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 filament
Trang 1Formation of aberrant phosphotau fibrillar polymers
in neural cultured cells
Mar Pe´rez1, Fe´lix Herna´ndez1, Alberto Go´mez-Ramos1, Mark Smith2, George Perry2and Jesu´s Avila1 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 are required for tau filament forma-tion Importantly, the in vitro formation of intracellular tau aggregates could be used as a model of 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, indicating that sulfated glycosaminoglycans or
other anionic compounds could favour tau polymerization
[9–11]
Another category of agents suggested to alter assembly
are fatty acids that can facilitate aggregation either directly
[12–15] and/or additionally through a reaction with the
highly reactive products of lipid oxidation [16,17]
Addi-tionally, proteolysis [18] and other tau modifications such as
phosphorylation, glycation or oxidation [19–23] could play
a role in the aberrant tau aggregation
The most studied mechanism of tau polymerization is
phosphorylation, although under many conditions,
hyper-phosphorylated tau does not show a high capacity for
in vitropolymerization as compared to unmodified tau [24]
This represents a pivotal paradox as mutations linked to
familiar Alzheimer’s disease (AD, the most common
tauopathy) like those found in presenilin-1 (PS1) and
amyloid b protein precursor (APP), result in an increase 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 product of lipid peroxidation [17] that is increased in AD [27] To extend these latter studies, in this work we investigated the effect of HNE on tau in different phosphorylation status within neuroblastoma cells As in cell free systems, tau phosphorylation is essential to HNE induced assembly
M A T E R I A L S A N D M E T H O D S
Materials Okadaic acid (OA) was purchased from Sigma 4HNE was prepared as described previously [28] PHF-1 antibody reacting with phosphotau [29] was a kind gift of P Davies (Albert Einstein College, Bronx, NY, USA); 7.51 and BR134 antibodies, reacting with tau protein, were a kind gift of C.M Wischik (MRC, Cambridge, UK) [30]; a polyclonal antibody 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 in chilled NaCl/Pi, resuspended and homogenized in buffer containing 50 mM Hepes, pH 7.4,
10 mMEDTA, 0.1% Triton X-100, 100 mMNaF, 0.1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 10 lgÆmL)1leupeptin, 10 lgÆmL)1pepstatin and
10 lgÆmL)1aprotinin Lysates were centrifuged at 10 000 g for 30 min at 4°C and boiled for 5 min in electrophoresis sample buffer The amount of protein in the samples was quantitated by the BCA protein assay SDS/PAGE was carried out using 10% gels, which were afterwards transferred to nitrocellulose to be tested with different antibodies Immunoreactive proteins were 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 3978440, E-mail: javila@cbm.uam.es
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 31 October 2001, revised 11 January 2002, accepted 18
January 2002)
Trang 2chemiluminiscence 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-ted with 10% fetal bovine serum and 2 mMglutamine plus
0,01% pyruvate in a humidified atmosphere with 7% CO2
The day before the experiment, the cells were subcultured,
and a cell suspension was placed into the wells After
overnight incubation in growth medium, the SH-SY5Y cells
were washed and incubated in DMEM without fetal bovine
serum containing vehicle, 0.25 lMOA, 10 lMHNE or OA
plus HNE for 45 min
Immunofluorescence analysis
Cells plated on polylysine-coated coverslips were fixed with
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 the incubation, the coverslips were washed with
NaCl/Pisupplemented with 0.1% Triton X-100 (NaCl/Pi/
Triton), for 10 min, then were incubated with 1% fetal
bovine serum in NaCl/Pi/Triton for additional 10 min
Incubation with primary antibodies was carried out in
NaCl/Pi/Triton for 45 min at room temperature Coverslips
were rinsed three times with NaCl/Pi/Triton and incubated
for 30 min with Oregon green or Texas-Red conjugated
secondary antibodies (1 : 400; Molecular Probes) Finally,
cells were rinsed with NaCl/Pi/Triton and mounted in
Fluoromount Coverslips were analyzed using a Zeiss
epifluorescence microscope Films were scanned in Filmscan
200 (EPSON), and images were processed in Adobe
PHOTOSHOP5.02 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 homogenized in buffer A (0.1M Mes, pH 6.5,
0.5 mM MgCl2, 2 mM EGTA, 0.5M NaCl, 1 mM
phenylmethanesulfonyl fluoride, 10 lgÆmL)1 leupeptin,
10 lgÆmL)1pepstatin and 10 lgÆmL)1aprotinin) by using
a Potter homogenizer provided with a loosely fitting Teflon pestle Homogenates 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 airfugue centrifuge (100 000 g) for 1 h, and the pelleted protein was tested Electron microscopy and immunoelectronmicroscopy
To test for the presence of intracellular aggregates,
untreat-ed or treatuntreat-ed cells were fixuntreat-ed with 4% paraformaldehyde and 2% glutaraldehyde in cacodylate buffer for 60 min at
4°C SH-SY5Y cells were collected and spun down at
1000 g for 5 min The pellet was postfixed in 1% osmium tetroxide for 1 h and, afterwards, in 1% uranyl acetate After dehydration 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, samples were placed on a carbon-coated grid for 2 min and then stained with 2% (w/v) uranyl acetate for 1 min Transmis-sion electron microscopy was performed in a JEOL Model 1200EX electron microscope operated at 100 kV Electron micrographs were obtained at a magnification of 40 000 on Kodak SO-163 film
Immunoelectron microscopy was performed after adsorption of the samples to electron microscopy grids and an incubation with the first antibody [(anti-HNE or anti-(tau BR134)], for 1 h at room temperature, was performed After extensive washing with NaCl/Pi, the grids were incubated with the secondary antibody conjugated with 5-nm diameter gold particles Finally, the samples were negatively stained and observed, as described above
R E S U L T S
Reaction of isolated PHF with an antibody raised against HNE
In an earlier report, we showed increased and selective adduction of lipid peroxidation products such as HNE in association with neurofibrillary tangles [27], the aberrant aggregates present in AD, and composed of bundles of paired helical filaments (PHF) [33] To extend this, here we determined that isolated PHF also contained protein-HNE conjugates (Fig 1A) suggesting that HNE could play an
in vivorole in the formation of tau aberrant aggregates As a
Fig 1 Reaction of an antibody raised against
HNE with isolated paired helical filaments
(PHF) PHF were isolated as indicated in
Methods and tested with Ab HNE (A) or with
PHF-1 (B) The result of that reaction is
shown Bar indicates 200 nm.
Trang 3positive control, the reaction of PHF 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 to the antibody because it could be partially
hidden in PHF structure Another possibility could be that
HNE modification may occur after PHF formation, being
that modification not recognized by the antibody
Although, some HNE molecules could be available to react
with the antibody This fact could also explain the relatively
weak reaction of HNE antibodies with neurofibrillary
tangles compared to that 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 inhibitor, results in the
hyperphosphorylation of tau protein, as determined by its
change in electrophoretic mobility and 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] The 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 SH-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 showed, in a cell-free system [17] that phosphorylated tau, in the presence of HNE, polymerizes into fibrillar polymers To test if, in a similar way, tau could form aggregates in cultured cells, neuroblastoma cells were incubated in the absence (Fig 3A), or the presence of OA (Fig 3B), HNE (Fig 3C), or a mixture of OA/HNE (Fig 3D) As clearly shown in Fig 3 cells treated with
OA show an increase in PHF-1 immunoreactivity with 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 in 9.2 ± 1.1% (n ¼ 4 independent experiments) of the cell treated with OA/HNE Thus, OA/HNE treatment 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 these aggregates, tau phosphorylation
is partially resistant to the action of alkaline phosphatase (data not shown), phenomenon that has been also observed
in cultured rat hippocampal neurons [32] These data suggest that tau is in a polymerized or aggregated form, similar to that observed in tauopathies, such as AD, where tau phosphoepitopes are masked and dephosphorylation by
AP is similarly restricted
Electron microscopy analysis of neuroblastoma 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 previous 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 SH-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 the presence of both (D) Then, the cells were lysed and the
presence of tau was analyzed by gel electrophoresis and Western blot
by using tau antibody 7.51 (I and II), or tau antibody PHF-1 (III) (I)
For 7.51 reaction, a decrease in electrophoretic mobility was found in
OA treated cells (II) No differences in electrophoretic mobility were
found for 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 cells) and in OA/HNE treated
cells (D, 4.20 ± 0.86-fold over control cells) compared to that found
in the absence of treatment (A, control cells) or the presence of HNE
( 0.80 ± 0.18-fold over control cells).
Fig 3 Formation of tau aggregates in neuroblastoma cells 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 both (D) The presence of aggregates is indicated in (D), after immunofluorescence by using PHF-1 tau antibody The arrow shows the aggregate, and the arrowhead the nucleus Bar indicates 15 lm.
Trang 4of fibrillar polymers Thus, to confirm the data, we tried to
further characterize those filaments from the cell
homogen-ate Figure 5A–C) shows the presence in OA/HNE treated
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 structure These filaments could be 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 compound
derived from oxidative stress
D I S C U S S I O N
Previous studies in cell-free systems 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 the
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 precedes amyloid plaque 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 tau and induces the Alz50 epitope in tau [35] It is important that the ability 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 epitope [35] In this study, we found that tau phosphorylation and HNE treatment of neuroblastoma cultured cells results in the assembly of tau into aberrant polymers similar to those found in human tauopathies This provides the foundation of a good model
to test different compounds that could prevent abnormal tau aggregation
The polymers assembled in OA/HNE treated neuro-blastoma cells are partially resistant to alkaline phospha-tase, a feature previously described [32] and that is also observed in tau filaments from some tauopathies These polymers from neuroblastoma cells have mainly a diameter
of 2–3 nm and are similar to those isolated from the brain
Fig 5 Presence of tau filaments in OA-plu-HNE treated
neuroblasto-ma cells Electron microscopy of negatively stained filaments (A,B,C) and immunogold electron microscopy (D,E,F) with tau antibody BR134 A secondary antibody conjugated with 5 nm diameter gold particle was used 20–30 filaments were found per carbon-coated grid loaded with protein obtained from OA/HNE treated cells No fila-ments were observed in control, OA- or HNE-treated cells Samples were obtained as described in Materials and methods Scale bar rep-resents 100 nm.
Fig 4 Aberrant aggregates in OA ± HNE treated neuroblastoma
cells The presence of intracellular filamentous aggregates were
observed in some cell sections after OA + HNE treatment (see
Materials and methods) Bar indicates 200 nm.
Trang 5of progressive supranuclear palsy (PSP) patients [36].
Nevertheless, wider polymers, similar to those described in
other tauopathies are also found It is not known if 2–3 nm
polymers could be precursors for the formation of wider
(10 nm) polymers Additionally, filaments present in
OA/HNE treated cells are not twisted suggesting that some
additional factors could be necessary to obtain twisted
filaments [37]
Recently, it was described in non-neural cells that
transfection with tau cDNA carrying some of the
muta-tions present in a tauopathy, frontotemporal dementia
linked to chromosome 17 (FTDP-17), results in the
expression of the mutated protein and in the formation
of aberrant tau aggregates, indicating that these aggregates
could be assembled in cultured cells [38] However, in other
tauopathies such as Alzheimer’s disease tau mutations are
not required for the formation of aberrant aggregates [1]
and only a post-translational modification,
phosphoryla-tion, has been proposed to play a role in tau assembly
[8,39] This role has been recently tested [40] and, in
agreement with our data, their results suggest that
phos-phorylated tau has a higher capacity for 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 mutated form) [26,42] could
induce tau phosphorylation through the activation of
GSK3 On the other hand, some other tau protein kinases
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 The residues modified by those
protein kinases in tau protein, could be dephosphorylated
by the action of some okadaic acid-sensitive phosphatases
such as PP2A or PP1 [45,46] Thus, we have treated neural
cells with OA to increase cellular phosphorylated tau A fact
that was tested by the 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 in tau) However, tau phosphorylation is not
sufficient for its aggregation This work shows that a second
element, HNE, is required for tau aggregation
HNE can easily pass through neuronal compartments
and bind to tau protein [32] If tau is phosphorylated, the
reaction with HNE modifies its conformation [35] and
promotes its assembly into fibrillar polymers [17] resembling
NFTs [47] Phosphorylation could facilitate a tau
conform-ational change that may allow the interaction of HNE with
those tau regions mainly involved in polymer formation
One of these regions is the third tubulin-binding motif
present in tau 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 filaments An additional
possibility could be that HNE may suffer a modification
after filament formation or that a twisted process would be
necessary to expose the HNE epitope
In summary, we demonstrate here that intraneuronal
hyperphosphorylated tau, in the presence 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 and lipid peroxidation are
concurrent features of disease
A C K N O W L E D G E M E N T S
This work was supported by grants from Spanish CICYT, Comunidad
de Madrid, Neuropharma and by an institutional grant from Fundacio´n R Areces and by the National Institutes of Health (to GP) A predoctoral fellowship from Gobierno Vasco was awarded to
A Go´mez-Ramos The help of R Cuadros and S Soto-Largo is acknowledged Also, we acknowledge to Dr J J Lucas for critical reading of the manuscript.
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