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Characteristics of pro-aggregant Tau transgenic mice

Inducible mice with constant expression of full-length pro-aggregant Tau^?K or repeat domain pro-aggregant TauRD^?K develop a progressive neuropathology including prominent cognitive deficits. Importantly,
cognitive deficits as well as synaptic impairments recover after switching off the
expression of human Tau 14],15], demonstrating that Tau-induced pathology can be reversed in principle. These studies
provide the rationale for treatment of pro-aggregant mice using Tau-directed drugs.

From 3 months (mo) of age onwards (or after 3mo of Tau expression), repeat domain
TauRD^?K mice show a pronounced neuropathology, especially in terms of Tau aggregation and
neuronal death. By comparison, the brain pathology is much less pronounced in full-length
Tau^?K mice. These mice develop a pre-tangle pathology indicated by the conformation-dependent
antibody MC1 49] but lack a silver-positive NFT pathology and neuronal loss in the hippocampus. The
difference is consistent with the fact that TauRD^?K lacks the flanking regions (Additional file 1: Figure S1), which leads to a higher ?-propensity, causing efficient aggregation
and a stronger neurotoxicity than full-length Tau^?K at comparable expression levels. Note that TauRD^?K cannot react with antibody MC1, since it lacks part of the epitope; the same holds
for other diagnostic antibodies (e.g. AT8) with epitopes outside the repeat domain.
Therefore, the appearance of MC1 or AT8 reactivity in TauRD^?K mice originates from endogenous mouse Tau, which has been transformed to a pathological
state, triggered by exogenous TauRD^?K. By contrast, the epitope of antibody 12E8 (pS262?+?pS356) lies within the repeats
and is present in all Tau variants.

Due to the specific design of the transgenic DNA the expression of human Tau^?K and TauRD^?K can be deduced from luciferase activity by bioluminescence imaging (BLI) in living
mice. This opens the possibility to preselect animals with comparable Tau expression
prior to experiments in order to reduce inter-individual variations, which is important
since Tau levels influence the severity of pathology 50].

Pharmacokinetic properties of MB in mice

To check bioavailability and lifetime of MB in mice, MB levels in plasma and brain
were measured after intravenous (i.v.) application of 30 mg/kg MB or oral application
of 45 mg/kg MB (Additional file 2: Figure S2) in wild-type C57BL/6 mice (n?=?3 per time point). MB levels were evaluated
by quantification of tetramethylthioninium-ion (TMT-ion) concentrations using liquid
chromatography – mass spectrometry (LC-MS/MS). TMT-ion equals MB without chloride
and 3*H₂O.

After i.v. administration, peak concentrations in plasma and brain were observed at
5 to 15 min post application and pharmacokinetic analyses over 24 h revealed MB half-lives
of t_1/2?=?4.4 h in plasma and t_1/2?=?3.0 h in brain (Additional file 2: Figure S2a). Intravenous application of MB led to a reduced general condition and
apathy of the mice, which may point to an acute toxic effect of the bolus. In contrast,
oral administration of MB was well-tolerated and led to peak concentrations in plasma
and brain within the first 2 hours post application. Pharmacokinetic analyses over
24 h exhibited MB half-lives of t_1/2?=?4.7 h in plasma and t_1/2?=?30.6 h in brain. The oral bioavailability was determined at ~18% (Additional file
2: Figure S2b). Furthermore, the results show a concentration of MB inside the brain,
with ~30-fold and ~60-fold higher MB levels in brain tissue compared to plasma at
24 h post i.v. or oral application, respectively.

MB application strategies

The goal of the study was to optimize MB treatment conditions in order to get the
maximum beneficial effect on learning and memory performance of Tau transgenic mice.
To this end we followed different treatment strategies (Figure 1). The major difference between the various treatment protocols is the initial time
point of intervention, namely before or after onset of cognitive impairments, which
start typically at ~12mo of age in Tau^?K mice and at ~10mo of age in TauRD^?K mice. The key question was, how much Tau pathology can be allowed while still preventing
cognitive decline by MB treatment.

Figure 1. Oral MB treatment strategies of mice. (a) Full-lengthTau^?K mice received MB for 14.5mo starting at 1.5mo of age, before onset of Tau pathological
changes and cognitive decline (protocol 1, 1.5 mo Tau pathology, 14.5 mo preventive
MB, 20 mg/kg), for 6mo starting at 9mo of age, shortly before the onset of cognitive
impairments (protocol 2, 9 mo Tau pathology, 6 mo preventive MB, 20 mg/kg) and for
3mo starting at 15mo of age, at a time point, when learning and memory deficits are
already present (protocol 3, 15 mo Tau pathology, 3 mo therapeutic MB, 20 mg/kg).
The constant expression of Tau^?K throughout the entire life-span in the absence of doxycycline is depicted as red
bar. The onset of progressive cognitive failure starting ~12 months of age is represented
by the dotted arrow. (b) TauRD^?K received MB for 14.5mo starting at the age of 1.5mo, before onset of Tau pathology
and cognitive impairment (protocol 1, 1.5 mo Tau pathology, 14.5 mo preventive MB,
20 mg/kg) and for 3mo starting at 15mo of age, after the onset of cognitive decline
(protocol 2, 15mo Tau pathology, 3mo therapeutic MB, 20 mg/kg). An excessive MB dose
of 40 mg/kg was applied for 3mo at the age of 12-15mo (protocol 3, excessive MB, 40 mg/kg).
The constant expression of TauRD^?K throughout life-span in the absence of doxycycline is depicted as orange bar. The
onset of cognitive deficits starting ~10mo of age is represented by the dotted arrow.
Periods and initiation of MB treatment are indicated by blue bars and red circles
on the time axis, including period of behavioral testing during the final month (green
box). The daily MB dose (20 or 40 mg/kg) for each treatment protocol is given in brackets.
MB was administered via the drinking water. MB: methylene blue; mo: months; B: behavior
tests; expr.: expression.

MB application strategies for pro-aggregant full-length Tau^?K mice

In preventive treatment protocol (1), MB application was initiated at 1.5mo of age
and continued for 14.5mo beyond the anticipated onset of cognitive deficits typically
around ~12mo of age in this mouse strain. The aim was to target Tau-induced changes
very early on, before any neuropathological and cognitive changes occur (Figure 1a, preventive MB for 14.5mo).

In preventive treatment protocol (2), MB administration was initiated at 9mo of age,
a time point ~3mo before the expected onset of cognitive impairment but with presence
of early Tau^?K-induced pathological changes such as conformational change, missorting and phosphorylation
of Tau (Figure 1a, preventive MB for 6mo).

Finally, therapeutic treatment protocol (3) aimed to test whether therapeutic intervention
by MB after onset of cognitive decline has the potential to rescue the existing neuropathology
and phenotype (Figure 1a, therapeutic MB for 3mo). Therefore MB application was initiated at 15mo of age
(~3mo after onset of cognitive impairments) and continued for 3mo. In this respect,
treatment protocol (3) was closely analogous to the aforementioned Tau switch-off
experiments, which resulted in a complete rescue of functional deficits 14],15]. All Tau^?K mice were treated using an oral daily dose of 20 mg/kg MB, which was administered
via the drinking water.

MB application strategies for pro-aggregant repeat domain TauRD^?K mice

Similar to Tau^?K animals, TauRD^?K mice received MB (20 mg/kg) for 14.5mo, starting at 1.5mo of age, long before the
appearance of Tau pathology and cognitive impairments (Figure 1b, protocol 1, preventive MB for 14.5mo).

In therapeutic treatment protocol (2), MB (20 mg/kg) application started at 15mo of
age and continued for 3mo (Figure 1b, therapeutic MB for 3mo). At the age of 15mo, TauRD^?K mice exhibit strong neuropathological changes as described above and show severe
learning and memory deficits.

Finally, TauRD^?K mice received an excessive MB dose of 40 mg/kg for 3mo at the age of 12-15mo to further
evaluate the effect of MB on TauRD^?K aggregation (Figure 1b, therapeutic protocol (3), excessive MB for 3mo).

After MB treatment, mice were tested for their general motor and exploration behavior
using an open field test and for their spatial reference learning and memory performance
in a Morris water maze (MWM) test. In the open field test, no differences between
MB-treated and untreated pro-aggregant Tau mice or wild-type (WT) mice were observed
for average velocity and distance covered throughout all experimental setups. In addition
swimming speed did not differ between these groups in the MWM test. Thus, we exclude
that motor deficiencies of pro-aggregant Tau mice account for the differences detected
in behavioral readouts (see below). This issue is of concern since some published
Tau-transgenic mice developed motor deficits caused by transgene expression in the
spinal cord due to Thy-1.2 or PrP promoters 11],13], which is avoided here by the use of the CaMKII? promoter.

MB treatment of pro-aggregant full-length Tau^?K mice

Preventive treatment starting 11mo before cognitive decline

In this protocol, MB treatment started shortly after birth and long before the expected
onset of cognitive decline (~12mo), and continued beyond this for a total of 14.5mo
(including the final month of behavioral testing, Figure 1a).

Regarding open field activity, the 14.5mo MB treatment resulted in significant group
differences, such that untreated Tau^?K mice were less active than WT animals or MB-treated Tau^?K mice (Figure 2a, p?=?0.004, Additional file 3: Figure S3). In contrast, no group differences were observed for anxiety-related
parameters (time in center, distance to wall), suggesting that preventive MB treatment
did not have anxiolytic effects (Additional file 4: Figure S4).

Figure 2. Exploration behavior of MB-treated Tau^?K mice. Total activity (%) within 15 min is analyzed by an open field test after various
MB treatment periods. (a) Preventive MB treatment for (a) 14.5mo and for (b) 6mo results in preservation of exploration behavior of Tau^?K mice similar to wild-type (WT) animals, whereas untreated Tau^?K mice show a significantly reduced activity. By contrast no group-differences are
observed after therapeutic MB treatment for 3mo (c). Bars represent mean values?±?SEM. Statistics: one-way analysis of variances with
post-hoc Newman-Keuls multiple comparisons test. Asterisks indicate significant differences
between groups; *: p??0.05; **: p??0.01; mo: months.

During MWM acquisition, WT mice and MB-treated pro-aggregant full-length Tau^?K mice showed a superior learning performance over untreated Tau^?K mice (WT vs. Tau^?K – MB: p?=?0.025). Importantly, MB-treated Tau^?K mice demonstrated a similar learning efficiency than WT animals (WT vs. Tau^?K?+?14.5mo MB: p?=?0.532) and performed significantly better than untreated Tau^?K at day 4 and day 5 (Tau^?K – MB vs. Tau^?K?+?14.5mo MB; day 4: p?=?0.025; day 5: p?=?0.018) (Figure 3a). In probe trials, MB-treated Tau^?K mice exhibited a higher preference for the target quadrant and a more precise localization
of the target platform position in comparison to untreated Tau^?K mice (Additional file 5: Figure S5). The results indicate a preservation of cognitive abilities upon preventive
MB treatment initiated before the onset of Tau pathological changes and cognitive
decline. Interestingly, WT mice receiving MB for 14.5mo, showed a slightly superior
learning performance over untreated WT mice (Figure 3a), pointing towards an additional beneficial effect of MB on brain metabolism.

Figure 3. Spatial learning of MB-treated Tau^?K mice. Morris water maze (MWM) acquisition shows spatial learning abilities as indicated
by path lengths (cm) of mice after MB application using different preventive and therapeutic
treatment paradigms. Significant group differences are observed between untreated
Tau^?K mice and WT animals throughout all experiments, demonstrating impaired learning abilities
upon expression of pro-aggregant Tau (a-c). By contrast Tau^?K mice treated with MB for 14.5mo (a) and 6mo (b) behave comparable to controls indicating preservation of cognitive functions. Short-term
MB treatment for 3mo has no beneficial impact on the learning performance of Tau^?K mice (c). Data shows mean path length?±?SEM. Statistics: two-way repeated measure analysis
of variances with post hoc Fishers LSD multiple comparisons test. Asterisks indicate
differences between MB-treated and untreated Tau^?K mice; *: p??0.05; n: number of mice.

MB application for 14.5mo (started at 1.5mo of age) reduced insoluble Tau^?K and endogenous mouse Tau, compared with untreated Tau^?K mice (Figure 4). Similarly, a decrease of conformationally-changed Tau (epitope MC1, 5-15?+?312-322,
Figure 5, Additional file 6: Figure S6) and a reduction of phosphorylated Tau (epitope AT180, pThr231?+?pSer235
and epitope PHF1, pSer396?+?pSer404) was observed after 14.5mo MB treatment (Figure 5, Figure 6, Additional file 6: Figure S6). By contrast, Tau phosphorylated at the KXGS motifs inside the repeat
domain (epitope 12E8, pSer262?+?pSer356) was increased in MB-treated animals (Figure 6). Note that Tau phosphorylation at the KXGS motifs causes detachment of Tau from
microtubules, but also protects Tau against aggregation 51].

Figure 4. MB treatment reduces detergent insoluble Tau^?K and mouse Tau. Sarcosyl-extraction of soluble and insoluble Tau from cortex tissue.
(a) Protein levels of soluble and insoluble Tau species in untreated Tau^?K mice compared to Tau^?K mice receiving MB for 14.5mo, 6mo and 3mo detected by the pan-Tau antibody K9JA and
the human Tau specific antibody TauY9. MB administration reduces levels of detergent-insoluble
human and mouse Tau. (b) Quantification of soluble Tau levels in cortex lysates shows a minor but non-significant
decrease of soluble Tau species upon MB treatment. (c) Quantification of insoluble Tau levels in cortex lysates demonstrates a clear decrease
of insoluble human and mouse Tau in MB-treated mice. Bars represent mean protein densities
(%)?±?SEM; statistics: one-way analysis of variances with post-hoc Newman-Keuls multiple
comparisons test. Asterisks indicate significant differences in comparison to untreated
Tau^?K mice; **: p??0.01; Ab: antibody; mTau: mouse Tau; n, number of samples.

Figure 5. Histological analysis of conformationally changed and phosphorylated Tau in MB-treated
mice. (a-e) MC1 immunoreactivity (epitope 5-15?+?312-322) indicates a pathological Tau conformation.
MC1 positive neurons are prominent in somatosensory cortex (SSCx) of untreated Tau^?K mice with missorting of Tau to cell soma (arrows) and apical dendrites (arrowheads).
In contrast MB treatment, especially preventive MB treatment for 14.5mo, clearly reduces
MC1 immunoreactivity in Tau^?K mice. (f-j) Histological analysis of phosphorylated Tau using the AT180 antibody (dual phosphorylation
epitope pThr231?+?pSer235). Untreated Tau^?K mice display a massive mislocalization of phosphorylated Tau to cell soma (arrows)
and apical dendrites (arrowheads) of SSCx neurons, whereas MB treatment diminishes
the extent of AT180 phosphorylation. Scale bar: 50 ?m.

Figure 6. MB application alters levels of phosphorylated Tau. (a) Levels of total Tau (pan-Tau antibody K9JA), PHF1 phosphorylated (epitope pSer396/pSer404)
and 12E8 (epitope pSer262/pSer356) phosphorylated Tau in cortex homogenates of MB-treated
and untreated Tau^?K mice. MB administration for 14.5mo, 6mo and 3mo results in a decrease of PHF1 phospho-Tau
as compared to untreated Tau^?K mice. By contrast an increase of 12E8 phospho-Tau inside the repeat domain of Tau
is observed after MB treatment, indicating detachment from microtubules. (b) Determination of relative protein densities by quantification of (a). Levels of phosphorylated Tau were normalized to total Tau levels. Bars represent
mean values?±?SEM. Statistics: one-way analysis of variances with post-hoc Newman-Keuls
multiple comparisons test. Asterisks indicate significant differences in comparison
to untreated Tau^?K mice; *: p??0.05; n, number of samples.

Previously, we described a close relationship between expression of pro-aggregant
Tau, synaptic failure inside the hippocampus formation and cognitive decline 14],15]. While untreated pro-aggregent Tau^?K mice showed a consistent reduction in pre- and postsynaptic proteins (i.e. synapsin
1, synaptophysin, PSD95), MB treatment for 14.5mo preserved pre- and postsynaptic
protein levels (Figure 7), suggesting a protective effect of MB on the synaptic integrity which likely contributes
to the preservation of cognition.

Figure 7. Recovery of synaptic marker proteins in hippocampus of MB-treated mice. (a) Levels of pre- and post-synaptic proteins synaptophysin, synapsin 1 and postsynaptic
density 95 (PSD95) in hippocampus lysates of age-matched untreated and MB-treated
Tau^?K mice. ?-actin serves as loading control. (b) Determination of relative protein densities by quantification of (a). Untreated Tau^?K mice show a constant decrease of synapsin 1, synaptophysin and PSD95 levels in comparison
to WT, due to constant expression of pro-aggregant Tau^?K. In contrast a recovery of pre- and postsynaptic markers is observed after preventive
MB application for 14.5mo and 6mo, whereas therapeutic MB treatment for 3mo causes
a partial rescue of post- but not of presynaptic markers. Protein levels were normalized
to ?-actin. Bars represent mean values?±?SEM. Statistics: one-way analysis of variances
with post-hoc Newman-Keuls multiple comparisons test. Asterisks indicate significant
differences in comparison to untreated Tau^?K mice; *: p??0.05; **: p??0.01; n, number of samples.

Clearance of proteins via autophagy is a major pathway to maintain neuronal homeostasis
and health. Preventive MB treatment of Tau^?K mice for 14.5mo increased levels of beclin, heat shock cognate protein 70 (HSC70)
and lysosome-associated membrane protein 2a (Lamp2a) (Figure 8), indicating an upregulation of autophagy. While beclin is involved in the initiation
of the autophagosome formation during macroautophagy, HSC70 (a constitutively expressed
molecular chaperone) and Lamp2a (lysosomal receptor) play a role in chaperone-mediated
autophagy (CMA) of Tau (Wang et al., 2009). Another prominent pathway for protein
degradation is the ubiquitin-proteasome system. As judged by an increased level of
PSMD13 (regulatory subunit of the 26S proteasome), 14.5mo MB treatment enhanced proteasome
function in comparison to WT and untreated Tau^?K mice (Figure 8). Taken together, the tendency of MB to preserve or enhance protein degradation may
counteract the continuous accumulation of toxic Tau species and contribute to maintenance
of protein homeostasis.

Figure 8. Preventive MB administration increases protein degradation. (a) Levels of autophagy- (beclin, HSC70), lysosome- (lamp2a), and proteasome-related
(PSMD13) proteins in hippocampus lysates of age-matched untreated Tau^?K mice compared to MB-treated Tau^?K mice for 14.5mo, 6mo and 3mo. ?-actin serves as loading control. (b) Quantification of (a). While both preventive MB treatment strategies (14.5mo and 6mo MB) increase levels
of beclin, HSC70, Lamp2a and PSMD13, therapeutic MB treatment for 3mo leads to a partial
increase of beclin and Lamp2a whereas levels of HSC70 and PSMD13 remain unaffected
in comparison to untreated Tau^?K mice. Protein levels were normalized to ?-actin. Bars indicate mean values?±?SEM.
Statistics: one-way analysis of variances with post-hoc Newman-Keuls multiple comparisons
test. Asterisks indicate significant differences in comparison to untreated Tau^?K mice; *: p??0.05; **: p??0.01; ***: p??0.001; n, number of samples.

MB’s role as electron carrier and thus redox-cycling compound is widely discussed
in the literature 52]. However, we did not detect obvious differences between MB-treated and untreated
Tau^?K mice regarding mitochondria distribution and proteins of the electron transport chain
(Additional file 7: Figure S7), indicating that MB did not alter this route of neuronal energy production.

In summary, if preventive treatment was started at an early age the cognitive abilities
of Tau^?K mice were essentially preserved, well beyond the expected onset of decline and in
spite of the continued expression of the toxic mutant full-length Tau^?K.

Preventive treatment starting 3mo before cognitive decline

Preventive MB administration (protocol 2, started ~3mo before onset of cognitive decline
and continued for 6mo) showed an increase of open field activity of Tau^?K mice in comparison to the reduced activity of untreated Tau^?K mice (Figure 2b, Additional file 3: Figure S3). No group differences concerning anxiety related parameters were observed
(Additional file 4: Figure S4). In MWM acquisition, both untreated Tau^?K and 6mo MB-treated Tau^?K mice performed less well than WT mice (WT vs. Tau^?K – MB: p?=?0.017; WT vs. Tau^?K?+?6mo MB: p?=?0.036, Figure 3b). However, Tau^?K mice with 6mo MB application showed a better learning performance compared to untreated
Tau^?K on day 4 (Tau^?K – MB vs. Tau^?K?+?6mo MB: p?=?0.042, Figure 3b). In addition they were able to localize the target quadrant and platform position
more precisely than untreated Tau^?K animals (Additional file 5: Figure S5), implying a minor beneficial effect of MB on cognition.

Neuropathological analysis revealed a reduction of insoluble Tau^?K and mouse Tau (Figure 4), a decrease of conformationally-changed Tau (MC1) and phosphorylated Tau (AT180,
PHF1) after 6mo of MB administration (Figure 5, Figure 6, Additional file 6: Figure S6). In addition, synaptic and autophagy marker proteins as well as the proteasome
marker PSMD13 were increased after 6mo MB (Figure 7, Figure 8) but mitochondria remained unaffected (Additional file 7: Figure S7).

Thus, if preventive treatment was started at adult age with progressive tau pathology
but still before the anticipated onset of cognitive decline, cognitive abilities were
protected to a certain extent, in spite of the continued expression of toxic Tau^?K.

Therapeutic treatment starting 3mo after cognitive decline

Finally, we tested the therapeutic potential of MB to reverse cognitive deficits ~3mo
after onset (Figure 1a, protocol 3). MB treatment for 3mo did not enhance open field activity of Tau^?K mice. Thus, treated Tau^?K mice behaved similarly to untreated Tau^?K animals (Figure 2c). WT mice showed a slight but non-significant increase in open field activity compared
to transgenic animals, which was most pronounced within the first 5 minutes of the
experiment (Additional file 3: Figure S3) and probably due to an advanced age of the mice during testing. Anxiety-related
parameters were unaffected (Additional file 4: Figure S4).

An MWM test indicated a severe learning impairment of 3mo MB-treated Tau^?K mice similar to untreated Tau^?K mice and in contrast to WT animals, which improved consistently on each acquisition
day (WT vs. Tau^?K – MB: p?=?0.001; WT vs. Tau^?K?+?3mo MB: p?=?0.011, Figure 3c). In addition, MB-treated mice did not show any preference for the target quadrant
(Additional file 5: Figure S5), underlining the persistence of cognitive deficits after 3mo MB.

Nevertheless, in spite of the failure in improving cognitive defects, MB treatment
was still able to reduce sarcosyl-insoluble Tau, although to a minor extent in case
of Tau^?K compared to preventive MB treatment paradigms (Figure 4). In addition, conformationally changed Tau (MC1) and phosphorylated Tau (AT180,
PHF1) were clearly reduced after 3mo MB treatment (Figure 5, Figure 6, Additional file 6: Figure S6).

By contrast, the 3mo MB application was not able to reverse the decline of presynaptic
markers synaptophysin and synapsin 1 and the protective effect on postsynaptic PSD95
was less pronounced than in preventive MB treatment protocols (Figure 7). These results point towards sustained synaptic malfunction, which may underlie
cognitive impairment independently of the reversal of biochemical Tau parameters.
In addition, 3mo MB treatment only partly affected protein degradation systems. Whereas
an increase in beclin and Lamp2a levels was detected, levels of HSC70 and PSMD13 were
comparable to untreated age-matched Tau^?K mice (Figure 8). As before, no effect on mitochondria was observed (Additional file 7: Figure S7).

Preventive and therapeutic MB treatments of TauRD^?K mice

In contrast to full-length Tau^?K mice, animals expressing the repeat domain TauRD^?K develop a pronounced brain pathology in terms of Tau aggregation (neurofibrillary
tangles) and neuronal loss and exhibit an earlier onset of cognitive decline ~10mo
of age. This is consistent with the fast aggregation of the protein in vitro caused
by the higher propensity for ?-structure, and the absence of the N-and C-terminal
flanking domains (Additional file 1: Figure S1). As in the case of full-length Tau^?K, we tested different treatment protocols for TauRD^?K mice (Figure 1b).

We performed preventive MB treatment for 14.5 mo, starting at 1.5mo of age, long before
onset of cognitive decline (Figure 1b, protocol 1), as well as therapeutic treatment for 3 mo, starting 5mo after onset
of cognitive decline (at age 15 mo) (Figure 1b, protocol 2). Both treatments failed to decrease the level of insoluble Tau (exogenous
and endogenous, Additional file 8: Figure S8) and did not protect synapses or induce protein degradation via autophagy
(data not shown).

Surprisingly, even long-term preventive treatment of TauRD^?K mice (14.5mo) failed to reverse Tau-induced cognitive deficits (Figure 9). MB-treated TauRD^?K mice did not learn the location of the hidden platform, similar to untreated TauRD^?K animals (Figure 9a), and did not show a significant preference for the target quadrant in subsequent
probe trials during MWM (Figure 9b). Thus, 20 mg/kg/day MB was not sufficient to suppress Tau aggregation and subsequent
neurotoxic processes initiated by TauRD^?K.

Figure 9. Preventive MB treatment does not rescue cognitive decline of TauRD^?K mice. MWM test shows cognitive abilities of TauRD^?K mice after preventive MB application for 14.5mo. (a) MB-treated as well as untreated TauRD^?K animals show impaired learning abilities similar to untreated TauRD^?K littermates, as indicated by increased path lengths to reach the hidden platform
in MWM acquisition in comparison to WT. Data shows mean path length?±?SEM. (b) Preventive MB treatment for 14.5mo does not result in a higher preference of the
target quadrant as compared to untreated TauRD^?K mice and controls. Bars represent mean values?±?SEM. Statistics: two-tailed one sample
t-test against chance level of 25%; *: p??0.05; **: p??0.01; n?=?number of animals;
T: target quadrant; R: right quadrant; O: opposite quadrant; L: left quadrant; LTPT:
long-term probe trial.

To investigate whether higher doses of MB would influence TauRD^?K pathology, the mice were treated with an excessive dose of MB (40 mg/kg/day MB) for
3mo (Figure 1b, protocol 3, ~12-15mo of age, after cognitive decline). This actually resulted in
an increase of aggregated Tau species in the hippocampus of TauRD^?K mice as detected by sarcosyl extraction and Gallyas silver staining (Figure 10). However, phosphorylated Tau was unaltered and autophagy partly reduced (data not
shown). This detrimental effect of high doses MB was further confirmed by cell culture
experiments. Inducible N2a cells expressing pro-aggregant TauRD^?K53] accumulated aggregated Tau species in response to high concentrations of MB (25-100nM)
in a dose-dependent manner (Additional file 9: Figure S9). Especially MB concentrations 100nM caused a massive loss of N2a cells,
indicating a toxic gain of function in cells.

Figure 10. High MB-doses increase Tau aggregation. (a) Sarcosyl-extraction of insoluble Tau from cortex tissue of TauRD^?K mice. MB application using a daily dose of 40 mg/kg for 3mo results in an increase
of sarcosyl-insoluble human TauRD^?K and endogenous mouse Tau (antibody K9JA). (b) Quantification of (a) shows a ~2-3 fold increase of insoluble Tau after high-dose MB treatment for 3mo.
Bars represent mean values?±?SEM. Statistics: two-tailed t-test; *: p??0.05; **:
p??0.01; n, number of samples. (c, d) Gallyas silver staining of Tau aggregates shows an increase of tangle-positive cells
inside the hippocampus formation of MB-treated TauRD^?K mice (40 mg/kg for 3mo). Scale bar: 200 ?m. Boxed areas indicate close-ups presented
in (e-j). Higher magnification of dentate gyrus (DG), CA3 and CA1 hippocampal areas of untreated
TauRD^?K mice (e-g) in comparison to corresponding areas of MB-treated TauRD^?K mice (h-j). CA3: region III cornus ammonis; CA1: region I cornus ammonis. Scale bar: 40 ?m.