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 Taglialatelagtaglial@utmb.edu

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:gtaglial@utmb.edu

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

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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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

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

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

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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

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

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

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.

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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