and S2¢. The pooled S2 and S2¢, designated S3,
was centrifuged for 2 h at 260 000 g, yielding cytosolic frac-
tion S3 and microsomal fraction P3. P2¢, representing the
crude synaptosomal fraction, was resuspended in homoge-
nization buffer to a final volume of 0.9 mL, and 4.2 mL of
ice-cold water was added and the suspension homogenized
to allow hypertonic lysis. The lysed synatosomes were
immediately mixed with 30 lL1m HEPES ⁄ NaOH buffer,
pH 7.4, and incubated on ice for 30 min. After incubation,
the lysed synaptosomes were centrifuged for 20 min at
33 000 g to yield the lysate pellet (LP1) and supernatant
LS1. LS1 was further fractionated by centrifugation for 2 h
at 260 000 g, yielding the supernatant LS2, containing the
cytosol-enriched fraction, and the pellet LP2, which is
enriched in synaptic vesicles. The protein concentrations
were measured using the bicinchoninic acid method
(Sigma). Equal amounts of protein (20 lg) from each frac-
tion were analyzed by Western blotting. The Western blot
was probed with antibodies for p25a, DAT, a-syn, the
endoplasmic reticulum marker glucose-regulated pro-
tein 78 ⁄ BIP, the trans-Golgi marker TGN38 and the synap-
tic vesicle marker synaptophysin.
Vesicle binding assay for recombinant p25a
proteins using flotation
Recombinant p25a in NaCl ⁄ P
i
, pH 8.0, was biotinylated
using NHS-PEO4-Biotin (Pierce, Rockford, IL, USA)
according to the manufacturer’s instructions, to allow it to
be distinguished from endogenous brain p25a. The biotiny-

lation reaction was quenched using 1 m Tris, pH 8.2, and
excess NHS-PEO4-Biotin was removed by fast desalting on
a PC3.2 ⁄ 10 column (Amersham, Denmark).
Vesicle isolation and binding were performed as
described previously [25]. Briefly, 1 g porcine brain was
homogenized in 2.5 mL of 5 mm dithiothreitol, 2 mm
EDTA, 9% sucrose, 25 m m MES, pH 7.0, in the presence
of protease inhibitors (Complete EDTA-free tablets;
Roche). Nuclei and debris were removed by centrifugation
at 500 g for 5 min at 4 °C, and a crude vesicle fraction was
isolated by ultracentrifugation of the supernatant at
100 000 g for 1 h at 4 °C. After ultracentrifugation, the
pellet was resuspended in 400 lL homogenization buffer,
and 100 lL of resuspended vesicles were incubated with
1 lm recombinant p25a protein (biotinylated p25a or unla-
beled truncated p25DN or p25DC) for 2 h at 4 °C. The
truncated peptides migrate faster than endogenous p25a,
and thus need no biotinylation. For the negative control,
1% Triton X-100 and 1% SDS were added to the sample
to solubilize the vesicles. The solution was brought to 55%
sucrose in a volume of 0.47 mL, and overlaid with 4 mL
48–20% sucrose gradient. The samples were then subjected
to ultracentrifugation at 100 000 g for 16 h at 4 °C. The
gradient was divided into nine fractions starting from the
top. The protein content of each fraction was precipitated
with 20% trichloroacetic acid and subjected to SDS ⁄ PAGE
followed by Western blotting. Endogenous p25a as well as
recombinant p25DN and p25DC were visualized using
anti-p25a1 IgG (rabbit) (1 : 1000) followed by horseradish
peroxidase-conjugated anti-rabbit IgG (1 : 1000) (DAKO).

Recombinant biotinylated p25a was visualized using horse-
radish peroxidase-conjugated streptavidin (1 : 1000) (GE
Healthcare).
Statistics
One-way ANOVA was performed with post hoc analysis
for comparison of the means of more than two groups,
with P < 0.05 considered significant. Student’s t test was
used to compare means between individual groups, and
P values < 0.05 were considered significant.
Acknowledgements
We thank Jette B. Lauritsen for technical assistance
with the SH-SY5Y cells and Anette Larsen for the art-
work. This work was supported by the Danish Council
for Strategic Research (Histoinformatics Centre), the
EU Marie Curie Molecular Neuroimmunology PhD
program (MNIEST, contact number MEST-CT-
514333), the Danish innovation consortium CureND,
the Lundbeck Foundation, the Danish Medical
Research Council (271-05-0166), the Augustinus Foun-
dation, the Familien Hede Nielsen Foundation and the
HistoInformatic Center.
References
1 Fearnley JM & Lees AJ (1991) Ageing and Parkinson’s
disease: substantia nigra regional selectivity. Brain 114,
2283–2301.
2 Hirsch E, Graybiel AM & Agid YA (1988) Melanized
dopaminergic neurons are differentially susceptible to
degeneration in Parkinson’s disease. Nature 334, 345–
348.
A. W. Fjorback et al. p25a regulates dopamine transporter function

FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 503
3 Jenner P & Olanow CW (1996) Oxidative stress and the
pathogenesis of Parkinson’s disease. Neurology 47,
S161–S170.
4 Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B &
Hong JS (2000) Regional difference in susceptibility to
lipopolysaccharide-induced neurotoxicity in the rat
brain: role of microglia. J Neurosci 20, 6309–6316.
5 Olanow CW, Jenner P & Brooks D (1998) Dopamine
agonists and neuroprotection in Parkinson’s disease.
Ann Neurol 44, S167–S174.
6 Smith LA, Jackson MJ, Al Barghouthy G, Rose S,
Kuoppamaki M, Olanow W & Jenner P (2005) Multiple
small doses of levodopa plus entacapone produce con-
tinuous dopaminergic stimulation and reduce dyskinesia
induction in MPTP-treated drug-naive primates. Mov
Disord 20, 306–314.
7 Vergo S, Johansen JL, Leist M & Lotharius J (2007)
Vesicular monoamine transporter 2 regulates the sensi-
tivity of rat dopaminergic neurons to disturbed cyto-
solic dopamine levels. Brain Res 1185, 18–32.
8 Zheng G, Dwoskin LP & Crooks PA (2006) Vesicular
monoamine transporter 2: role as a novel target for
drug development. AAPS J 8, E682–E692.
9 Goedert M, Spillantini MG & Davies SW (1998)
Filamentous nerve cell inclusions in neurodegenerative
diseases. Curr Opin Neurobiol 8, 619–632.
10 Wersinger C, Prou D, Vernier P & Sidhu A (2003)
Modulation of dopamine transporter function by
a-synuclein is altered by impairment of cell adhesion

and by induction of oxidative stress. FASEB J 17,
2151–2153.
11 Wersinger C & Sidhu A (2003) Attenuation of dopa-
mine transporter activity by a-synuclein. Neurosci Lett
340, 189–192.
12 Song YJ, Lundvig DM, Huang Y, Gai WP, Blumbergs
PC, Hojrup P, Otzen D, Halliday GM & Jensen PH
(2007) p25a relocalizes in oligodendroglia from myelin
to cytoplasmic inclusions in multiple system atrophy.
Am J Pathol 171, 1291–1303.
13 Hlavanda E, Kovacs J, Olah J, Orosz F, Medzihradszky
KF & Ovadi J (2002) Brain-specific p25 protein binds
to tubulin and microtubules and induces aberrant
microtubule assemblies at substoichiometric concentra-
tions. Biochemistry 41, 8657–8664.
14 Baker KG, Huang Y, McCann H, Gai WP, Jensen PH
& Halliday GM (2006) P25a immunoreactive but a-syn-
uclein immunonegative neuronal inclusions in multiple
system atrophy. Acta Neuropathol 111, 193–195.
15 Jellinger KA (2006) P25 a immunoreactivity in multiple
system atrophy and Parkinson disease. Acta Neuropa-
thol 112, 112.
16 Lindersson E, Lundvig D, Petersen C, Madsen P,
Nyengaard JR, Hojrup P, Moos T, Otzen D, Gai WP,
Blumbergs PC et al. (2005) p25a stimulates a- synuclein
aggregation and is co-localized with aggregated a-synuc-
lein in a-synucleinopathies. J Biol Chem 280, 5703–
5715.
17 Kragh CL, Lund LB, Febbraro F, Hansen HD, Gai
WP, El-Agnaf O, Richter-Landsberg C & Jensen PH

(2009) a-synuclein aggregation and Ser-129 phosphory-
lation-dependent cell death in oligodendroglial cells.
J Biol Chem 284, 10211–10222.
18 Chen R, Tilley MR, Wei H, Zhou F, Zhou FM, Ching
S, Quan N, Stephens RL, Hill ER, Nottoli T et al.
(2006) Abolished cocaine reward in mice with a
cocaine-insensitive dopamine transporter. Proc Natl
Acad Sci USA 103, 9333–9338.
19 Otzen DE, Lundvig DM, Wimmer R, Nielsen LH,
Pedersen JR & Jensen PH (2005) p25a is flexible but
natively folded and binds tubulin with oligomeric stoi-
chiometry. Protein Sci 14, 1396–1409.
20 Aramini JM, Rossi P, Shastry R, Nwosu C, Cunning-
ham K, Xiao R, Liu J, Baran MC, Rajan PK, Acton
TB et al. (2008) Solution NMR structure of tubulin poly-
merization-promoting protein family member 3 from
Homo sapiens. />explore.do?structureId=2JRF. Accessed September 16
2008.
21 Kobayashi N, Koshiba S, Inoue M, Kigawa T &
Yokoyama S (2005) Solution structure of mouse CGI-38
protein. />structureId=1WLM.
22 Monleon D, Chiang Y, Aramini JM, Swapna GV,
Macapagal D, Gunsalus KC, Kim S, Szyperski T &
Montelione GT (2004) Backbone
1
H,
15
N and
13
C

assignments for the 21 kDa Caenorhabditis elegans
homologue of ‘brain-specific’ protein. J Biomol NMR
28, 91–92.
23 Wersinger C, Jeannotte A & Sidhu A (2006) Attenua-
tion of the norepinephrine transporter activity and traf-
ficking via interactions with a-synuclein. Eur J Neurosci
24, 3141–3152.
24 Wersinger C, Rusnak M & Sidhu A (2006) Modulation
of the trafficking of the human serotonin transporter by
human alpha-synuclein. Eur J Neurosci 24, 55–64.
25 Jensen PH, Nielsen MS, Jakes R, Dotti CG & Goedert
M (1998) Binding of a-synuclein to brain vesicles is
abolished by familial Parkinson’s disease mutation.
J Biol Chem 273, 26292–26294.
26 Lotharius J & Brundin P (2002) Impaired dopamine
storage resulting from a-synuclein mutations may
contribute to the pathogenesis of Parkinson’s disease.
Hum Mol Genet 11, 2395–2407.
27 Langston JW (1989) Mechanisms underlying neuronal
degeneration in Parkinson’s disease: an experimental
and theoretical treatise. Mov Disord 4, S15–S25.
28 Takahashi N, Miner LL, Sora I, Ujike H, Revay RS,
Kostic V, Jackson-Lewis V, Przedborski S & Uhl GR
(1997) VMAT2 knockout mice: heterozygotes display
reduced amphetamine-conditioned reward, enhanced
p25a regulates dopamine transporter function A. W. Fjorback et al.
504 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
amphetamine locomotion, and enhanced MPTP toxic-
ity. Proc Natl Acad Sci USA 94, 9938–9943.
29 Kova

´
cs GG, La
´
szlo
´
L, Kova
´
cs J, Jensen PH,
Lindersson E, Botond G, Molna
´
r T, Perczel A, Hudecz
F, Mezo G et al. (2004) Natively unfolded tubulin
polymerization promoting protein TPPP ⁄ p25 is a
common marker of alpha-synucleinopathies. Neurobiol
Dis 17, 155–162.
30 Kova
´
cs GG, Gelpi E, Lehotzky A, Ho
¨
ftberger R, Erdei
A, Budka H & Ova
´
di J (2007) The brain-specific
protein TPPP ⁄ p25 in pathological protein deposits of
neurodegenerative diseases. Acta Neuropathol 113, 153–
161.
31 Skjoerringe T, Lundvig DM, Jensen PH & Moos T
(2006) P25a ⁄ Tubulin polymerization promoting protein
expression by myelinating oligodendrocytes of the
developing rat brain. J Neurochem 99, 333–342.

32 Acevedo K, Li R, Soo P, Suryadinata R, Sarcevic B,
Valova VA, Graham ME, Robinson PJ & Bernard O
(2007) The phosphorylation of p25 ⁄ TPPP by LIM
kinase 1 inhibits its ability to assemble microtubules.
Exp Cell Res 313, 4091–4106.
33 Wersinger C, Vernier P & Sidhu A (2004) Trypsin dis-
rupts the trafficking of the human dopamine transporter
by a-synuclein and its A30P mutant. Biochemistry 43,
1242–1253.
34 Wersinger C & Sidhu A (2005) Disruption of the inter-
action of a-synuclein with microtubules enhances cell
surface recruitment of the dopamine transporter.
Biochemistry 44, 13612–13624.
35 Vincze O, To
¨
ke
´
si N, Ola
´
h J, Hlavanda E, Zotter A,
Horva
´
th I, Lehotzky A, Tiria
´
n L, Medzihradszky KF,
Kova
´
cs J et al. (2006) Tubulin polymerization promot-
ing proteins (TPPPs): members of a new family with
distinct structures and functions. Biochemistry 45 ,

13818–13826.
36 Gu HH, Ahn J, Caplan MJ, Blakely RD, Levey AI &
Rudnick G (1996) Cell-specific sorting of biogenic
amine transporters expressed in epithelial cells. J Biol
Chem 271, 18100–18106.
37 Mortensen OV, Kristensen AS, Rudnick G & Wiborg
O (1999) Molecular cloning, expression and character-
ization of a bovine serotonin transporter. Brain Res
Mol Brain Res 71, 120–126.
38 Jensen LD, Vinther-Jensen T, Kahns S, Sundbye S &
Jensen PH (2006) Cellular parkin mutants are soluble
under non-stress conditions. Neuroreport 17, 1205–1208.
39 Kleinnijenhuis AJ, Hedegaard C, Lundvig D, Sundbye
S, Issinger OG, Jensen ON & Jensen PH (2008) Identifi-
cation of multiple post-translational modifications in
the porcine brain specific p25a. J Neurochem 106,
925–933.
40 Kubo S, Kitami T, Noda S, Shimura H, Uchiyama Y,
Askawa S, Minoshima S, Shimizu N, Mizuno Y & Hat-
tori N (2001) Parkin is associated with cellular vesicles.
J Neurochem 78, 42–54.
41 Larsen MB, Fjorback AW & Wiborg O (2006) The
C-terminus is critical for the functional expression of
the human serotonin transporter. Biochemistry 45,
1331–1337.
A. W. Fjorback et al. p25a regulates dopamine transporter function
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 505