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Neuron-to-neuron wild-type Tau protein transfer through a trans-synaptic
mechanism: Relevance to sporadic tauopathies
Acta Neuropathologica Communications 2014, 2:14doi:10.1186/2051-5960-2-14
Simon Dujardin (firstname.lastname@example.org)
Katia Lécolle (email@example.com)
Raphaëlle Caillierez (firstname.lastname@example.org)
Séverine Bégard (email@example.com)
Nadège Zommer (firstname.lastname@example.org)
Cédrick Lachaud (email@example.com)
Sébastien Carrier (firstname.lastname@example.org)
Noëlle Dufour (email@example.com)
Gwennaëlle Aurégan (firstname.lastname@example.org)
Joris Winderickx (email@example.com)
Philippe Hantraye (firstname.lastname@example.org)
Nicole Déglon (Nicole.Deglon@chuv.ch)
Morvane Colin (email@example.com)
Luc Buée (firstname.lastname@example.org)
19 December 2013
16 January 2014
30 January 2014
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Neuron-to-neuron wild-type Tau protein transfer
through a trans-synaptic mechanism: Relevance to
1 Inserm, UMR837, Place de Verdun, 59045 Lille, France
2 Faculté de Médecine, JPArc, Université Lille 2, Place de Verdun, 59045 Lille,
3 CMRR, CHR, 59037 Lille, France
4 Atomic Energy Commission (CEA), Institute of Biomedical Imaging (I2BM),
Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses,
5 CNRS, URA2210, Molecular Imaging Research Center (MIRCen), F-92265
6 Functional Biology, KU Leuven, Kasteelpark Arenberg 31, box 2433, B-3001
7 Present address: Department of Clinical Neurosciences (DNC), Laboratory of
Cellular and Molecular Neurotherapies (LMCN), Lausanne University Hospital
(CHUV), CH-1011 Lausanne, Switzerland
In sporadic Tauopathies, neurofibrillary degeneration (NFD) is characterised by the
intraneuronal aggregation of wild-type Tau proteins. In the human brain, the hierarchical
pathways of this neurodegeneration have been well established in Alzheimer’s disease (AD)
and other sporadic tauopathies such as argyrophilic grain disorder and progressive
supranuclear palsy but the molecular and cellular mechanisms supporting this progression are
yet not known. These pathways appear to be associated with the intercellular transmission of
pathology, as recently suggested in Tau transgenic mice. However, these conclusions remain
ill-defined due to a lack of toxicity data and difficulties associated with the use of mutant
Using a lentiviral-mediated rat model of hippocampal NFD, we demonstrated that wild-type
human Tau protein is axonally transferred from ventral hippocampus neurons to connected
secondary neurons even at distant brain areas such as olfactory and limbic systems indicating
a trans-synaptic protein transfer. Using different immunological tools to follow phospho-Tau
species, it was clear that Tau pathology generated using mutated Tau remains near the IS
whereas it spreads much further using the wild-type one.
Taken together, these results support a novel mechanism for Tau protein transfer compared to
previous reports based on transgenic models with mutant cDNA. It also demonstrates that
mutant Tau proteins are not suitable for the development of experimental models helpful to
validate therapeutic intervention interfering with Tau spreading.
AD is the most common neurodegenerative disorder. It results from an accumulation of
extracellular amyloid deposits and a neurodegenerative process called NFD, which is
characterised by the intraneuronal aggregation of the microtubule-associated Tau proteins.
This Tau pathology has been associated with a number of neurodegenerative disorders,
referred to as tauopathies. In contrast to AD, where mutations have not been identified on the
Tau gene (MAPT), patients presenting fronto-temporal dementia with parkinsonism,
associated with chromosome 17 (FTDP-17), exhibit Tau mutations . These mutations have
been used to develop animal models with Tau aggregation and NFD to decipher the role of
Tau in tauopathies [2,3]. However, the relevance of this approach remains unknown, as
mutant Tau proteins show a higher nucleation process than WT Tau and fibrillogenesis, often
leading to rapid neuronal death [4,5]. Moreover, in FTDP-17, there is no specific neural
network affected by the Tau pathology. Conversely, certain sporadic tauopathies display
hierarchical pathways of NFD. For example, in AD, neurodegeneration begins in the trans-
entorhinal cortex, spreads to the hippocampal formation, anterior temporal cortex, and
polymodal and unimodal association areas and eventually invades the entire cerebral cortex
[6-8]. In progressive supranuclear palsy, another tauopathy, a specific pathway of NFD has
also been identified leading from subcortical structures to the primary motor cortex through
the pedunculopontine nucleus and eventually to other frontal regions . The pathways of
NFD have also been described for other tauopathies, such as argyrophilic grain disease, in
which Tau aggregation starts in the vicinity of the ambient gyrus, then spreads to the
temporal lobe and subiculum and entorhinal cortices and eventually reaches the septum,
insular cortex and cingulate gyrus . In addition to differences in these specific pathways,
different Tau phosphorylation, isoforms, species and aggregates have also been identified
among tauopathies . Taken together, these characteristics are consistent with the presence
of different Tau strains that might transfer Tau pathology from cell to cell .
Recent data suggest that Tau pathology may be induced and propagated after the injection of
Tau oligomers and/or aggregates in either wild-type (WT) or mutated Tau transgenic mice
[11-14]. Moreover, there is evidence that Tau aggregates can be transferred from cell to cell
in vitro [15-19]. The hierarchical pathways of NFD in tauopathies might be associated with
the trans-synaptic transfer of Tau pathology, as recently suggested in vivo [20,21]. However,
these conclusions could be hampered by at least two factors: the use of a leaky inducible
system that may induces the weak expression of the transgene in the hippocampus, and the
use of mutated Tau protein to study propagation, as the spreading of Tau pathology is only
observed in sporadic tauopathies, where no mutation on MAPT has been identified. In these
models, Tau diffusion was consistently observed in close vicinity to the expression region,
and no definite evidence of a direct cell-to-cell transfer of pathological Tau proteins was
To address these two weaknesses, we took advantage of a recently developed lentiviral-
mediated rat model of hippocampal NFD  to demonstrate that 1) WT Tau protein is
transferred in a trans-synaptic manner from primary neurons located in the CA1 region of the
hippocampal formation to many anatomically connected secondary neurons in different brain
areas including the most distant ones (10 mm away from the injection site (IS)), 2) Tau
species found in secondary connected neurons are mainly in a dephosphorylated form and 3)
WT and mutated Tau species display differential spreading of Tau pathology.
Monoclonal antibody AT8 (Thermo Scientific MN1020 - 1:400 for immunolabelling)
recognises phosphorylated residues serine 202 and threonine 205 of Tau, and monoclonal
antibody AT100 (Thermo Scientific MN1060 - 1:400 for immunolabelling) recognises
phosphorylated residues threonine 212 and serine 214 of Tau. Tau C-ter is a polyclonal rabbit
antibody, which recognises the carboxyl terminal region of Tau . The monoclonal
antibody MC1 was a generous gift from Peter Davis and recognises conformational changes
in residues seven to nine and 313-322 (1:1000 for immunolabelling). ADx215 is a human
specific anti-Tau antibody that recognizes Tau only when Tyr18 residue is dephosphorylated
. Mouse monoclonal (Invitrogen P/N-0705; 1:10000 for immunolabelling) and rabbit
polyclonal antibodies (1:10000 for immunolabelling) to V5 recognise the V5 epitope of
The packaging construct pCMV∆R8.92 was used. The Rev gene was inserted into the pRSV-
Rev plasmid to minimise the risk of recombination and the production of replication-
competent lentiviruses. Viral particles were pseudotyped with the vesicular stomatitis virus
G-protein encoded in the previously described pMD2.G plasmid . cDNAs encoding the 2
+ 3-10+ 1N4R isoforms of human WT Tau and mutant P301L Tau were first cloned into the
Gateway Entry pCR8/GW/TOPO vector (Invitrogen) using TOPO TA cloning methodology.
The Gateway LR clonase (Invitrogen) catalysed the in vitro recombination between the
Gateway Entry pCR8/GW/TOPO vector (containing the Tau cDNA flanked by attL sites) and
the lentiviral destination vector (containing homologous attR sites). For specific constructs,
the sequence of the epitope tag V5, previously validated in vivo using immunohistochemical
analysis (14 aa, GKPIPNPLLGLDST) , was inserted into the cDNA encoding the 2+3-
10+ isoform of the human WT Tau between the sequences encoding exons two and four.
Production and assay of recombinant lentiviral vectors (LVs)
LVs vectors were amplified as previously described  and encode either for V5-hTau46WT,
hTau46WT, hTauP301L or eGFP proteins. Human 293 T cells (4 × 106) were plated onto 10-cm
plates and transfected the following day with 13 µg of human Tau cDNA, 13 µg of
pCMV∆R8.92, 3 µg of pRSV-Rev and 3.75 µg of pMD.2G using the calcium phosphate
DNA precipitation procedure. Four to six hours later, the medium was removed and replaced
with fresh medium. Forty-eight hours later, the supernatant was collected and filtered. High-
titre stocks were obtained through two successive ultracentrifugation steps at 19,000 rpm
(Beckman Coulter SW 32Ti and SW 60Ti rotors) and 4°C. The pellet was resuspended in
PBS with 1% bovine serum albumin (BSA) and stored frozen at -80°C until further use. Viral
concentrations were determined through ELISA for the HIV-1 p24 antigen (Gentaur BVBA).
The p24 protein is a lentiviral capsid protein that is commonly used in ELISA assays to
determine the physical titre of lentiviral batches per ml. All viral batches were produced in
appropriate areas in compliance with institutional protocols for genetically modified
organisms according to the ‘Comité Scientifique du Haut Conseil des Biotechnologies’
(Identification Number 5258).
Neuronal cultures, microfluidic chamber system and assays for the neuron-to-
cell transfer of Tau
Briefly, glass coverslips were coated overnight at 4°C with 0.5 mg/ml of poly-D-Lysine
(SIGMA). The microfluidic chamber (AXISTM, Temecula, CA) was subsequently placed on
coated glass coverslips and sealed to the glass. Rat Primary embryonic neuronal cultures were
performed as previously described , and approximately 30,000 cells each were plated in
the two wells of the somatodendritic compartment. The cultures were maintained at 37°C for
15 days for differentiation. All chambers had a microgroove length of 450 µm and a width of
10 µm. One week post-plating, after axonal growth across the microgrooves, a second rat
primary embryonic neuronal cultures were plated in the axonal compartment in the
appropriate medium. Twenty-four hours later, the V5-Tau-LVs or eGFP-LVs (200 ng of LVs
per well) were added to the somatodendritic compartment after first reversing the volume
gradient between the compartments to counteract viral diffusion. Forty-eight hours later,
eGFP fluorescence and V5 immunolabelling were analyzed. The compartments
(somatodendritic and axonal) were washed once with phosphate-buffered saline (PBS) and
fixed with 4% paraformaldehyde (PFA) for 20 min. After removing the fixative, the cells
were washed three more times with 50 mmol/L NH4Cl and processed as described in the
Testing of fluidic isolation
Isolation of the different compartments in microfluidic conditions without cells was also
assessed using either Coomassie Blue or LVs particles. Coomassie Blue (2%) or LVs
particles (400 ng p24) diluted in PBS were added to the somatodendritic compartment of
microfluidic device and PBS was added to the axonal compartment. The volume gradient
between both compartments was adapted to avoid or to mediate diffusion across the
microgrooves. 5 min, 1, 2, 24 or 48 h later, optical density at 595 nm.
When LVs particles were used instead of Coomassie Blue, the medium in both compartments
was recovered and viral RNA extracted (Nucleospin RNA Virus, MACHEREY-Nagel,
Düren, Germany). cDNAs were generated by RT-PCR and PCR done using the following
oligonucleotides specific to the viral WPRE (Woodchuck Hepatitis Post-transcriptional
Regulatory Element) (forward: 5′-TAC-GCT-ATG-TGG-ATA-CGC-TGC-3′ and reverse: 5′-
The animals were purchased from Janvier Laboratories and housed in a temperature-
controlled room maintained on a 12 h day/night cycle with food and water provided ad
libitum. The present experimental research has been performed with the approval of an ethics
committee (‘Comité d’éthique en expérimentation animale du Nord Pas-de-Calais’-CEEA
342012) and follows internationally recognized guidelines.
Stereotaxic injections and sacrifice procedures
Intracerebral injections of viral particles into the brain of anesthetised 2-month-old Wistar
rats (Ketamine 100 mg/kg, Xylazine 10 mg/kg i.p.) were performed using classic stereotaxic
procedures at the following coordinates relative to bregma: posterior, -5.3 mm; lateral, +/- 6.2
mm; ventral, -7 mm and -6.2 mm; depth. The injections were performed bilaterally. The
standard injection procedure consisted of the delivery of 400 ng of p24 using a 10 µL glass
syringe with a fixed needle (Hamilton). After injection at a rate of 0.25 µl per min, the needle
was left in place for 1 min before reaching the second depth; the second injection was
performed after 5 min. Control groups (n = nine) consisted of rats injected with PBS instead
of LVs. For anterograde tracing, 5% biotinylated dextran amines (molecular weight 10 000
kilodaltons (kD); BDA 10 000, Invitrogen) were injected using 10 µl glass syringes with
fixed needles (Hamilton).
For immunohistochemical analyses, animals were anesthetised (8% chloride hydrate) at two
(n = 13), four (n = 13) or eight (n = 13) months post-injection and transcardially perfused first
with cold 0.9% NaCl followed by 4% PFA for 20 min before beheading. The brains were
immediately removed, fixed overnight in 4% PFA, placed in 20% sucrose for one week and
frozen until further use. Free-floating coronal cryostat sections (40 µm thickness) were used
for immunohistochemical analysis.
For RNA extraction, rats (n = eight) were deeply anesthetised using 8% chloride hydrate.
Brains were dissected, and 1-mm-thick coronal sections were generated using an acrylic rat
brain matrix (Electron Microscopy Sciences). The sections were immediately frozen on dry
ice and stored at -80°C until further use.
For anterograde transport studies, rats were sacrificed 1 week post-dextran-injection and
transcardially perfused with 0.9% NaCl and 4% PFA in 0.1 mol/L phosphate-buffered saline
(pH 7.4). Brains were post-fixed for 24 hours in 4% PFA and cryoprotected before freezing
for storage. Coronal sections (40 µm thickness) were washed three times in 0.1 mol/L PBS
containing 0.2% Triton X-100 and incubated with fluorescein streptavidin (Vector) for 1
hour. After three washes, sections were mounted onto gelatine-coated slides and coverslipped
with Vectashield Mounting Medium (Vector).
The sections from the entire brain were washed in PBS-0.2% Triton and treated for 30 min
with H2O2 (0.3%). Non-specific binding was then blocked using goat serum (1:100 in PBS,
Vector) for 60 min. Incubation with the primary antibody in PBS-0.2% Triton was performed
overnight at 4°C. After several washes, labelling was amplified by incubation with an anti-
mouse biotinylated IgG (1:400 in PBS-0.2% Triton, Vector) for 60 min followed by the
application of the ABC kit (1:400 in PBS, Vector) prior to visualisation with 0.5 mg/ml DAB
(Vector) in Tris-HCl 50 mmol/L, pH 7.6, containing 0.075% H2O2. Brain sections were
mounted onto gelatine-coated slides, stained for 1 min in a cresyl violet solution (0.5%),
washed in water with 2% acetic acid, dehydrated by passage through a graded series of
alcohol and toluene and mounted with Vectamount (Vector) for microscopic analysis.
For brain sections: sections from the entire brain were washed in PBS-0.2% Triton and
blocked with goat serum (1:100 in PBS, Vector) for 60 min. Incubation with a primary
antibody in PBS-0.2% Triton was performed overnight at 4°C. After several washes, the
primary antibody against the second antigen was added in PBS-0.2% Triton, and the sections
were again incubated at 4°C overnight. Incubation with the two Alexa Fluor secondary
antibodies (1:1000 in PBS) was performed for 60 min at room temperature. The slides were
mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) to label the
nuclei (Vector). For cell cultures: sections were rinsed once in 50 mmol/L NH4Cl, and cells
were permeabilised with Triton X-100 (0.1%, 10 min at room temperature). Subsequently,
the slides were incubated with primary antibodies at 4°C overnight, and labelling was
performed by a reaction with the appropriate Alexa Fluor secondary antibodies (1:400-
Invitrogen) for 45 min at room temperature. The cells were mounted with Vectashield
medium containing DAPI (Vector, Burlingame, USA).
Confocal microscopy was performed using a Zeiss LSM 710 inverted confocal microscope.
For each optical section, two fluorescence images were obtained. The signal was subjected to
line averaging to integrate the signal collected over two or four lines to reduce noise. The
confocal pinhole was adjusted to facilitate a minimum field depth. A focal series was
collected for each specimen. The focal step between sections was typically 1 µm.
RNA extraction from brain sections and RT-PCR/PCR
RT-PCR of lentiviral mRNA was performed using total RNA. The brain slices were lysed,
and total RNA was extracted using the RNeasy Lipid Tissue kit (Qiagen, France) according
to the manufacturer’s instructions. The RNA (1 µg) was denatured for 10 min at 68°C, and
cDNA was generated using reverse transcription with 200 nmol/L of dNTPs, 1 ng/µl of
random primers, 1 ng/µl of oligo dT, 5 mmol/L of dithiothreitol (DTT), 2 units/µl of RNase
Out and 10 units/µl of M-MLV reverse transcriptase. The viral cDNAs were then amplified
using oligonucleotides specific to human Tau (forward: 5′-TGG-GGG-ACA-GGA-AAG-A-
3′ and reverse: 5′-CCT-CAG-ATC-CGT-CCT-CAG-TG-3′). The following pair of primers
was used to amplify murine Tau for calibration: forward: 5′-CAC-AAT-GGA-AGA-CCA-
GGC-C-3′ and reverse: 5′-TAA-GCC-ATG-GCT-CAT-GTC-TCC-3′. PCR was performed
using 2 µl of the previously obtained RT products, reverse and forward primers (0.5 µmol/L),
dNTPs (1 µmol/L) and 0.02 unit/µl of DNA polymerase in a commercial reaction buffer
(GoTaq Green Master Mix, Promega). The PCR products were electrophoresed on an 8%
acrylamide gel stained with 1 µg/ml ethidium bromide.
A mechanism of trans-synaptic transfer of WT Tau protein can be
demonstrated in vitro
We first determined whether the human WT Tau protein could be transferred between
neuronal cells. Using a microfluidic device [27-29] comprising two compartments connected
through embedded microgrooves, we studied the potential transfer of WT Tau from the
somatodendritic compartment seeded with rat primary neurons to an axonal compartment
seeded with N1-E115 murine neuroblastoma cells a second rat primary embryonic neuronal
culture. Microfluidic devices have already been validated for the axonal transport of
recombinant α-synuclein or Tau [29,30]. To address this question in our LVs assay and to
characterise the human Tau protein, we designed a new LV encoding a WT Tau with a V5
epitope (See Additional file 1). First of all, we performed several assays to control that LVs
are not able to diffuse in the microgroove or to traffic along the microtubule (See Additional
After differentiation of rat brain primary neuronal cultures , the hydrostatic pressure
difference was reversed to avoid viral vector diffusion, and an LV was added to the
somatodendritic compartment to facilitate the expression of the WT V5-Tau in rat primary
neurons. Simultaneously, a second rat primary embryonic neuronal culture was seeded in the
axonal compartment. After 48 hours, V5-immunoreactivity was primarily observed in the
somatodendritic compartment and in axons located in the microgrooves. Interestingly, V5
immunoreactivity was also detected in secondary neurons, indicating the cell-to-cell transfer
of WT V5-Tau protein via axonal transport from the primary neurons (Figure 1). This
transfer was specific to Tau since GFP was not found in secondary neurons (See Additional
file 2). Finally, all controls in the axonal compartment were negative. It is not surprising
since, due to the speed of HIV axonal transport and its time of uncoating, LVs are not able to
reach the axonal endings in the present microfluidic devices where the length of
microgrooves is 450 µm [31,32]. These results demonstrate in vitro that donor neurons
overexpress human WT V5-Tau, which is transported along axons, secreted and taken up by
Figure 1 Neuron-to-cell spread of WT Tau in a microfluidic device. The microfluidic
device used in our study comprised two compartments separated by a physical barrier
containing microgrooves that facilitate the passage of axons, but not neuronal cell bodies,
during neuronal differentiation. (a) Primary culture of embryonic rat cortical neurons seeded
in the first compartment (somatodendritic) was infected at DIV 7 with LVs encoding V5-
hTau46WT. The flow was then reversed and a second rat primary embryonic neuronal culture
cells was seeded in the axonal compartment. (b) Forty-eight hours post-infection, the cells
were processed for immunofluorescence analysis using anti-V5 antibodies and an Alexa
Fluor 488-labeled secondary antibody (green). The nuclei were counterstained with DAPI
(blue). The scale bar is indicated on the figure. These data showed that V5 is found in axons
in primary neurons and in cell bodies of secondary neurons in the axonal compartment.
A mechanism of trans-synaptic transfer of WT Tau protein can be
demonstrated in vivo
The first aim of this study was to identify the IS in the rat brain that would facilitate not only
the monitoring of cell-to-cell transfer of Tau protein in distant brain areas but also to
discriminate between free protein diffusion and active transfer through anatomically
organised neuronal pathways. Using published data on the rat brain neuroanatomy , we
selected stereotactic coordinates in the caudal region of the CA1 layer, which is associated
with brain regions remote from the injected area and is anatomically connected through well-
defined neural networks. First, we assessed these regions using an anterograde tracer ,
and as expected, these areas were identified among projecting fibres throughout the brain,
i.e., those in rostral areas, such as limbic regions, olfactory/orbital systems and caudal areas,
such as the subiculum and entorhinal cortex (Figure 2a). Using the LV encoding WT V5-Tau
protein, we injected rats in the CA1 region of the hippocampus. Five months post-injection,
V5-immunohistochemistry was performed and V5-immunoreactivity was found in different
regions of the whole brain (Figure 2b). More importantly, both axonal tracer labelling and
V5-were found associated with the same regional pattern supporting that WT Tau protein is
transferred in each area connected to the IS (see Figure 2a and b). Most of the V5-
immunoreactive cell bodies were observed in several connected regions in caudal but also in
rostral brain areas (Figure 2c). For instance, some cell bodies were found as far as 10 mm
from the IS in the granular layer of the olfactive bulb (GrO, Bregma +5.2). Altogether, these
analyses strongly suggest that the spatio-temporal transfer of WT Tau is progressing through
Figure 2 The transfer of V5-hTau46WT protein is correlated to brain area connected to
the IS (caudal part of the CA1 layer). (a) Dextran amines (10 kD) are anterograde tracers:
they were injected into the rat brain (n = 3), and one week later, the animals were sacrificed
and their brains were processed to reveal fluorescent axons efferent from the IS. Brains were
virtually divided into five sections: bregma +5.20 to +1.40, bregma +1.20 to -1.40, bregma -
1.80 to -4.30, bregma -4.52 to -6.04 and bregma -6.30 to -7.80. (b) LVs encoding V5-
hTau46WT were bilaterally injected into the CA1 layer (IS; bregma -5.3) of rat brains (n = 3).
Five months later, the animals were sacrificed, and the whole brain was processed for
immunohistochemical analysis using a rabbit polyclonal anti-V5 antibody. The brains were
virtually divided into five sections as in (a). The bar scale is indicated on the figure. The
drawing showing the extension of WT V5-Tau protein transfer from the IS to extreme rostral
and caudal positions is illustrated in the lower part of the figure. These data showed that V5-
hTau46WT Tau is transported throughout the brain using neural networks. (c) The V5-
immunoreactivity is summarized in a cartoon drawing of several coronal sections at different
bregma coordinates. The different blue intensities (level 1 to 3) indicate the density of fibres
and the red stars indicate the presence of V5-immunopositive cellular bodies indicating a
trans-cellular transfer of V5-hTau46WT Tau.
A follow-up study of V5-immunoreactivity in the rat brain was also performed at 1, 3 and 5
months post-injection to assess the time-dependent distribution of the tagged WT human Tau
protein. As an exemple, we focused on two of the most rostral distant systems: the olfactory
and the limbic areas. At 1 month post-injection, V5-immunoreactivity was observed at the IS
and in fibres projecting in both areas. No labelled neuronal cell bodies were observed at this
stage. At 3 months post-injection, in addition to fibres, some V5-immunoreactive cell bodies
(50 < cell bodies < 100, N = 3 rats, bregma +5.2) were visualized in the olfactory area,
whereas more fibres, but no cell bodies, were observed in the limbic cortex. At five months
post-injection, numerous V5-immunoreactive cell bodies were observed mainly in the
olfactory area (cell bodies > 100, N = 3 rats, bregma +5.2) but also in the limbic regions (5 <
cell bodies < 10, N = 3 rats, bregma +5.2) (Figure 3). Such dynamic process over time
supports that WT Tau is transported through axons and then trans-synaptically transferred to
secondary neurons in different brain regions. To further demonstrate that this WT Tau
transport results from an intrinsic property of the Tau species and not from our model used to
induce local overexpression of the transgene i.e. the LVs, different controls were used.
Indeed, one explanation for the occurrence of Tau proteins far from the IS could be the
diffusion of LVs during the injection process, although this is unlikely to be the case since 1)
LVs delivery remains spatially restricted to the injected area when a reporter green
fluorescent protein (GFP) is used instead of WT V5-Tau protein. Indeed, GFP protein was
observed only in neurites, never in secondary neurons in connected areas (ie : the prelimbic
structures and olfactory bulb) (Figure 4a) and 2) human Tau RNA was never detected in
areas distant from the IS (Figure 4b). Polymerase chain reaction was used to detect human
Tau RNA in sections covering the entire brain. The presence of the human Tau RNA was
restricted to the area around the IS, where it followed a Gaussian distribution for both forms
of Tau. It was neither detected in PBS-injected rats nor in areas distant from the IS in LVs-
injected animals, whereas V5 immunoreactivity in WT V5-LVs-injected rats was found in
cell bodies in connected secondary regions (Figure 2b), supporting that the mechanism of
cell-to-cell protein transfer is likely specific to WT Tau protein.
Figure 3 In vivo trans-synaptic transfer of WT Tau protein. LVs encoding V5-hTau46WT
were bilaterally injected into the CA1 layer (IS; bregma -5.3) of rat brains (n = 3 rats per
group). One, three and five months later, the animals were sacrificed, and the whole brains
were processed for immunohistochemical analysis using an antibody to total V5-Tau.
Sections from the prelimbic or orbital cortex (bregma +4.7), the olfactory bulb (bregma +5.2)
and the CA1 (bregma -5.3, IS) are shown. The scale bars are indicated on the figure. These
data showed that V5-hTau46WT is transported from primary to secondary neurons in a time-
Figure 4 Cell-to-cell protein transfer is specific to Tau protein WT. (a) eGFP is not
transported in secondary connected neurons. LVs encoding eGFP were bilaterally injected
into the CA1 layer of rat brains (IS bregma -5.3, n = 3). Eight months later, the animals were
sacrificed, and brains sections processed for immunofluorescence assays to detect eGFP.
Sections from the prelimbic (bregma +4.7), external capsule (bregma -1.8), CA1 (IS, bregma
-5.3) and ectorhinal cortex (bregma -7.8) are shown. The scale bars are indicated on the
figure. (b) Restricted lentiviral insertion in the vicinity of the IS. PBS (left panel, n = 3) or
LVs encoding hTau46WT (middle panel, n = 3) or hTau46P301L (right panel, n = 3) were
bilaterally injected into the CA1 layer of rat brains. One month later, the brains were
dissected, and coronal sections of 1 mm thickness (indicated as the position relative to
bregma) were prepared using an acrylic rat brain matrix. Total RNA was extracted from these
sections to generate cDNA using RT-PCR. cDNAs were amplified using oligonucleotides
specific to human Tau (hTau, 117 base pairs) or murine Tau (70 base pairs). The positive
control was prepared by amplifying the plasmid containing hTau sequence. The lower parts
represent the mean +/-SEM of the relative density (hTau/mTau) coming from the three rats.
These data showed that Tau transport is a specific mechanism since neither eGFP nor viral
genome is found associated to distant secondary brain areas.
We therefore concluded that WT V5-Tau protein, expressed in CA1 neurons, can be 1)
transported through normal CA1-efferent projections even to distant brain regions, such as
the olfactory and limbic systems and 2) secreted and transferred intercellularly to secondary
neurons located in CA1 efferent regions.
To confirm that the V5 tag does not affect the behaviour of Tau proteins in the trans-synaptic
transfer, we compared the observed effects to that of non-tagged Tau proteins. The
progression of Tau pathology was followed using anti-Tau antibodies specific for different
pathological states of the Tau protein: a hyperphosphorylated state (AT8 ), a
conformational change (MC1 [36,37]) and an aggregated form (AT100 ) already
validated in this model . Antibody mappings of the whole rat brain showed differential
gradients of Tau pathology spreading (Figure 5). These data also supports that the V5 tag
does not interfere with the progression Tau pathology throughout the brain (compared Figure
5a and b). More interestingly, in a two points kinetic study, we observed that two months
post-injection, Tau pathology related to WT Tau was only weakly detected at the IS, as
described by Caillierez et al.  whereas the extended spreading of the Tau pathology was
observed throughout the brain 8 months post-injection (Figure 5a).
Figure 5 Differential Tau spreading between WT and mutated Tau species. LVs
encoding hTau46WT (n = 5, sacrificed at 2 months; n = 5, sacrificed at 8 months) (a), V5-
hTau46WT (n = 3, sacrificed at 8 months) (b) or hTau46P301L (n = 5, sacrificed at 8 months)
(c) were bilaterally injected into the CA1 layer of rat brains. After sacrifice, the whole brain
was processed for immunohistochemical analysis using AT8, MC1 or AT100 phosphorylated
Tau antibodies. Among the positive rats, the rostralmost and caudalmost brain coordinates
(from bregma) labeled by each antibody were determined for each brain. The figures
represent the mean values +/-SEM of the rostralmost of caudalmot brain coordinates. These
data showed that whereas hTau46P301L diffusion is restricted to the vicinity of the IS,
hTau46WT has spread throughout the brain. Numbers of immunopositive rats per group are
indicated on the right of each mapping.
To determine the influence of Tau species in the propagation process, we then compared Tau
pathology induced by non-tagged Tau proteins with or without a P301L mutation (Figure 5c).
P301L exhibited a severe Tau pathology (primarily immunoreactive for all antibodies) with a
narrow diffusion (up to 2 mm from the IS on both the rostral and caudal sides). Conversely,
WT Tau showed Tau pathology with a wider spatial propagation in both the rostral (10 mm
ahead of the IS with AT8-immunoreactivity; 4 mm ahead with MC1-immunoreactivity) and
caudal (greater than 2 mm from the IS) CA1-projecting regions (Figure 5a, Additional files 3
and 4). The pathological spreading clearly progressed through the brain neural networks, as
evidenced by the co-localisation between phosphorylated Tau (pTau) and anterograde tracer
as well as the lack of pTau in dextran-negative regions (Figure (comments: I removed the ‘s’
as it is removed in the other sentence containing two figures) 2a and 6).
Figure 6 Spatiotemporal progression of the Tau pathology through neural networks.
LVs encoding hTau46WT were bilaterally injected into the CA1 layer of rat brains (n = 5).
Eight months later, the animals were sacrificed, and the whole brain was processed for
immunohistochemical analysis to show AT8-related pTau. The brains were virtually
separated into five sections: (a) bregma +5.20 to +1.40, (b) bregma +1.20 to -1.40, (c)
bregma -1.80 to -4.30, (d) bregma -4.52 to -6.04 and (e) bregma -6.30 to -7.80. The bar scale
is indicated on the figure. These data showed that phospho-hTau46WT is found all over the
brain eight months post-LVs delivery.
Transferred Tau species are mainly in a dephosphorylated state
It remains unknown whether the AT8-immunoreactivity observed in distant brain areas is
associated with endogenous rat Tau or human Tau. To address this question, we performed
additional co-labeling assays using WT V5-Tau five months post-injection. In distant brain
regions, the majority of the V5-immunoreactive neurons was not AT8-immunoreactive
(Figure 7a and b) suggesting that serine residues 202 and 205 are dephosphorylated. We also
tested the phosphorylation state of tyrosine 18 using additional antibody, ADx215, which
recognized Tau only when this residue is dephosphorylated (ADx215 ). The
immunoreactivities of both ADx215 (Figure 7c) and V5 (Figure 7d) look like similar;
suggesting that most Tau associated to secondary neurons in the GrO is dephosphorylated on
tyrosine 18. More interestingly, a few secondary neurons displayed both AT8 and V5
immunoreactivities (7%), indicating the hyperphosphorylation of the transferred human V5-
Tau protein (Figure 7e and f). It should be noted that less than 1% of the secondary neurons
in the olfactory system were AT8-immunoreactive without any V5 labelling suggesting a
prion-like conversion process. Altogether, these data suggest that only a few pathological Tau
species are present in secondary neurons consistent with the slow kinetics of Tau spreading in
Figure 7 Transferred Tau species are mainly in a dephosphorylated state. LVs encoding
V5-hTau46WT were bilaterally injected into the CA1 layer (IS; bregma -5.3) of rat brains (n =
3). Five months later, the animals were sacrificed, and the rostral areas of the brain were
processed for immunofluorescence analysis using a rabbit polyclonal antibody anti-V5
antibody to detect total Tau and a mouse monoclonal antibody to pTau (AT8). The V5-Tau
proteins were visualised in green using the corresponding secondary antibody Alexa Fluor-
488 labelled goat anti-rabbit IgG (a and e) and the phospho-Tau in red using Alexa Fluor-
568 labelled goat anti-mouse IgG (b and f). The scale bars are indicated on the figure.
Adjacent brain slides from the rostral areas were assessed by immunohistochemistry using an
antibody directed against the N-terminal portion of Tau containing a dephosphorylated
tyrosine 18 residue (ADx215) (c) or an antibody to total V5-Tau (d). Most of hTau46WT Tau
species found in secondary neurons are dephosphorylated.
In the present study, we demonstrated in vivo that WT Tau proteins are transferred through
primary neurons from the IS to secondary neurons in distant rat brain areas to initiate Tau
pathology. Recent studies have shown that the Tau pathology might spread in vivo through
mono-synaptic connections in the hippocampal formation [20,21]. Nevertheless, the transfer
of Tau proteins from neuron to neuron has not been conclusively demonstrated. For example,
hippocampal NFD might result from either trans-synaptic transfer or signal transduction
associated with neuronal processes, such as receptor activation through Tau
In vitro, there is now considerable evidence demonstrating that Tau is secreted into the
extracellular medium [15,17,18,30,41-44] and that Tau aggregates are internalised in
different cell lines [15-19,41,45,46]. Nevertheless, to date there has been no evidence of
trans-synaptic protein transfer.
In vivo, Tau aggregates from P301S transgenic mice and human brains, injected into Alz17
mice (mouse strain overexpressing the longest Tau isoform), were captured/internalised by
neurons at the IS. Some degree of diffusion was observed at the vicinity of the IS,
reminiscent of Tau spreading [11,12]. In contrast, an inducible mouse line, in which the tTA
activator is driven through a neuropsin promoter specific for the entorhinal cortex, was cross-
bred with the tetracycline-inducible Tau Tg mouse line, rTg4510, expressing human four-
repeat Tau with the P301L mutation. In the resulting rTgTauEC mice, neurons expressing
P301L Tau species in the trans-entorhinal cortex underwent NFD [20,21]. Associated
hippocampal neurons also degenerated with time, although there was no demonstration of a
trans-synaptic transfer, as hippocampal NFD might also result from signal transduction
related to receptors activation through Tau aggregation/oligomerisation. Moreover, the
authors reported that hippocampal neurons also express mutated Tau species that may result
from the non-specific activity of the neuropsin promoter [21,47]. To conclude, until now,
only in vivo models of Tau capture [12,48] and/or Tau secretion have been developed .
The present study represents the first clear demonstration of the in vivo transfer of Tau
proteins between anatomically connected neurons at distant brain (d > 10 mm). Within five
months, Tau proteins spread to connected areas, such as the limbic and olfactory regions,
where it appeared within secondary neurons. These V5-immunoreactive Tau species were
primarily dephosphorylated (AT8 negative, ADx215 positive), suggesting that normal soluble
Tau might be secreted and transferred into secondary neurons. Altogether these observations
were further supported through studies in microfluidic chambers, where WT V5-Tau was
identified, after 48 hours, in the axonal compartment in primary neurons. These results
support previous data showing that Tau is secreted in a dephosphorylated state [41,43,49] and
may activate muscarinic receptors (M1 and M3) leading to intracellular dysfunctions [40,49].
In this present study, we also demonstrated differential Tau spreading between WT Tau and
mutant P301L Tau. Most studies of Tau propagation have used mutant forms, although a
majority of tauopathies are sporadic and involve only the WT species and whether WT
human Tau spreads as readily as the FTDP-17 Tau mutant is unknown. In the present model,
Tau pathology associated with WT Tau was observed in brain areas connected to the IS,
further supporting a trans-synaptic mechanism. In contrast, the Tau pathology induced
through mutant P301L Tau protein remained near the LV injection site. Because P301L Tau
mutant shows better nucleation and more rapid fibrillogenesis than does WT Tau , it is
likely that the mutant protein might aggregate more readily, as observed using AT100-
immunoreactivity, leaving fewer soluble species to migrate through axons. Moreover,
consistent with the results of Caillierez et al. , neuronal death is higher with the P301L
Tau mutant than with the WT Tau species. Such differential behaviour between the two Tau
species also supports the in vivo specific trans-synaptic transfer of soluble Tau species rather
than aggregates, as suggested using WT V5-Tau.
These findings also support the concept that Tau transmission occurs in sporadic diseases,
such as AD, whereas Tau toxicity, leading to neuronal loss, more readily occurs in aggressive
Tauopathies (FTDP-17) where all neurons have the mutant Tau, causing damage without
spreading . Taken together, the data derived from this in vivo model suggest that
soluble/oligomeric forms of Tau protein drive spreading as suggested by Kayed’s group
Finally, from data obtained from our in vivo model, one may argue that only a few secondary
neurons displayed AT8-immunoreactivity and that it remains unclear whether this effect
reflects the transfer of hyperphosphorylated
hyperphosphorylation occurring in secondary neurons. In any way, a few pathological Tau
species are present in secondary neurons. Such observations are consistent with the slow
kinetics of Tau spreading observed in sporadic Tauopathies. For in instance, in AD, the
hierarchical spreading of Tau pathology, defined by the Braak stages, may last for 20 years
. Moreover, results generated from our model are of great relevance for future clinical
investigations. Indeed, the discovery of distinct secreted/non secreted Tau species will define
new perspectives in diagnosis of neurodegenerative diseases. Selection of patients at the
beginning of the NFD spreading will then allowed the test of new therapeutics that would
block this spreading and subsequently slow down the development of sporadic disease such
as AD at the asymptomatic stages of disease. In this context for instance, Tau immunotherapy
may be relevant for interfering with NFD in AD and related disorders referred to as
Tauopathies. We showed previously than active immunotherapy allows for Tau clearance in
the periphery and improves cognitive deficits promoted by Tau pathology in a well-defined
mutant Tau mutant model . More recently, passive immunotherapy further supports the
targeting of extracellular Tau .
Tau or a subsequent V5-Tau
In conclusion, independent of the mechanisms involved in Tau spreading, we demonstrated
for the first time in vivo that the specific trans-synaptic transfer of Tau protein from
degenerating neurons might lead to the preliminary steps of Tau pathology in secondary
neurons. These data are of great interest considering that most tauopathies reflect the
aggregation of Tau devoid of mutation. In the future, this model will facilitate a better
understanding of Tau spread throughout the brain in sporadic tauopathies.
AcbSh, Accumbens nucleus shell; Amy, Amygdala; AOL, Anterior olfactory nucleus lateral
part; Au1, Primary auditory cortex; AuD, Secondary auditory cortex dorsal area; AuV,
Secondary auditory cortex ventral area; CA1-CA2-CA3, Fields of the hippocampus; cc,
Corpus callosum; Den, Dorsal endopiriform nucleus; DG, Dendate gyrus; DP, Dorsal
peduncular cortex; ec, External capsule; Ect, Ectorhinal cortex; Ent, Entorhinal cortex; fi,
Fimbria of the hippocampus; fmi, Forceps minor of the corpus callosum; fmj, Forceps major
of the corpus callosum; GrO, Granular layer of olfactory bulb; IL, Infralimbic cortex; Lent,
Lateral entorhinal cortex; LO, Lateral orbital cortex; LSD, Lateral septal nucleus dorsal part;
LSI, Lateral septal nucleus intermediate part; LSV, Lateral septal nucleus ventral part; PaS,
Parasubiculum; Pir, Piriform cortex; PRh, Perirhinal cortex; PrL, Prelimbic cortex; PrS,
Presubiculum; PtA, Parietal association cortex; S, Subiculum; SFi, Septofimbrial nucleus;
TeA, Temporal association cortex; V2L, Secondary visual cortex lateral area; Ven, Ventral
The authors declare no competing interests in this research.
MC and LB designed the study. SD, KL, RC, SB, NZ, CL, SC and MC performed the
research. MC, SD, PH, NiD and LB analysed the data. NoD, PH, NiD, GA and JW
contributed new reagents, materials and analyses. LB, MC, NiD and PH wrote the paper. All
authors read and approved the final manuscript.
This work was developed and supported through the LabEx DISTALZ (Laboratory of
Excellence, Development of Innovative Strategies for a Transdisciplinary approach to
ALZheimer’s disease), the FUI MEDIALZ and CPER DN2M (VICTAUR grant). This study
was also supported by Inserm, CNRS, University of Lille 2, LMCU (Lille Métropole
Communauté Urbaine), Région Nord/Pas-de-Calais, FEDER and grants from the European
Community: MEMOSAD (FP7 contract 200611) and the ‘Fondation Plan Alzheimer’
(PRIMATAU project). JW acknowledges the funding provided through the KU Leuven-IOF
programme and the IWT-Baekeland programme for the development of the ADx215/YT1.15
antibody. We thank the Institut de Médecine Prédictive et de Recherche Thérapeutique
(IMPRT, Lille) for access to core facilities (confocal microscopy, M. Tardivel and animal
facility, Delphine Tailleu). We also would like to thank Dr. Eugeen Vanmechelen (Ghent,
Belgium) for advice.
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Additional_file_1 as JPEG
Additional file 1 In-vitro functionality of V5-hTau46WT. (a) Schematic representation of the
full-length 4R Tau isoform (2+3-10+) tagged with V5 epitope (GKPIPNPLLGLDST).
HEK293 cells were infected with LVs encoding either V5-hTau46WT or hTau46WT and
processed 48 hours later for immunofluorescence analysis using a rabbit polyclonal antibody
to total V5-Tau (IF) (b), a polyclonal rabbit antibody to Tau C-Ter , which recognises the
carboxyl terminal region of Tau, or a polyclonal rabbit antibody to GAPDH for biochemical
assays (c) to demonstrate that the V5 tag did not alter transgene expression nor the
electrophoretic mobility. Tau proteins were visualised using the corresponding secondary
antibodies: Alexa Fluor-488 labelled goat anti-rabbit IgG antibody (green) for IF and
peroxidase goat anti-Rabbit IgG antibody for biochemical assays. In (b), nuclei were labelled
with DAPI are appear in blue. The scale bars are indicated on the figure. In (c), NI is related
to the control non-infected cells.
Additional_file_2 as JPEG
Additional file 2 LVs particles are not diffusing in the microgrooves or trafficking along the
microtubules. (a) LVs were added to the somatodendritic compartment of a microfluidic
device in the absence of cells. The flow was reversed to isolate compartments and then
prevent viral diffusion. Viral RNA was extracted from somatodendritic and axonal
compartments 5 min, 1, 12, 24 and 48 hours post-lentiviral delivery. cDNAs were generated
and amplified using oligonucleotides specific to viral WPRE (540 bp). In the positive control,
PCR was done from purified LVs batches and in the negative one without cDNA. (b)
Coomassie Blue (2%) was added to the somatodendritic compartment in the absence of cells.
The flow was reversed (upper panels) or not (lower panels) and the presence of colorant
analysed 5 min, 1, 12, 24 and 48 later. The medium was recovered from both compartments
and the optical density (O.D. 595 nm) analysed to determine the Coomassie Blue
concentration. (c) Primary culture of embryonic rat cortical neurons seeded in the
somatodendritic compartment was infected at DIV 7 with eGFP-LVs. The flow was then
reversed and a second primary culture of embryonic rat cortical neurons was seeded in the
axonal compartment. Forty-eight hours post-infection, cells were processed for visualizing
eGFP fluorescence (green). The nuclei were counterstained with DAPI (blue). The scale bar
is indicated on the figure. These data showed that LVs are not able to diffuse in the
microgrooves compartment. It also showed that they are not trafficking along the
microtubule. Tau transport in the microfluidic device is a specific mechanism since eGFP
was not found associated to secondary connected neurons.
Additional_file_3 as JPEG
Additional file 3 Mutant-Tau-associated Tau pathology progression. LVs encoding
hTau46P301L were bilaterally injected into the CA1 layer of rat brains (n = 5). Eight months
later, the animals were sacrificed, and the whole brain was processed for
immunohistochemical analysis using the AT8 antibody. In the middle upper panel, the total
area positive for AT8 is represented from bregma -3.30 to bregma -7.47. The brain was
virtually separated into three parts: bregma -2.30 to -4.30, bregma -4.52 to -6.04 and bregma -
6.30 to -7.64. In each part, certain regions were selected to illustrate the Tau pathology
(images on the left), and their location in the brain is shown on the right. The distribution of
AT8 labelling (cell bodies (C) vs. neurites (N)) is presented in the tables. The scale bars are
indicated on the figure. PhosphoTau (AT8 imunoreactivity) related to mutant Tau is restricted
to the vicinity of the IS.
Additional_file_4 as JPEG
Additional file 4 WT Tau-associated Tau pathology progression. LVs encoding hTau46WT
were bilaterally injected into the CA1 layer of rat brains (n = 5). Eight months later, the
animals were sacrificed, and the whole brain is processed for immunohistochemical analyses
using AT8. In the middle upper panel, the total AT8 immunopositive area is represented from
bregma +5.20 to bregma -7.60. The brain was virtually separated into six parts: bregma +5.20
to +2.70, +2.20 to +1.40, bregma +1.20 to -1.40, bregma -1.80 to -4.30, bregma -4.52 to -6.04
and bregma -6.30 to -7.80. For each part, certain regions were selected to illustrate the Tau
pathology (part left of the brain), and the total brain overlay is shown in the right part of the
brain. The distribution of AT8 labelling (cellular bodies (C) vs. neurites (N)) is presented in
the table. The scale bars are indicated on the figure. PhosphoTau (AT8 imunoreactivity)
related to WT Tau is found all over the rat brain even in distant areas.
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Additional file 1: 1941955291113178_add1.jpeg, 336K
Additional file 2: 1941955291113178_add2.jpeg, 996K
Additional file 3: 1941955291113178_add3.jpeg, 1433K
Additional file 4: 1941955291113178_add4.jpeg, 1129K