Formation, release, and internalization of stable tau oligomers in
cells
Susanne Wegmann
1,#
, Samantha Nicholls
1,#
, Shuko Takeda
1
, Zhanyun Fan
1
, and Bradley T.
Hyman
1,*
1
Massachusetts General Hospital, Dept. of Neurology, Charlestown, MA and Harvard Medical
School, Boston, MA, Massachusetts Institute of Neurodegenerative Diseases (MIND)
Abstract
Tau is a neuronal microtubule binding protein that, in Alzheimer’s disease and other
neurodegenerative diseases, can form oligomeric and large fibrillar aggregates, which deposit in
neurofibrillary tangles. Tau’s physiological state of multimerization appears to vary across
conditions, and a stable dimeric form of soluble tau has been suggested from experiments using
recombinant tau in vitro. We tested if tau dimerization, or oligomerization, also occurs in cells,
and if soluble tau oligomers are relevant for the release and internalization of tau. We developed a
sensitive tau split-luciferase assay to show the rapid intracellular formation of stable tau dimers
that are released and taken up by cells. Our data further suggest that tau dimerization can be
accelerated slightly by aggregation catalysts. We conclude that tau oligomers are a stable
physiological form of tau, and that tau oligomerization does not necessarily lead to tau
aggregation.
Graphical Abstract
Tau dimers and oligomersare intermediates
in vitro
, but their formation in cells is not established.
To study the formation of tau oligomers, we designed a split-luciferase assay enabling the sensitive
detection of tau oligomerization in cells: dimerization or oligomerization of full-length tau fused
corresponding authors: Bradley T. Hyman (bhyman@mgh.harvard.edu) and Susanne Wegmann (swegmann@mgh.harvard.edu).
#
authors contributed equally
HHS Public Access
Author manuscript
J Neurochem
. Author manuscript; available in PMC 2017 December 01.
Published in final edited form as:
J Neurochem
. 2016 December ; 139(6): 1163–1174. doi:10.1111/jnc.13866.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
to either hemisphere of Gaussia luciferase reconstitutes luciferase activity. We found that upon
expression in cells, tau rapidly forms stable intracellular oligomers that are released and can be
internalized by cells. These oligomeric species may or may not be on-pathway with tau
aggregation.
Keywords
tau protein; dimerization; oligomers; Gaussia Luciferase assay
BACKGROUND
The progressive accumulation of tau proteins is a pathological hallmark of different
neurodegenerative diseases including Alzheimer's diseases (AD) and frontotemporal
dementia (FTD), collectively termed tauopathies. In the human central nervous system, six
tau isoforms are generated by alternative splicing (Goedert
et al.
1989), of which the longest
isoform (2N4R tau) contains 441 amino acids with two N-terminal inserts and four pseudo-
repeats in the C-terminus. Tau is a neuronal protein that regulates microtubule stability and
dynamics (Weingarten
et al.
1975, Lindwall & Cole 1984, Drechsel
et al.
1992, Gigant
et al.
2014) and axonal transport (Trinczek
et al.
1999). The binding of tau to microtubules is
mediated by the C-terminal part of tau and regulated by phosphorylation in regions adjacent
to the microtubule binding sites (Preuss
et al.
1997). Tau aggregation is facilitated by
hexapeptide motifs in the C-terminal repeat domain (TauRD), and the repeat domain is
sufficient to form the core of amyloid-like paired helical filaments
in vitro
(von Bergen
et al.
2005). The long N-terminal part of tau projects from the surface of both microtubules
(Preuss et al. 1997) and tau fibril (Wegmann
et al.
2013, Sillen
et al.
2005).
In AD, tau aggregates intracellularly into paired helical filaments, accumulating in
neurofibrillary tangles and neuropil threads, and forms high molecular weight (HMW)
soluble oligomeric species containing hyper-phosphorylated tau (Lasagna-Reeves
et al.
2012, Takeda
et al.
2015). In FTDs, tau aggregates deposit in neurofibrillary tangles, Pick
bodies, glial fibrillary tangles, and/or in small silver grains, although the majority of
misfolded tau remains soluble in these diseases. While a substantial number of studies have
explored the biophysical properties of paired helical filaments from recombinant tau that
were assembled
in vitro
with the help of poly-anionic pro-aggregation molecules such as
heparin (Daebel
et al.
2012, Jeganathan
et al.
2008, von Bergen
et al.
2006, Bibow
et al.
2011, Mukrasch
et al.
2007, Kumar
et al.
2014, Wegmann et al. 2013, Wegmann
et al.
2010),
the biology and biochemical characterization of soluble multimeric tau species in a cellular
context is far less studied, in part because of the lack of tools available for such studies.
Importantly, soluble tau also is released by neurons
in vitro
and
in vivo
(Pooler
et al.
2013,
Yamada
et al.
2011), and is found even in normal healthy people in the cerebrospinal fluid
(CSF) (Yamamori
et al.
2007). Oligomeric HMW tau has also been identified in the
interstitial fluid of tau overexpressing animals (Takeda et al. 2015), raising the possibilities
that soluble tau species can aggregate in the extracellular space, or that aggregated tau
species can be secreted from tau expressing cells.
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The very low concentration of extracellular tau in the ng/ml-range (for example ~0.16 ng/ml
tau in CSF of healthy controls and ~0.85 ng/ml tau in CSF of AD patients (Johnson
et al.
1997); ~30–40 ng/ml in wild-type mouse ISF (Yamada et al. 2011) is near the detection limit
of most tau ELISA assays, and the detection with such sandwich ELISA assays takes several
hours to days. But most importantly, measuring total tau levels by ELISA does not access
the oligomerization state of tau, and thus does not inform about the functional state of the
protein. These confounds make it difficult to study the cellular release, uptake, and the
aggregation state of tau at physiological concentrations and in real-time. To overcome these
limitations, we developed an ultrasensitive tau-luciferase assay, in which full-length human
tau (2N4R) is fused to either the N- or the C-terminal part of Gaussia luciferase (gLuc), and
tau oligomer formation allows for complementation of split-gLuc parts tau-L1 and tau-L2,
which then reconstitutes gLuc activity (Remy & Michnick 2006, Hashimoto
et al.
2011,
Kalia
et al.
2011). The detected luciferase activity thus derives from tau dimers or
multimers; to simplify we refer to all these species as oligomers. One benefit of the split-
gLuc assay over split-fluorescent protein (split-FP) assays is the dynamic reversibility of the
complementation (Remy & Michnick 2006). Using this tau-gLuc assay, we were able to
study the generation, release, and uptake properties of oligomeric soluble tau species. For
example, the ultrasensitive assay can detect tau concentrations of 0.01–1000 ng/ml and show
that oligomeric tau can be actively released and taken up by cells and neurons
in vitro
.
Furthermore, agents seeding the aggregation of tau did barely increase gLuc activity,
suggesting that factors that promote tau aggregation of recombinant species may differ from
those that govern the formation of soluble oligomeric tau species in cells. Naturally
occurring tau dimerization and oligomerization may thus not necessarily be “on pathway”
with tau aggregate formation.
METHODS
Plasmids
The split luciferase constructs (L1, aa 1–92; L2, aa 93–163), as previously described
(Hashimoto et al. 2011), were each amplified and ligated into the HindIII (5’) and EcoRV
(3’) restrictions sites in an AAV-CBA-4RTau-WPRE vector previously described (de
Calignon
et al.
2010), adding the luciferase fragments in frame to the C-terminus of full-
length (2N, 4R) human Tau. The luciferase fragments were also added to the N-terminus of
the Tau using amplification of each fragment and ligating into the XhoI site of the AAV-
CBA-4RTau-WPRE vector. All plasmids were confirmed by sequencing.
HEK293 cells culture
HEK293 cells (ATCC) were maintained in OPTI-MEM+ 5% fetal bovine serum (FBS)
following standard cell culture procedures. For experiments, cells were plated ~30%
confluent in opaque white 96-well tissue culture plates with glass bottom. Next day, cells
were transfected for 3 hours with tau-gLuc constructs, or with GFP or full-length tau as
controls, using ~1% Lipofectamine 2000 (Life technologies), and subsequently cultivated in
serum-free OPTI-MEM. At designated time points post transfection, 80 µl culture medium
was separated into fresh culture plates, cells were rinsed once and then overlayed with 80 µl
fresh pre-warmed OPTI-MEM for immediate luciferase activity measurement; for
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immunostaining, cells were fixed with PFA, for protein analysis, cells were harvested in
RIPA buffer. Extracellular vesicles were enriched by ultracentrifugation of conditioned
medium at 100,000g for 1 hour at 4°C and subsequent resuspension of pelleted membranes
in PBS.
Immunostaining of cells
After removal of culture medium, cells were rinsed with warm PBS once and then fixed in
4% PFA/PBS for 15 min at room temperature, permeabilized with 0.2% TritonX-100/PBS,
blocked with 3–5%normal goat serum (NGS)/PBS, and incubated in primary antibodies
(mouse anti-human Tau13, Biolegends, 1:1000, and rabbit anti-β-actin, 1:1000, Abcam) in
3%NGS/PBS overnight at 4 °C. Secondary antibodies (Alexa488-anti-mouse and Cy3 anti-
rabbit, Life technologies, 1:1000) in 3%NGS/PBS were applied for 1 h at room temperature.
After washing in PBS, and incubation with DAPI, cells were imaged with a Zeiss Axiovert
200 inverted confocal microscope.
Primary cortical neurons
Primary cortical neurons were prepared from cerebral cortices of embryonic day E14–E15
CD1 mice embryos (Charles River Laboratories) as described previously (Wu
et al.
2012). In
brief, cortices were mechanically dissociated in Neurobasal medium (Life Technologies)
including with 10%FBS, 2 mM Glutamate, 100 U ml
−1
penicillin, and 100 g ml
−1
streptomycin, then pelleted at ~150 g for 5 min, resuspended in the same medium, and
plated in 96-well or 6-well plates coated with poly-D-lysine (50 µg/ml Sigma) for 1 hour.
Cultures were maintained at 37 °C with 5%CO
2
in Neurobasal medium with 2%(v/v) B27
nutrient, 2 mM Glutamate, 100 U ml
−1
penicillin and 100 g ml
−1
streptomycin. Neuronal
transfection was done as described for HEK293 cells at day 7
in vitro
(DIV7). All
experiments were performed under national (United States National Institutes of Health) and
institutional (Massachusetts General Hospital Subcommittee for Research Animal Care and
the Institutional Animal Care and Use Committee at Harvard Medical School) guidelines.
All animal experiments were approved by the Massachusetts General Hospital and
McLaughlin Research Institute Institutional Animal Care and Use Committees.
Luciferase assay
Gaussia luciferase
(gLuc) activity of adherent cells were measured directly in opaque plastic
glass bottom 96-well culture plates, culture medium, or cell lysates and centrifugation
fractions were added to the same plates and measured by adding luciferase substrate, 50 µl
per well of 10 µM coelenterazine (Nanolight) diluted in culture medium or PBS, and
counting emitted photons 1 s after substrate injection for the duration of 2 s. Measurements
and substrate injections were performed on a semi-automated plate reader (Wallac) and raw
values were transformed into photons per second.
Immunoblot analysis and human tau ELISA
For Western blot analysis, proteins in cell lysates and centrifugation fractions were separated
by SDS-PAGE in 4–12% Bis-Tris gels (NuPAGE, Invitrogen) and blotted onto nitrocellulose
membrane (Amersham) for 2 hours at 90 V. After blocking the membrane in blocking buffer
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(LICOR PBS-based blocking buffer) for 1 hour at room temperature, primary antibodies
mouse anti-human tau specific Tau13 (Biolegends) and rabbit anti-tubulin (Abcam) were
diluted 1:1000 in blocking buffer and applied over night at 4°C. After three washes with
TBS+0.2% Tween-20 (TBS-T), secondary antibodies (anti-mouse-800, anti-rabbit-700,
LICOR) diluted 1:5000 in blocking buffer were applied for 2 hours at room temperature,
then the membrane was washed three times in TBS-T and imaged using a LICOR infrared
imaging setup. Human tau levels in conditioned medium of HEK293 cells and primary
neurons and in size exclusion chromatography (SEC) fractions were measured by total
Human Tau ELISA Kit (Life Technologies).
Size exclusion chromatography (SEC)
HEK tau-L1/L2 conditioned medium (500–600 µl), rTg4510 brain PBS-extracts (300–400
µl), and monomeric/oligomeric recombinant human tau (hTau-441, 500–600 µl, ~3 mg/ml in
PBS with 2 mM DTT) were separated by SEC using a Superdex200 10/300GL column
(#17-5175-01, GE Healthcare) mounted on an AKTA purifier 10 (GE Healthcare) in
phosphate buffered saline (Sigma-Aldrich) at a flow rate of 0.5 ml/min; all samples were
filtered through a membrane filter (pore size 0.2 µm) before loading onto the column.
Human tau concentrations in the collected SEC fractions (0.5 ml) were determined by
human total tau ELISA (Life Technologies).
Atomic force microscopy (AFM)
Isolation of tau from HEK tau-L1/L2 conditioned medium for AFM analysis was performed
as described previously (Takeda et al. 2015). Briefly, tosyl-activated magnetic Dynabeads
(Life Technologies) were coated with human tau-specific Tau13 antibody (Biolegend).
Beads were washed (0.2 M Tris, 0.1%bovine serum albumin, pH 8.5) and incubated with
HEK tau-L1/L2 conditioned medium for 1 h at room temperature. After the beads were
washed three times with PBS, human tau was eluted using 0.1 M glycine, pH 2.8 for ~1 min,
and the pH of the eluted fraction was immediately adjusted using 1 M Tris pH 8.0. For AFM
imaging, the isolated tau was adsorbed onto freshly cleaved muscovite mica and imaged
using oscillation mode AFM (Nanoscope III, Di-Veeco, Santa Barbara, CA) and Si
3
N
4
cantilevers (NPS series, Di-Veeco) in PBS, as described previously (Wegmann et al. 2010).
Most probable particle sizes (=AFM heights) of tau-L1/L2 monomers and oligomers were
determined from Gaussian fits to AFM height distribution histograms of 269 particles
imaged in six randomly picked areas (1.0 × 1.0 µm).
Thioflavine-T assay
To test the presence of fibrillar tau aggregates, 20 µl of HEK tau-L1/L2 conditioned medium
were transferred into opaque glass bottom 96-well plates and incubated with 180 µl of
Thioflavne-T solution (50 mM Sodium Acetate, pH 6.8, containing 3 µM Thioflavine-T) for
15 min in the dark. The amount of Thioflavne-T bound to fibrillar tau was measured as
emission at 510 nm after excitation at 430 nm using a plate reader (Wallac). Samples were
measured in duplicates and pre-aggregated recombinant huTau-441 was used as positive
control.
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