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.. We d
Trang 1presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity
Anja W Fjorback1,2,*, Sabrina Sundbye2, Justus C Da¨chsel2,, Steffen Sinning1, Ove Wiborg1 and Poul H Jensen2
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: phj@biokemi.au.dk
*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 ) by fluorescence 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 ) by cosedimentation in solution ( MI:0028 )
l MINT-8055878 : Synaptophysin (uniprotkb: Q62277 ), bip (refseq:GI:194033595) and p25-alpha (uniprotkb: O94811 ) colocalize ( MI:0403 ) by cosedimentation 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.
Trang 2Parkinson’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-transfectnon-glycosylat-ed cells lacknon-glycosylat-ed 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
Trang 3(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 [3H]-DA in HEK-293-MSR cells, and this was enhanced by co-expression with p25a As a control, we also
A
C
D
E
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).
Trang 4co-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 Vmax without affecting the Km
(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 Vmax
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 Vmax
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 Vmax 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
Vmaxand Kmvalues are given as means ± 1 SEM (n = 3).
V max
(pmolÆmin)1per well)
K m
(l M )
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.
Trang 5but 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 fracconcentra-tion, 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 concentrafrac-tions 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 synapsynap-tosomal 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.
Trang 6cytosolic 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 vesves-icles float-ing in the density gradient and the cytosolic proteins remainfloat-ing 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.
Trang 7dissolve 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 detecbiotinyla-tion 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).
Trang 8extracellular 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
Trang 9cannot 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 resiresi-dues
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
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 NaH2PO4, 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¢-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
(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
100 UÆmL)1penicillin at 37C and 5% CO2 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
NaCl⁄ Pi, the transfected cells were stained with the respec-tive primary antibodies, diluted in NaCl⁄ Pi 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
Probes⁄ Invitrogen) The cells were investigated using a Zeiss
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⁄ Pi:
Trang 10137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm
Tri-ton X-100, 0.1% SDS and protease inhibitor cocktail
30 min at 4C under gentle shaking The cell lysate was
cleared by centrifugation at 12 000 g at 4C for 15 min,
and the remaining supernatant was mixed with SDS sample
brom-ophenol blue, 25% glycerol) and left at 37C for 30 min
Fractions of the cell samples were analyzed by 8–16%
The membrane was blocked for 2 h in 5% dry milk in
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⁄ Pi(137 mm NaCl, 2.7 mm
KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.4)
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 [3H]-dopamine or [3
H]-serotonin, respectively For Kmand Vmaxdeterminations, the
cells were incubated with increasing concentrations of [3
H]-dopamine or [3H]-serotonin diluted 20 times with unlabeled
dopamine or serotonin, respectively The final concentration
of dopamine and serotonin ranged from 0–10 lm For single
Vmaxdeterminations, only one concentration (5 lm) of [3
H]-dopamine diluted 20 times with unlabeled H]-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 soluscintilla-tion (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
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 37C, 5% CO2, 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
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 lL of 20 mm dithioerythrei-tol-containing SDS sample buffer, and incubated for 30 min
at 37C 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