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Near Infrared Light Treatment Reduces Synaptic Levels of Toxic
TauOligomers in Two Transgenic Mouse Models of Human
Tauopathies
Michele M. Comerota1 & Batbayar Tumurbaatar1 & Balaji
Krishnan1 & Rakez Kayed1 & Giulio Taglialatela1
Received: 8 May 2018 /Accepted: 15 July 2018 /Published online:
17 August 2018# The Author(s) 2018
AbstractTau oligomers are emerging as a key contributor to the
synaptic dysfunction that drives cognitive decline associated with
theclinical manifestation and progression of Alzheimer’s disease
(AD). Accordingly, there is ample consensus that interventions
thattarget tau oligomers may slow or halt the progression of
AD.With this ultimate goal in mind, in the present study, we
investigatedtau oligomer accumulation and its synaptic and
behavioral consequences after an in vivo treatment with near
infrared (NIR) light(600–1000 nm) in two transgenic mouse models,
overexpressing human tau either alone (hTau mice) or in combination
withamyloid beta (3xTgAD mice). We found that a 4-week exposure to
NIR light (90 s/day/5 days a week) significantly reducedlevels of
endogenous total and oligomeric tau in both synaptosomes and total
protein extracts from the hippocampus and cortex ofhTau mice and
improved deteriorating memory function. Similar results were
observed in the 3xTgAD mice, which furtherdisplayed reduced
synaptic Aβ after NIR light treatment. On the other hand, ex vivo
binding of tau oligomers in isolatedsynaptosomes as well as tau
oligomer-induced depression of long-term potentiation (LTP) in
hippocampal slices from NIRlight-treated wt mice were unaffected.
Finally, levels of proteins critically involved in two mechanisms
associated with clearanceof misfolded tau, inducible HSP70 and
autophagy, were upregulated in NIR light treated mice.
Collectively, these results showthat NIR light decreases levels of
endogenous toxic tau oligomers and alleviate associated memory
deficits, thus furthering thedevelopment of NIR light as a possible
therapeutic for AD.
Keywords Near infrared light . Tau oligomers . hTaumouse .
3xTgADmouse . Autophagy
Introduction
Alzheimer’s disease (AD) is the most frequent age-related
de-mentia, for which there is currently no resolving cure. The
mul-tifactorial nature of AD has contributed to the ongoing
challengeof developing effective disease-modifying therapeutics.
The ac-cumulation of plaques and neurofibrillary tangles
(NFTs)consisting of amyloid beta (Aβ) and hyperphosphorylated
tauprotein, respectively, are two quintessential hallmarks of
AD.However, many factors such as mitochondrial dysfunction,
neu-roinflammation, impaired clearance of dysfunctional
proteins,and synaptic retraction contribute to the disease
progression[1–4]. Among those factors, synaptic dysfunction is
believed tounderlie the onset and progression of the cognitive
impairment
that characterizes the symptomatic phase of AD [5].
Emergingevidence suggests that the small soluble oligomeric
aggregateforms of tau contribute to the dysfunction of the synapses
byacting both intracellularly and extracellularly [6, 7].
Indeed,while accumulations of NFTs have been found to strongly
cor-relate with the decline of cognitive function [8, 9],
compellingevidence points at tau oligomers as the most toxic form
of tauaggregates [10, 11]. After the hyperphosphorylation of the
tauprotein, aggregates mislocalize from the axonal to
thesomatodendritic region [12]. This increased concentration
ofsynaptic tau oligomers interferes with the process of
synaptictransmission [6]. In addition, tau oligomers released to
the extra-cellular space have also been suggested to act on the
postsynapticregions resulting in increased intracellular calcium
[13] and im-paired long-term potentiation (LTP) [7]. Further, tau
oligomersact in a prion-like manner, seeding the misfolding and
aggrega-tion of cellular monomeric tau and resulting in the
spreading ofthe disease from cell to cell [14]. Together, this
evidence suggeststhat the targeting of tau oligomer accumulations,
specifically atthe synapses, will mitigate the synaptic dysfunction
and the pro-gression of clinical manifestation of AD.
* Giulio [email protected]
1 University of Texas Medical Branch, 301 University
Blvd.,Galveston, TX 77555-1045, USA
Molecular Neurobiology (2019)
56:3341–3355https://doi.org/10.1007/s12035-018-1248-9
http://crossmark.crossref.org/dialog/?doi=10.1007/s12035-018-1248-9&domain=pdfmailto:[email protected]
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With this ultimate goal in mind, we investigated the
adminis-tration of non-invasive, transcranial near infrared (NIR;
600–1000 nm) light as a potential treatment for AD in relevant
trans-genic mouse models. Previous studies have found a reduction
ofhyperphosphorylated tau and NFTs in NIR light-treated K369Imice
[15], a model of frontotemporal dementia [16]. In addition,we and
others have previously shown a direct effect of NIR lightin
reducing pathological Aβ accumulations in transgenic mousemodels of
APP overexpression and further described increasedsynaptic
resilience to the binding and dysfunctional impact Aβoligomers in
NIR light-treated mice [15, 17–19]. Despite thissuggestive initial
evidence, however, the effect of NIR light onlevels of total and
synaptic tau oligomers as well as synaptic tauoligomer-induced
functional changes remains unknown. In thepresent study, we aimed
to determine whether NIR light couldpromote the reduction of
synaptic accumulation of tau in two tgmouse models of human
tauopathies (hTau and 3xTgAD) invivo and thus offer neuroprotection
against the dysfunctionaltau oligomer synaptic binding. We further
investigatedautophagy-related proteins and the chaperone inducible
heatshock protein 70 (HSP70) as possible mechanisms
mediatingclearance of tau in response to NIR light treatment. We
showthat, when applied to either of these transgenic mouse models
ofhuman tauopathies, NIR light effectively clears toxic tau
oligo-mers, both from the CNS parenchyma and the synapse,
andrestores memory functions in these impairedmice. Overall,
thesenovel results illustrate a direct effect of NIR light in
clearing theCNS from toxic tau oligomers, thus supporting the
notion thatNIR light should be further explored as a non-invasive
therapeu-tic strategy in AD and related tauopathies.
Results
Reduced Tau Pathology in Cortical and HippocampalTotal Protein
Extracts and Synaptosomal Fractionsof NIR Light-Treated
13-Month-Old hTau Mice
We aimed to determine if NIR light reduces the in vivo
synapticaccumulation of endogenous tau oligomers by utilizing the
trans-genic human tau mouse model, hTau. This
well-characterizedmouse model develops tau aggregates, including
abundant tauoligomers, around 9–10 months of age [20, 21].
Therefore, toensure adequate tau accumulation prior to our
intervention, webegan NIR light treatment at 12months of age (n=7;
per group).The total protein extracts and the isolated synaptosomal
fractionsof the cortex and hippocampus regions were analyzed
byWestern blot (Fig. 1a–c, Fig. 2a–c; respectively), ELISA (Fig.1d,
Fig. 2d; respectively), and fluorescence immunohistochem-istry
(IHC) (Fig. 1e, f) to qualitatively and quantitatively deter-mine
levels of tau oligomers and total tau (including both mono-meric
and oligomeric species). Tau oligomerswere quantitativelymeasured
by densitometry analysis of Western blot bands of
110 kDa and higher (Fig. 1a–c) as detected by the total
tauantibody, tau5. In the cortical and hippocampal total protein
ex-tracts, we found a decrease in tau oligomers in NIR
light-treatedhTau mice compared to sham-treated control mice
(cortexp=0.016, hippocampus p=0.049). We further analyzed the
totaltau levels in the total protein extracts utilizing a tau5
ELISAanalysis. As shown in Fig. 1d, there was a statistically
significantdecrease in the total tau in both the cortex and
hippocampus(cortex p=0.049, hippocampus p=0.049). Finally, these
resultswere confirmed by fluorescence IHC analysis of the
hippocam-pus and cortex regions using antibodies specific for tau
oligo-mers, T22 and total tau, tau5. The immunofluorescence
furtherverified a reduction of both total tau (cortex p=0.002,
hippocam-pus p=0.032) and tau oligomers (cortex p=0.003,
hippocampusp=0.045) in both regions (Fig. 1e, f). We then analyzed
thelevels of total and oligomeric tau in the synaptosomal
fractionsby Western blot and ELISA analysis to determine whether
thereduction of tau observed in the total protein extracts
wasparalleled by a similar reduction at the synaptic
compartment.Due to technical challenges in imaging simultaneously
all formsof tau in the synaptosomal regions of the hTau mice on the
sameWestern blot, two different exposures were taken to
properlyvisualizemonomeric tau (low exposure) and oligomeric tau
(highexposure) (Fig. 2a, b). Tau oligomer levels were reduced in
syn-aptosomal fractions of both cortex and hippocampus of
NIRlight-treated hTau mice compared to the sham-treated mice
(cor-tex p=0.049, hippocampus p=0.049) (Fig. 2c). Further, levelsof
total tau, asmeasured by tau5 ELISA,were also reduced in
thecortical and hippocampal synapses of thesemice (Fig. 2d)
(cortexp =0.024, hippocampus p = 0.003). Collectively, these
resultssuggest a systemic, as well as, synaptic reduction of total
andoligomeric tau in hTau transgenic mice treated with NIR
light.
Rescue of Impaired Long-Term Memory in Aged NIRLight-Treated
hTau Mice
Because of the reduction in tau oligomers observed after
NIRlight treatment in hTau mice, we next aimed to determine ifsuch
reduction would translate into a functional benefit in thesemice.
To determine if the cognitive function improved in hTaumice treated
with NIR light, we performed the novel objectrecognition (NOR)
paradigm immediately following the lastNIR light treatment. During
the training phase, the mice wereallowed to freely explore for 10
min two identical objectsplaced in the testing arena. We found no
difference betweenhTau mice treated or not with NIR light in the
time spent ex-ploring the objects during the training phase, which
was equallysplit between the two identical objects (Fig. 3a). To
determinelong-termmemory, 24 h after the training phase was
completed,one of the two objects was replaced by a novel object and
themice were again allowed to freely explore the objects. Based
onthe propensity of mice to spend more time exploring an objectthey
have not explored before, an extended amount of time
3342 Mol Neurobiol (2019) 56:3341–3355
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exploring the novel object reflects memory of the familiar
ob-ject. The object discrimination ratio (ODR) was calculated
todetermine the percentage of time spent with the novel object(Fig.
3). We found the animals treated with NIR light had anODR of about
0.8 (one sample t test, p=0.0001) of the novelobject indicating an
increased exploration of the novel objectcompared to the familiar
object. On the other hand, the hTaumice receiving no treatment had
a ODR of about 0.48 (onesample t test, p=0.765), indicating no
difference in the explo-ration time between the novel and familiar
object and thusreflecting memory impairments as previously
described in agedhTau mice [22]. This suggests a recovery of
impaired memoryfunction hTau mice that received the NIR light
treatment.
Reduced Tau and Aβ Pathology in the Cortexand Hippocampus of NIR
Light-Treated 13-Month Old3xTgAD Mice
In order to determine if NIR light treatment results in
areduction of both Aβ and tau when they are co-expressedin a
combined endogenous system, we investigated chang-es in both Aβ and
tau oligomers at the synapses and in totalprotein extracts of
3xTgAD mice. The 3xTgAD mice
model exhibit overexpression of three AD-relevant humangenes;
human APP bearing the Swiss mutation, human tauwith a P301L
mutation, and presenilin-1 with the M146Vmutation, resulting in
extensive aggregated Aβ and taupathology around 12 months of age
[23]. We thereforebegan the 4 week-long NIR light treatment at 12
monthsof age to ensure that accumulations of both toxic proteinshad
already occurred when treatment was applied (n = 7;per group). We
first employed Western blot, ELISA, andfluorescence IHC analyses to
determine levels of total andoligomeric tau in total protein
extracts from the corticaland hippocampal regions of 3xTgAD mice
receiving NIRlight treatment (Fig. 4). Densitometry analysis of the
bands> 110 kDa (tau oligomers) detected in Western blots by
thetau5 antibody (Fig. 4a, b) revealed a statistically
significantdecrease of tau oligomers in both brain regions of the
NIRlight-treated mice as compared to the sham treated
animals(cortex p = 0.001, hippocampus p = 0.047) (Fig. 4c).
Wefurther measured total tau levels using the tau5 ELISAand found a
similar decrease in both brain regions of theNIR light-treated mice
(cortex p = 0.038, hippocampusp = 0.049) (Fig. 4d). We further used
fluorescence IHC toconfirm the reduction of tau oligomers, using
the tau
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oligomer specific antibody T22, and total tau, using thetotal
tau antibody tau5 (Fig. 4e, f). We found a decreaseof both
oligomeric and total tau in the hippocampus(p = 0.001, p = 0.045)
and cortex (p = 0.007, p = 0.045) ofNIR light-treated mice as
compared to the sham treated.We next measured total and oligomeric
tau levels in thesynaptosomal fractions of NIR light-treated 3xTgAD
miceby Western blot and ELISA. Western blot analysis revealeda
decrease in the levels of oligomeric tau in the cortex(Fig. 5a, c)
and hippocampus regions (Fig. 5b, c) (cortexp = 0.009, hippocampus
p = 0.038). In addition, a signifi-cant decrease of total tau
levels in the synaptosomal frac-tions was observed in the cortex
and the hippocampus(cortex p = 0.039, hippocampus p = 0.015) of NIR
light-treated mice, as measured by tau5 ELISA (Fig. 5d).Finally, we
employed a Aβ1–42-specific ELISA to measurethe levels of Aβ in the
synaptosomal fractions and totalprotein extracts from the cortex
and hippocampus. Asshown in Fig. 6a, Aβ levels were reduced in the
synapto-somal fractions from the cortex and hippocampus regionsof
NIR light-treated mice as compared to sham animals(cortex p =
0.002, hippocampus p = 0.040). However, thelevels of Aβ in the
total protein extracts were unchangedin the NIR light-treated group
compared to the sham-
treated group (cortex p = 0.495, hippocampus p = 0.145)(Fig.
6b). Collectively, these results show a similar de-crease in the
tau and Aβ levels induced by the NIR lighttreatment in the 3xTgAD
mice.
NIR Light Treatment Does Not Affect Ex Vivo SynapticBinding of
Tau Oligomers
Recent studies have indicated that extracellular tau
oligomersplay a key role in the induction of synaptic dysfunction
[7]. Inthe current study, we aimed to determine if NIR light
induces areduction of synaptic vulnerability to tau oligomers thus
pre-serving cognitive function. We performed an ex vivo tau
bind-ing study in which synaptosomes isolated from wild-typemice
that received either NIR light or sham treatment (n= 7,per group)
were exposed to 50 nM of tau oligomers for 1 h, asdescribed in the
BMethods^ section. After washing off un-bound oligomers, the
remaining levels of tau protein boundto synaptosomes were measured
by ELISA analysis. Wefound that both cortical and hippocampal
synaptosomes fromNIR light-treated mice bound similar levels of
exogenouslyadded tau oligomers as compared to the sham-treated
mice(cortex p = 0.514, hippocampus p = 0.867) (Fig. 7). This
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tau oligomer levels
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(total tau)
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(total tau)
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suggests that NIR light treatment does not affect the
suscepti-bility of the synapses to the association with tau
oligomers.
Suppression of Hippocampal Long-Term Potentiationby Tau
Oligomers Is Not Reversed by NIR LightTreatment
Although we found that NIR light treatment induced nochanges in
synaptic susceptibility to tau oligomer binding,we aimed to
determine if NIR light provides protection againsttau
oligomer-induced synaptic dysfunction. Hippocampalslices prepared
from wild-type mice that were exposed toNIR light or sham treatment
were added with 50 nM of tauoligomers (n=5 per treatment group, two
slices per conditionfrom each animal), a concentration known to
impair long-termpotentiation (LTP) [7] and determine the extent of
LTP expres-sion after high-frequency stimulation in the Schaffer
collateralpathway (Fig. 8). The last 10 min of LTP were averaged
foreach treatment group (Fig. 8b) and evaluated for
statisticaldifferences among groups. Consistent with the
literature, wefound a statistically significant reduction in the
magnitude ofLTP in the slices from sham-treated mice exposed to 50
nM oftau oligomers (p= 0.001). We also found a reduction in
themagnitude of LTP in slices prepared from the NIR light-treated
group exposed to tau oligomers compared to similarslices that
however were not exposed to tau oligomers(p=0.01). We further found
that there was no difference be-tween the NIR light-treated and
untreated groups in the extentof the reduction of LTP induced by
tau oligomer exposure(p>0.05). Lastly, the basal synaptic
strength, as measure byinput-output curves, was not changed in any
of the treatmentgroup (data not shown). Collectively, these results
suggest thatNIR light treatment does not affect vulnerability of
synapsesto tau oligomer-induced LTP impairments.
Increased Inducible HSP70 at the Synapses of NIRLight-Treated
3xTgAD, hTau, and Wild-Type Mice
The results of our experiments suggested that NIR light
effec-tively induces a significant reduction of the levels of both
totaland oligomeric tau, suggesting increased clearance. To
inves-tigate a potential mechanism for this putative clearance,
wemeasured heat shock protein 70 (HSP70) levels in
wild-type,3xTgAD, and hTau mice after NIR light treatment. We
electedto investigate HSP70 because previous studies have
describedthe intimate relationship between upregulation of
inducibleHSP70 and the reduction of tau in animal models [24].
Weutilized Western blot analysis to measure the levels of
induc-ible HSP70 as well as its constitutive isoform, heat
shockcognate 70 (HSC70), in the synaptosomal fractions and
totalprotein extracts from brains of mice exposed to a
4-weektreatment with NIR light. We found that in all three
animalmodels, there was an increase of HSP70 levels in the
synap-tosomal fractions (Fig. 9a–c) ((a) p= 0.049, (b) p=0.034,
(c)p=0.029) but no change in the total protein extract (Fig.
9d–f)((d) p=0.651, (e) p=0.127, (f) p=0.844). On the other
hand,there was no change in the levels of the constitutively
activeHSC70 in either the total protein or the synaptosome
fractionsof the NIR light treated mice ((a) p=0.664, (b) p=0.143,
(c)p= 0.268, (d) p= 0.401, (e) p= 0.084, (f) p= 0.275).
Theseresults indicate a selective increase of the inducible HSP70at
the synapse in NIR light-treated mice.
Increased Levels of Autophagy Markers in NIRLight-Treated
3xTgAD
To further gain an understanding of the contributing mech-anisms
to the NIR light-induced reduction of tau in thetransgenic mice
models, we measured the protein andmRNA levels of LC3A and B, and
Atg5, proteins that areknown to be key in the initiation of
autophagy [25]. Wefound that the levels of LC3B were increased (p =
0.001) inthe NIR light-treated mice (n = 8) compared to
sham-treated mice (n = 10), whereas the levels of LC3Aremained
unchanged (0.992) between the treatment groups(Fig. 10a). The
resulting increased ratio between LC3Band LC3A (p = 0.008) in the
NIR light-treated group sug-gests an increased promotion of
autophagosome formation[26]. In addition, the mRNA levels of LC3
were also in-creased (Fig. 10b). The protein expression levels of
Atg5showed a trend of increase in NIR light-treated mice
thathowever did not reach statistically significance (Fig. 10c).On
the other hand, Atg5 mRNA levels were significantlyincreased (p =
0.044) in the NIR light-treated 3xTgAD(Fig. 10d). These results
suggest NIR light-treated3xTgAD have increased expression of
proteins that con-tribute to the induction of autophagy.
Fig. 7 Synaptic binding of tau oligomers is not altered in NIR
light-treatedWTmice. ELISA analysis was conducted on the ex vivo
challengeof synaptosomes isolated from the cortex and hippocampus
of NIR light-and sham-treated wild-type mice with 50 nM of tau
oligomers (n=8; pergroup). Statistical significance was determined
by Student’s two-tailed ttest analysis. Error bars represent
standard deviation. n.s. represents notstatistically
significant
Mol Neurobiol (2019) 56:3341–3355 3347
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Discussion
Impairment of synaptic function is a key event that initiatesthe
progressive cognitive decline that is associated with clin-ically
manifest AD [5, 27]. The oligomeric form of
themicrotubule-associated protein tau is emerging as a key
con-tributor to the disruption of synaptic function in AD
[28–30].The main goal of our study was to determine if NIR
lightinduces neuroprotection against the synaptic association
andaccumulation of the toxic tau oligomers, as well as the
subse-quent oligomer-driven dysfunction. We first aimed to
deter-mine if the administration of NIR light treatments in
twomouse models of tauopathies, hTau (accumulation of tau)and
3xTgAD (accumulation of Aβ and tau), would reducethe synaptic
accumulation of tau oligomers. Because of thecomplex relationship
between Aβ and tau with extensive ev-idence that these proteins can
influence the pathology of each
other, it is critical to examine the proteins in a combinedmouse
model, as well as, in a simpler mouse model wheretau is singly
overexpressed. Previous research has found ageneral reduction of
hyperphosphorylated tau in theParkinsonian mouse model K369I upon
exposure to NIR light[15]. However, whether this NIR light-induced
reduction oftau would hold true for tau oligomers and their
synaptic accu-mulation (a major determinant of tau neurotoxicity)
remainedunexplored. In physiological conditions, tau is localized
to theaxonal region of neurons with low levels in the synaptic
re-gions contributing to the stability of protein
scaffolding.However, in AD, tau relocalizes to the somatodendritic
region.The elevated levels of tau oligomers at the synapses are
be-lieved to contribute to the interference of synaptic
function[12, 31, 32]. The hTau mice, a mouse that expresses a
humanMAPT transgene and knockout of mouse MAPT, served as amodel
that exclusively expresses the accumulation of tau
Fig. 8 Tau oligomer induced impairment of LTP is not altered in
NIRlight-treated mice. The long-term potentiation (LTP) was
measured bySchaffer collateral field recordings to determine the
impact of NIR lighttreatment on tau oligomer induced impairments in
wild-type mice. a ThefEPSP amplitude was calculated for the four
groups; NIR light treatedwith and without tau oligomers and sham
treated with and without tau
oligomers. (n= 5; per group, two slices per condition). b For
each group,the calculated fEPSP of the final 10 min of recording
was averaged. One-way ANOVAwith Dunn’s post hoc analysis was used
to determine sta-tistical significance. Error bars represent ±
standard error of mean.*p
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protein [20]. Our results showed not only a reduction of
tauoligomer levels at the cortex and hippocampus synapses ofNIR
light-treated animals but also in the total protein extracts.We
further found that total tau levels, which include mono-mers, as
well as all aggregated tau species, were also de-creased in both
the synapses and total protein extracts.While previous studies have
suggested the knockout of tauin mice can result in degeneration of
the axons [33], the de-creased observed in this study does not
eliminate all tau sug-gesting that an impairment to the structural
system of theneurons will not be greatly impacted. In addition,
this decreaseseems to include monomeric tau, which can act as seeds
forthe formation of oligomeric tau suggesting this decrease intotal
tau may be beneficial [34]. Thus, unlike our previousresults in
which we found that NIR light induces a selectivereduction of Aβ
oligomers at the synapses in the human
amyloid precursor protein (APP) overexpressing mouse mod-el,
Tg2576 [19], the tau reduction in the hippocampus andcortex of NIR
light-treated hTau mice that we observed inthe present study is not
limited to the synaptic region, butrather occurs throughout the
brain parenchyma. Nonetheless,this molecular phenomenon of overall
reduced tau oligomerstranslates into a functional benefit as
illustrated by the ob-served memory improvement in the novel object
recognition(NOR) test of hTaumice that received the NIR light
treatment.This mouse model is known to have memory deficits as
mea-sured by NOR at 10 months of age [22]. The NIR
light-treatedhTau displayed an improved performance compared to
thesham-treated hTau mice, as measured by the increased timespent
with the novel object. This test indicates that the NIRlight
treatment ameliorated the cognitive impairment typicallydisplayed
by hTau mice at this age. Collectively, our data
HSC70
3xTgAD synaptosome fraction 3xTgAD total protein extract
Wild type synaptosome fraction Wild type total protein
extract
htau synaptosome fraction htau total protein extract
HSP70 HSP70
β tubulin β tubulin
HSC70HSC70
HSP70
HSP70
HSC70
β tubulin
β tubulin
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HSP70
HSP70
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HSC70HSC70
NIR Light treatment
NIR Light treatment
NIR Light treatment
NIR Light treatment
NIR Light treatment
Sham treatment
Sham treatment
Sham treatment
Sham treatment
Sham treatment
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synaptosomes
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treatment
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HSC70 levels in WT
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xp
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HSP70 levels in WT total
protein extract
Sham
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NIR Light
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HSC70 levels in WT
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SP
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HSP70 levels in htau
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treatment
0
500
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0 e
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HSC70 levels: htau
synaptosomes
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treatment
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treatment
0
100
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HS
P7
0 e
xp
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HSP70 levels in htau
total protein extract
Sham
treatment
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treatment
0
700
1400
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HS
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0 e
xp
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HSC70 levels in htau
total protein extract
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treatment
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treatment
0
200
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600
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HS
P7
0 e
xp
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U)
HSP70 levels: 3xTgAD
synaptosomes
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treatment
NIR Light
treatment
0
1000
2000
HS
C7
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xp
re
ssio
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leve
l (A
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HSC70 levels: 3xTgAD
synaptosomes
Sham
treatment
NIR Light
treatment
0
1000
2000
HS
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ssio
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l (A
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HSC70 levels: 3xTgAD
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HSP70 levels: 3xTgAD
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Sham
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*
*
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show that NIR light treatment reduced the tau pathology andthe
corresponding memory deficits in hTau mice.
The second mouse model we utilized, 3xTgAD,
exhibitsoverexpression of three genes associated with familial
AD;presenilin 1, human tau with the P301L mutation, and humanAPP
with the Swedish mutation. These mice display depositsof both
aggregated Aβ and tau by 12 months of age, servingas a model for
the simultaneous presence of these toxic pro-teins as observed in
AD [23]. We found that both total andoligomeric tau was reduced in
the synaptic compartment aswell as in the total protein extract in
3xTgAD mice, whereasthe Aβ was reduced exclusively at the synapses.
This is sim-ilar to what we observed here in the hTau mice, as well
as wepreviously reported in the Tg2576 mice [19]. Our results
thussuggest that the NIR light-induced mitigation of Aβ and
taupathology is sufficient and equally effective when the
twoamyloid proteins coexist in a system, supporting NIR lightas a
promising treatment for AD.
We next aimed to determine if NIR light induces neuropro-tective
synaptic resistance to the association of extracellulartau
oligomers and tau oligomer-induced synaptic dysfunction.We utilized
an ex vivo binding approach to determine if syn-aptosomes isolated
from NIR light-treated wt mice displayedan altered affinity to
exogenously applied tau oligomers. Theresults showed an equivalent
tau association to the synapsesisolated from the NIR light-treated
group compared to thesham-treated mice.
We previously reported that NIR light treatment in wt
micereduced synaptic vulnerability to Aβ oligomers, both in termsof
binding and Aβ oligomer-induced suppression of hippo-campal LTP
expression [19]. Therefore, in the present studies,we further
measured LTP in the Schaffer collateral pathway of
the hippocampus after the application of tau oligomers tobrain
slices prepared from these NIR light-treated wt mice.Application of
tau oligomers to brain slices has previouslybeen shown to induce a
significant impairment of hippocam-pal LTP induction/expression
[7]. As in the case of the tauoligomer synaptic binding experiments
described earlier, in-cubation of brain slices with tau oligomers
evoked a similarimpairment in LTP expression in the NIR
light-treated groupand the sham. Contrary to what we previously
observed forAβ oligomers, these results suggest that NIR light does
notinduce mechanisms that reduce the synaptic vulnerability toand
functional impact of tau oligomers. This could indicatethat tau and
Aβ oligomers act on the synapses in differingways. While Aβ
oligomers are known to have multiple syn-aptic binding partners
such as mGluR5 and α7-nicotinic ace-tylcholine receptors among
others [35, 36], the mechanismsby which extracellular tau oligomers
associate with the syn-apses remain elusive. Previous studies have
implied that thesimilar structures of Aβ and tau oligomers
contribute to sim-ilar mechanisms of toxicity [37]. However, our
combinedstudies suggest that NIR light alters mechanisms that
exclu-sively contribute to Aβ oligomer synaptic association.
While the primary mechanisms of action of the NIR light
instimulating bioenergy output and efficiency of mitochondriahas
been well established, the secondary mechanisms that leadto
neuroprotection remains poorly understood [38]. The sys-temic
reduction of both total and oligomeric tau in NIR light-treated
hTau mice that we report here suggests that NIR lightinitiates
mechanisms that contribute to the regulation and theclearance of
tau. In the current study, we investigated the chap-erone HSP70 and
proteins involved the induction of autophagyas two known pathways
that have been reported to promote the
Fig. 10 Upregulation of autophagy markers in total protein
extracts afterNIR light treatment. Protein and mRNA levels of
autophagy markers in thetotal protein extracts of NIR light (filled
columns) (n=9) and sham treated(open columns) (n=7) 3xTgAD mice as
determined by Western blot
analysis and RT-PCR. a Representative Western blot detecting
LC3A andLC3B proteins and quantitative densitometry analysis of the
LC3B/A ration.b LC3 and cAtg5 mRNA expression levels as measured by
RT-PCR. Errorbars represent standard deviation. *p
-
degradation of dysfunctional tau [2, 39]. The HSP70 family
ofproteins are chaperones that are involved in the refolding
andshuttling of dysfunctional proteins to degradation pathways[40].
There is extensive literature describing the intimate rela-tionship
between inducible HSP70 and the clearance of thetoxic tau proteins.
Particularly, evidence demonstrates the re-duction of aggregated
tau after stimulating the activity of oroverexpressing endogenous
inducible HSP70 or following ad-ministration of exogenous inducible
HSP70 [39, 41, 42]. Onthe other hand, an opposite correlation
exists between tau andheat shock cognate 70 (HSC70), a
constitutively expressedmember of the HSP70 family, whereby HSC70
overexpressionslows the clearance of misfolded tau deposition [24].
The ob-served exclusive synaptic increase of inducible HSP70 and
un-changed levels of HSC70 after NIR light treatment that
weobserved in all animal models surveyed here suggests specific-ity
of the phenomenon. Such increase could indicate a specificinduction
of HSP70 in neurons or possibly reflect therelocalization of
inducible HSP70 to the synapses as a meansof protecting the
compartment from future insults. We furtherinvestigated the
autophagy pathway, one of the pathways thatHSP70 has been shown to
shuttle dysfunctional tau to [43]. Theincrease in expression levels
of the autophagy-related proteinAtg5 and the raised ratio between
LC3A and LC3B in the totalprotein extracts in NIR-treated 3xTgAD
mice suggests an in-creased induction of autophagy as reflected by
increased pro-duction of autophagosomes. Atg5 is associated with
the elon-gation of the autophagosomal membrane and the LC3B
proteinis necessary for the formation and closure of
theautophagosome [25]. The increased expression of these pro-teins
thus demonstrates the increased availability of importantmachinery
involved in the degradation of dysfunctional tau inNIR
light-treated animals, providing the opportunity for theincreased
clearance of the toxic protein. Together, these resultsprovide
insight into chaperone-mediated clearance and inducedautophagy
mechanisms that may be contributing to NIR light-induced reduction
of tau. Many future studies can be performedto further the
understanding of the application of NIR lighttreatment for AD. The
investigation of other characteristics ofAD pathology including
neuroinflammation can provide agreater insight into the beneficial
effects of NIR light treat-ments. In addition, alternative
strategies for administration ofNIR light can also be investigated
to optimize the use of thetreatment in human patients.
In conclusion, this study provided valuable evidence of
thebeneficial effects of NIR light treatments on the reduction
oftau pathology and related cognitive dysfunction. The
noveldemonstration of this reduction of toxic tau species in
thehTau mice, combined with the decrease of synaptic Aβ pa-thology
in the Aβ/tau co-expressing 3xTgAD mice, furthersupport the
effectiveness of NIR light as a non-invasive treat-ment to reduce
AD-related neuropathology and encourages itsfuture clinical
development.
Methods
Animals
Male and female hTau mice were utilized to measure in vivolevels
of total tau and tau oligomers at the synapses and in thetotal
protein extracts of the cortex and hippocampus regions(n=6, per
experimental group). Female 3xTgAD (hAPP, tauP301L, presenilin-1)
mice were used to measure Aβ and taulevels at the synapses and
total protein extracts (n= 7, perexperimental group). Further,
3xTgAD mice were used todetermine the induction of autophagy after
NIR light treat-ment. Male 3xTgAD were not included in this study
due tothe known extensive variation in pathology development inthe
3xTgAD male mice. The transgenic mice were 12-month-old at the
initiation of the NIR light treatment schedule.Biochemical analysis
was completed at the conclusion of themonth-long treatment when the
mice were 13-month-old.C57BL/6 wild-type male and female mice were
employed todetermine the ex vivo synaptic binding of tau oligomers,
aswell as the electrophysiological properties of brain slices
fromNIR light-treated mice challenged with ex vivo administrationof
tau oligomers (n=5, per experimental group, two slices peranimal
per experimental condition).
The experimental protocols performed in this study wereapproved
and performed in accordance with the InstitutionalAnimal Care and
Use Committee of the University of TexasMedical Branch. All animals
were housed under USDA stan-dards (12:12 h light dark cycle, food
and water ad libitum) atthe UTMB vivarium.
NIR Light Treatments
The NIR light treatments were performed in the same manneras
previously described [19]. Briefly, a 90-s preprogramed,670 nm
wavelength light-emitting diode (LED) device,WARP10
(QuantumDevices, Barneveld,WI, USA), was heldapproximately 1 cm
over from the top of the head while thebody of the animal was
covered with aluminum foil to localizetreatment to the head. The
control sham treatment group washeld in the same manner with the
LED device remaining off.The transgenic mice received one dose per
day, 5 days a weekfor four consecutive weeks [15]. The wild-type
mice receiveda condensed treatment schedule of 4 treatments per day
over5 days (20 total treatments in one week). As we
previouslydescribed, the condensed treatment in wild-type mice
resultedin similar alterations in binding properties as the 4-week
treat-ment schedule [19]. Mice were utilized (behavior or
sacrificefor tissue collection or brain slice preparation)
immediatelyafter the final NIR light treatment. At sacrifice,
brains werequickly removed and used to prepare brain slices. The
hippo-campus and frontal cortex were collected and stored at − 80
°Cuntil further analyses were performed.
Mol Neurobiol (2019) 56:3341–3355 3351
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Synaptosome Isolation
To isolate synaptosomes, the collected regional tissue was
sep-arately homogenized in Syn-PER synaptic protein
extractionreagent (ThermoFisher) and underwent serial
centrifugations,as per manufacturer’s instructions. Briefly, the
homogenatewas centrifuged at 1200xg for 10 min at 4 °C. A small
fractionof the supernatant, consisting of the total protein
extract, wascollected for further biochemical analysis. The
supernatant wastransferred to a new tube and further centrifuged at
15,000xgfor 20 min at 4 °C. The pellet formed after the second
centrifu-gation contains the synaptosomal fraction. The pellet was
re-suspended in either HEPES-buffered Krebs-like (HBK) buffer,for
binding experiments or radioimmunoprecipitation assay(RIPA) buffer,
for Western blot and ELISA analysis. The syn-aptosome fractions are
routinely analyzed by Western blot andelectron microscopy to
guarantee the quality of the preparation,as we have previously
reported [44].
ELISA Analysis of Total Tau
Total tau levels were measured by ELISA analysis using thetotal
tau antibody, tau5 (ThermoFisher). For the ELISA, sam-ples were
incubated at 4 °C overnight on an ELISA plate withthe coating
buffer 0.1 M sodium bicarbonate (pH 9.6). Theplates were then
washed with Tris-buffered saline with lowTween 20 (0.01%) (TBS-low
T) followed by blocking with10% nonfat milk. The plates received
another washing stepfollowed by an incubation with tau5 antibody
(1:1000 in 5%nonfat milk in TBS-low T; ThermoFisher) for 1 h at
roomtemperature. Following a washing step,
horseradishperoxidase-conjugated anti-rabbit IgG (1:10,000 in 5%
nonfatmilk in TBS-low T; Promega) was added to the plate
andincubated for 1 h at room temperature. The plates were
againwashed with TBS-low T and 3,3,5,5-tetramethylbenzidine(TMB-1
component substrate; Sigma-Aldrich) was added.After 15 min, 1 M HCl
was added to stop the reaction andthe plate was read at 450 nm.
Fluorescence Immunohistochemistry of Total Tauand Oligomeric
Tau
Immunofluorescence was performed on post fixed (4%
para-formaldehyde in 0.01 M PBS, pH 7.4) cryosectioned brainslices
of the 3xTgAD and hTau mice that received NIR lightor sham
treatment. First, the slices were washed in phosphate-buffered
saline (PBS) followed by permeabilized with 5%normal goat serum,
0.3% Triton X-100, and 0.05% Tween-20 in PBS for 1 h at room
temperature. After a wash with PBS,the slides were incubated
overnight at 4 °C with primary an-tibodies. The primary antibodies
used were the tau oligomerspecific antibody, anti-T22 (1:500;
produced by Dr. RakezKayed [10]) and the total tau antibody,
anti-tau 5 (1:1000,
ThermoFisher). The slices were then washed with PBS andincubated
with Alexa-conjugated secondary antibodies(1:400; Life
Technologies) for 1 h at room temperature.Finally, the slices were
washed in PBS and coverslips weremounted using Vectashield mounting
medium containingDAPI (Vector Laboratories).
Western Blot Analysis
Western blot analysis was performed on the total protein
extractsand synaptosome fractions. Separation of the proteins in
the sam-ples obtained was done by 12% gradient
SDS-polyacrylamidegel (HSP70 proteins) or 4–20% gradient gel (tau5,
LC3A&B)electrophoresis. The separated proteins were transferred
to a ni-trocellulosemembrane (Bio-Rad) and incubatedwith the
specificantibody such as Tau5 (total tau; ThermoFisher),
HSP70/HSP72,and HSC70 (Enzo Life Sciences) or LC3A&B (Cell
Signaling)antibody overnight. The nitrocellulose membrane was then
incu-bated with the appropriate fluorescent secondary antibody
andimaged with an Odyssey infrared imager. The band densitieswere
analyzed using Image J software, normalizing using thedensities of
the loading control obtained by reprobing the mem-branes for
β-tubulin.
RT-qPCR
Total RNA was isolated from the hippocampus of the sham-(n=7)
and NIR light-treated (n= 9) 3xTgAD mice utilizingthe RNA isolation
kit (QIAGEN). Real-time quantitative po-lymerase chain reaction
(RT-qPCR) was conducted to deter-mine mRNA levels of Atg5, as
previously described [45]. Thefold expression was calculated
relative to the beta-actin gene.
Tau Oligomer Preparation
Prepared recombinant tau oligomers were obtained by Dr.Rakez
Kayed’s laboratory. The tau oligomers were pro-duced as previously
described [46]. Briefly, recombinanttau monomer protein was added
to 1xPBS to obtain a con-centration of 0.3 mg/ml. Aβ42 oligomers
seeds were addedto the tau mixture and incubated on an orbital
shaker for1 h at room temperature. The produced tau oligomers
wereused as seeds in a second batch of tau monomers to pro-duce a
new batch of tau oligomers. This protocol was re-peated three times
to ensure the elimination of the originalAβ seeds resulting in the
production of tau oligomers.Each batch of oligomers is tested using
dot blot withT22, a tau oligomer-specific antibody, Western blot
analy-sis, and atomic force microscopy (AFM) to verify the qual-ity
of the tau oligomer preparation.
3352 Mol Neurobiol (2019) 56:3341–3355
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Ex Vivo Tau Oligomer Binding to Synaptosomes
Synaptosomes isolated frommice receiving NIR light or
shamtreatment were resuspended in HBK buffer. Using
thebicinchoninic acid (BCA) assay, 50 mg of total protein inour
synaptosomal preparations was determined and aliquotedfrom each
individual animal. Then, 50 nM of tau oligomerswas then added to
each synaptosome preparation and allowedto incubate for 1 h at room
temperature. The samples werethen centrifuged and washed with HBK
buffer three times tothoroughly remove any unbound tau oligomers.
The total pro-tein levels were again measured by BCA and equal
amountsof protein was analyzed by tau5 ELISA analysis, as
describedabove.
Electrophysiology
Long-term potentiation (LTP) measurements were performedas
described previously [19]. Briefly, NIR light- and sham-treated
wild-type mice were deeply anesthetized withisoflurane and
transcardially perfused with 25–30mL of roomtemperature
carbogenated (95% O2 and 5% CO2 gas mixture)NMDG-ACSF. Further,
350μm transverse brain sections con-taining Schaffer Collateral
(SC) synapses were generatedusing Compresstome VF-300 (Precisionary
Instruments,Greenville, NC). An initial protective recovery was
done inthe cutting solution at 32–34 °C for < 12 min, then
transferredto carbogen bubbling HEPES-ACSF recovery solution atroom
temperature. After recovery, slices were perfused incarbogen
bubbling room temperature nACSF at a rate of ap-proximately 3
mL/min. Treatments with oligomers occurredin the recovery phase
where the slices were isolated to a sep-arate chamber and incubated
with the desired concentration ofthe oligomers for one hour. After
the treatment, the slices werebriefly washed by placing them in
drug-free, oligomer-freerecovery ACSF for 5 min before placing them
on the record-ing stage. Using a horizontal P-97 Flaming/Brown
micropi-pette puller (Sutter Instruments, Novato, CA),
borosilicateglass capillaries were used to pull electrodes and
filled withnACSF to get a resistance of 1–2MΩ. Evoked field
excitatorypost synaptic potentials (fEPSPs) in the CA1 by
stimulatingSC were measured using HFS (3X100 Hz, 20 s) as
describedin our previous studies [19], digitized with Digidata
1550B(Molecular Devices, Sunnyvale, CA) and collected using anAxon
MultiClamp 700B differential amplifier (MolecularDevices) connected
to a Windows 7 computer (DellInstruments, Round Rock, TX) running
Clampex 10.6 soft-ware (Molecular Devices). Current stimulation was
deliveredthrough a digital stimulus isolation amplifier
(A.M.P.I,ISRAEL) and set to elicit a fEPSP approximately 30%
ofmaximum for synaptic potentiation experiments
usingplatinum-iridium tipped concentric bipolar electrodes
(FHCInc., Bowdoin, ME). A stable baseline was obtained by
delivering single pulse stimulation at 20 s interstimulus
inter-vals. All data are represented as percentage change from
theinitial average baseline fEPSP slope, which was defined as
theaverage slope obtained for the 10 min prior to HFS.
Novel Object Recognition
After the conclusion of NIR light treatment regimen, the
hTaumice underwent cognitive testing using the novel object
rec-ognition (NOR) paradigm. NOR consisted of three phases—(1) a
habituation phase, (2) a training phase, and (3) a testingphase
[47]. The habituation phase (two sessions of 10 minseparated by 24
h) acclimated the animals to the experimenterand the environment.
This was followed by the training phase,where the habituated
animals were exposed to two identicalobjects placed in two
quadrants at specific locations. After theanimals explored the
objects for 10 min, they were placedback in their home cages. After
a retention interval of 24 h,novel object memory was tested, where
the mice were ex-posed to one familiar and a novel object
(different color andshape but sharing a common size and volume).
The time spentexploring each object was recorded using
ANY-Maze(Stoelting. Inc.) where an area 2 cm2 surrounding the
objectwas defined such that nose entries within 2 cm of the
objectwere recorded as time exploring the object. The percent
timeexploring each object (familiar versus novel) was reported asan
object discrimination ratio (ODR) calculated by the follow-ing
formula: ODR= (time exploring specified object) / (timeexploring
novel object + time exploring familiar object).
Statistical Analysis
Data were statistically analyzed using SPSS software.Student’s t
test was used to determine statistical significancein the tau level
experiments. The calculated object discrimi-nation ratios of the
NOR behavior test were analyzed by theone-sample t test to
determine the statistical variation fromchance (0.50). The one-way
ANOVA with Dunn’s post hoctest was used to determine statistical
significance between thecalculated fEPSP percentage of each
condition in the electro-physiology experiment.
Acknowledgements We would like to thank TJ Goble for his
contribu-tion of preliminary immunostaining of NIR light-treated
3xTgAD.
Funding This work was supported by a National Institutes of
Health/National Institute on Aging grant 5R01AG042890 and a grant
from theAmon Carter Foundation to GT.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no
conflict ofinterest.
Mol Neurobiol (2019) 56:3341–3355 3353
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Open Access This article is distributed under the terms of the
CreativeCommons At t r ibut ion 4 .0 In te rna t ional License (h t
tp : / /creativecommons.org/licenses/by/4.0/), which permits
unrestricted use,distribution, and reproduction in any medium,
provided you give appro-priate credit to the original author(s) and
the source, provide a link to theCreative Commons license, and
indicate if changes were made.
References
1. Shankar GM, Bloodgood BL, Townsend M et al (2007)
Naturaloligomers of the Alzheimer amyloid- protein induce
reversible syn-apse loss by modulating an NMDA-type glutamate
receptor-dependent signaling pathway. J Neurosci 27:2866–2875.
https://doi.org/10.1523/JNEUROSCI.4970-06.2007
2. Orr ME, Oddo S (2013) Autophagic/lysosomal dysfunction
inAlzheimer’s disease. Alzheimers Res Ther 5:53.
https://doi.org/10.1186/alzrt217
3. Swerdlow RH, Burns JM, Khan SM (2014) The Alzheimer’s
dis-ease mitochondrial cascade hypothesis: progress and
perspectives.Biochim Biophys Acta (BBA) - Mol Basis Dis
1842:1219–1231.https://doi.org/10.1016/j.bbadis.2013.09.010
4 . Heneka MT, Ca r son MJ , Khou ry JE e t a l ( 2015
)Neuroinflammation in Alzheimer’s disease. Lancet Neurol
14:388–405. https://doi.org/10.1016/S1474-4422(15)70016-5
5. Selkoe DJ (2002) Alzheimer’s disease is a synaptic failure.
Science298:789–791. https://doi.org/10.1126/science.1074069
6. Zhou L, McInnes J, Wierda K et al (2017) Tau association
withsynaptic vesicles causes presynaptic dysfunction. Nat Commun
8:15295. https://doi.org/10.1038/ncomms15295
7. Fá M, Puzzo D, Piacentini R et al (2016) Extracellular tau
oligo-mers produce an immediate impairment of LTP and memory.
SciRep 6. https://doi.org/10.1038/srep19393
8. Giannakopoulos P, Herrmann FR, Bussière T et al (2003)
Tangleand neuron numbers, but not amyloid load, predict cognitive
statusin Alzheimer’s disease. Neurology 60:1495–1500
9. Braak H, Braak E (1991)Neuropathological stageing of
Alzheimer-related changes. Acta Neuropathol (Berl) 82:239–259.
https://doi.org/10.1007/BF00308809
10. Lasagna-Reeves CA, Castillo-Carranza DL, Sengupta U et
al(2012) Identification of oligomers at early stages of tau
aggregationin Alzheimer’s disease. FASEB J 26:1946–1959.
https://doi.org/10.1096/fj.11-199851
11. Maeda S, Sahara N, Saito Yet al (2006) Increased levels of
granulartau oligomers: an early sign of brain aging and Alzheimer’s
disease.Neurosci Res 54:197–201.
https://doi.org/10.1016/j.neures.2005.11.009
12. Li C, Götz J (2017) Somatodendritic accumulation of tau
inAlzheimer’s disease is promoted by Fyn-mediated local
proteintranslation. EMBO J 36:3120–3138.
https://doi.org/10.15252/embj.201797724
13. Usenovic M, Niroomand S, Drolet RE et al (2015) Internalized
tauoligomers cause neurodegeneration by inducing accumulation
ofpathogenic tau in human neurons derived from induced
pluripotentstem cells. J Neurosci 35:14234–14250.
https://doi.org/10.1523/JNEUROSCI.1523-15.2015
14. Sanders DW, Kaufman SK, DeVos SL et al (2014) Distinct
tauprion strains propagate in cells and mice and define
differentTauopathies. Neuron 82:1271–1288.
https://doi.org/10.1016/j.neuron.2014.04.047
15. Purushothuman S, Johnstone DM, Nandasena C et al
(2014)Photobiomodulation with near infrared light
mitigatesAlzheimer’s disease-related pathology in cerebral cortex
–
Evidence from two transgenic mouse models. Alzheimers ResTher
6:2. https://doi.org/10.1186/alzrt232
16. Ittner LM, Fath T, Ke YD et al (2008) Parkinsonism and
impairedaxonal transport in a mouse model of frontotemporal
dementia.Proc Natl Acad Sci 105:15997–16002.
https://doi.org/10.1073/pnas.0808084105
17. Grillo SL, Duggett NA, Ennaceur A, Chazot PL (2013)
Non-invasive infra-red therapy (1072nm) reduces β-amyloid
proteinlevels in the brain of an Alzheimer’s disease mouse
model,TASTPM. J Photochem Photobiol B 123:13–22.
https://doi.org/10.1016/j.jphotobiol.2013.02.015
18. De Taboada L, Yu J, El-Amouri S et al (2011) Transcranial
lasertherapy attenuates amyloid-β peptide neuropathology
inamyloid-β protein precursor transgenic mice. JAlzheimeraposs Dis
23:521–535. https://doi.org/10.3233/JAD-2010-100894
19. Comerota MM, Krishnan B, Taglialatela G (2017) Near
infraredlight decreases synaptic vulnerability to amyloid beta
oligomers.Sci Rep 7:15012.
https://doi.org/10.1038/s41598-017-15357-x
20 . Ando r f e r C , K r e s s Y, E s p i n o z a M e t a l ( 2
003 )Hyperphosphorylation and aggregation of tau in mice
expressingnormal human tau isoforms. J Neurochem 86:582–590
21. Duff K, Knight H, Refolo LM et al (2000) Characterization
ofpathology in transgenic mice over-expressing human genomicand
cDNA tau transgenes. Neurobiol Dis 7:87–98.
https://doi.org/10.1006/nbdi.1999.0279
22. Castillo-Carranza DL, Gerson JE, Sengupta U et al (2014)
Specifictargeting of tau oligomers in Htau mice prevents cognitive
impair-ment and tau toxicity following injection with brain-derived
tauOligomeric seeds. J Alzheimers Dis 40:S97–S111.
https://doi.org/10.3233/JAD-132477
23. Oddo S, Caccamo A, Shepherd JD et al (2003)
Triple-transgenicmodel of Alzheimer’s disease with plaques and
tangles: intracellu-lar Abeta and synaptic dysfunction. Neuron
39:409–421
24. Jinwal UK, Akoury E, Abisambra JF et al (2013) Imbalance
ofHsp70 family variants fosters tau accumulation. FASEB J
27:1450–1459. https://doi.org/10.1096/fj.12-220889
25. Friedman LG, Qureshi YH, Yu WH (2015) Promoting
Autophagicclearance: viable therapeutic targets in Alzheimer’s
disease.Neurotherapeutics 12:94–108.
https://doi.org/10.1007/s13311-014-0320-z
26. Mizushima N, Yoshimori T, Levine B (2010) Methods in
mamma-lian autophagy research. Cell 140:313–326.
https://doi.org/10.1016/j.cell.2010.01.028
27. Terry RD, Masliah E, Salmon DP et al (1991) Physical basis
ofcognitive alterations in alzheimer’s disease: synapse loss is the
ma-jor correlate of cognitive impairment. Ann Neurol
30:572–580.https://doi.org/10.1002/ana.410300410
28. Lasagna-Reeves CA, Castillo-Carranza DL, Sengupta U et
al(2011) Tau oligomers impair memory and induce synaptic and
mi-tochondrial dysfunction in wild-type mice. Mol Neurodegener
6:39. https://doi.org/10.1186/1750-1326-6-39
29. Tai H-C, Wang BY, Serrano-Pozo A et al (2014) Frequent
andsymmetric deposition of misfolded tau oligomers within
presynap-tic and postsynaptic terminals in Alzheimer’s disease.
ActaNeuropathol Commun 2:146.
https://doi.org/10.1186/s40478-014-0146-2
30. Fontaine SN, Nation G, Meier SE, Abisambra JF (2017) Tau
olig-omers mediate ribosomal dysfunction at the synapse.
AlzheimersDement 13:P603.
https://doi.org/10.1016/j.jalz.2017.07.249
31. Delacourte A, Flament S, Dibe EM et al (1990) Pathological
pro-teins tau 64 and 69 are specifically expressed in the
somatodendriticdomain of the degenerating cortical neurons during
Alzheimer’sdisease. Demonstration with a panel of antibodies
against tau pro-teins. Acta Neuropathol (Berl) 80:111–117
3354 Mol Neurobiol (2019) 56:3341–3355
https://doi.org/10.1523/JNEUROSCI.4970-06.2007https://doi.org/10.1523/JNEUROSCI.4970-06.2007https://doi.org/10.1186/alzrt217https://doi.org/10.1186/alzrt217https://doi.org/10.1016/j.bbadis.2013.09.010https://doi.org/10.1016/S1474-4422(15)70016-5https://doi.org/10.1126/science.1074069https://doi.org/10.1038/ncomms15295https://doi.org/10.1038/srep19393https://doi.org/10.1007/BF00308809https://doi.org/10.1007/BF00308809https://doi.org/10.1096/fj.11-199851https://doi.org/10.1096/fj.11-199851https://doi.org/10.1016/j.neures.2005.11.009https://doi.org/10.1016/j.neures.2005.11.009https://doi.org/10.15252/embj.201797724https://doi.org/10.15252/embj.201797724https://doi.org/10.1523/JNEUROSCI.1523-15.2015https://doi.org/10.1523/JNEUROSCI.1523-15.2015https://doi.org/10.1016/j.neuron.2014.04.047https://doi.org/10.1016/j.neuron.2014.04.047https://doi.org/10.1186/alzrt232https://doi.org/10.1073/pnas.0808084105https://doi.org/10.1073/pnas.0808084105https://doi.org/10.1016/j.jphotobiol.2013.02.015https://doi.org/10.1016/j.jphotobiol.2013.02.015https://doi.org/10.3233/JAD-2010-100894https://doi.org/10.3233/JAD-2010-100894https://doi.org/10.1038/s41598-017-15357-xhttps://doi.org/10.1006/nbdi.1999.0279https://doi.org/10.1006/nbdi.1999.0279https://doi.org/10.3233/JAD-132477https://doi.org/10.3233/JAD-132477https://doi.org/10.1096/fj.12-220889https://doi.org/10.1007/s13311-014-0320-zhttps://doi.org/10.1007/s13311-014-0320-zhttps://doi.org/10.1016/j.cell.2010.01.028https://doi.org/10.1016/j.cell.2010.01.028https://doi.org/10.1002/ana.410300410https://doi.org/10.1186/1750-1326-6-39https://doi.org/10.1186/s40478-014-0146-2https://doi.org/10.1186/s40478-014-0146-2https://doi.org/10.1016/j.jalz.2017.07.249
-
32. Zempel H, Mandelkow E (2014) Lost after translation:
missortingof tau protein and consequences for Alzheimer disease.
TrendsNeurosci 37:721–732.
https://doi.org/10.1016/j.tins.2014.08.004
33. Dawson HN, Cantillana V, Jansen M et al (2010) Loss of tau
elicitsaxonal degeneration in a mouse model of Alzheimer’s
disease.Neuroscience 169:516–531.
https://doi.org/10.1016/j.neuroscience.2010.04.037
34. Mirbaha H, Chen D, Morazova OA et al (2018) Inert and
seed-competent tau monomers suggest structural origins of
aggregation.eLife 7. https://doi.org/10.7554/eLife.36584
35. Parri HR, Hernandez CM, Dineley KT (2011) Research
update:alpha7 nicotinic acetylcholine receptor mechanisms
inAlzheimer’s disease. Biochem Pharmacol 82:931–942.
https://doi.org/10.1016/j.bcp.2011.06.039
36. Um JW, Kaufman AC, Kostylev M et al (2013) Metabotropic
glu-tamate receptor 5 is a coreceptor for Alzheimer Aβ oligomer
boundto cellular prion protein. Neuron 79:887–902.
https://doi.org/10.1016/j.neuron.2013.06.036
37. Kayed R (2003) Common structure of soluble amyloid
oligomersimplies common mechanism of pathogenesis. Science
300:486–489. https://doi.org/10.1126/science.1079469
38. Karu T (2010) Mitochondrial mechanisms of
photobiomodulationin context of new data about multiple roles of
ATP. Photomed LaserSurg 28:159–160.
https://doi.org/10.1089/pho.2010.2789
39. Evgen’ev MB, Krasnov GS, Nesterova IV et al (2017)
Molecularmechanisms underlying neuroprotective effect of intranasal
admin-istration of human Hsp70 in mouse model of Alzheimer’s
disease. JAlzheimers Dis 59:1415–1426.
https://doi.org/10.3233/JAD-170398
40. Leak RK (2014) Heat shock proteins in neurodegenerative
disor-ders and aging. J Cell Commun Signal 8:293–310.
https://doi.org/10.1007/s12079-014-0243-9
41. Young ZT, Rauch JN, Assimon VA et al (2016) Stabilizing
theHsp70-tau complex promotes turnover in models of Tauopathy.Cell
Chem Biol 23:992–1001.
https://doi.org/10.1016/j.chembiol.2016.04.014
42. Kundel F, De S, Flagmeier P et al (2018) Hsp70 inhibits the
nucle-ation and elongation of tau and sequesters tau aggregates
with highaffinity. ACS Chem Biol 13:636–646.
https://doi.org/10.1021/acschembio.7b01039
43. Demand J, Alberti S, Patterson C, Höhfeld J (2001)
Cooperation ofa ubiquitin domain protein and an E3 ubiquitin ligase
duringchaperone/proteasome coupling. Curr Biol CB 11:1569–1577
44. Franklin W, Taglialatela G (2016) A method to determine
insulinresponsiveness in synaptosomes isolated from frozen brain
tissue. JNeurosci Methods 261:128–134.
https://doi.org/10.1016/j.jneumeth.2016.01.006
45. Ali YO, Allen HM, Yu L et al (2016) NMNAT2:HSP90
complexmediates Proteostasis in Proteinopathies. PLoS Biol
14:e1002472.https://doi.org/10.1371/journal.pbio.1002472
46. Lasagna-Reeves CA, Castillo-Carranza DL, Guerrero-MuñozMJ
etal (2010) Preparation and characterization of neurotoxic tau
oligo-mers. Biochemistry (Mosc) 49:10039–10041.
https://doi.org/10.1021/bi1016233
47. Dineley KT, Kayed R, Neugebauer Vet al (2010) Amyloid-β
olig-omers impair fear conditioned memory in a
calcineurin-dependentfashion in mice. J Neurosci Res.
https://doi.org/10.1002/jnr.22445
Mol Neurobiol (2019) 56:3341–3355 3355
https://doi.org/10.1016/j.tins.2014.08.004https://doi.org/10.1016/j.neuroscience.2010.04.037https://doi.org/10.1016/j.neuroscience.2010.04.037https://doi.org/10.7554/eLife.36584https://doi.org/10.1016/j.bcp.2011.06.039https://doi.org/10.1016/j.bcp.2011.06.039https://doi.org/10.1016/j.neuron.2013.06.036https://doi.org/10.1016/j.neuron.2013.06.036https://doi.org/10.1126/science.1079469https://doi.org/10.1089/pho.2010.2789https://doi.org/10.3233/JAD-170398https://doi.org/10.3233/JAD-170398https://doi.org/10.1007/s12079-014-0243-9https://doi.org/10.1007/s12079-014-0243-9https://doi.org/10.1016/j.chembiol.2016.04.014https://doi.org/10.1016/j.chembiol.2016.04.014https://doi.org/10.1021/acschembio.7b01039https://doi.org/10.1021/acschembio.7b01039https://doi.org/10.1016/j.jneumeth.2016.01.006https://doi.org/10.1016/j.jneumeth.2016.01.006https://doi.org/10.1371/journal.pbio.1002472https://doi.org/10.1021/bi1016233https://doi.org/10.1021/bi1016233https://doi.org/10.1002/jnr.22445
Near...AbstractIntroductionResultsReduced Tau Pathology in
Cortical and Hippocampal Total Protein Extracts and Synaptosomal
Fractions of NIR Light-Treated 13-Month-Old hTau MiceRescue of
Impaired Long-Term Memory in Aged NIR Light-Treated hTau
MiceReduced Tau and Aβ Pathology in the Cortex and Hippocampus of
NIR Light-Treated 13-Month Old 3xTgAD MiceNIR Light Treatment Does
Not Affect Ex Vivo Synaptic Binding of Tau OligomersSuppression of
Hippocampal Long-Term Potentiation by Tau Oligomers Is Not Reversed
by NIR Light TreatmentIncreased Inducible HSP70 at the Synapses of
NIR Light-Treated 3xTgAD, hTau, and Wild-Type MiceIncreased Levels
of Autophagy Markers in NIR Light-Treated 3xTgAD
DiscussionMethodsAnimalsNIR Light TreatmentsSynaptosome
IsolationELISA Analysis of Total TauFluorescence
Immunohistochemistry of Total Tau and Oligomeric TauWestern Blot
AnalysisRT-qPCRTau Oligomer PreparationEx Vivo Tau Oligomer Binding
to SynaptosomesElectrophysiologyNovel Object RecognitionStatistical
Analysis
References