AAV-tau mediates pyramidal neurodegeneration by cell-cycle re-entry without neurofibrillary tangle formation in wild-type mice.
ABSTRACT In Alzheimer's disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the definition of their respective contributions.Transgenic mouse models do not resolve this problem conclusively, i.e. the relative hierarchy of amyloid and tau pathology depends on the actual model and the genes expressed or inactivated. Here, we approached the problem in non-transgenic models by intracerebral injection of adeno-associated viral vectors to express protein tau or amyloid precursor protein in the hippocampus in vivo. AAV-APP mutant caused neuronal accumulation of amyloid peptides, and eventually amyloid plaques at 6 months post-injection, but with only marginal hippocampal cell-death. In contrast, AAV-Tau, either wild-type or mutant P301L, provoked dramatic degeneration of pyramidal neurons in CA1/2 and cortex within weeks. Tau-mediated neurodegeneration proceeded without formation of large fibrillar tau-aggregates or tangles, but with increased expression of cell-cycle markers.We present novel AAV-based models, which demonstrate that protein tau mediates pyramidal neurodegeneration in vivo. The data firmly support the unifying hypothesis that post-mitotic neurons are forced to re-enter the cell-cycle in primary and secondary tauopathies, including Alzheimer's disease.
- SourceAvailable from: Anna Kremer[Show abstract] [Hide abstract]
ABSTRACT: The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer's disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3β with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer's disease.PLoS ONE 02/2014; 9(2):e87605. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Abnormal levels of mammalian target of rapamycin (mTOR) signaling have been recently implicated in the pathophysiology of neurodegenerative diseases, such as Alzheimer's disease (AD). However, the implication of mTOR in diabetes mellitus (DM)-related cognitive dysfunction still remains unknown. In the present study, we found that phosphorylated mTOR at Ser2448, phosphorylated p70S6K at Thr421/Ser424 and phosphorylated tau at Ser396 were significantly increased in the hippocampus of streptozotocin (STZ)-induced diabetic mice when compared with control mice. A low dose of rapamycin was used to elucidate the role of mTOR signaling in DM-related cognitive deficit. Rapamycin restored abnormal mTOR/p70S6K signaling and attenuated the phosphorylation of tau protein in the hippocampus of diabetic mice. Furthermore, the spatial learning and memory function of diabetic mice significantly impaired compared with control mice, was also reversed by rapamycin. These findings indicate that mTOR/p70S6K signaling pathway is hyperactive in the hippocampus of STZ-induced diabetic mice and inhibiting mTOR signaling with rapamycin prevents the DM-related cognitive deficits partly through attenuating the hyperphosphorylation of tau protein.Metabolic Brain Disease 03/2014; · 2.40 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder for which no effective treatment is available. Increased insight into the disease mechanism in early stages of pathology is required for the development of a successful therapy. Over the years, numerous studies have shown that cell cycle proteins are expressed in neurons of AD patients. Traditionally, neurons are considered to be post-mitotic, which means that they permanently retract from the cell cycle. The expression of cell cycle proteins in adult neurons of AD patients has therefore been suggested to promote or even instigate pathomechanisms underlying AD. Interestingly, expression of cell cycle proteins is detected in post-mitotic neurons of healthy controls as well, albeit to a lesser extent than in AD patients. This indicates that cell cycle proteins may serve important physiological functions in differentiated neurons. Here, we provide an overview of studies that support a role of cell cycle proteins in DNA repair and neuroplasticity in post-mitotic neurons. Aberrant control of these processes could, in turn, contribute to cell cycle-mediated neurodegeneration. The balance between regenerative and degenerative effects of cell cycle proteins in post-mitotic neurons might change throughout the different stages of AD. In the early stages of AD pathology, cell cycle protein expression may primarily occur to aid in the repair of sublethal double-strand breaks in DNA. With the accumulation of pathology, cell cycle-mediated neuroplasticity and neurodegeneration may become more predominant. Understanding the physiological and pathophysiological role of cell cycle proteins in AD could give us more insight into the neurodegenerative process in AD.Acta neuropathologica. 01/2015;
AAV-Tau Mediates Pyramidal Neurodegeneration by
Cell-Cycle Re-Entry without Neurofibrillary Tangle
Formation in Wild-Type Mice
Tomasz Jaworski1, Ilse Dewachter1, Benoit Lechat1, Sophie Croes1¤, Annelies Termont1¤, David
Demedts1, Peter Borghgraef1, Herman Devijver1, Robert K. Filipkowski2, Leszek Kaczmarek2, Sebastian
Ku ¨gler3, Fred Van Leuven1*
1Experimental Genetics Group, Department of Human Genetics, KULeuven-Campus, Leuven, Belgium, 2Lab of Molecular Neurobiology, Nencki Institute, Warszawa,
Poland, 3Center of Molecular Physiology of the Brain (CMPB), Department of Neurology, University Medicine Go ¨ttingen, Go ¨ttingen, Germany
In Alzheimer’s disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the
definition of their respective contributions.Transgenic mouse models do not resolve this problem conclusively, i.e. the
relative hierarchy of amyloid and tau pathology depends on the actual model and the genes expressed or inactivated. Here,
we approached the problem in non-transgenic models by intracerebral injection of adeno-associated viral vectors to
express protein tau or amyloid precursor protein in the hippocampus in vivo. AAV-APP mutant caused neuronal
accumulation of amyloid peptides, and eventually amyloid plaques at 6 months post-injection, but with only marginal
hippocampal cell-death. In contrast, AAV-Tau, either wild-type or mutant P301L, provoked dramatic degeneration of
pyramidal neurons in CA1/2 and cortex within weeks. Tau-mediated neurodegeneration proceeded without formation of
large fibrillar tau-aggregates or tangles, but with increased expression of cell-cycle markers.
models, which demonstrate that protein tau mediates pyramidal neurodegeneration in vivo. The data firmly support the
unifying hypothesis that post-mitotic neurons are forced to re-enter the cell-cycle in primary and secondary tauopathies,
including Alzheimer’s disease.
We present novel AAV-based
Citation: Jaworski T, Dewachter I, Lechat B, Croes S, Termont A, et al. (2009) AAV-Tau Mediates Pyramidal Neurodegeneration by Cell-Cycle Re-Entry without
Neurofibrillary Tangle Formation in Wild-Type Mice. PLoS ONE 4(10): e7280. doi:10.1371/journal.pone.0007280
Editor: Ashley I. Bush, Mental Health Research Institute of Victoria, Australia
Received July 9, 2009; Accepted August 28, 2009; Published October 1, 2009
Copyright: ? 2009 Jaworski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The investigations were made possible by funding and support from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO-Vlaanderen, www.
fwo.be), the Instituut voor Wetenschap en Techniek (IWT, www.iwt.be), the EEC-6th and 7th Framework Programs, the Rooms-fund, the KULeuven-Research Fund
(BOF) and KULeuven-Research&Development. These funders had no rule in the study design, data collection, analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: reMYND nv, Bio-Incubator #1, Gaston Geenslaan1, Leuven, Belgium
Aggregation of hyper-phosphorylated protein tau into filaments and
eventually neurofibrillary tangles (NFT) is characteristic for tauopa-
thies, a large and diverse group of neurodegenerative disorders,
including Alzheimer’s disease (AD) [1–11]. Primary tauopathies
present as clinically variable entities, e.g. Pick’s disease, progressive
supranuclear palsy, corticobasal degeneration and frontotemporal
dementia, among others . Tauopathy is defined by fibrillar and
tangled aggregates of phosphorylated protein tau, which is normally a
stability and spacing [6–12]. Tau3R and Tau4R isoforms have
different affinity for microtubules and their relative abundance is
regulated by alternative mRNA splicing . Post-translational,
dynamic regulation of microtubule-binding is thought to occur by
phosphorylation of tau at various serine/threonine residues by various
kinases, including GSK3, cdk5, and MARK, among others. In adult
ageing brain, in primary tauopathies and in AD, protein tau becomes
excessively phosphorylated, eventually changing its conformation to
induce aggregation resulting in tauopathy [1–12]. Interestingly, both
are dominantly associated with various tauopathies [4,5] implying that
neurotoxicity results from mutant tau protein, but as well from wild-
type tau by isoform imbalances.
Alzheimer’s disease (AD) is the most prominent secondary
tauopathy, wherein intracellular tau inclusions combine with
amyloid deposits [1–3]. Amyloid peptides are normal constituents
in human brain at any age, stemming from amyloid precursor
protein (APP) by a complex set of proteinases . With ageing,
amyloid peptides accumulate and aggregate, eventually becoming
deposited in parenchym and vasculature, even in cognitive normal
individuals as is emerging from clinical imaging studies. How and
why accumulating amyloid peptides cause tauopathy, and thereby
AD in some individuals and not in others, remains to be explained
by genetic and environmental factors acting at the cellular, i.e.
neuronal level. The relation between the two defining pathologies
in AD, and their relative contribution to cognitive defects, clinical
symptoms, neurodegeneration, brain atrophy and dementia
remains subject to academic debate, obscures early diagnosis
and hinders development of effective therapy.
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Transgenic mice have been invaluable for understanding
molecular mechanisms underlying amyloid peptide generation,
but amyloid mice lack two major pathological features of AD, i.e.
tauopathy and neuro-degeneration [14–18]. Tauopathy is patho-
diagnostically linked to all AD-cases, including early-onset cases
due to mutations in APP or presenilins that are by definition
caused by amyloid overproduction. In an experimental model,
absence of protein tau alleviated the cognitive defects inflicted by
amyloid , while expressing human wild-type tau causes no or
minimal tauopathy [14–17]. Conversely, mice expressing mutant
tau associated with familial fronto-temporal dementia (FTD)
recapitulate robust tauopathy [14–17]. Bigenic and multiple
transgenic mice expressing various combinations of mutant APP
and mutant tau recapitulate the combined amyloid and tau-
pathology of AD, but lack neurodegeneration and brain-atrophy
typical for AD [14–18].
Here we expressed Tau or APP, both wild-type and mutants, by
adeno-associated viral vectors (AAV) injected directly into the
hippocampus of wild-type mice. The observed dramatic pyramidal
neuro-degeneration inflicted by wild-type Tau4R and by mutant
Tau-P301L within weeks, contrasted with mutant APP that
provoked amyloid pathology after 6 months but with only minor
neurodegeneration. Importantly, tau-mediated neurodegeneration
was not caused by fibrillar tau-aggregates. Most prominent were
cell-cycle markers, indicating that degenerating neurons were
attempting to re-entry the cell-cycle. The in vivo AAV-based
models firmly support the unifying hypothesis that protein tau
mediates neurodegeneration by forcing post-mitotic neurons to re-
enter the cell-cycle in primary and secondary tauopathies.
AAV vectors to express EGFP, APP and Tau in pyramidal
neurons in vivo
Initial experiments were performed with triple mutant APP.-
SLA, described in the next paragraph, and mutant Tau.P301L,
both packaged in AAV-vectors with hybrid serotype-1/2 .
Intracerebral injection of these vectors into the hippocampal
complex of wild-type mice, expresses the embedded cDNA under
control of the human synapsin-1 promoter, specifically in
pyramidal neurons of hippocampus and cortex (Figure S1).
The generated triple mutant APP.SLA construct contained the
Swedish, London and Austrian mutations that are associated with
early-onset familial AD [20–22]. Transient expression in neuro-
blastoma cells demonstrated APP.SLA to produce highest levels of
Ab42 (Figure S2 A,B,C). Tau.P301L is associated with FTDP-17
and produced experimentally robust tauopathy in single and
bigenic mice by us [23,24] and others [15–17].
Initially, brains were analyzed 12 weeks after intracerebral
injection of AAV-vectors in wild-type mice (age 3–6 months).
Expression of APP.SLA was pronounced in pyramidal neurons in
CA and cortex (Figure 1A). Antibody 6E10  revealed intense
intraneuronal accumulation of APP metabolites (Figure 1B), while
antibody 3D6, specific for amyloid peptides  showed granular
intracellular amyloid deposits (Figure 1C). Amyloid plaques were
not detectable at 12 weeks p.i. of APP.SLA (Figure 1D).
In sharp contrast, the intracerebral injection of AAV-
Tau.P301L presented at first a very surprising outcome at 12
weeks p.i., because expression of human tau was detected in cortex
but was hardly detectable in the hippocampus, i.e. where the virus
was injected (Figure 2 A–C). Counterstaining for nuclei and in
depth analysis resolved the apparent contradiction: expression of
human protein Tau was low in pyramidal neurons in the
hippocampus because of the nearly complete loss of pyramidal
neurons in CA1/2 (Figure 2 A–C; compare injected to contra-
lateral hemisphere). The dramatic nearly complete elimination of
pyramidal CA neurons was confirmed by Nissl staining (data not
shown) and by IHC for NeuN as marker for neuronal nuclei
(Figure 3A, lower panels).
Human protein tau was more clearly expressed in cortical
neurons, as well as in apparently intact hippocampal neurons that
were located adjacent to the degenerated CA regions (Figure 2
A,C,D; Figure S4B). These remaining pyramidal neurons
expressed typical phospho-epitopes of tau, i.e. AT8 (pS202/
pT205), AT180 (pT231), AT270 (pT181) (Figure 2, Figure S4).
Importantly, the CA1/2 regions that were devoid of neurons
following AAV-Tau.P301L injection did not contain any immu-
noreactive remains of tau-aggregates or ghost tangles (Figure 2,
Figure 3A upper panel, Figure S4B).
Tau-mediated neurodegeneration is rapid and closely
associated with microgliosis
Lower doses of AAV-Tau-P301L resulted in less neurodegen-
eration with marked thinning of CA1/2 in injected mice
(Figure 3A), coinciding spatially with expression of protein tau.
The relation was actually inverse: more extensive neuron-loss
contrasted with less human tau (Figure 3A), which is attributed to
diminished or abolished protein synthesis in degenerating neurons.
Temporal progression of neurodegeneration, analyzed at 1.5, 3,
6, 9 and 12 weeks p.i. of AAV-Tau.P301L was evident in CA2
already at 1.5 weeks p.i., progressing to CA1 at 3 weeks p.i. and
evolving into nearly complete pyramidal neuron-loss at 6–12
weeks p.i. (Figure 2A–C, Figure 3). At the later time-points neuro-
degeneration was extensive also in the cortex (Figure 3B, panels
FluoroJadeB (FJB)  strongly labeled degenerating neurons in
CA2 at 1.5 weeks (not shown) and in CA1 at 3 weeks p.i. of AAV-
Tau.P301L injected mice (Figure 3B, middle panels). At later time-
points, neuronal FJB staining decreased in parallel with NeuN
(Figure 3B), while reaction was also noted with micro- and
astroglia, that were clearly activated and indicative of inflamma-
tion (Figure 3B). FJB is hereby confirmed as convenient, but not
specific marker for degenerating neurons, as observed also in our
inducible p25 mice, a model for hippocampal sclerosis .
Inflammation was confirmed by IHC for activated microglia
and astroglia (Figure 3B). Microgliosis was transient and most
intense at the time-points of onset and of active neurodegenera-
tion, while fading later to even disappear completely at 12 weeks
p.i. (Figure 3B, utmost right panels marked MHCII). Conversely,
some astrogliosis was evident throughout the observation period
(Figure 3B, panels marked GFAP). Parallel control experiments
with intracerebral injection of AAV-EGFP in age- and sex-
matched groups of wild-type mice did neither show neuronal loss
nor microgliosis (Figure S1; results not shown).
We conclude that intracerebral injection of AAV-Tau.P301L,
but not of AAV-APP.SLA or of AAV.EGFP, caused pyramidal
neurons to degenerate in CA and cortex. Moreover, microgliosis is
closely linked to Tau-mediated neurodegeneration, but not to
amyloid production, nor to reaction to viral particles or proteins.
Biochemical analysis of levels of expression and tau-
Biochemical analysis was performed in parallel, on four separate
cohorts of wild-type mice injected intracerebrally with AAV-
APP.SLA or AAV-Tau.P301L, analyzed at 1.5 weeks p.i. which
marks the beginningintense
Tau.P301L mice (Figure 3B). We analyzed hippocampal extracts,
PLoS ONE | www.plosone.org2 October 2009 | Volume 4 | Issue 10 | e7280
Figure 1. AAV-mediated expression of APP.SLA in wild-type mouse brain. Intracerebral injection of 10E8 transducing units (t.u.) AAV-
APP.SLA vector in wild-type mice (n=3) analyzed 12 weeks p.i. A, B: IHC for APP and its metabolites with Mab 6E10 on brain sections after antigen
retrieval with formic acid . Red square in panel B (middle) is enlarged in right panel. C: IHC for amyloid peptides with Mab 3D6; red square in
middle panel is enlarged in right panel. D: histochemical staining with compound X-34 for protein aggregates  of AAV-APP.SLA injected mouse
(left) and from an APP.V717I transgenic mouse (age 22 months) (right panel) as positive control for amyloid pathology as described [24,29]. Scale bars
panel A 1 mm, others as indicated.
PLoS ONE | www.plosone.org3 October 2009 | Volume 4 | Issue 10 | e7280
Figure 2. AAV-mediated expression of Tau.P301L in wild-type mouse brain. Intracerebral injection of 10E8 t.u. AAV-Tau.P301L vector in
wild-type mice (n=4) analyzed 12 weeks p.i. A: IHC for human Tau with HT7 (panel A). B, C: IHC for phospho-epitopes AT8 and AT180 respectively. D:
detail of panel C at higher magnification to show pyramidal neurons on the border of CA1 and subiculum, which appear intact despite high levels of
phosphorylated tau (AT180). E: IHC of AAV-Tau.P301L injected mouse at 3 weeks p.i., without primary antibody as negative control. Note the severe
neurodegeneration in panels A–C and the absence of any indication of pronounced protein tau-aggregates in the degenerated regions. Scale bars A–
C 1 mm, D 50 mm, E 0,5 mm.
PLoS ONE | www.plosone.org4 October 2009 | Volume 4 | Issue 10 | e7280
and not total brain extracts to avoid dilution by regions that were
not transduced (Figure S3).
Western blotting of hippocampal protein extracts demonstrated
relative levels of APP.SLA or Tau.P301L to be about two-fold
higher than endogenous murine APP or murine protein tau,
respectively (Figure 4A,B respectively). We conclude that neuro-
degeneration inflicted by Tau.P301L was not attributable to
massive over-expression. Actually, pyramidal degeneration oc-
Figure 3. Dose-dependence and time-line of AAV-Tau.P301L mediated neurodegeneration. A. Intracerebral injection of AAV-Tau.P301L
at doses indicated in wild-type mice (n=3 per dose) analyzed at 4 weeks p.i. by IHC for total human Tau with HT7 and counterstained with
hematoxylin (upper panels). IHC for NeuN visualized neuronal nuclei without counterstaining (lower panels). Scale bar 0.5 mm. Note thinning of CA1/
2 already with lowest dose of APP-Tau.P301L. B. Time line of AAV-Tau.P301L mediated neurodegeneration. Intracerebral injection of 10E8 t.u. of AAV-
Tau.P301L in wild-type mice analyzed at 1.5 weeks (n=4), 3 weeks (n=6), 6 weeks (n=6), 9 weeks (n=6) and 12 weeks (n=6) p.i.. Control mice were
intracerebrally injected with AAV-EGFP (10E8 t.u.) sacrificed at same time-points p.i. (all n=4). Analysis by IHC with HT7 for total human tau, NeuN for
neuronal nuclei, GFAP for astroglia and MHCII for activated microglia as indicated above the panels (scale bars 1 mm). Histological staining with
FluoroJadeB for degenerating neurons and activated glia (see text for details) (scale bar 0.1 mm). Note that expression of human Tau is highest at 1.5
week p.i. and subsides later, paralleling the loss of NeuN immunoreactivity. The FJB signals in CA1 mark degenerating pyramidal neurons at 3 weeks
p.i. but changes to an astroglial pattern at later time-points (see text for details). Note that at 3 weeks p.i. the loss of neurons and FJB positive signals
in CA concurs with intense microgliosis, which subsides completely at 12 weeks p.i. while astrogliosis is much less specific in time and spatial
distribution (see text for details).
PLoS ONE | www.plosone.org5 October 2009 | Volume 4 | Issue 10 | e7280
Figure 4. Protein levels of APP.SLA and Tau.P301L in hippocampal extracts. Biochemical analysis by western blotting of hippocampal extracts
from AAV injected mice, as indicated. A: western blotting for total human and mouse APP with antibody B10/4 and for human APP with Mab WO2 on
hippocampal extracts from AAV-APP.SLA injected mice at 1.5 weeks p.i. Quantitative data are from measurements with B10/4 following densitometric
scanning (mean+/2SD; n=3). B: western blotting for total human and mouse Tau with Mab Tau5 and for human Tau with Mab HT7 on hippocampal
2SD; n=3). C: western blotting reveals aggregated Tau oligomers in AAV-TauP301L mice. Protein extracts from AAV-Tau.P301L and AAV-EGFP injected
mice (1.5 week p.i.) were separated on 8% Tris-Glycine gel under non-reducing and under reducing conditions. When blots were probed first with the
secondary antibody only, non-specific bands denoted by asterisks were also revealed. Note the smears in the non-reduced samples (see text for details).
PLoS ONE | www.plosone.org6 October 2009 | Volume 4 | Issue 10 | e7280
curred at near-physiological levels of protein tau that were similar
to those in our transgenic Tau.P301L mice [23,24], which make
the AAV models even more interesting.
Biochemical evidence for tau-aggregates in the brain of AAV-
Tau injected mice was deduced from Western blots following
SDS-PAGE under non-reducing conditions of hippocampal
protein extracts. Protein-smears in the high Mr regions reacted
with antibodies specific for human Tau (Figure 4C). Smears
disappeared upon disulfide bond reduction concomitant with
increased monomeric ,64 kDa Tau (Figure 4C, arrowheads).
Wild-type Tau inflicts neurodegeneration as effectively as
Additional AAV-vectors were constructed to express wild-type
APP and wild-type Tau4R, in first instance as extra controls for
the degeneration provoked by mutant Tau.P301L. Surprisingly,
wild-type Tau4R inflicted very similar dramatic and rapid
neurodegeneration in CA1/2 as mutant Tau.P301L at 3 weeks
p.i. (Figure 5 upper panels, Figure S4 B). Wild-type Tau4R
appeared even more deleterious at lowering protein tau levels,
although these would result from more intense neurodegeneration
with evident less protein synthesis (Figure 5 upper panels, Figure
S4B, utmost right panels). Importantly, the findings considerably
extend the potential of the paradigm, because in many
tauopathies, and definitely in all AD cases, wild-type Tau4R is
Conversely, wild-type APP and mutant APP.SLA compared
with respect to expression of APP and the nearly unaffected
appearance of pyramidal neurons in CA1/2 up to 3 months p.i..
Occasionally, NeuN appeared reduced in some AAV-APP.SLA
mice (Figure 5 lower panels; results not shown). Obviously, any
neuronal loss inflicted by APP.SLA is much less, or occurs much
slower than the intense, rapid pyramidal neurodegeneration
provoked by wild-type and mutant tau in this paradigm.
Amyloid plaques develop in AAV-APP.SLA mice at 6
months p.i. with only marginal neuron loss
The impotency of AAV-APP.SLA in provoking neurodegener-
ation relative to AAV-Tau was not compensated for by time, as
demonstrated in a fourth large series of experiments. We
performed intracerebral injections in 4 cohorts of wild-type mice
with AAV-APP.SLA or AAV-Tau.P301L, with AAV-EGFP as
independent control next to the sham-injected group. All mice
were analyzed for brain histology and immunohistochemistry at 6
months p.i., which is the longest time-point studied so far.
In AAV-Tau.P301L mice the pyramidal neurons were again
lost completely in CA and also in deep cortical layers (Figure 6A,
in between red arrowheads in panel marked Tau.P301L).
Conversely, in AAV-APP.SLA mice the CA regions were still
largely intact at 6 months p.i., very similar to AAV-EGFP injected
and sham-operated mice. Nevertheless, typical amyloid plaques
were evident in hippocampus and cortex of all AAV-APP.SLA
injected mice at 6 months p.i. (Figure 6B–D). This is much earlier
than in APP.V717I mice and in bigenic biAT mice, which we
attribute to the triple mutant APP.SLA in the current AAV model,
which produces more Ab42 than APP.V717I (Figure S2)
expressed in our amyloid model [24,29].
As extra parameter for neurodegeneration, we measured the
CA blade thickness in all AAV-injected mice of the four cohorts at
6 months p.i.. Relative to the nearly annihilated CA in AAV-
Tau.P301L mice, these hippocampal region were hardly affected
in AAV-APP.SLA mice (Figure 7). The combined data prove that
mutant APP is only marginally potent and on a much longer time-
scale, relative to protein Tau in provoking damage to pyramidal
neurons, under very comparable experimental conditions. More-
over, IHC with AT180 demonstrated considerable tau-phosphor-
ylation in the pyramidal neurons (Figure 6G, H) which tempts us
to speculate that it is actually the beginning tauopathy that is
detrimental to these neurons.
Relation of neurodegeneration to phosphorylation and
aggregation of protein tau
Intense and painstaking analysis by histological, immunohisto-
logical and biochemical methods failed to detect larger or marked
aggregates of protein tau in pyramidal neurons at any time-point
after injection of AAV-Tau vectors, i.e. before, during or after the
documented dramatic neurodegeneration. Pathological phospho-
tau epitopes were conspicuous by IHC in neurons that expressed
human tau (Figure 2, Figure S4; results not shown) and were also
evident in AAV-Tau mice by western blotting with specific
monoclonal antibodies, e.g. AT180, AT270 and AT8 (Figure S4).
Because in biAT mice, our bigenic AD model, the amyloid
pathology synergistically promotes tau phosphorylation and
tauopathy in limbic regions by activating GSK3 , we analyzed
AAV-APP.SLA mice for endogenous mouse tau phosphorylation.
Specified epitopes AT270 (Figure 6E, F) and AT180 (Figure 6 G,
H) were evident particularly in dystrophic neurites around
plaques, as in AD patients and in our APP.V717I transgenic
mice . Because the AT270 antibody had to be used at higher
concentrations than normal to reveal reaction with endogenous
mouse protein Tau, the background staining was also increased
and higher than normal (Figure 6E, compare to Figure S4B lower
The biochemical and immunohistochemical data-set of phos-
pho-epitope analysis that we have compiled is too extensive to be
included here, while on the other hand it failed to reveal a direct
relation of any particular phospho-epitope to the actual neurode-
generation (cfr discussion).
Neurotoxicity of AAV-Tau depends on the microtubule
The hypothesis that microtubule binding of tau was involved in
the observed neurodegeneration was supported by AAV-Tau255
that we generated to express C-terminally truncated Tau4R
lacking the microtubule binding domain (Figure S5A). At 3 weeks
p.i. AAV-Tau255 was efficiently expressed at similar levels as full-
length Tau in limbic and cortical regions (Figure S5A). In contrast
to full-length Tau, truncated Tau255 did not induce appreciable
neurodegeneration, nor microgliosis (Figure S5B, C). Interestingly,
Tau255 appeared more localized to neuronal somata whereas full-
length Tau distributed also to somatodendritic compartments of
pyramidal neurons (Figure S5D). Intriguingly, although Tau255
carried amino acid sequence 181–205 (numbering of Tau441),
which upon phosphorylation constitutes the AT8 (pS202/pT205)
and AT270 (pT181) epitopes, these phospho-epitopes were hardly
detectable in AAV-Tau255 injected mice (Figure S5A). In
contrast, phospho-epitope AT180 (pT231) is located in the same
region and equally evident in pyramidal neurons in AAV-Tau255
as in AAV-Tau.4R and AAV-Tau.P301L mice (Figure S5A).
Search for mechanisms contributing to tau-mediated
The unequivocal demonstration that wild-type and mutant
protein tau are equally neurotoxic for hippocampal and cortical
pyramidal neurons in vivo, instigated a search for molecular
markers in these novel mouse models for tau-mediated neurode-
PLoS ONE | www.plosone.org7October 2009 | Volume 4 | Issue 10 | e7280
Figure 5. Wild-type and mutant Tau, but not APP.SLA or APP.WT, cause neurodegeneration in vivo. Intracerebral injection of 10E8 t.u.
AAV-Tau.WT, AAV-Tau.P301L, AAV-APP.SLA and AAV-APP.WT vectors as indicated, in wild-type mice (n=4) analyzed at 3 and 12 weeks p.i. by IHC for
NeuN as marker for neuronal nuclei, without counterstaining. Note the dramatic neurodegeneration inflicted by wild-type and mutant Tau already at
3 weeks p.i. in contrast to AAV-APP.SLA. Scale bars 1 mm.
PLoS ONE | www.plosone.org8October 2009 | Volume 4 | Issue 10 | e7280
Figure 6. Amyloid plaques 6 months after intracerebral injection of AAV-APP.SLA. Intracerebral injection of 10E8 t.u. AAV-APP.SLA vector
in wild-type mice (n=6) analyzed 6 months p.i. A: IHC for NeuN as marker for neuronal nuclei, comparing 4 different AAV-constructs (see text for
details). Note only in panel Tau.P301L the loss of pyramidal cells in CA and also in the deep cortical layers, indicated by the red arrowheads. B: X34
staining for amyloid plaques and protein aggregates . C, D: IHC for amyloid peptides with Mab 3D6; plaque indicated with arrowhead enlarged in
panel D. E, F: IHC with AT270 for phosphorylated endogenous mouse tau, highlights also phosphorylated tau in dystrophic neurites around amyloid
plaque (panel F). AT270 was used at higher concentration than normal to reveal endogenous mouse protein Tau, which increases also the
background staining (panel E, compare to Figure S4B lower panels). G, H: IHC with AT180 for phosphorylated endogenous mouse tau in CA pyramidal
neurons expressing APP.SLA, with magnified detail (square in panel G in panel H). Scale bars A, B, C, E, G 0.5 mm; D 25 mm; F, H 30 mm.
PLoS ONE | www.plosone.org9 October 2009 | Volume 4 | Issue 10 | e7280
generation. We analyzed a broad panel of markers and
characteristics of degenerating neurons (Table S1, S2) based on
known or suspected mechanisms that might be responsible for, or
contribute to tau-mediated neurodegeneration.
Apoptotic markers were evident in AAV-Tau injected mice,
albeit outside the actual degenerating regions, i.e. particularly in
dentate gyrus granular cells and sparingly in CA1 adjacent to the
subiculum (Figure S6A).
Beclin and Atg8/LC3 are essential regulators of autophagy that
mark early and mature steps of autophagosomes . The overall
decrease in expression of both markers in AAV-Tau.P301L mice
was evident, but also without direct temporal and spatial
association with degenerating neurons (Figure S6B; results not
shown). Lipofuscin was evident as intra- and extra-cellular puncta
in AAV-Tau injected mice in CA pyramidal neurons and region
from 3 weeks p.i. onwards, correlating with neurodegeneration
and persisting in hippocampal regions after the neurons were
annihilated (Figure S7; results not shown). These apparent
remnant cellular debris of degenerated hippocampal neurons
might support a contribution for autophagy, but also demonstrate
that the complete absence of tau-aggregates in these areas is not a
technical problem, and therefore conspicuous and informative.
Ultrastructurally, degenerating CA neurons presented with
nuclear and cytoplasmic condensation and vacuolization, clumped
chromatin and indentated or blebbing nuclear membranes (Figure
S6C). These features closely resembled degenerating hippocampal
neurons in our mice that model hippocampal sclerosis by inducible
expression of p25, the truncated activator of cdk5 . Other
pathological features like tangles, spheroids and axonal dilatations
were revealed by various staining methods, but only sporadically
and not consistently in all AAV-Tau injected mice (Figure S6D).
Neither their localization nor distribution nor abundance could
explain the observed massive pyramidal cell-death.
Truncated AAV-Tau255, lacking the microtubule binding
domain and failing to provoke neurodegeneration, favored the
hypothesis that microtubular networks, and associated synaptic
architecture, are affected which was supported by marked changes
in tubulin and actin, as well as in synaptophysin (Figure S8). These
features were widespread and correlated with actual degenerating
neurons, although they can equally well be consequences of the
Cell-cycle and signal transduction
Expression of cell cycle-related proteins fueled the hypothesis
that post-mitotic neurons attempting to reactivate cell-cycling
contribute to neurodegeneration in AD [31–35]. We analyzed
selected markers to pinpoint an eventual role in tau-mediated
neurodegeneration (Table S2).
Among cell-cycle and related markers, cyclinD2 and cyclinB1
were strongly up-regulated 3 weeks p.i. in degenerating neurons in
AAV-Tau mice (Figure 8A) corroborated by other markers, e.g.
PCNA and phosphorylated retinoblastoma protein (Figure 8A).
Conversely, cell-cycle inhibitor p27KIP1 was strongly down-
regulated, while markers like Ki67 and cdk2 were hardly affected
by tau-mediated neurodegeneration (Figure 8A; results not
shown). Effects on these and other markers were restricted to
sub-regions and/or sub-sets of neurons in AAV-Tau mice, while
most were unaffected in AAV-EGFP or AAV-APP.SLA injected
mice (results not shown).
Ras-dependent mitogen-activated protein (MAP) kinases control
cell-proliferation and stress responses. Expression of activated,
phosphorylated forms of three members of the MAPK family by
IHC revealed no signal for phospho-SAPK/JNK, whereas
phospho-p44/42 MAPK was elevated mainly in glia cells. Only
phospho-p38 MAPK was observed in a subset of degenerating
neurons (Figure 8B).
The strong expression of cyclinD2, i.e. the only D-type cyclin
expressed in dividing cells derived from neuronal precursors in
adult hippocampus  prompted us to inject cyclinD2 deficient
mice intracerebrally with AAV-Tau.P301L. Neurodegeneration in
cyclin D2 deficient mice was, however, similar to that in wild-type
littermates at 3 weeks p.i. of AAV-Tau.P301L (Figure 8C),
excluding cyclinD2 on its own as decisive factor in the observed
Here we provide direct in vivo experimental evidence for
protein tau-mediated hippocampal neuro-degeneration using
intracerebral injection of specified adeno-associated viral vectors.
The salient features of the model, based on extensive character-
ization, qualify them as innovative and unique in several aspects:
protein tau at near-physiological protein levels invokes rapid and
specific degeneration of pyramidal neurons in limbic regions.
Moreover, wild-type Tau4R is as effective as mutant Tau.P301L
which is unique, to our knowledge. Thereby AAV-tau model
recapitulates, and is informative, for the majority of tauopathies
that are caused by wild-type Tau4R, including all AD cases.
We discuss three important aspects that are directly concerned
by the AAV-models: (i) the mechanism by which protein tau
destroys pyramidal neurons; (ii) the information content of the
experimental models for human disease, i.e. primary tauopathies
and AD; (iii) the marked difference with classical transgenic mice
that have amyloid deposits and/or tangles with minimal
The mechanism underlying AAV-Tau neurodegeneration
Importantly, the mechanism whereby AAV-Tau inflicts neuro-
degeneration does not depend on the formation of large, fibrillar
tau-aggregates. Conversely, surviving pyramidal neurons flanking
Figure 7. Hippocampal CA1/2 in AAV injected mice. The
thickness of CA1/2 was measured at three sites along the CA region,
in the four cohorts of AAV and sham-injected mice indicated (mean+/
2SD; sham and APP.SLA, n=6; EGFP and Tau.P301L, n=5). Statistical
analysis (Mann-Whitney test) versus sham, *p=0.01, **p=0.006.
PLoS ONE | www.plosone.org10October 2009 | Volume 4 | Issue 10 | e7280
Figure 8. Tau-mediated neurodegeneration relates to cell cycle. A: IHC for indicated markers in brain of wild-type mice injected with 10E8 t.u.
AAV-Tau.P301L analyzed 3 weeks p.i. comparing injected (left panels) to non-injected (right panels) hemispheres. Note the predominant nuclear
localization of cyclinB1 and cytoplasmic expression of cyclinD2 as well as the strong phosphorylation of Retinoblastoma protein. Marker PCNA was
typical for a subset of neurons in a different stage of degeneration. Scale bars 30 mm. B: IHC for active members of MAPK family: phospho-SAPK/JNK,
phospho-p44/42 MAPK, phospho-p38 MAPK. Compared injected (left) to non-injected (right) hemispheres. Scale bars 30 mm. C: CyclinD2 deficient
mice were injected with 10E8 t.u. AAV-Tau.P301L and analyzed 3 weeks p.i. compared to wild-type littermates (n=4 each) by IHC for total human tau
with HT7 (lower panel; scale bar 100 mm) and for NeuN as marker for neuronal nuclei (upper panel; scale bars 1 mm). Note the similar level of
neurodegeneration in wild-type and CyclinD2 deficient mice injected with AAV-Tau.P301L.
PLoS ONE | www.plosone.org 11October 2009 | Volume 4 | Issue 10 | e7280
the degenerated neurons in CA1/2, contained hyper-phosphory-
lated tau aggregates, but nevertheless appeared morphologically
intact. This remarkable outcome corroborates our previous
observation in bigenic mice with massive forebrain tauopathy,
i.e. fibrillar tau is not neurotoxic per se . Moreover, the
combined data support the hypothesis that fibrillar tau-aggregates
function as a sink, to protect neurons against smaller tau-
aggregates or -oligomers that are the real neurotoxic species
[24,36]. The biochemical and physical properties of the postulated
neurotoxic tau-oligomers can eventually be defined in the AAV-
model, and compared to those in transgenic models. Nevertheless,
that might prove a non-conclusive exercise, as discussed below.
Particularly mice with inducible tau.P301L expression showed
neurodegeneration and tangles but at much higher expression
levels of the mutant tau.P301L . To our knowledge, models
based on wild-type Tau.4R have not been reported to suffer
appreciable neurodegeneration in the hippocampus.
We analyzed the phosphorylation status of tau and collected
extensive biochemical and immuno-histochemical data-sets on tau
phospho-epitopes in the AAV models, as in our transgenic mice
[23,24]. No specific single or complex phospho-epitope on tau is
identified as essential for, or directly related to the neurodegen-
eration. This is not unexpected, and in line with many studies on
human brain and in experimental models, supporting the
conviction that variable combinations of phosphorylations of
protein tau underlie its neurotoxicity [1–11,15–17,31]. By logical
extension, not a single kinase but combined actions of several
kinases are to be hold responsible for phosphorylating tau to make
it eventually harmful to neurons. Moreover, the sets of phospho-
epitopes and of kinases most likely will vary pending the affected
brain region, i.e. the disorder. Because tauopathies vary widely in
their clinical, pathological and biochemical characteristics, the
molecular identification of a unique, unifying neurotoxic tau-
species becomes more and more unlikely.
We first and foremost consider important the only known
physiological function of protein tau, i.e. binding to microtubules.
If phosphorylated protein tau fails to stabilize microtubules, or
affects microtubule dynamics, a dysfunctional cytoskeleton with
axonal, dendritic and synaptic defects as results. The observed
changes in cytoarchitecture in degenerating neurons, support the
hypothesis that they contribute to the overall process. The
experimental data obtained with truncated Tau255 most strongly
imply that microtubule binding of protein tau is essential in
inflicting neurodegeneration and involve the microtubular net-
work as a structural and transport scaffold. The alternative
explanation, i.e. that wild-type and mutant full-length Tau but not
truncated Tau255 interact with cellular proteins other than
microtubuli, remains open for experimental verification.
We went on to define the underlying mechanism of tau-
mediated neurodegeneration by analysis of a large and wide panel
of molecular targets and pathways, conform the hypothesis that
neurons do not die by a single mechanism . Although the
outcome did not yield a single mechanism to be responsible for
tau-mediated neuronal cell-death, the indications for attempted
cell-cycle reentry were most marked.
Cell cycle re-entry
In AD and unrelated tauopathies, cell-cycle events aresuggested to
correlatewith,ifnot cause neuro-degeneration. Specifically,cell-cycle
activation caused neurodegeneration in a Drosophila tauopathy
that support theconclusionthat cell-cycle re-entryisprominent inthe
AAV-Tau model. Supporting data and many remaining problems of
this hypothesis are reviewed elsewhere [31–35].
Cyclins B1 and D2 were most prominent in degenerating CA1
neurons of AAV-Tau mice. Cyclins D1, D2, D3 with cdk4 and
cdk6, regulate G1/S transition by releasing E2F transcription
factors via increased phosphorylation of retinoblastoma protein.
The latter is here also demonstrated in degenerating pyramidal
neurons expressing protein Tau. Upregulation of cyclinB1 also
points to the G2/M checkpoint, which depends on cdk2/cyclinB1
activity. Both cyclinD2 and cyclinB1 have been shown to mark
cell-cycle events in AD brain . The proliferating cell nuclear
antigen (PCNA) marked a subset of CA1 pyramidal neurons, but
these were, surprisingly, not marked by proliferation marker Ki67.
This is particularly interesting in view of the role of PCNA in DNA
The fact that cyclinD2 deficient mice that have problems with
adult neurogenesis  did suffer no less neurodegeneration
induced by AAV.Tau than wild-type mice, corroborates that cell
cycle control, and/or induction of the post-mitotic state, is not left
to one component or complex. Rather, or alternatively, the
contribution of the cell-cycle to neurodegeneration would be by
multi-point or by fragmented reentry attempts, whereby cyclinD2
is not the decisive factor, or is made redundant by cyclins D1 and
D3 . Among candidate factors that could force post-mitotic
neurons to re-enter the cell-cycle are mitogenic factors from the
MAPK family, e.g. p44/42 MAPK (Erk1/2), p38 MAPK and
SAPK/JNK. While JNK/SAPK was not detected and p44/42
MAPK was observed in glial cells, only p38 MAPK was markedly
increased in degenerating neurons in AAV-tau mice. Similarly,
whereas Akt/PKB is a central regulator of metabolism, apoptosis,
transcription and cell-cycle, this kinase appeared not activated in
the AAV-Tau model.
From the many markers analyzed (Tables S1, S2) we conclude
that the strongest indications support the hypothesis that neurons
degenerate because of their attempts to re-enter the cell-cycle.
Nevertheless, other markers are observed and proposed to
contribute, not in the least microgliosis, as discussed below.
Activated microglia are spatially and temporally closely associated
with degenerating neurons in the AAV-tau models, strongly
resembling the pathological characteristics of mice with hippo-
campal and cortical sclerosis .
The relevance for human disease: tauopathies and AD
The rapid and dramatic neurodegeneration inflicted by protein
tau contrasts with the minimal neurotoxic effects sorted by the
mutant APP.SLA under the same experimental conditions. Only
marginal neuro-degeneration resulted despite important accumu-
lation of amyloid peptides that led even at 6 months p.i. to amyloid
plaques in hippocampus and cortex. This marked dissociation in
outcome by the same experimental approach, corroborates the
growing awareness that neurodegeneration in AD is not mediated
primarily or directly by amyloid. The essential contribution of
protein tau to pyramidal cell-death is thereby joined seamlessly to
the primary tauopathies. Consequently, tau pathology is essential
and decisive, together with amyloid, in the overall pathogenesis of
AD, notwithstanding its pathological classification as a ‘secondary
We adhere the hypothesis that in AD the accumulation of
amyloid is the trigger, but that protein tau executes specified
neurons. The transgenic models for amyloid and tau pathology
have not, however, substantiated this hypothesis to the fullest,
although they have been invaluable for understanding molecular
mechanisms of amyloid peptide generation, amyloid pathology,
repercussions on cognition and behavior [13–17]. Even multigenic
mice that express various combinations of mutant APP and
mutant tau to recapitulate the combined amyloid and tau-
PLoS ONE | www.plosone.org12 October 2009 | Volume 4 | Issue 10 | e7280
pathology of AD, still lack the specific regional neurodegeneration
that leads to the typical brain atrophy in AD [13–17].
Remarkably, transgenic mouse models now even support the
reverse hypothesis, i.e. that tangle deposition is protective, similar
to amyloid plaques in amyloid pathology. Both are sinks for mis-
folded, aggregated, indigestible peptides or proteins. Whereas such
detoxification by aggregation and deposition can be effective as a
short-term strategy, it eventually becomes futile and then
aggravates the pathology, particularly in humans with their long
life-span. Of note, the view on tauopathy is evolving very
analogous as that on amyloid pathology in AD: the emphasis
shifts from large visible deposits, i.e. amyloid plaques and
neurofibrillary tangles, to smaller molecular entities, i.e. amyloid-
and tau-oligomers. A similar evolution is evident in other
The combined transgenic and AAV-models allow us to propose
confidently that the link between amyloid, tau pathology and
neurodegeneration does not involve directly the formation or
repercussions of amyloid plaques and neurofibrillary tangles.
Rather, these are the products of dead-end escape-routes whereby
cells, in the case of AD pyramidal neurons in limbic brain regions,
attempt to limit the negative impact of accumulating misfolded
and aggregated proteins that cannot be properly handled by
normal cellular proteolytic degradation mechanisms, i.e. endoso-
mal, lysosomal, auto-phagosomal, proteasomal, … It is then not
surprising to see many of these involved in, or attained by, the
amyloid or tau pathology.
Although tauopathy with tangles is and remains the post-
mortem pathological hallmark that is co-diagnostic for AD, the
genetic evidence of Tau mutations in familial primary tauopathies
has promoted tauopathy to be mechanistically relevant and
important. Experimental evidence in vivo that was largely lacking
is convincingly provided by the current AAV-models.
AAV versus transgenic models
A final important point concerns the question why AAV-based
models are more powerful in producing neuro-degeneration than
transgenic models expressing the same wild-type Tau4R isoform
 or Tau.P301L mutant  at similar near-physiological
levels. These transgenic models suffer axonopathy and tauopathy,
respectively, but without appreciable neurodegeneration.
Although data to answer this problem do not abound, we
consider as major difference the observed microgliosis that is much
more intense in the AAV-Tau model than in the AAV-APP mice.
Microgliosis is spatially and temporally most closely associated
with degenerating neurons in the AAV-tau models. This is strongly
reminiscent of our observations in inducible p25 mice that suffer a
profound hippocampal and cortical sclerosis with pathological
characteristics very similar to the AAV-Tau mice . A recent
study described wild-type tau to mediate some neurodegeneration
with combined microgliosis by AAV gene-transfer . Therein,
degeneration of dopaminergic neurons in the substantia nigra of
aged rats was also directly associated with microgliosis, lending
support to our assumption that microgliosis contributes essentially
to neurodegeneration. Whereas also the viral vectors used as
delivery tool, can be of some importance, they are evidently not
decisive because neurodegeneration is specific for AAV-tau, wild-
type and mutant, and was lacking in AAV-APP and AAV-EGFP
mice, observed here and as observed in other models [19,42].
Aspects that are important to explain the apparently contra-
dictory outcome in different models, relate to differences in time-
scale and/or kinetics whereby neurons either degenerate or
whereby tau aggregates and ‘sinks’ into tangles. It is evident that
various post-translational modifications, e.g. phosphorylation,
ubiquitinylation, glycation, O-glucosylation, … can and will
contribute to either mechanism. The overall process is complex
and implies enzymes, i.e. proteinases, kinases, phosphatases, …
but also cellular factors like chaperones and heat-shock proteins.
Their structural features and dynamic actions will tilt the balance
to either slow death by progressive accumulation of aggregated,
undigested or undigestible amyloid and/or protein tau, or to faster
death by cell-cycle re-entry, accelerated by microglia derived pro-
inflammatory neurotoxic factors .
Moreover, the tau-species that are responsible for aggregation
and neurotoxicity are proposed to differ at the molecular level. We
refer here also to a most recent report on the transmission and
spreading of tauopathy in transgenic mouse brain, following intra-
cerebral injection of tau-aggregates . Those findings are
relevant for the possible cell-to-cell spreading of tauopathy in brain
and imply an extracellular route, which is to be defined for the
cytoplasmic protein tau. Nevertheless, the time-scale of spreading
was very slow and resulted in typical tauopathy with aggregates
and tangles, while neuro-degeneration was minimal or absent .
Thereby, that model conforms to the tauopathy as observed in the
parental tau.P301S transgenic mice that have no neurodegener-
ation in limbic regions.
In conclusion, we present in vivo experimental evidence for a
major problem in tauopathies: effective modeling of pyramidal
neurodegeneration that is mediated by protein tau.4R, which is
responsible for the majority of human tauopathies, including all
Alzheimer patients. We further delineate two major mechanisms
that contribute to the rapid neurodegeneration mediated by AAV-
Tau: attempted cell-cycle re-entry by the post-mitotic neurons,
and microgliosis. We are confident that these innovative models
will contribute considerably to unravel the molecular factors and
mechanistic details. Importantly, the ease whereby the AAV-
vectors and the models can be implemented widely in research-
projects on neurodegeneration is a further strong point of this
report. The wider distribution of the model, and its application for
analysis of mechanisms that will untangle tau-mediated neurode-
generation, is hoped to help meet the needs of patients suffering
from primary tauopathies or Alzheimer’s disease.
Materials and Methods
Generation of AAV-vectors
Human cDNA of wild-type and mutant P301L in the longest
tau isoform (Tau4R/2N) was used as described [23,24,41,45].
Human APP695 cDNA was used as wild-type or as triple mutant,
containing the Swedish, London and Austrian mutations (denoted
APP.SLA). The mutations were introduced by site directed
mutagenesis by standard techniques.
For cloning into the pAAV vectors  the cDNA was
amplified from the plasmids by polymerase chain reaction
(Phusion DNA polymerase; New England Biolabs), with primers
GCCCCGCCAGGAGTTCGAAGTGATGG – 39
AGGGAGGCAGACACCTCGTCAGC – 39.
and primers for APP
CATGCTGCCCGGTTTGGCACTGCTCCTGCT - 39
AAGAACTTGTAGGTTGG – 39
These primers contain suitable restriction sites for cloning into
the pAAV-constructs. Amplified Tau cDNA was inserted into
BamHI-NotI sites while amplified APP cDNA was inserted into
PLoS ONE | www.plosone.org 13October 2009 | Volume 4 | Issue 10 | e7280
NheI-NotI sites of pAAV. Integrity of the ITR was confirmed by
restriction with SmaI and by sequencing. All molecular cloning
procedures were performed in SURE bacteria (Stratagene, La
Jolla, California) to minimize recombination events.
Recombinant AAV vectors of hybrid serotype 1/2 were
produced essentially as described [45,46] to express either EGFP,
human Tau (WT or mutant P301L) or human APP (WT or SLA
triple mutant) under control of the human synapsin1 gene
promoter . Vectors were purified from cell lysates by iodixanol
step gradient centrifugation followed by heparin affinity chroma-
tography. Eluted virus was dialyzed against PBS and stored in
single use aliquots at 280uC. Vector genomes were titrated by
quantitative PCR and purity validated by SDS-PAGE and silver
Animals and stereotaxic injection
Adult wild-type FVB/N mice were used for most studies, except
for the cyclin D2 deficient mice  that were maintained in the
C57Bl background. Intracerebral injection of viral particles in the
left hemisphere of anesthetized mice (Nembutal, i.p. 0.7 mg/kg)
was performed stereotactically at coordinates posterior 1.94 mm,
lateral 1.4 mm, ventral 2.2 mm relative to bregma . Standard
injection was 2 ml of a viral suspension containing 10E8
transducing units (t.u.) using 10 ml glass syringes with a fixed
needle (Hamilton, Reno, Nevada). After injection at a rate of
0.5 ml/min, the needle was left in place for 5 min before
All animal experiments were performed by certified researchers
conforming to regional, national and European regulations
concerning animal welfare and animal experimentation, autho-
rized and supervised by the university animal welfare commission
(Ethische Commissie Dierenwelzijn, KULeuven). We formally
declare that we comply to the European FP7-Decision 1982/
2006/EC, Article 611, i.e. all research activities is carried out in
compliance with fundamental ethical principles and all experi-
ments are approved and overlooked by the respective Animal
At the indicated time periods post-injection (p.i.) mice were
anesthetized and perfused transcardiacally with ice-cold saline for
2 min. Brains were removed rapidly and fixed overnight in 4%
paraformaldehyde for subsequent immunohistochemical analysis
on free-floating coronal vibratome sections (40 mm). Primary
antibodies were either affinity-purified polyclonal antibodies or
mouse monoclonal antibodies that were biotinylated (Table S1).
Immune reactions were developed with streptavidin-HRP com-
plex for monoclonal antibodies or by a three-step method for
polyclonal antibodies [18,26]. Sections were counterstained with
hematoxylin, except after NeuN staining, dehydrated by passage
through a graded series of alcohol and xylol and mounted in
DepeX for microscopic analysis.
In separate experiments, the brain of injected mice was
processed for biochemistry. Following anesthesia and perfusion
as above, the brain was removed, dissected into hippocampus and
cortex on ice and snap-frozen in liquid nitrogen. Hippocampi were
homogenized in 6 volumes of homogenization buffer, i.e. 25 mM
Tris?HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
20 mM sodium fluoride, 1 nM okadaic acid, 0.2 mM sodium
vanadate, 1 mM phenylmethylsulfonyl fluoride, and complete
protease inhibitor cocktail and stored at 220uC until analysis. For
SDS-PAGE total homogenate was loaded onto 8% Tris-glycine
gels (Invitrogen, Merelbeke, Belgium). Following separation,
180 mA for 2.5 hr and membranes were blocked for 1 hr with
5% non-fat milk-powder in TBST before incubation with primary
antibodies (Table S1) at 4uC overnight, followed by incubation
with secondary antibody at RT for 1 hr. Immune-reactions were
visualized (ECL, GE-Health, Amersham, UK) and signals
captured on photographic film for scanning and densitometric
analysis (Lab-scan and ImageQuant TL, GE-Health, Amersham,
Compound X34 is a derivative of Congo Red binding to beta-
pleated proteins . Sections were washed in PBS and incubated
in 10 mM X34 in 40% ethanol, 50 mM Tris-HCl pH 9.5 for
10 min. Sections were rinsed in tap water, treated with 0.2%
NaOH in 80% ethanol, washed in tap water and mounted on
silanised glass-slides, dried at 40uC for 2 hr before mounting in
Depex. FluoroJadeB was used as described [24,28]. Sections were
mounted on gelatin-coated glass-slides and sequentially treated
with 1% NaOH, 80% ethanol (5 min), 70% ethanol (2 min) and
water (2 min). Sections were oxidized by 0.06% KMnO4 solution,
2 min, and washed in deionized water. After incubation with
FluoroJadeB (0.0004% in 0.1% acetic acid, 10 min) sections were
rinsed in deionized water, dried at 50uC (10 min) and cleaned in
xylol before mounting in Depex.
Anesthetized mice (Nembutal, i.p. 1.7 mg/kg) were perfused
transcardiacally with ice-cold saline (2 min) followed by Kar-
novsky fixation for 10 min. Brains were removed and stored in
Karnovsky solution at 4uC. Thick vibratome sections (300 mm)
were cut and incubated with 1% osmium tetroxide (60 min) before
dehydration through a graded series of ethanol and impregnation
with Agar100 resin. Ultrathin sections (80 nm) were stained with
uranyl acetate and lead citrate by standard procedures before
analysis by transmission electron microscopy (160 kV; JEM-2100;
Jeol, Tokyo, Japan).
injection of 10E8 t.u. AAV-EGFP in wild-type mice analyzed 12
weeks months p.i. by fluorescence microscopy for EGFP in
neurons in the hippocampal formation and in layers 5 and 6 of the
cortex (upper panel). Confocal microscopy of CA1 (lower left
panel) and CA2 (lower right panel) pyramidal neurons with EGFP
expressed in soma and apical dendrites. Scale bar 40 mm.
Found at: doi:10.1371/journal.pone.0007280.s001 (8.88 MB TIF)
AAV1/2-mediated expression of EGFP. Intracerebral
peptides in transfected N2a. Mouse neuroblastoma N2a cells
transiently transfected with pcDNA3 vectors encoding human
APP695 containing no, Swedish (K670M/N671L), London
(V717I) or Austrian (T714I) mutations, alone and in the
combinations indicated. Panels A:. cellular growth media were
collected after 48 hours of culture, immunoprecipitated with Mab
6E10 and protein G-agarose beads before Western blotting with
Mab WO2, after microwave heating of the filters, as recommend-
ed for the WO2 antibody. Panel B: Cell extracts analyzed directly
by Western blotting with WO2. Panel C: acid-urea SDS-PAGE to
separate amyloid peptides with synthetic amyloid peptides as
standards (Ab-mix). Quantification by densitometric scanning
using synthetic peptides as standards. Note the highest ratio Ab42/
Effect of APP-mutations on generation of amyloid
PLoS ONE | www.plosone.org14 October 2009 | Volume 4 | Issue 10 | e7280
Ab40 for APP.SLA triple mutant, which was used in the AAV-
Found at: doi:10.1371/journal.pone.0007280.s002 (1.40 MB TIF)
Tau.P301L injection. Compilation of 40 sections (each 40 mm)
spaced each about 3–4 sections apart throughout the brain of a
wildtype mouse injected with 10E8 t.u. AAV-TauP301L and
analyzed at 1.5 weeks p.i. for human protein tau by IHC with Mab
Found at: doi:10.1371/journal.pone.0007280.s003 (10.05 MB
Distribution ofhuman taufollowingAAV-
rebral injection of 10E8 t.u. of the indicated AAV-vectors in wild-
type mice analyzed 1.5 week (panel A) and 3 weeks p.i. (panel B)
with indicated antibodies in Western blotting and IHC, respec-
tively. Note some minor cross-reaction of the polyclonal antibody
against Tau.P301L (ref. 46) with wild-type Tau in IHC (right
Found at: doi:10.1371/journal.pone.0007280.s004 (10.17 MB
Comparison of wild-type and mutant Tau. Intrace-
is not neurotoxic. Intracerebral injection of 10E8 t.u. AAV-
Tau255 vector in wild-type mice (n=8) analyzed 3 weeks p.i. A:
representation of Tau.255 and Tau4R constructs and represen-
tative IHC for human Tau with HT7, AT180, AT8, AT270. Note
that Tau.255 lacks phosphorylation at AT8 and AT270 epitopes.
B: IHC for NeuN (upper panels) and histological staining with FJB
(lower panels) of injected (left) and noninjected (right) hemispheres.
C: IHC for MHCII for microgliosis (upper panel) and for GFAP
for astrogliosis (lower panel) in injected (left) and non-injected
(right) hemispheres. D: IHC with HT7 for human tau in AAV-
Tau.255 injected mice (left panel) compared to AAV-TauP301L
injected mice (right panel). Note the lack of neurodegeneration
inflicted by Tau255 (panels A, B, C, D) and the different
subcellular localization ofTau255
Tau.P301L (panel D, right). Scale bars: A, C 0.5 mm; B
0.5 mm (upper panel) and 50 mm (lower panel); D 40 mm
Found at: doi:10.1371/journal.pone.0007280.s005 (10.29 MB
Protein Tau255 lacking microtubuli binding domains
(panel D,left) versus
mediated neurodegeneration. Intracerebral injection of 10E8 t.u.
AAV-Tau.P301L vector in wild-type mice analyzed 3 weeks p.i. A.
IHC for active caspase-3 and quantification of apoptotic cells in
ipsilateral and contralateral hemispheres (mean, p,0.05, ANOVA
single factor). Note the distribution of presumed apoptotic neurons
Morphological and pathological aspects of Tau-
(arrowheads) in regions that do not correlate with degenerating
neurons. B. IHC for LC3 and Beclin as mediators of autophagy.
Scale bar 40 mm. C. Histological and ultra-structural analysis of
brain sections stained with toluidin-blue a–d: shrunken dark
neurons (a,b red arrows) absent at contralateral side (c, d). e:
vacuolization of cytoplasm (green arrow) and condensed chroma-
tin (blue arrow) f: indentations of nuclei (red arrowheads). Scale
bars: a–d 20 mm, e–f 2 mm. D. IHC with AT8 and AT270 reveal
sporadic tangles, spheroids and axonal dilatations.
Found at: doi:10.1371/journal.pone.0007280.s006 (9.31 MB TIF)
lipofucsin-like deposits in brain of mice injected with 10E8 t.u. of
AAV-Tau.P301L analyzed at different periods p.i. as indicated,
with enlarged view at higher magnification (utmost right panel).
The efficient elimination with a proprietary reagent is illustrated
(panel marked ‘‘Autofluo-eliminator’’).
Found at: doi:10.1371/journal.pone.0007280.s007 (2.21 MB TIF)
Lipofucsin in degenerating neurons. Autofluorescent
rebral injection of 10E8 t.u. of AAV-TauP301L in wild-type mice
3 weeks p.i. analyzed by IHC for tubulin, actin and synaptophysin
in injected (left panels) and non-injected (right panels) hemi-
Found at: doi:10.1371/journal.pone.0007280.s008 (9.50 MB TIF)
Defects in AAV-Tau.P301L injected mice. Intrace-
Found at: doi:10.1371/journal.pone.0007280.s009 (0.08 MB
Antibodies used in this study
Found at: doi:10.1371/journal.pone.0007280.s010 (0.04 MB
Markers tested by IHC on AAV-Tau.P301L mice.
We sincerely thank collaborators and scientists for technical assistance,
advice, materials and scientific or moral support, particularly Monika
Zebski for assistance with virus production, Peter Davies (New York) and
Eugeen Van Mechelen (Innogenetics, Gent) for generous gifts of
monoclonal antibodies, and Annick Vogels for assistance with administra-
tion. TJ received an EU-Marie Curie doctoral fellowship (MEST-CT-
2005-020589 - EURON PhD School for Neurosciences). IDW is senior
scientist at FWO-Vlaanderen.
Conceived and designed the experiments: TJ ID FVL. Performed the
experiments: TJ ID BL SC AT DD PB HD SK. Analyzed the data: TJ ID
BL SC AT DD PB RKF LK SK FVL. Contributed reagents/materials/
analysis tools: RKF LK SK FVL. Wrote the paper: TJ SK FVL.
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PLoS ONE | www.plosone.org 15October 2009 | Volume 4 | Issue 10 | e7280