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Aggregation of highly phosphorylated tau is a hallmark of Alzheimer's disease and other tauopathies. Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase glycogen-synthase-kinase-3beta (GSK-3β). Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure. This may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others. Whether these are inert or actively protective remains to be established. Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.
GSK-3β inhibition rescued microtubule number in hTau0N3RDrosophila, but increased total hTau protein and caused formation of electron-dense granules a-l) Electron micrographs of transverse sections of peripheral nerves in L3 Drosophila (scale bar 200 nm).: In hTau-expressing (elavC155-Gal4/Y; UAS-hTau0N3R/+) animals treated with either 20 mM LiCl (hTau-Li, a–c) or with 20 μM AR-A01448 (hTau-AR, d-f), some axons exhibited small electron-dense globular structures of approximately 20–50 nm in size (black arrows). These structures were extremely rare in control larvae expressing elavC155-Gal4 driver alone (WT, g–i) or untreated hTau0N3R-expressing neurons (j–l). In WT larvae the axon profiles showed numerous regularly-spaced, correctly-aligned transverse microtubule profiles (black arrowheads in g-i; 8.1 ± 0.2/axon profile). As we have previously shown29, in hTau0N3R-expressing axons the microtubules were dramatically disrupted, with fewer correctly-aligned transverse microtubule profiles (black arrowheads in j-l; 5.3 ± 0.3/axon profile), and evidence of disorganised microtubules in the same axon profiles (white arrowheads in j–l). Indeed, approximately 30% of hTau0N3R-expressing axons displayed no visible microtubule profiles (Figure S1). In hTau0N3R-expressing larvae fed with Li (a–c) or AR (d–f), there were significantly more correctly-aligned transverse microtubule profiles (black arrowheads in a-l; 9.2 ± 0.3/axon) and fewer misaligned microtubules. Microtubule numbers per axon are quantified in m (**p < 0.01, unpaired Students t test). Representative Western blots of hTau0N3R-expressing fly head lysates showed that tau phosphorylation was decreased (at T231/S235 detected by AT180) whilst total tau levels were increased by 40–60% (o–r) by 20 mM lithium treatment (hTau-Li, o), 20 μM AR-A01448 treatment (hTau-AR, p), co-expression of dominant negative shaggy(hTau;sggDN, q) {elavC155-Gal4/Y; UAS- hTau0N3R/ + ; UAS-sggDN/ + }. Conversely, total tau levels were decreased by approximately 50% by co-expression of constitutively active shaggy (hTau;sgg*, r) {elavC155-Gal4/Y; UAS- hTau0N3R/ + ; UAS-sgg*/ + }. This is quantified in s (error bars are standard error of mean; *p < 0.05 by Students t-test).
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Insoluble tau oligomers can be purified from hTau0N3R-expressing Drosophila and were increased dramatically after treatment with GSK-3β inhibitors.: TEM of immuno-gold labelling for anti-hTau of S3 insoluble fractions show granular tau oligomers comprised of hTau in all conditions expressing hTau {elavC155-Gal4/Y; UAS-hTau0N3R/ + }: (a) hTau, (b) hTau-Li, (c) hTau-AR. No such structures were detected in controls: d) wild-type. (See also Supplementary , for additional controls of no sample labelled with anti h-Tau; and hTau labelled for an irrelevant rabbit polyclonal antibody, anti-v-glut). Scale bar in a (applicable to a–f) = 100 nm. (g–h); hundreds of such structures were observed in preparations from 18 pooled flies) Atomic Force Microscopy of material immuno-precipiated from fly head lysates using anti-hTau antibody shows the appearance of numerous granular tau oligomers present after LiCl treatment (f) but only very sparse in untreated hTau0N3R flies (e). Oligomer sizes were determined by cross-sectional height analysis of individual oligomers. The heights of the oligomers ranged between 15 and 30 nm, with a mean height of 17.07 nm (SD = 8.86). The widths of the majority of oligomers are between 20 and 40 nm and the average width was calculated to be 20.6 nm (SD = 11.4). A minority of oligomers have a larger width than 30 nm but the height of the oligomers was consistently 30 nm or below. Scale bar in i = 1 μm. (g–i)’) Immuno-gold labeling for hTau in situ in sections of peripheral nerves from hTau-Li flies demonstrates labeled granular tau oligomers (arrows) within axons. Examples are given at lower magnifications (g–i) in which axonal profiles are clearer, and at higher magnifications (g’–i’) in which GTOs can be seen more clearly. Scale bars = 100 nm.
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1
Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
www.nature.com/scientificreports
Rescue from tau-induced neuronal
dysfunction produces insoluble tau
oligomers
Catherine M. Cowan1, Shmma Quraishe1, Sarah Hands1, Megan Sealey1, Sumeet Mahajan2,
Douglas W. Allan3 & Amritpal Mudher1
Aggregation of highly phosphorylated tau is a hallmark of Alzheimer’s disease and other tauopathies.
Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require
large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small
tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble
tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows
that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with
increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase
glycogen-synthase-kinase-3beta (GSK-3β). Moreover, contrary to common belief, these tau oligomers
were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure. This
may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-
toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others.
Whether these are inert or actively protective remains to be established. Nevertheless, this has
wide implications for emerging therapeutic strategies such as those that target dissolution of tau
oligomers as they may be ineective or even counterproductive unless they act on the relevant toxic
oligomeric tau species.
All tauopathies, including Alzheimer’s disease (AD), are characterized by the accumulation of insoluble,
hyper-phosphorylated aggregates of the microtubule-associated protein tau. Both tau aggregation and
hyper-phosphorylation are implicated in tau-mediated dysfunction and toxicity1. Hence, research focuses
on developing therapies to inhibit aggregation or hyper-phosphorylation1,2.
Tau can be phosphorylated at a large number of sites, and many of these sites are abnor-
mally hyper-phosphorylated in AD3. Various serine-threonine kinases have been implicated in tau
hyper-phosphorylation including glycogen synthase kinase 3β (GSK-3β )4–6. We have previously shown
that soluble tau that is highly phosphorylated at GSK-3β sites causes neuronal dysfunction by desta-
bilizing cytoskeletal integrity, impairing axonal transport and disrupting synaptic function7–9. Others
have similarly reported phospho-tau mediated neuronal dysfunction in various animal models of
tauopathy10–12. As well as causing dysfunction, soluble hyper-phosphorylated tau has been shown to
be directly toxic triggering degeneration and neuronal loss13–16. Some studies have also reported that
hypo-phosphorylation of tau may also be toxic17, perhaps due to dysregulation of microtubules, which
will have the same eect as hyper-phosphorylated tau by impacting axonal transport and synaptic func-
tion18. Overall, the causal pathogenic role played by soluble hyper-phosphorylated tau is well docu-
mented by many studies and thus largely undisputed.
In contrast, the case for tau aggregates as a primary toxic species is less clear. Indeed the toxicity of
aggregates has been challenged for other aggregating proteins in other proteinopathies as well19–22. In
1Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK. 2Institute of Life
Sciences and Department of Chemistry, University of Southampton, Southampton SO17 1BJ, UK. 3Department
of Cellular and Physiological Sciences, Life Science Institute, University of British Columbia, Vancouver, V6T 1Z3,
Canada. Correspondence and requests for materials should be addressed to A.M. (email: a.mudher@soton.ac.uk)
Received: 13 March 2015
Accepted: 12 October 2015
Published: 26 November 2015
OPEN
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
AD brains and animal models, a wide range of tau aggregates of varying size, morphology and solubility
have been identied. ese range from soluble dimers and small oligomers23, to larger insoluble granular
tau oligomers (GTOs) of approximately 40 tau units24 that are assumed to be precursors of the proto-
brils which ultimately form neurobrillary tangles. ough tangle pathology correlates with cognitive
decline in AD, results from animal models have raised questions about their toxicity25–27. For example
in inducible tau transgenic mice, both memory decits28 and neuronal loss29 are rescued by switching
o tau transgene expression and yet tangle pathology persists. Following such ndings, the search for
the toxic tau aggregates deviated from tangles to their precursors, the tau oligomers. Tau oligomers have
been described in early stages in AD brains30,31 and in transgenic models of tauopathy32,33. Several studies
imply that they mediate tau toxicity in tauopathies34. For example tau oligomerisation closely correlates
with memory loss in a transgenic model of tauopathy32 and stereotaxic injection of recombinant tau
oligomers but not monomers or brils impairs learning and memory in wild-type mice35. In the latter
study, the tau oligomers also caused signicant neuronal death around the injection site. us oligomeric
tau species are now seriously being considered as targets of tau-based therapeutic strategies34,36.
ough the ever-increasing studies on tau oligomers clearly describe a variety of oligomers that dif-
fer in size (and number of tau protein constituents), shape and solubility, these dierences are rarely
acknowledged or discussed. Consequently their contribution to the pathogenic potential of oligomeric
tau species is not fully appreciated26. Instead tau oligomers are generally considered to be a toxic species
of tau comprised of highly phosphorylated and aggregated tau. e results we describe in this paper
challenge this view and thus highlight the need for scientists of future studies to more clearly character-
ize and describe the oligomeric tau species they are working on. We show that insoluble tau oligomers,
comprising of non-phosphorylated tau can form in vivo in situations where tau-mediated neuronal dys-
function is rescued. us tau oligomers are not necessarily made up of hyper-phosphorylated tau and
they are not necessarily associated with tau toxicity.
Results
Rescue of tau-induced phenotype led to formation of structures resembling tau oli-
gomers. We have previously shown that reduction of GSK3β -mediated tau phosphorylation (using
LiCl or a more specic GSK-3β inhibitor, AR-A01448) rescues phenotypes induced by human tau
(hTau 0N3R) in Drosophila. ese phenotypes include locomotor impairment and disrupted axonal trans-
port7–9,37,38. While examining the ultrastructure of hTau0N3R-expressing neurons in these animals, we
made an unexpected observation: treatment with either drug led to the formation of 20–50 nm elec-
tron-dense granules in axonal EM sections (Fig.1, arrows in 1a-f and quantied in Supplementary Fig. 1).
ese structures bear a striking resemblance to granular tau oligomers (GTOs) rst described by the
Takashima group in AD brains30. Subsequent in vitro characterization by their group proved that AD
brain GTOs weigh 1800 kDa and contain on average 40 molecules of tau24,39. Here, we tested the hypoth-
esis that the electron-dense granules we observed in hTau-expressing Drosophila aer GSK-3β inhibition
are indeed GTO-like structures.
We rst ascertained whether these GTO-like structures were associated with rescue of
phospho-tau-mediated phenotypes. erefore we examined whether like LiCl8,9, AR-A01448, which also
produces GTOs and rescues axonal transport and locomotion (Supplementary Fig. 2), similarly restores
cytoskeletal integrity. Figure1 and Supplementary Fig. 3 show that microtubules in AR-A01448-treated
hTau0N3R larvae (black arrowheads in Fig. 1d–f) were indistinguishable from control in number and
integrity (black arrowheads in Fig.1g–i). In marked contrast, axons of untreated hTau0N3R animals dis-
played reduced numbers and misaligned microtubules, as previously reported8,9 (white arrowheads in
Fig.1j–l. Quantied in Fig. 1m and Supplementary Fig. 3). is demonstrates that functional rescue by
GSK-3β inhibition (both LiCl and AR-A01448) is sucient to restore microtubule integrity thus rescuing
tau-induced neuronal dysfunction. Remarkably, rescuing tau-induced phenotypes by this mechanism
also resulted in formation of GTO-like structures.
Inhibition of tau phosphorylation led to increased tau levels. Since LiCl and AR-A01448 treat-
ments produce GTO-like structures, which we hypothesize contain tau, we assessed human tau levels in
animals treated with these drugs. We observed an intriguing increase of total hTau in response to GSK-3β
inhibition. Treatment with either LiCl (Fig.1o) or AR-A01448 (Fig. 1p) not only predictably reduced
tau phosphorylation (Fig. 1n), but also signicantly increased hTau levels by 50% (Fig. 1s). Genetic
manipulation of GSK-3β conrmed this. Co-expression of hTau0N3R with a dominant-negative allele of
shaggy (sggDN), the Drosophila homolog of GSK-3β , similarly reduced tau phosphorylation (Fig.1n) and
signicantly increased hTau levels (Fig. 1q,s; for representative loading controls of blots in Fig. 1o–n
see Supplementary Fig. 4). Conversely, expression of constitutively-active shaggy (sgg*; which increases
tau phosphorylation and exacerbates the behavioural phenotype8,9) reduced hTau levels (Fig.1r,s). is
shows that GSK-3β inhibition increases hTau levels, whilst GSK-3β activation decreases it. is eect is
specic to GSK-3β because treatment with a microtubule-stabilising peptide davunetide/NAP rescues tau
phenotypes but does not alter tau levels38. Also, inhibition of GSK-3β does not non-specically increase
levels of other transgenes (Supplementary Fig. 5) showing that this is a human tau-specic eect. Our
result is counter-intuitive, since many individuals with tauopathies exhibit elevated tau levels40. In con-
trast, we observed elevated tau levels in conditions where tau phenotypes were rescued, and lower levels
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
Figure 1. GSK-3β inhibition rescued microtubule number in hTau0N3R Drosophila, but increased total
hTau protein and caused formation of electron-dense granules a-l) Electron micrographs of transverse
sections of peripheral nerves in L3 Drosophila (scale bar 200 nm). In hTau-expressing (elavC155-Gal4/Y;
UAS-hTau0N3R/+) animals treated with either 20 mM LiCl (hTau-Li, a–c) or with 20 μ M AR-A01448
(hTau-AR, d-f), some axons exhibited small electron-dense globular structures of approximately 20–50 nm
in size (black arrows). ese structures were extremely rare in control larvae expressing elavC155-Gal4 driver
alone (WT, gi) or untreated hTau0N3R-expressing neurons (jl). In WT larvae the axon proles showed
numerous regularly-spaced, correctly-aligned transverse microtubule proles (black arrowheads in g-i;
8.1 ± 0.2/axon prole). As we have previously shown29, in hTau0N3R-expressing axons the microtubules were
dramatically disrupted, with fewer correctly-aligned transverse microtubule proles (black arrowheads
in j-l; 5.3 ± 0.3/axon prole), and evidence of disorganised microtubules in the same axon proles
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
where the phenotypes were exacerbated. We propose that high tau levels are not detrimental when low
levels of tau phosphorylation are maintained. Elevated tau levels could conceivably cause GTO formation
in our model, as aggregation of tau has been shown to be critically dependent upon tau concentration41.
Multiple techniques conrmed that GTO-like structures were authentic tau oligomers. We
tested our hypothesis that the GTO-like structures formed following GSK-3β inhibition are indeed com-
posed of insoluble tau using several approaches including biochemical assays, immuno-gold EM and
Atomic Force Microscopy (AFM).
First, we hypothesized that if GTO-like structures were forming then we would identify them
using commonly used biochemical assays used to detect insoluble tau oligomers. Indeed the presence
of tau immuno-positive material within the stacking gel of brain lysates from drug treated hTau0N3R
ies suggested that insoluble tau species were present but were unable to enter the SDS-resolving gel
(Supplementary Fig. 6). To explore this further, we fractionated brain lysates into aqueous-soluble (S1),
detergent-soluble (S2) and detergent-insoluble (S3) fractions using established protocols for identifying
insoluble tau oligomers24,39 (Supplementary Fig. 7 shows that this protocol detects insoluble tau). In all
hTau0N3R ies, whether drug treated or not, the majority of hTau was in the S1 aqueous-soluble (Fig.2a)
or S2 detergent-soluble fractions (Fig.2b). e fraction of tau (as a percentage of total tau) found in
either S1 or S2 was the same for all hTau0N3R ies, whether drug treated or not (Fig.2a’,b’). In contrast,
the amount of tau found in the insoluble S3 fraction changed signicantly following drug treatment.
Whereas only a very small amount of tau was detected in the insoluble S3 fraction in untreated ies
(Fig. 2c htau lane and Fig.2c’ white bar), this increased dramatically, almost by three-fold, aer LiCl
or AR-A01448 treatment (Fig.2c htau-Li and htau-AR lanes and Fig2c’ light gray and dark gray bars).
To conrm that the protocol used to generate the S3 fraction was indeed enriching for insoluble aggre-
gated tau, it was used to fractionate tau from transgenic mice where such tau is found in abundance. As
expected, insoluble tau was detected with our protocol from tau transgenic brain homogenate (Fig.2c
3xT lane). To verify that the increase in insoluble tau was caused by GSK-3β inhibition and not any other
drug action, we assessed tau solubility in hTau0N3R;SggDN ies. Co-expression of SggDN led to a signicant
increase in insoluble tau, proving that this eect was mediated by GSK-3β inhibition (Fig.2dandd’).
ese results were corroborated using a second anti-tau antibody, which conrmed that the material
picked up in the S3 fraction of drug treated hTau0N3R ies was indeed insoluble tau (Supplementary
Fig. 6b). As mentioned above, this eect is specic to GSK-3β because davunetide/NAP treatment nei-
ther produced GTO-like structures38 nor increased insoluble tau levels (Supplementary Fig. 6c). is
data shows that pharmacological or genetic reduction of GSK-3β -mediated tau phosphorylation leads to
increased tau levels and formation of structures containing insoluble tau.
To further conrm that these structures contain tau, we performed immuno-gold EM on insoluble
fractions. In all hTau0N3R-expressing conditions we detected 20–50 nm granular oligomeric structures
decorated with anti-hTau antibody (Fig.3a–c). No labeling was evident in non-transgenic or antibody
controls (Fig.3d and Supplementary Fig. 8). In line with the results from the EM (Fig.1) and biochem-
ical analyses (Fig.2), many more such structures were evident in the drug treated hTau0N3R animals than
in untreated controls. ese data strongly imply that insoluble tau granules, GTO-like structures, are
forming in drug-treated hTau0N3R ies.
To corroborate this and to accurately measure the size of the granular structures, we subjected hTau
immuno-precipitated from y head lysates to atomic force microscopy (AFM), a procedure routinely
used to visualise tau oligomers24. Abundant granular spherical structures were observed in samples from
LiCl-treated hTau0N3R brains (Fig.3f), but were extremely sparse in untreated hTau ies (Fig.3e). e
dimensions of these structures were strikingly similar to GTOs identied from AD brains24,30, varying
in width from 5–50 nm, with an average width and height of 20 nm. is data strongly indicates that
inhibition of GSK-3β in hTau0N3R ies produces insoluble GTO-like structures similar to those found in
AD brain.
To conrm that these GTO-like structures isolated biochemically represent the same structures as
the electron-dense granules observed in situ, we performed in situ immuno-gold labeling for hTau on
(white arrowheads in jl). Indeed, approximately 30% of hTau0N3R-expressing axons displayed no visible
microtubule proles (Figure S1). In hTau0N3R-expressing larvae fed with Li (ac) or AR (df), there were
signicantly more correctly-aligned transverse microtubule proles (black arrowheads in a-l; 9.2 ± 0.3/axon)
and fewer misaligned microtubules. Microtubule numbers per axon are quantied in m (**p < 0.01,
unpaired Students t test). Representative Western blots of hTau0N3R-expressing y head lysates showed that
tau phosphorylation was decreased (at T231/S235 detected by AT180) whilst total tau levels were increased
by 40–60% (or) by 20 mM lithium treatment (hTau-Li, o), 20 μ M AR-A01448 treatment (hTau-AR, p), co-
expression of dominant negative shaggy ( hTa u;sggDN, q) {elavC155-Gal4/Y; UAS- hTau0N3R/ + ; UAS-sggDN/ + }.
Conversely, total tau levels were decreased by approximately 50% by co-expression of constitutively active
shaggy (hTau;sgg*, r) {elavC155-Gal4/Y; UAS- hTau0N3R/ + ; UAS-sgg*/ + }. is is quantied in s (error bars are
standard error of mean; *p < 0.05 by Students t-test).
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
Figure 2. e amount of insoluble tau detected from hTau0N3R-expressing Drosophila is increased
dramatically aer treatment with GSK-3β inhibitors. Western blots of aqueous-soluble fraction (S1),
detergent-soluble fraction (S2) and insoluble fraction (S3) probed for anti-hTau. Samples in lanes 1–3 are
from heads of hTau0N3R ies (hTau – {elavC155-Gal4/Y ; UAS-hTau0N3R/ + }), hTau0N3R ies treated with 20 mM
lithium (hTau-Li), hTau0N3R ies treated with 20 μ M AR-A01448 (hTau-AR), and ies co-expressing hTau0N3R
with dominant negative shaggy (hTau;sggDN {elavC155-Gal4/Y; UAS- hTau0N3R/+; UAS-sggDN/ + }). e fourth
lane in the third panel (labelled “3xT mouse”) is a 10-fold dilution of sample from triple-transgenic mouse,
used as a positive control for insoluble tau. Bar charts in a’–d’ are quantications of blots in a – d presented
as a percentage of total tau {S3/(S1 + S2 + S3)} in each genotype. Treatment with Li or AR-AR01448 did not
alter the amount of tau detected in either the S1 (a,a’) or S2 fractions (b,b’). However treatment with Li,
AR-AR01448 (c,c’) or co-expression of dominant negative shaggy (hTau;sggDN) (d,d’) signicantly increased
the amount of tau detected in the insoluble S3 fraction. (error bars are standard error of mean and n = 5 for
each genotype/treatment; *p < 0.05 by Students t-test).
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
sections of peripheral nerve. In conditions in which electron-dense granules were observed, such as
Li-treated hTau ies, we found granules decorated with gold particles within axons (arrows in Fig.3g–i).
Collectively, the results from all the above assays prove our hypothesis by demonstrating that inhibi-
tion of GSK-3β produces insoluble oligomers showing GTO-like structure.
hTau in GTO-like structures was not phosphorylated. It is oen presumed that tau aggregates
including oligomers are composed of phosphorylated tau. However we show here that decreased phos-
phorylation leads to formation of insoluble tau oligomers. erefore we determined whether these
Figure 3. Insoluble tau oligomers can be puried from hTau0N3R-expressing Drosophila and were
increased dramatically aer treatment with GSK-3β inhibitors. TEM of immuno-gold labelling for anti-
hTau of S3 insoluble fractions show granular tau oligomers comprised of hTau in all conditions expressing
hTau { elavC155-Gal4/Y; UAS-hTau0N3R/ + }: (a) hTau, (b) hTau-Li, (c) hTau-AR. No such structures were
detected in controls: d) wild-type. (See also Supplementary Fig. 8, for additional controls of no sample
labelled with anti h-Tau; and hTau labelled for an irrelevant rabbit polyclonal antibody, anti-v-glut). Scale bar
in a (applicable to af) = 100 nm. (gh); hundreds of such structures were observed in preparations from 18
pooled ies) Atomic Force Microscopy of material immuno-precipiated from y head lysates using anti-hTau
antibody shows the appearance of numerous granular tau oligomers present aer LiCl treatment (f) but only
very sparse in untreated hTau0N3R ies (e). Oligomer sizes were determined by cross-sectional height analysis
of individual oligomers. e heights of the oligomers ranged between 15 and 30 nm, with a mean height of
17.07 nm (SD = 8.86). e widths of the majority of oligomers are between 20 and 40 nm and the average
width was calculated to be 20.6 nm (SD = 11.4). A minority of oligomers have a larger width than 30 nm
but the height of the oligomers was consistently 30 nm or below. Scale bar in i = 1 μ m. (gi)’) Immuno-gold
labeling for hTau in situ in sections of peripheral nerves from hTau-Li ies demonstrates labeled granular
tau oligomers (arrows) within axons. Examples are given at lower magnications (gi) in which axonal
proles are clearer, and at higher magnications (g’–i’) in which GTOs can be seen more clearly. Scale
bars = 100 nm.
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
Figure 4. Tau within GTO-like structures is largely unphosphorylated Western blots of the soluble
fraction (S1) and the insoluble fraction (S3) from hTau {elavC155-Gal4/Y; UAS- hTau0N3R/ + }, hTau-Li and
hTau – AR-A01448 treated y brain lysates probed with an antibody that detects total tau (a and a”)
and those that detect various phospho-tau epitopes (b-g and b”-g”). ough there is a signicant amount
of tau in the insoluble fraction of drug treated brain lysates (a”), it is largely unphosphorylated at many
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
insoluble oligomers are in fact phosphorylated by assessing the phosphorylation status of hTau in soluble
and insoluble fractions from y heads. We found that tau in the soluble fraction was highly phospho-
rylated in hTau0N3R ies, at many of the sites hyper-phosphorylated in AD (htau lanes in Fig.4b–g). As
expected, GSK-3β inhibition signicantly reduced tau phosphorylation at several of these sites except
at the ser262 site (htau-Li and htau-AR lanes in Fig. 4b–g and Fig. 4b’–g’ where reduction in phos-
phorylation in drug-treated hTau0N3R ies is quantied as a percentage of phosphorylation in untreated
hTau0N3R ies). However, unlike the soluble tau fraction, the hTau in the insoluble (GTO) fraction was
largely un-phosphorylated in all animals in which GTOs were produced (Fig.4b”–f”). ough there is
signicantly less tau protein in the S3 fractions, the data in Fig.4a” implies that the lack of phospho-tau
immunoreactivity in this fraction, was not due to undetectable total tau levels. is is because, in line
with the biochemical analyses presented in Fig.2, a non-phosphorylation dependent anti-tau antibody
detected signicant amounts of tau in this S3 fraction (Fig.4a”). Additionally, equating the total tau levels
of the S1, S2 and S3 fractions (by decreasing the amount of tau protein loaded in S1 and S2 by several
fold), still gave a positive signal with phospho-tau antibodies in these fractions without any signal in
S3 (Supplementary Fig. 9), further implying that the lack of signal in S3 was not due to inadequate tau
protein in S3. us, the tau proteins contained within the GTOs formed following GSK-3β in hibition
are unlikely to be phosphorylated. ese ndings imply that tau oligomers formed in vivo don’t always
need to comprise of phosphorylated tau molecules; they can also be made up from non-phosphorylated
tau molecules.
GTO-like structures displayed oligomerisation and lack of β-pleated sheet structure. To fur-
ther investigate the chemical nature of these GTO-like structures we carried out Raman spectroscopy. is
technique interrogates the vibrations of bonds in molecules generating characteristic spectral proles. It
allows insight into the chemical structure and interactions between dierent groups and side chains in
proteins, providing an analysis of their secondary structure42 and aggregated state43. We hypothesized
that the spectral prole of insoluble fractions generated from hTau ies in which the tau is primarily
monomeric, would dier from that of Li-treated hTau ies in which tau oligomers form. Indeed, we
found that the insoluble GTO-like structure containing samples (which we know only contain tau and
no other proteins – see Supplementary. Fig. 10) provided several spectral markers to conrm their oli-
gomeric nature. For instance specic peaks such as the one at 1382 cm1 that is indicative of disorder43
in untreated Tau spectrum disappears while new peaks such as the one at ~540 cm1 characteristic of
disulde linkages appear conrming their formation as one would expect in Tau oligomerisation44. e
change of state from monomer to oligomeric species is further reinforced by the evolution of the amide
II vibration from 1544 cm1 to 1578 cm1. Furthermore, several peaks of reduced bandwidth (i.e. greater
sharpness) and greater intensity than those from untreated hTau ies (arrows – Fig.5) can be observed
in the spectra of treated ies. e increased intensity signies an increase in corresponding bond num-
ber, while increased peak sharpness indicates less conformational freedom (more rigidity)45 which
will occur upon oligomerisation. Such oligomer related peak-changes have been observed by others43.
Furthermore the Raman spectra of Lithium treated Tau samples shows that not only do the Tau-Li sam-
ples have sharper peaks, but that these are also slightly shied indicating a species with an oligomeric
conformation.
All these observations imply that tau in the hTau-Li sample is oligomeric, conrming our biochemical
data. Ponceu staining of the P2 fraction, from which the insoluble S3 oligomer fraction is generated for
this spectroscopic analysis shows that there is primarily only one band corresponding to the tau protein
in that fraction (S3) so the only signal possible is from the tau oligomers (Supplementary. Fig. 10).
Phosphorylation has previously been measured by Raman spectroscopy, and it has been clearly
demonstrated that, under basic conditions, a strong peak is evident at around 980 cm1 46,47. Our Raman
spectra recorded on the S3 fraction, which were prepared under basic conditions, do not show the pres-
ence of any signicant peak at ~980 cm-1 (Fig.5). e absence of such a peak in our spectra is highly
suggestive of the lack of phosphorylation in the tau species that constitute these oligomers.
To probe the secondary structure of our tau oligomers, we studied the spectroscopic traces within
regions 1625–1700 cm1 (amide I) and 1220–1300 cm1 (amide III). ese are typically used for deter-
mining the secondary structure of proteins, although the former is more reliable as it is free from con-
tributions from side-chain vibrations42,45,48. In the Raman spectrum of hTau-Li, there was a near absence
sites (b”–g”). Signal at these sites in the soluble fractions (bg and b’–g’) provides a positive control for the
antibodies, and shows that treatment with GSK-3β inhibitors LiCl and AR-A01448 reduced phosphorylation
at many of these sites: quantied in graphs (b’–g’). Graphs show the average of 6 independent experiments;
error bars are SEM; * p < 0.05. Blots were probed with (a) dako polyclonal anti-tau (total tau), (b) anti-tau-1
(unphosphorylated at 192–204), (c) anti-PHF-1 (pS396, pS404), (d) anti-AT8 (pS202, pT205), (e) anti-AT180
(pT231, pS235), (f) anti-MC1 (it is curious that this supposedly conformation specic anti-body picks up
its epitope aer SDS-PAGE denaturation; it is likely that the epitope recognized here is largely denatured
but nonetheless is disease predictive since others, like us have also shown similar WB immunoreactivity in
another Drosophila model of tauopathy10), (g) anti-pS262.
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of any spectral feature around 1665–1690 cm1 (arrowhead – Fig. 5), indicating the lack of a β -pleated
sheet structure. e very weak presence of a peak at ~1650 cm1 (and more so in the untreated hTau
spectrum) suggests a helical arrangement of the protein backbone. is is also consistent with the slight
increase in intensity of the peak at ~1250 cm1 observed, which indicates an increase in the non-β -sheet
structures (such as α -helices or random secondary structures), in the oligomeric protein compared to the
monomer49. Taken together, these data imply that the GTO-like structures generated following GSK-3β
inhibition lack β -pleated sheet structures. Since it is generally presumed that β -pleated sheet structures
are toxic, the lack of such structure in our insoluble tau oligomers is consistent with their apparent
non-toxicity.
Discussion
We present here novel data that genetic or pharmacological inhibition of GSK-3β -mediated tau phos-
phorylation rescues phospho-tau phenotypes, and unexpectedly increases total tau levels and produces
insoluble tau oligomers resembling granular tau oligomers (GTO)s. ese insoluble tau oligomers are
not phosphorylated, and do not contain β -pleated sheet structure. Contrary to prevailing opinion, this
demonstrates that oligomeric tau is not necessarily toxic.
Not all aggregated proteins are toxic. In all proteinopathies there are a variety of soluble and
insoluble misfolded proteins. It is now generally accepted that the largest insoluble structures in these
diseases (such as neurobrillary tangles in tauopathies) are not the most toxic species. In the case of tau
protein, suspicion has now fallen on small soluble and insoluble oligomers25,26. However, there is prece-
dent from many aggregate-prone proteins such as beta-amyloid, huntingtin and alpha-synuclein that the
smallest insoluble form is relatively protective, while the toxic species is soluble19–21. In the case of tau
protein this idea is in its infancy, though supportive circumstantial evidence is beginning to emerge. Our
data are the rst to show that treatments which render tau less soluble rescue tau-induced toxicity, thus
demonstrating that small insoluble tau oligomers are associated with neuroprotection in vivo. ough
our ndings do not necessarily imply that tau oligomers are directly neuroprotective, they clearly shows
that they are certainly not always toxic when formed in vivo.
As we, and others have previously shown, tau phosphorylated at GSK-3β sites is associated with toxicity
in both Drosophila9,50 and rodent models51. e fact that the insoluble tau oligomers we describe here are
not phosphorylated and do not contain β -pleated sheet structure may reconcile the present results with
other reports suggesting that tau oligomers are toxic24,30. We speculate that tau oligomers comprised of
non-phosphorylated tau may be non-toxic, whereas those comprised primarily of highly-phosphorylated
tau in β -pleated sheet conformation may be toxic26.
Reduction of tau phosphorylation unexpectedly promotes tau aggregation. It is intriguing
that reduction of tau phosphorylation in this study promotes tau aggregation, because conventional opin-
ion dictates that hyper-phosphorylation of tau precedes and promotes aggregation. Although one other
study is consistent with ours in showing that decreasing GSK-3β activity leads to increased levels of insol-
uble tau oligomers33, many other studies, mostly in rodent models, have conversely found that increasing
GSK-3β activity promotes tau aggregation6,14,52, and that inhibition of GSK-3β reduces insoluble tau51,53.
Figure 5. Raman spectroscopy indicates oligomeric structure and lack of β-pleated sheet Raman
spectroscopy was carried out on the P2 (detergent-insoluble GTO) fraction prepared from y heads.
e spectrum of Li-treated hTau {elavC155-Gal4/Y; UAS- hTau0N3R/ + } P2 fraction (black) contained several
peaks (arrows) of reduced bandwidth and greater intensity than that from untreated hTau fractions (red),
indicating more oligomeric structure in hTau-Li. No peaks are observed at 1665–1690 cm1 (arrowhead)
indicating a lack of β -pleated sheet structure or at 980–1000 cm1 indicating lack of phosphorylated species.
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ough it is conceivable that our ndings, like that of Blard et al.33, are specic to Drosophila models of
tauopathy, we do not believe this to be the case. Instead we speculate that the discrepancy between our
results and those of others is due to two reasons. Firstly, in studies in which the eect of GSK-3β inhib-
itors on tau aggregation was analysed51,53, the insolubility fractionation protocols employed enrich for
tau laments and tangles and actually loose small insoluble tau oligomers. As discussed in26 insoluble tau
oligomers are too small to sediment in the standard high-spin sedimentation spins used in these proto-
cols and yet are too large to enter a SDS-PAGE resolving gel. Hence they would not be detectable unless
the insolubility fractionation was specically chosen to enrich for small insoluble tau oligomers24. us,
though the studies using GSK-3β inhibitors report a reduction of large tau aggregates (such as laments)
it is not possible to conclude whether or not insoluble tau oligomer levels were aected. Perhaps like us,
they too may have detected increased insoluble tau oligomer levels following GSK-3β inhibitors if they
had used the insolubility fractionation protocols employed for detecting such small insoluble tau species.
e second explanation for the discrepancy between our ndings and those of others is the increased
total amount of tau that we observe aer GSK-3β inhibition. Since tau aggregation (at least in vitro) is
critically dependent upon tau concentration41, we suggest that it is the increased tau concentration that
drives formation of insoluble tau oligomers in our model. We would argue that increased phospho-tau
levels (by inhibiting degradation of phospho-tau) would also lead to tau oligomer formation but in that
case these oligomers may be toxic because they would be composed of phosphorylated tau. Indeed this
was recently demonstrated in a rodent model of tauopathy54.
It will be interesting to investigate the mechanism by which GSK-3β inhibition increases total tau.
Like us, others have also shown GSK-3β mediated regulation of tau turnover in both invertebrate and
vertebrate models33,55–57. It is plausible that phosphorylation at some of the GSK-3β sites may be targeting
the tau for degradation, and thus under GSK-3β inhibition there is less tau protein turnover, leading to
tau accumulation. Other GSK-3β substrates such as β -catenin do indeed signal their own degradation in
this manner58, and there is precedence for preferential degradation of phosphorylated tau over unphos-
phorylated tau55,56.
ough phosphorylation of tau is believed to precede tangle formation, it is intriguing that not all tau
proteins derived from paired helical laments in AD are phosphorylated59. Indeed it is speculated that
tau proteins found at the core of paired helical laments are not phosphorylated (either that or phospho-
rylated epitopes are cleaved) whilst those located at the periphery are59. In light of this, it is conceivable
that non-phosphorylated tau oligomers, such as the ones we describe in our study, may actually form at
an early stage in human brain, and though may not be toxic themselves, they could seed aggregation of
more toxic phosphorylated tau species.
Implications for therapeutic approaches to tauopathy. e failure of amyloid-based thera-
pies in clinical trials has stimulated research into tau-based targets for the treatment of tauopathies
like Alzheimer’s disease. Two major tau-based strategies are directed at inhibiting GSK-3β -mediated tau
phosphorylation, and reducing tau aggregation/oligomerisation by various means34,60. Examples of the
latter include chemicals such as rhodanines, N-phenylamines, phenylthiazolhydrazides, and methylene
blue that can inhibit or dissolve tau aggregates61. Alternatively, tau might be cleared by tau vaccination
approaches62.
Our nding that inhibition of tau phosphorylation leads to production of insoluble tau oligomers
raises a cautionary note regarding the therapeutic strategies aimed at reducing tau phosphorylation. Our
results suggest the outcome of this strategy may conict with the aim of the other key therapy if, as we
show, inhibition of tau phosphorylation leads to formation of tau oligomers.
Additionally, our nding that tau oligomers are not always toxic has implications for the eectiveness
of strategies aimed at reducing tau aggregation. Such strategies would be ineective if the tau oligomers
that they are clearing are not toxic. Furthermore, these approaches might even be counter-productive
if solubilising aggregated tau releases the more toxic species. Our results underscore the importance of
establishing which species of oligomeric tau are toxic, neutral or protective to determine the most appro-
priate target for eective tau-based therapies.
Conclusion
e mechanism(s) by which abnormal tau causes toxicity in tauopathies is not clear. Although it is
established that abnormal tau phosphorylation plays a critical role, the role of tau aggregation, and in
particular oligomerisation, is less well understood. We have found that reduction of tau phosphorylation
promotes tau aggregation, and that this is, surprisingly, associated with rescue of neuronal dysfunction
in vivo. is has profound implications for therapeutic approaches aimed both at inhibiting tau phos-
phorylation and reducing tau aggregation.
Materials and Methods
Flies. Drosophila melanogaster expressing the GAL4 drivers elavC155-Gal4 or D42-Gal4 were crossed
with either wild-type Oregon-R ies (wt) as a control, or with ies transgenic for human 3-repeat tau
(UAS-hTau0N3R from Bloomington Stock centre), with or without UAS-sgg* (UAS-sggS9A from Bloomington
Stock centre) or UAS-sggDN (Dr. D Allan UBC, Canada). Flies were raised at 25 °C on standard y food
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with or without 20 mM LiCl or 20 μ m AR-A01448 (Sigma-Aldrich). For all experiments, equivalent num-
bers of males and females were utilized for each condition compared.
Solubility fractionation of granular tau oligomers. 10 y heads (0-3d ies) were pooled and
homogenized in 40 μ l aqueous buer (50 mM Tris-HCl pH 7.4, 175 mM NaCl, 1 M sucrose, 5 mM EDTA)
at 4 °C, and centrifuged at 1,000 g for 2 minutes to remove unhomogenized debris. e supernatant was
then centrifuged for 2 h at 200,000 g (4 °C). e resulting supernatant (S1) is the aqueous-soluble frac-
tion. Pellet (P1) was resuspended in SDS buer (50 mM Tris-HCl pH 7.4, 175 mM NaCl, 5% SDS) and
centrifuged for 2 h at 200,000 g (25 °C). Supernatant (S2) is the detergent-soluble fraction. P2 was washed
by repeating this spin and, aer discarding the supernatant, P2 was resuspended in buer containing
8 M urea (50 mM Tris-HCl pH 7.4, 175 mM NaCl, 8% SDS, 8 M urea) with agitation for 18 h at room
temperature. S3 is the detergent-insoluble fraction.
Western blotting. Samples of pooled y heads (0-3d ies) were either prepared for solubility frac-
tionation, as above, or homogenized in buer containing protease, kinase and phosphatase inhibitors
(150 mM NaCl, 50 mM MES, 1% triton-X 100, 1% SDS, 2 μ g/ml leupeptin, 2 μ g/ml aprotinin, 100 μ g/ml
PMSF, 30 mM NaF, 40 mM 2-glycerophosphate, 20 mM sodium pyrophosphate, 3.5 mM sodium orthov-
anadate, 10 μ M staurosporine). Samples were heated for 5 minutes at 95 °C in Laemmli buer, separated
by 10% PAGE, and transferred to PVDF membrane (Amersham). Blots were probed with the following
primary antibodies: anti-human-tau antibody (Dako, 1:15,000), anti-human-tau N-terminal (Abcam,
1:1000), tau-1 (Millipore, 1:2,000), PHF-1 (Peter Davies, 1:500), AT8 (Source Biosciences, 1:800), AT180
(Source Biosciences, 1:100), MC1 (Peter Davies, 1:200), or anti-pS262 (Invitrogen, 1:1,000), followed
by HRP-conjugated anti-rabbit secondary antibody (Cell signalling) and Chemiluminescent substrate
(Amersham). Band densities were measured using Image J.
Transmission Electron Microscopy, in situ. Filleted L3 larvae were xed (3% glutaraldehyde,
4% formaldehyde in 0.1 M PIPES buer, pH 7.2) for 1 hour. Specimens were rinsed in 0.1 M PIPES
buer, post-xed in 1% buered osmium tetroxide (1 hour), block-stained in 2% aqueous uranyl acetate
(20 min), dehydrated in ethanol and embedded in Spurr resin (EM Science). Ultra-thin sections were
cut through the nerves at the base of the ventral-cord on a Leica OMU 3 ultramicrotome. e sections
were stained with Reynolds lead stain and viewed on a Hitachi H7000 transmission electron microscope
with SIS Megaview-III digital camera. e number of microtubule proles per axon was counted in cross
sections of peripheral nerves. Comparable regions of peripheral nerves were studied and all animals were
processed in parallel. All visible axons were analysed in 5 animals per condition (approximately 240
axons per animal), with the experimenter blinded to condition.
Transmission Electron Microscopy with in situ immuno-gold labeling. Filleted CNS prepara-
tions from L3 larvae were xed in 4% PFA (15 min), then the anterior half of the llet was cut away and
post-xed overnight in fresh 4% PFA. Samples were cryoprotected by incubation in a glycerol series up
to 30%. Samples were briey plunge-frozen in liquid ethane ( 170 °C). Frozen samples were incubated
in 1.5% uranyl acetate in methanol at-90 °C for 30 h, then at 45 °C for11 h. is was followed by inl-
tration with a series of resin:methanol mixtures at 45 °C, culminating in pure Lowicryl HM-20 resin
(EM Science) overnight. Specimens were polymerized by UV light (24 h, 45 °C), then the temperature
increased to 0 °C over 9 h. Gold/silver sections were cut through the proximal peripheral nerves on a
Leica ultramicrotome, and transferred to formvar-coated Ni grids (EM Science). Immunochemistry was
carried out as described28 using anti-tau (Sigma, polyclonal T6402 1:100), and secondary goat-anti-rabbit
10 nm gold (EM Sciences). e specimens were viewed on a FEI Tecnai G2 Spirit Transmission Electron
Microscope.
Transmission Electron Microscopy with immuno-gold labelling of fractionated protein. e
insoluble protein fraction (P2 from the solubility fractionation method) was resuspended in 20 μ l dis-
tilled water, and 5 μ l spotted onto carbon-formvar copper grids (EM Science) for 2 min. Grids were
blocked in 1% milk in PBS; incubated in anti-human-tau (Sigma) or anti-v-glut at 1:200 in 1% milk in
PBS (60 min room temperature); washed in PBS; incubated in gold-conjugated anti-rabbit secondary
antibody (EM Sciences - 1:30) in 1% milk in PBS (60 min room temperature); washed in PBS then water;
negative stained with 2% uranyl acetate (10 s). Grids were viewed as above.
Atomic force microscopy. 20 brains of L3 larvae per sample were dissected on ice and homogenized
in 500 μ l buer (50 mM MES, 150 mM NaCl, 1% triton-X, protease inhibitor cocktail), centrifuged (2 min
2,000 g) and unhomogenized material discarded. Samples were immuno-precipitated as follows: preclear
in 20 μ l protein A/G beads (Sigma) at 4 °C 1 hour; incubate with 1 μ l anti human-tau (Dako) at 4 °C
1 hour; add 60 μ l beads and incubate at 4 °C 1 hour; wash beads 3 times in buer; reduce precipitated
sample in 20 μ l Laemlli buer for 5 min 90 °C. Precipitates were spotted onto freshly cleaved mica discs
(Agar Scientic), incubated for 2 minutes, rinsed with 200 μ l ultrapure water and dried with compressed
air. Samples were imaged in air with a digital multimode Nanoscope III AFM (www.veeco.com) operating
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in tapping mode with an uncoated silicon cantilevers (FM-W, Nanoworld Innovative Technologies,
Switzerland, nominal spring constant 2.8 N/m) with set points of 0.6–0.8 at a scan frequency of 3 Hz.
Oligomer sizes were determined by cross-sectional height analysis of individual aggregates.
Raman Spectroscopy. Insoluble protein fractions (P2 from solubility fractionation protocol above),
from hTau and hTau-Li brains were placed on slides. An InVia Renishaw Raman microscope system
was used for obtaining spectra. A 633 nm laser was used for excitation with an acquisition consisting of
3 exposures of 10 s each. Spectra were processed with Wire 3.1 soware: uorescence background was
subtracted and spectra were smoothed.
Locomotor contractions assay. Ht au0N3R expression was driven by the motor neuron specic drive
D42-GAL4. ird instar L3 larvae were allowed to crawl freely on an agarose plate under standardized
lighting conditions. eir crawling behavior was recorded using standard video recording equipment and
body wall contractions undertaken in one min were counted.
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Acknowledgements
is project was funded by the International Specialist Grant on Dementia from Bupa and the Alzheimer’s
Society. anks to Dr. Julian orpe (University of Sussex), Fergil Mills and Andrea Globa (University of
British Columbia) for assistance and advice on optimizing the in situ immuno-gold protocol.
www.nature.com/scientificreports/
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Scientific RepoRts | 5:17191 | DOI: 10.1038/srep17191
Author Contributions
C.M.C. carried out experiments and contributed to scientic design and manuscript writing, S.H.
carried out A.F.M., S.Q. and M.S. contributed to biochemistry experiments, S.Q. and S.M. carried out
Raman Spectroscopy, DA contributed to scientic design, A.M. led the scientic design and wrote the
manuscript. All authors reviewed the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Cowan, C. M. et al. Rescue from tau-induced neuronal dysfunction produces
insoluble tau oligomers. Sci. Rep. 5, 17191; doi: 10.1038/srep17191 (2015).
is work is licensed under a Creative Commons Attribution 4.0 International License. e
images or other third party material in this article are included in the article’s Creative Com-
mons license, unless indicated otherwise in the credit line; if the material is not included under the
Creative Commons license, users will need to obtain permission from the license holder to reproduce
the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
... Three sequential fractions, S1, S2 and S3, were extracted from each 3D sample processed (1 culture well) to study the differential solubility of Tau in the 3D cultures [34,35]. Halts Protease Inhibitor Cocktail (78430, Thermo Fisher Scientific, UK), 0.1 mM Phenylmethylsulfonyl fluoride (PMSF, P20270, Thermo Fisher Scientific, UK) was added along with a mixture of phosphatase inhibitors (P52102-1, Melford, UK) to each of the three buffers. ...
... In AD brains, Tau undergoes conformational changes to transition from soluble monomers to increasingly insoluble oligomers and fibrils, that finally coalesce to form insoluble aggregates. In this experiment, soluble and insoluble Tau levels were assessed by enriching for insoluble Tau fractions obtained by differential centrifugation following standardised techniques [34,35]. The TBS soluble fraction (S1) shows the presence of monomeric Tau. ...
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Purpose Alzheimer’s disease (AD) early pathology needs better understanding and models. Here, we describe a human induced pluripotent stem cells (iPSCs)-derived 3D neural culture model to study certain aspects of AD biochemistry and pathology. Method iPSCs derived from controls and AD patients with Presenilin1 mutations were cultured in a 3D platform with a similar microenvironment to the brain, to differentiate into neurons and astrocytes and self-organise into 3D structures by 3 weeks of differentiation in vitro. Results Cells express astrocytic (GFAP), neuronal (β3-Tubulin, MAP2), glutamatergic (VGLUT1), GABAergic (GAD65/67), pre-synaptic (Synapsin1) markers and a low level of neural progenitor cell (Nestin) marker after 6 and 12 weeks of differentiation in 3D. The foetal 3R Tau isoforms and adult 4R Tau isoforms were detected at 6 weeks post differentiation, showing advanced neuronal maturity. In the 3D AD cells, total and insoluble Tau levels were higher than in 3D control cells. Conclusion Our data indicates that this model may recapitulate the early biochemical and pathological disease features and can be a relevant platform for studying early cellular and biochemical changes and the identification of drug targets.
... This size is compatible with formation of large granular Tau oligomers (GTO) [57]. It was suggested in a Drosophila model that GTO's are not toxic and may be neuroprotective [58] [59]. Reduction of ThT signal suggests a reduction of Tau β-sheet content, but this method cannot detect the presence of oligomers. ...
... ThT and Drosophila data was analysed using one factor repeated measures ANOVA with Tukey post hoc testing through IBM SPSS Statistics 23. For the Drosophila survival data, a Kaplan-Meier survival curve was plotted and a Log-rank (Mantel-Cox) test was performed on the data using GraphPad Prism [58]. Cells were fixed using 4% formaldehyde, washed with TBS and mounted to microscope slides using ProLong™ Gold Antifade Mountant. ...
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There are currently no disease altering drugs available for Tauopathies such as Alzheimer’s disease, which alone is predicted to affect ~88 million people worldwide by 2050. As Tau aggregation underpins its toxicity, aggregation inhibitors are likely to have disease-modifying potential. Guided by in-silico mutagenesis studies, we developed a potent retro-inverso peptide inhibitor of Tau aggregation, RI-AG03 [Ac-rrrrrrrrGpkyk(ac)iqvGr-NH2], based on the 306VQIVYK311 hotspot. Aggregation of recombinant Tau was reduced by >90% with equimolar RI-AG03 and no fibrils were observed by EM. When added during the growth phase, RI-AG03 blocked seeded aggregation. Fluorescein-tagged RI-AG03 efficiently penetrated HEK-293 cells over 24 hours and was non-toxic at doses up to 30 μM. In transgenic Drosophila, RI-AG03 significantly improves neurodegenerative and behavioural phenotypes caused by expression of human Tau. Collectively this shows that RI-AG03 can effectively reduce Tau aggregation in vitro and block aggregation-dependent phenotypes in vivo, raising possibilities for exploring its translational potential.
... Next, in order to investigate likely insoluble forms of tau, the other hippocampus was used to obtain solubility fractionation of granular tau oligomers [21]. Similar to the results found in 2% SDS extracts, in a first TBS soluble fraction, despite inter-animal differences within hTauP301L-injected mice, an increase of soluble total tau was found in injected animals (data not shown). ...
... To obtain total protein extracts, half mice hippocampi were homogenized in lysis buffer with protease inhibitors (10 mM Tris-HCl ph = 7.5, 1 mM NaF, 0.1 mM Na 3 VO 4 , 2% SDS), sonicated for 2 min, left 20 min on ice and centrifuged at 13,000 rpm for 13 min at 8 • C. The supernatant was stored at −80 • C. Total protein concentrations were determined using the Pierce TM BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA). The other half mice hippocampi were used to obtain fractionation of granular tau oligomers [21]. Tissues were homogenized in aqueos buffer (50 mM Tris-HCl pH 7.4, 175 mM NaCl, 2% SDS) and centrifuged first at 1000 g for 2 min to remove unhomogenized debris and then for 2 h at 200,000× g (4 • C). ...
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Despite the well-accepted role of the two main neuropathological markers (β-amyloid and tau) in the progression of Alzheimer’s disease, the interaction and specific contribution of each of them is not fully elucidated. To address this question, in the present study, an adeno-associated virus (AAV9) carrying the mutant P301L form of human tau, was injected into the dorsal hippocampi of APP/PS1 transgenic mice or wild type mice (WT). Three months after injections, memory tasks, biochemical and immunohistochemical analysis were performed. We found that the overexpression of hTauP301L accelerates memory deficits in APP/PS1 mice, but it did not affect memory function of WT mice. Likewise, biochemical assays showed that only in the case of APP/PS1-hTauP301L injected mice, an important accumulation of tau was observed in the insoluble urea fraction. Similarly, electron microscopy images revealed that numerous clusters of tau immunoparticles appear at the dendrites of APP/PS1 injected mice and not in WT animals, suggesting that the presence of amyloid is necessary to induce tau aggregation. Interestingly, these tau immunoparticles accumulate in dendritic mitochondria in the APP/PS1 mice, whereas most of mitochondria in WT injected mice remain free of tau immunoparticles. Taken together, it seems that amyloid induces tau aggregation and accumulation in the dendritic mitochondria and subsequently may alter synapse function, thus, contributing to accelerate cognitive decline in APP/PS1 mice.
... This size is compatible with formation of large granular Tau oligomers (GTO) [57]. It was suggested in a Drosophila model that GTO's are not toxic and may be neuroprotective [58] [59]. Reduction of ThT signal suggests a reduction of Tau β-sheet content, but this method cannot detect the presence of oligomers. ...
... ThT and Drosophila data was analysed using one factor repeated measures ANOVA with Tukey post hoc testing through IBM SPSS Statistics 23. For the Drosophila survival data, a Kaplan-Meier survival curve was plotted and a Log-rank (Mantel-Cox) test was performed on the data using GraphPad Prism [58]. Cells were fixed using 4% formaldehyde, washed with TBS and mounted to microscope slides using ProLong™ Gold Antifade Mountant. ...
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There are currently no disease altering drugs available for Tauopathies such as Alzheimer's disease, which alone is predicted to affect ~88 million people worldwide by 2050. As Tau aggregation underpins its toxicity, aggregation inhibitors are likely to have disease-modifying potential. Guided by in-silico mutagenesis studies, we developed a potent retro-inverso peptide inhibitor of Tau aggregation, RI-AG03 [AcrrrrrrrrGpkyk( ac)iqvGr-NH2], based on the 306VQIVYK311 hotspot. Aggregation of recombinant Tau was reduced by >90% with equimolar RI-AG03 and no fibrils were observed by EM. When added during the growth phase, RI-AG03 blocked seeded aggregation. Fluorescein-tagged RI-AG03 efficiently penetrated HEK-293 cells over 24 hours and was non-toxic at doses up to 30 μM. In transgenic Drosophila, RI-AG03 significantly improves neurodegenerative and behavioural phenotypes caused by expression of human Tau. Collectively this shows that RI-AG03 can effectively reduce Tau aggregation in vitro and block aggregationdependent phenotypes in vivo, raising possibilities for exploring its translational potential.
... The evidence for neurotoxic assemblies formed from tau remains unclear [5]. Toxic tau oligomers have been described as having β-sheet secondary structure [35] while nontoxic, β-sheet negative tau oligomers have also been described [44,45]. To investigate and compare the toxicity of non-oxidised and DiY dGAE, we utilised differentiated human neuroblastoma SHSY5Y cells [14,46] and assessed the cell death using the ReadyProbes Cell Viability Imaging Kit. ...
... This may indicate that the DiY cross-linking facilitates the formation of non-toxic, off-pathway tau oligomers. Previous work has shown that the rescue of tau toxicity in Drosophila results in the formation of non-toxic tau oligomers lacking β-sheet [44]. Similarly, it has also been demonstrated that the inhibition of tau aggregation using phthalocyanine tetrasulfonate (PcTS) results in the formation of β-sheet negative, soluble tau oligomers [68]. ...
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The self-assembly of tau into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs) is a significant event in Alzheimer’s disease (AD) pathogenesis. Numerous post-translational modifications enhance or inhibit tau assembly into NFTs. Oxidative stress, which accompanies AD, induces multiple post-translational modifications in proteins, including the formation of dityrosine (DiY) cross-links. Previous studies have revealed that metal-catalysed oxidation (MCO) using Cu2+ and H2O2 leads to the formation of DiY cross-links in two misfolding proteins, Aβ and α-synuclein, associated with AD and Parkinson’s disease respectively. The effect of MCO on tau remains unknown. Here, we examined the effect of MCO and ultra-violet oxidation to study the influence of DiY cross-linking on the self-assembly of the PHF-core tau fragment. We report that DiY cross-linking facilitates tau assembly into tau oligomers that fail to bind thioflavin S, lack β-sheet structure and prevents their elongation into filaments. At a higher concentration, Cu2+ (without H2O2) also facilitates the formation of these tau oligomers. The DiY cross-linked tau oligomers do not cause cell death. Our findings suggest that DiY cross-linking of pre-assembled tau promotes the formation of soluble tau oligomers that show no acute impact on cell viability.
... The evidence for neurotoxic assemblies formed from tau remains unclear (5). Toxic tau oligomers have been described as having β -sheet secondary structure (37) while non-toxic, β -sheet negative tau oligomers have also been described (45). To investigate and compare the toxicity of non-oxidised and DiY dGAE, we utilised differentiated human neuroblastoma SHSY5Y cells (15,46) and assessed the effect on cell survival using the ReadyProbes cell viability assay. ...
... This may indicate that the DiY cross-linking facilitates the formation of non-toxic, off-pathway tau oligomers. Previous work has shown that the rescue of tau toxicity in Drosophila results in the formation of non-toxic tau oligomers lacking β -sheet (45). Similarly, it has also been demonstrated that the inhibition of tau aggregation using phthalocyanine tetrasulfonate (PcTS) results in the formation of β -sheet negative, soluble tau oligomers (67). ...
Preprint
The self-assembly of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs) is a significant event in Alzheimer s disease (AD) pathogenesis. Oxidative stress, which accompanies AD, induces multiple post-translational modifications in proteins including the formation of dityrosine (DiY) cross-links, previously observed for Abeta. Here, metal catalysed- and ultra-violet oxidation were used to study the influence of DiY cross-linking on the self-assembly of the PHF-core tau fragment. We report that DiY cross-linking facilitates tau assembly into tau oligomers and prevents their elongation into filaments. The DiY cross-linked tau oligomers were not toxic to cells. Our findings suggest that DiY cross-linking of pre-assembled tau, promotes the formation of soluble tau oligomers that show no acute toxicity.
... There are multiple other factors such as the role of phosphorylation [142,143] and liquid-liquid phase separation in tau oligomerization [144][145][146] that have to be taken into account to work towards achieving the identification of the true toxic tau species. While targeting toxic tau oligomers has been demonstrated to be effective in several pre-clinical studies [68,69,147], a caveat in specific targeting lies in need to disrupt or eliminate toxic tau oligomers, but not the non-toxic forms [148], which may aid in sequestering the toxic species. Advanced visualization and computational techniques such as machine learning-based classification [149] could further be developed and utilized to segregate these distinct tau species for more specific targeting. ...
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Tauopathies, including Alzheimer’s disease (AD), are a group of neurodegenerative disorders characterized by pathological aggregation of microtubule binding protein tau. The presence of tau neurofibrillary tangles, which are insoluble β-sheet fibrils, in the brain has been the histopathological hallmark of these diseases as their level correlates with the degree of cognitive impairment. However, recent studies suggest that tau oligomers, which are soluble proteins that are formed prior to insoluble fibrils, are the principal toxic species impairing neurons and inducing neurodegeneration. Targeting toxic tau oligomers is challenging, as they are mostly unstructured and adopting multiple conformations. The heterogeneity of tau oligomers is further illustrated by the different oligomeric species formed by various methods. The current models and technologies to study tau oligomerization represent important resources and avenues to push the forefront of elucidating the true toxic tau species. In this review, we will summarize the distinct tau oligomers generated using different strategies and discuss their conformational characteristics, neurotoxicity, relevance to pathological phenotypes, as well as their applications in drug discovery. This information will provide insights to understanding heterogeneous tau oligomers and their role as molecular targets for AD and related tauopathies.
... Aβ, Monomeric tau and Monomeric Aβ) that are commonly known to be present in brain slides are gathered from literature. [51][52][53] As demonstrated in Figure 5, Raman peak ranges are constructed by granting a shift of 5 wavenumbers for each biomarker representing important feature regions, the sum of which is utilized as the denominator for further calculations. Similarly, pinpointing the important feature ranges for the feature map as numerator is also desired. ...
... In light of these findings, it seems likely that the transition from toxic soluble tau forms into less toxic or inert insoluble aggregates may rescue the cell. Cowan et al. [208] found that inhibiting tau GSK-3β not only rescued neurons from tau-induced dysfunction but also produced insoluble tau oligomers similar to granular tau oligomers (GTOs). Importantly, these GTOs-like structures were present in neurons in which cytoskeletal integrity was preserved, proving that this form is non-toxic. ...
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Although the mechanisms of toxic activity of tau are not fully recognized, it is supposed that the tau toxicity is related rather not to insoluble tau aggregates but to its intermediate forms. It seems that neurofibrillar tangles (NFTs) themselves, despite being composed of toxic tau, are probably neither necessary nor sufficient for tau-induced neuronal dysfunction and toxicity. Tau oligomers (TauOs) formed during the early stages of tau aggregation are the pathological forms that play a key role in eliciting the loss of neurons and behavioral impairments in several neurodegenerative disorders called tauopathies. They can be found in tauopathic diseases, the most common of which is Alzheimer’s disease (AD). Evidence of co-occurrence of b-amyloid, α-synuclein, and tau into their most toxic forms, i.e., oligomers, suggests that these species interact and influence each other’s aggregation in several tauopathies. The mechanism responsible for oligomeric tau neurotoxicity is a subject of intensive investigation. In this review, we summarize the most recent literature on the damaging effect of TauOs on the stability of the genome and the function of the nucleus, energy production and mitochondrial function, cell signaling and synaptic plasticity, the microtubule assembly, neuronal cytoskeleton and axonal transport, and the effectiveness of the protein degradation system.
... It is interesting that, although blocking Cys-291 does not appear to affect the conformation or aggregation propensity of Tau, the mutant is less toxic than wt protein, and its accumulation in the insoluble fraction does not result in memory deficits. A possible explanation will be that the C291A mutation leads to aggregated forms of the protein with distinct structural characteristics and reduced toxicity compared with wt Tau (Cowan et al., 2015;Shammas et al., 2015). This again demonstrates the complex interplay of Tau PTMs (i.e., differences in phosphorylation) underpinning the differences in toxicity and dysfunction described here. ...
Article
Although Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease (AD) and other Tauopathies, the mechanism that initiates the aggregation of this highly soluble protein in vivo remains largely unanswered. Interestingly, in vitro Tau can be induced to form fibrillar filaments by oxidation of its two cysteine residues, generating an intermolecular disulfide bond that promotes dimerization and fibrillization. The recently solved structures of Tau filaments revealed that the two cysteine residues are not structurally equivalent since Cys-322 is incorporated into the core of the fibril whereas Cys-291 projects away from the core to form the fuzzy coat. Here, we examined whether mutation of these cysteines to alanine affects differentially Tau mediated toxicity and dysfunction in the well-established Drosophila Tauopathy model. Experiments were conducted with both sexes, or with either sex. Each cysteine residue contributes differentially to Tau stability, phosphorylation status, aggregation propensity, resistance to stress, learning and memory. Importantly, our work uncovers a critical role of Cys-322 in determining Tau toxicity and dysfunction.SIGNIFICANCE STATEMENTCysteine-291 and Cysteine-322, the only two cysteine residues of Tau present in only 4-Repeat or all isoforms respectively, have competing functions: as the key residues in the catalytic center, they enable Tau auto-acetylation, and as residues within the microtubule-binding repeat region are important not only for Tau function but also instrumental in the initiation of Tau aggregation. In this study, we present the first in vivo evidence that their substitution leads to differential consequences on Tau's physiological and pathophysiological functions. These differences raise the possibility that cysteine residues play a potential role in determining the functional diversity between isoforms.
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Background: As tau aggregation pathology correlates with clinical dementia in Alzheimer's disease (AD), a tau aggregation inhibitor (TAI) could have therapeutic utility. Methylthioninium (MT) acts as a selective TAI in vitro and reduces tau pathology in transgenic mouse models. Objective: To determine the minimum safe and effective dose of MT required to prevent disease progression on clinical and functional molecular imaging outcomes. Methods: An exploratory double-blind, randomized, placebo-controlled, dose-finding trial of MT (69, 138, and 228 mg/day) was conducted in 321 mild/moderate AD subjects. The primary outcome was change on the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) at 24 weeks relative to baseline severity. Effect of treatment on regional cerebral blood flow decline was determined in a sub-study in 135 subjects. After 24 weeks, subjects were re-consented to enter sequential 6- and 12-month blinded extension phases. Registered with ClinicalTrials.gov (NCT00515333). Results: At 24 weeks, there were significant treatment benefits in two independent populations at the 138 mg/day dose: in moderate subjects on the ADAS-cog scale (treatment effect: -5.42 units, corrected p = 0.047) and two other clinical scales; in mild subjects on the more sensitive regional cerebral blood flow measure (treatment effect: 1.97%, corrected p < 0.001). With continued treatment for 50 weeks, benefit was seen on the ADAS-cog scale in both mild and moderate subjects. The delivery of the highest dose was impaired due to dose-dependent dissolution and absorption limitations. Conclusion: The minimum safe and effective daily MT dose is 138 mg and suggests that further study of MT is warranted in AD.
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