Báo cáo Y học: Formation of aberrant phosphotau fibrillar polymers in neural cultured cells doc

Formation of aberrant phosphotau fibrillar polymers
in neural cultured cells
Mar Pe
´
rez
1
,Fe
´
lix Herna
´
ndez
1
, Alberto Go
´
mez-Ramos
1
, Mark Smith
2
, George Perry
2
and Jesu
´
s Avila
1
1
Centro de Biologı
´
a Molecular (CSIC/UAM), Facultad de Ciencias. Universidad Auto
´
noma de Madrid, Spain;
2

Institute of Pathology, Case Western Reserve University, Cleveland, OH, USA
Here we show, for the first time, the in vitro formation of
filamentous aggregates of phosphorylated tau protein in
SH-SY5Y human neuroblastoma cells. The formation of
such aberrant aggregates, similar to those occurring in vivo in
Alzheimer’s disease and other tauopathies, requires okadaic
acid, a phosphatase inhibitor , to increase the level of phos-
phorylated tau, and hydroxynonenal, a product of oxidative
stress that selectively adducts and modifies phosphorylated
tau. Our findings suggest that both phosphorylation and
oxidative modification a re r equired f or tau filament forma-
tion. I mportantly, the in vitro formation of intracellular tau
aggregates could be used as a model o f tau polymerization
and facilitate the development of novel therapeutic
approaches.
Keywords: Alzheimer’s disease; tauopathies; oxidative stress;
tau phosphorylation; aberrant aggregates.
Tauopathies are a heterogeneous group of dementias
sharing a common pathological hallmark, the presence of
aberrant tau filaments or forms of tau [1,2]. Tau is a
microtubule-associated protein [3,4] that in pathological
situations, and in a hyperphosphorylated form [5–8]
assembles into fibrillar polymers. The mechanism for that
aberrant tau assembly has been widely analyzed by several
groups, i ndicating that sulfated glycosaminoglycans or
other anionic compounds could favour tau polymerization
[9–11].
Another category of agents suggested to alter assembly
are fatty acids t hat can facilitate a ggregation e ither dire ctly
[12–15] and/or additionally through a reaction with the

highly reactive products of lipid oxidation [16,17]. Ad di-
tionally, proteolysis [18] and o ther tau modifications such as
phosphorylation, glycation or oxidation [19–23] could p lay
a role in the aberrant tau aggregation.
The most studied mechanism of tau polymerization is
phosphorylation, although under m any conditions, hyper-
phosphorylated tau does not show a high c apacity f or
in vitro polymerization as compared t o unmodified tau [24].
This represents a pivotal paradox as mutations linked t o
familiar A lzheimer’s disease (AD, the mo st common
tauopathy) like those f ound in presenilin-1 (PS1) a nd
amyloid b protei n precursor ( APP), result in an i ncrease in
both the level of phosphorylation of tau protein [ 25,26] and
in its aggregation leading to neurofibrillary tangles. None-
theless, it is clear that phosphorylated, but not unmodified
tau, is able to polymerize in vitro in the presence of 4-
hydroxynonenal (HNE), a naturally occurring p roduct of
lipid peroxidation [17] that is increased in AD [27]. T o
extend these latter studies, in this work we investigated the
effect of HNE on tau in d ifferent phosphorylation status
within neuroblastoma cells. As in cell f ree systems, t au
phosphorylation is essential to HNE induced assembly.
MATERIALS AND METHODS
Materials
Okadaic a cid ( OA) was purchased f rom Sigma. 4HNE wa s
prepared as described previously [28]. P HF-1 antibody
reacting with phosphotau [29] was a kind gift of P. Davies
(Albert E instein College, Bronx, NY, USA); 7.51 a nd
BR134 antibodies, reacting w ith t au protein, were a kind
gift of C.M. Wischik (MRC, Cambridge, UK) [30]; a

polyclonal a ntibody specific for the lysine-derived pyrrole
adducts formed by HNE was used [27]. Alkaline phospha-
tase was purchased from Roche.
Gel electrophoresis and Western blot
Cells were harvested i n chilled N aCl/P
i
, resuspended and
homogenized in buffer c ontaining 50 m
M
Hepes, pH 7.4,
10 m
M
EDTA, 0.1% Triton X -100, 100 m
M
NaF, 0.1 m
M
sodium orthovanadate, 1 m
M
phenylmethanesulfonyl
fluoride, 10 lgÆmL
)1
leupeptin, 1 0 lgÆmL
)1
pepstatin a nd
10 lgÆmL
)1
aprotinin. Lysates were centrifuged at 10 000 g
for 30 min at 4 °C a nd boiled f or 5 min in electrophoresis
sample buffer. The amount of protein in the samples was
quantitated by the BCA protein a ssay. SDS/PAGE was

carried out using 10% gels, w hich were afterwards
transferred to n itrocellulose to be tested with different
antibodies. Immunoreactive proteins w ere visualized by
Correspondence to J. Avila, Centro de Biologı
´
a Molecular (CSIC/
UAM), Facultad de Ciencias. Universidad Auto
´
noma de Madrid,
Cantoblanco 28049, Madrid, Spain. Fax: + 34 91 3974499,
Tel.: + 34 91 39 78440, E-mail:
Abbreviations: AD, Alzheimer’s disease; APP, amyloid precursor
protein; DMEM, Dulbecco’s modified Eagle’s medium; HNE,
4-hydroxynonenal; NFT, neurofibrillary tangles; OA, okadaic acid;
PHFs, paired helical filaments; PP1, protein phosphatase 1; PP2A,
protein phosphatase 2A; PS1, presenilin-1; PSP, progressive
supranuclear palsy.
(Received 3 1 October 2 001, revised 1 1 January 20 02, accepted 18
January 2 002)
Eur. J. Biochem. 269, 1484–1489 (2002) Ó FEBS 2002
chemiluminiscence detection (ECL kit from Pierce). Quan-
titation of immunoreactivities was performed by densito-
metric scanning.
Cell culture SH-SY5Y neuroblastoma cells
Human neuroblastoma SH-SY5Y cells [31] were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supplemen-
tedwith10%fetalbovineserumand2m
M
glutamine plus
0,01% pyruvate i n a humidified atmosphere wit h 7 % CO

2
.
The day before the experiment, the cells were subcultured,
and a cell s uspension was placed into the w ells. A fter
overnight incubation in growth medium, the SH-SY5Y cells
were washed and incubated in DMEM w ithout fetal bovine
serum c ontaining vehicle, 0.25 l
M
OA, 10 l
M
HNE or OA
plus HNE for 45 min.
Immunofluorescence analysis
Cells plated on polylysine-coated coverslips w ere fi xed w ith
4% paraformaldehyde for 30 min. Dephosphorylation of
phosphotau in fixed cultured cells using alkaline phospha-
tase were carried out as described by Mattson et al. [32].
After t he incubation, the c overslips w ere w ashed with
NaCl/P
i
supplemented with 0.1% Triton X-100 (NaCl/P
i
/
Triton), for 10 min, then were incubated with 1 % fetal
bovine serum in NaCl/P
i
/Triton for additional 1 0 min.
Incubation with primary antibodies was carried out in
NaCl/P
i

/Triton for 45 min at room temperature. Coverslips
were rinsed three t imes with NaCl/P
i
/Triton and incuba ted
for 30 min with Oregon green or Texas-Red conjugated
secondary antibodies (1 : 400; Molecular Probes). F inally,
cells were r insed with N aCl/P
i
/Triton a nd mounted i n
Fluoromount. Coverslips were analyzed using a Zeiss
epifluorescence microscope. Films were scanned in Filmscan
200 (EPSON), and images were processed in Adobe
PHOTOSHOP
5.02 on a PC workstation.
Isolation of PHF and tau filaments
Brain samples, supplied by R. Ravid (Netherlands Brain
Bank), from AD patients, were used as a source to isolate
PHFs, by following the procedure of Greenberg and Davies
[29]. To obtain filaments from SH-SY5Y cells, cells
were homogen ized in buffer A (0.1
M
Mes, pH 6.5,
0.5 m
M
MgCl
2
,2m
M
EGTA, 0.5
M

NaCl, 1 m
M
phenylmethanesulfonyl fluoride, 10 lgÆmL
)1
leupeptin,
10 lgÆmL
)1
pepstatin and 10 lgÆmL
)1
aprotinin) by using
a P otter homogenizer provided with a loosely fitting Teflon
pestle. H omogenates were analyzed by direct adsorption of
the samples to electron microscopy grids. Western blots
studies, using Ab 7.51, homogenates from SH-SY5Y neuro-
blastoma cells cultured in a P100 dish, were centrifuged at
the highest speed of a Beckman a irfugue centrifuge
(100 000 g) for 1 h, and the pelleted protein was tested.
Electron microscopy and immunoelectronmicroscopy
To t est for the presence of intracellular aggregates, untreat-
ed or treated cells were fixed with 4% paraformaldehyde
and 2 % g lutaraldehyde in cacodylate buffer for 60 min at
4 °C. SH-SY5Y cells were co llected a nd spun down at
1000 g for 5 min The pellet was postfixed i n 1% osmium
tetroxide for 1 h and, afterwards, in 1 % u ranyl a cetate.
After d ehydration with graded alcohols, the pellets were
embedded in Epon and polymerized at 60 °C for 48 h.
Ultrathin sections were observed by electron microscopy.
To test for the presence of isolated filaments, s amples
were placed on a carbon-coated grid fo r 2 min and then
stained with 2% (w/v) uranyl acetate for 1 min. Transmis-

sion electron microscopy was performed in a JEOL M odel
1200EX electron microscope operated at 100 kV. Electron
micrographs were obtained at a magnification of 40 000 on
Kodak SO-163 film.
Immunoelectron m icroscop y was perform ed after
adsorption of the s amples to electron microscopy grids
and an incubation with the first antibody [(anti-HNE or
anti-(tau BR134)], for 1 h a t room temperature, was
performed. After extensive washing w ith NaCl/P
i
,thegrids
were incubated w ith the secondary antibody conju gated
with 5-nm diame ter gold particles. Finally, the samples were
negatively stained and observed, as described above.
RESULTS
Reaction of isolated PHF with an antibody raised
against HNE
In an earlier report, we showed increased a nd selective
adduction of lipid peroxidation products such as HNE in
association w ith n eurofibrillary tangles [27], the aberrant
aggregates present in AD, and composed of bundles of
paired helical filaments (PHF) [33]. To extend this, here w e
determined that isolated PHF also c ontained p rotein-HNE
conjugates (Fig. 1A) suggesting that HNE could play an
in vivo role in the formation of tau aberrant aggregates. As a
Fig. 1. Reaction of an antibody raised against
HNE with isolated paired h elical filaments
(PHF). PHF w ere isolated a s indicated in
Methods and tested with Ab HNE (A) or with
PHF-1 (B). T he result of that reaction is

shown. B ar indicates 200 n m.
Ó FEBS 2002 Tau aggregates in cultured cells (Eur. J. Biochem. 269) 1485
positive c ontrol, the r eaction of P HF with a tau antibody is
shown in Fig. 1B. That HNE immunoreactivity is only seen
on some regions of PHF may be due to HNE induced
facilitation of tau–tau interaction. If so, HNE may be not
easily accessible t o the antibody because it could be partially
hidden in PHF structure. Another possibility could be t hat
HNE modification may occur after PHF formation, being
that modification not recognized by the antibody.
Although, some HNE molecules could b e available to react
with the antibody. This fact could also explain the relatively
weak reaction of HNE antibodies with neurofibrillary
tangles c ompared to t hat found in neuronal cytoplasm [ 27].
Tau in okadaic acid treated cells
is in hyperphosphorylated form
Treatment of SH-SY5Y human neuroblastoma cells with
okadaic acid (OA), a phosphatase inh ibitor, results in the
hyperphosphorylation of tau protein, as determined by its
change in electrophoretic mobility a nd its reaction with Ab
7.51, an antibody that recognizes all tau isoforms inde-
pendently of its phosphorylation status (Fig. 2, part I) or by
the reaction with tau antibodies that specifically recognize
phosphoepitopes, such as PHF-1 (Fig. 2, part III). This
modified tau resembles that found in the brain of patients
with different tauopathies [1]. T he OA-induced phosphory-
lation of tau could be reversed by alkaline phosphatase
treatment (data not shown). HNE treatment did not alter
the level of tau phosphorylation found in OA treated cells as
a similar pattern to that observed with OA alone (Fig. 2,

part IIB) was observed in S H-SY5Y human neuroblastoma
cells treated with OA/HNE (Fig. 2, part IID).
Tau forms aberrant aggregates in neuroblastoma cells
treated with okadaic acid and 4-hydroxynonenal
Recently, we s howed, in a cell-free system [17] that
phosphorylated tau, in the presence of H NE, polymerizes
into fibrillar polymers. To test if, i n a similar way, tau could
form aggregates in cultured cells, neuroblastoma cells were
incubated in the absence (Fig. 3A), or the presence of O A
(Fig. 3 B), HNE (Fig. 3C), or a mixture of OA/HNE
(Fig. 3 D). As c learly shown in Fig. 3 cells treated with
OA show an increase in PHF-1 immunoreactivity w ith a
diffuse pattern (Fig. 3B) while in cells treated with OA/
HNE the pattern of PHF-1 immunoreactivity was clearly
present in patches (Fig. 3D). Patches were present i n
9.2 ± 1.1% (n ¼ 4 i ndependent experiments) of the cell
treated w ith OA/HNE. T hus, OA/HNE t reatment may
result in the formation of aberrant aggregates (patches)
distributed through the cytoplasm. These aggregates could
be stained with antibodies raised against phosphotau
(PHF-1). Notably, in thes e aggregates, tau phosphorylation
is partially resistant to t he action of alkaline phosphatase
(data not shown), pheno menon that has been also observed
in cultured rat hippocampal n eurons [32]. T hese data
suggest that tau is in a polymerized or aggregated f orm,
similar to that observed i n t auopathies, such as AD , where
tau phosphoepitopes are masked and dephosphorylation by
AP is similarly restricted.
Electron microscopy analysis of neu roblastoma cell
sections also suggests the existence of aberrant filamentous

aggregates in OA/HNE treated neuroblastoma cells
(Fig. 4 ). These filamentous aggregates were not found in
control, OA or HNE treated cells.
Tau filaments are assembled in OA/HNE treated
neuroblastoma cells
The p revious results suggest that tau aggregates found in
OA/HNE treated neuroblastoma cells could be composed
Fig. 2. Okadaic acid (OA) treatment results in an increase of tau
phosphorylation. S H -SY5Y neuroblastoma cells were treated for
45 min in the absence ( A), or presence of 0.25 l
M
OA (B), 10 l
M
HNE
(C) or in th e presence o f both (D). Then, the cells were lysed and the
presence of tau w as analyzed by gel electrophoresis and Western blot
by using tau antibody 7.51 (I and II), or tau antib ody PHF-1 (III). (I)
For 7.51 reaction, a decrease i n electrophoretic mobility was found in
OA treated c ells. ( II) No differences in electrophoretic mobility w ere
found f or samples treated with OA (B) or OA/HNE ( D). (III) For
PHF-1 reaction, an increase in that reaction was found in the presence
of OA (B, 4.55 ± 0.46-fold over control c ells) and in OA/HNE treated
cells (D, 4.20 ± 0.86-fold over control c ells) compared to t hat found
in the absence of treatment ( A, control c ells) or t he presence o f HNE
( 0. 80 ± 0.18-fold ove r control cells).
Fig. 3. Formation of t au aggregates in neuroblastoma c ells. SH -SY5Y
neuroblastoma cells were incubated in the absence (A), or presence of
0.25 l
M
OA (B), 10 l

M
HNE (C), or b oth (D). The presence of
aggregates is indicated i n (D), after i mmuno fluorescence by using
PHF-1 t au antibody. T he arro w shows th e aggregate, an d t he
arrowhead the nucleus. Bar indicates 15 lm.
1486 M. Pe
´
rez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
of fibrillar polymers. Thus, to confirm t he data, we tried to
further c haracterize t hose fi laments from t he cell homogen-
ate. Figure 5A–C) shows the presence in OA/HNE treate d
cells of fibrillar polymers of 2–3 nm width, and, in some
cases, wider 10 nm polymers were also found (not shown).
These filaments were straight, and they did not presented a
twisted s tructure. These filaments could b e stained with tau
antibody BR134 (Fig. 5D–F) but a weak, if any, reaction
with anti-HNE Ig was observed. These filaments were not
found in control, OA or HNE treated cells. These results
indicate that phosphotau protein could form aberrant
filaments, in cultured cells, in the presence of a c ompound
derived from oxidative stress.
DISCUSSION
Previous studies i n cell-free s ystems had suggested that
phosphorylation and HNE binding act synergistically to
promote tau aggregation [17].
In this study, we further extend those observations but in
a context that mimics those conditions in which wild-type
phosphotau forms polymers in tauopathies. We have also
found here that HNE -adducts are associated with PHF th e
component of neurofibrillary tangles ( NFT).

HNE, a product of lipid peroxidation has been found
associated in vivo with NFT [27] and it is able to modify in a
way that results in the in vitro assembly of PHF like filaments
[17]. Interestingly, it has been recently published that lipid
peroxidation also precede s amyloid p laque formation [ 34]
giving a strong support to a possible role of HNE in the
formation of the aberrant structures found in AD.
The second essential element for tau assembly is its
phosphorylation. We have previously found that HNE
reacts with normal t au and induces the A lz50 epitope in tau
[35]. It is important that the a bility of HNE to create the
Alz50 epitope not only is dependent on lysine residues of tau
but also requires tau phosphorylation because neither
methylated, recombinant, nor dephosphorylated tau reacts
with HNE to create the Alz50 epit ope [35]. In this study, w e
found that tau phosphorylation and HNE treatment of
neuroblastoma cultured cells results in the assembly of tau
into aberrant polym ers similar t o those found in human
tauopathies. This provides the f oundation of a g ood m odel
to test different compounds that could prevent abnormal
tau aggregation.
ThepolymersassembledinOA/HNEtreatedneuro-
blastoma cells are partially resistant t o alkaline phospha-
tase, a feature previously described [ 32] a nd that is also
observed in tau filaments from some tauopathies. These
polymers f rom neuroblastoma cells have mainly a diameter
of 2–3 n m and are similar to t hose isolated from the brain
Fig. 5. Presence of tau filaments in OA-plu-HNE t reated ne uroblasto-
ma cells. E lectron microscopy o f negatively stained filaments (A,B ,C)
and i mmunogold electron mic roscopy (D,E,F) with tau antibod y

BR134. A secondary antibody conjugated with 5 n m diameter gold
particle was u sed. 20–30 filaments w ere found per c arbon-coated grid
loaded with protein obtained fro m OA/HN E t reated cells. No fi la-
ments were observed in control, OA- or HNE-treated cells Samples
were obtained as described in M aterials and m ethods. S cale bar rep-
resents 100 nm.
Fig. 4. Aberrant aggregates in OA ± H NE treated neuroblastoma
cells. The presence of intracellular filamentous aggregates were
observed in some cell sections after OA + HNE t reatment (see
Materials a nd methods). Bar indic ates 200 nm.
Ó FEBS 2002 Tau aggregates in cultured cells (Eur. J. Biochem. 269) 1487
of progressive supranuclear palsy (PSP) patients [36].
Nevertheless, wider polymers, similar to those described in
other tauopathies a re also found. It is not known if 2 –3 nm
polymers could be p recursors for the f ormation of wider
(10 n m) polymers. Additionally, fi laments p resent in
OA/HNE treated cells are not twisted s uggesting that some
additional factors could be n ecessary to obtain twisted
filaments [37].
Recently, it was described in non-neural cells that
transfection with tau cDNA carrying some of t he muta-
tions present in a tauopathy, frontotemporal d ementia
linked t o chromosome 17 (FTDP-17), r esults in the
expression of the mutated protein and in the f ormation
of aberrant tau aggregates, indicating that these a ggregates
couldbeassembledinculturedcells[38].However,inother
tauopathies such a s Alzheimer’s disease tau mutations are
not required f or the formation of aberrant aggregates [1]
and only a post-translational modification, phosphoryla-
tion, has been proposed to play a r ole in tau assembly

[8,39]. This r ole h as bee n recently t ested [ 40] and, in
agreement with our data, their results suggest that phos-
phorylated tau has a high er c apacity f or self-assembly than
unmodified tau.
It is not well known how tau phosphorylation is
promoted. It has been suggested that proteins such as beta
amyloid [ 41] or presenilin 1 (in m utated form) [26,42] c ould
induce tau phosphorylation through the activation of
GSK3. On the other hand, some other tau protein k inases
could be activated by other ways, such as by oxidative stress
[43,44]. These kinases could play a role in tauopathies such
as Alzheimer’s disease. T he residues modified b y those
protein kinases in tau protein, c ould be dephosphorylated
by the action of some okadaic acid-sensitive phosphatases
such as PP2A or PP1 [45,46]. Thus, we have t reated neural
cells with OA to increase cellular phosphorylated tau. A fact
that was tested by t he use of an antibody (PHF-1) that
recognized tau in phosphorylated form (also an increase in
tau phosphorylation was found by testing with two other
antibodies, AT8 and 12E8, that recognize other phospho-
epitopes i n tau). H owever, tau phosphorylation is n ot
sufficient for its aggregation. This work shows that a second
element, HNE, is required for tau aggregation.
HNE c an easily pass through neuronal c ompartments
and bind t o tau protein [32]. If tau is phosphorylated, the
reaction with HNE modifies i ts conformation [35] and
promotes its assembly into fibrillar polymers [17] resembling
NFTs [47]. Phosphorylation could facilitate a tau conform-
ational c hange that may allow t he interaction of HNE with
those tau regions mainly involved in polymer formation.

One o f t hese regions is the third tubulin-binding motif
present i n t au molecule [11]. Thus, HNE binding domain is
inside the filament structure and an anti-HNE Ig may not be
able to label the OA/HNE treated fi laments. An additional
possibility could b e t hat HNE may suffer a modification
after filament formation or t hat a twisted process would be
necessary to expose the HNE epitope.
In sum mary, we demonstrate here t hat intraneuronal
hyperphosphorylated t au, i n the p resence of a natural
peroxidation product, HNE, forms fibrillar polymers. This
process likely resembles the mechanism responsible for the
formation of aberrant tau aggregates present in tauopathies
where both phosphorylation a nd lipid peroxidation are
concurrent features of disease.
ACKNOWLEDGEMENTS
This work was supported by grants from Spanish CICYT, Comunidad
de Madrid, Neuropharma a nd by an institutional grant from
Fundacio
´
n R. Areces and by the National Institutes of Health (to
GP). A predoctoral fellowship from Gob ierno Vasco was awarded to
A. Go
´
mez-Ramos. The help of R. Cuadros and S. Soto-Largo i s
acknowledged. Also, we a cknowledge to Dr J. J . Lucas for c ritical
reading of t he manuscript.
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Ó FEBS 2002 Tau aggregates in cultured cells (Eur. J. Biochem. 269) 1489